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Issue 4

December 2017

80 TL

ISSN 1300-7777

Volume 34

Review The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance Şadiye Mehtat Ünlü, et al.; İzmir, Turkey

Research Articles Retrospective Evaluation of Hairy Cell Leukemia Patients Treated with Three Different First-Line Treatment Modalities in the Last Two Decades: A Single-Center Experience Şeniz Öngören, et al.; İstanbul, Turkey

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics Narges Rezaei, et al.; Shiraz, Iran

Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia Ahmet Menteşe, et al.; Trabzon, Rize, Turkey

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment Emine Begüm Gençer, et al.; Ankara, Turkey

Effectiveness of Visual Methods in Information Procedures for Stem Cell Recipients and Donors Çağla Sarıtürk, et al.; Adana, Turkey

Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma Carl Hsieh and Vani Rajashekharaiah, et al.; Bangalore, India

Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats Hong Qian, et al.; Nanjing, China

Cover Picture: Yasushi Kubota et al. Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia

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International Review Board

Editor-in-Chief Reyhan Küçükkaya

İstanbul, Turkey rkucukkaya@hotmail.com

Associate Editors Ayşegül Ünüvar

İstanbul, Turkey aysegulu@hotmail.com

Cengiz Beyan TOBB University of Economics and Technology, Ankara, Turkey cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey haznedar@yahoo.com

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey mcemar68@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey sepay@cu.edu.tr

Assistant Editors A. Emre Eşkazan

İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul, Turkey

Ali İrfan Emre Tekgündüz

Dr. A. Yurtaslan Ankara Oncology Training and Research Hospital, Ankara, Turkey

Claudio Cerchione

University of Naples Federico II Napoli, Campania, Italy

Elif Ünal İnce

Ankara University, Ankara, Turkey

İnci Alacacıoğlu

Nejat Akar Görgün Akpek
 Serhan Alkan
 Çiğdem Altay
 Koen van Besien
 Ayhan Çavdar M. Sıraç Dilber
 Ahmet Doğan
 Peter Dreger
 Thierry Facon Jawed Fareed
 Gösta Gahrton
 Dieter Hoelzer
 Marilyn Manco-Johnson Andreas Josting Emin Kansu
 Winfried Kern
 Nigel Key
 Korgün Koral Abdullah Kutlar Luca Malcovati
 Robert Marcus
 Jean Pierre Marie Ghulam Mufti Gerassimos A. Pangalis Antonio Piga Ananda Prasad Jacob M. Rowe Jens-Ulrich Rüffer Norbert Schmitz Orhan Sezer
 Anna Sureda Ayalew Tefferi Nükhet Tüzüner Catherine Verfaillie Srdan Verstovsek Claudio Viscoli

TOBB Economy Technical University Hospital, Ankara, Turkey Maryland School of Medicine, Baltimore, USA
 Cedars-Sinai Medical Center, USA
 Ankara, Turkey Chicago Medical Center University, Chicago, USA Ankara, Turkey
 Karolinska University, Stockholm, Sweden
 Mayo Clinic Saint Marys Hospital, USA Heidelberg University, Heidelberg, Germany Lille University, Lille, France
 Loyola University, Maywood, USA
 Karolinska University Hospital, Stockholm, Sweden Frankfurt University, Frankfurt, Germany Colorado Health Sciences University, USA
 University Hospital Cologne, Cologne, Germany
 Hacettepe University, Ankara, Turkey
 Albert Ludwigs University, Germany
 University of North Carolina School of Medicine, NC, USA Southwestern Medical Center, Texas, USA Georgia Health Sciences University, Augusta, USA
 Pavia Medical School University, Pavia, Italy
 Kings College Hospital, London, UK
 Pierre et Marie Curie University, Paris, France
 King’s Hospital, London, UK
 Athens University, Athens, Greece
 Torino University, Torino, Italy
 Wayne State University School of Medicine, Detroit, USA Rambam Medical Center, Haifa, Israel
 University of Köln, Germany
 AK St Georg, Hamburg, Germany
 Memorial Şişli Hospital, İstanbul, Turkey
 Santa Creu i Sant Pau Hospital, Barcelona, Spain
 Mayo Clinic, Rochester, Minnesota, USA
 İstanbul Cerrahpaşa University, İstanbul, Turkey
 University of Minnesota, Minnesota, USA The University of Texas MD Anderson Cancer Center, Houston, USA San Martino University, Genoa, Italy

Past Editors Erich Frank Orhan Ulutin Hamdi Akan Aytemiz Gürgey

Language Editor Leslie Demir

Senior Advisory Board Yücel Tangün Osman İlhan Muhit Özcan Teoman Soysal Ahmet Muzaffer Demir

Editorial Office İpek Durusu Bengü Timoçin

Dokuz Eylül University, İzmir, Turkey

Müge Sayitoğlu

İstanbul University, İstanbul, Turkey

Nil Güler

Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay

Koç University, İstanbul, Turkey

Şule Ünal

Hacettepe University, Ankara, Turkey

Veysel Sabri Hançer

İstinye University, İstanbul, Turkey

Zühre Kaya

Gazi University, Ankara, Turkey

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Publishing Services

Statistic Editor Hülya Ellidokuz

GALENOS PUBLISHER Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr


Contact Information Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya E-mail : rkucukkaya@hotmail.com

All Inquiries Should be Addressed to TURKISH JOURNAL OF HEMATOLOGY Address Phone Fax E-mail

: İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No: 8 06550 Çankaya, Ankara / Turkey : +90 312 490 98 97 : +90 312 490 98 68
 : info@tjh.com.tr

ISSN: 1300-7777

Publishing Manager Sorumlu Yazı İşleri Müdürü Muhlis Cem Ar

Management Address Yayın İdare Adresi

Publishing House / Yayınevi

Türk Hematoloji Derneği İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No: 8 06550 Çankaya, Ankara / Turkey

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093 Fındıkzade, İstanbul, Turkey Tel: +90 212 621 99 25 Faks: +90 212 621 99 27 E-posta: info@galenos.com.tr Baskı: Özgün Ofset Ticaret Ltd. Şti.

Online Manuscript Submission

Yeşilce Mah. Aytekin Sk. No: 21 34418 4. Levent / İstanbul

http://mc.manuscriptcentral.com/tjh

Printing Date / Basım Tarihi 20.11.2017

Web page www.tjh.com.tr

Cover Picture Owner on behalf of the Turkish Society of Hematology Türk Hematoloji Derneği adına yayın sahibi Güner Hayri Özsan Üç ayda bir yayımlanan İngilizce süreli yayındır. International scientific journal published quarterly. Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi tarafından yayınlanmasına karar vermiştir.

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Yasushi Kubota et al., Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia Peripheral blood smear showed not only RBC agglutination but also neutrophil aggregates, eosinophil aggregates, and monocyte aggregates.


AIMS AND SCOPE The Turkish Journal of Hematology is published quarterly (March, June, September, and December) by the Turkish Society of Hematology. It is an independent, non-profit peer-reviewed international English-language periodical encompassing subjects relevant to hematology. The Editorial Board of The Turkish Journal of Hematology adheres to the principles of the World Association of Medical Editors (WAME), International Council of Medical Journal Editors (ICMJE), Committee on Publication Ethics (COPE), Consolidated Standards of Reporting Trials (CONSORT) and Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). The aim of The Turkish Journal of Hematology is to publish original hematological research of the highest scientific quality and clinical relevance. Additionally, educational material, reviews on basic developments, editorial short notes, images in hematology, and letters from hematology specialists and clinicians covering their experience and comments on hematology and related medical fields as well as social subjects are published. As of December 2015, The Turkish Journal of Hematology does not accept case reports. Important new findings or data about interesting hematological cases may be submitted as a brief report. General practitioners interested in hematology and internal medicine specialists are among our target audience, and The Turkish Journal of Hematology aims to publish according to their needs. The Turkish Journal of Hematology is indexed, as follows: - PubMed Medline - PubMed Central - Science Citation Index Expanded - EMBASE - Scopus - CINAHL - Gale/Cengage Learning - EBSCO - DOAJ - ProQuest - Index Copernicus - Tübitak/Ulakbim Turkish Medical Database - Turk Medline Impact Factor: 0.686 Open Access Policy Turkish Journal of Hematology is an Open Access journal. This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge. Open Access Policy is based on the rules of the Budapest Open Access Initiative (BOAI) http://www.budapestopenaccessinitiative.org/. Subscription Information
 The Turkish Journal of Hematology is sent free-of-charge to members of Turkish Society of Hematology and libraries in Turkey and abroad. Hematologists, other medical specialists that are interested in hematology, and academicians could subscribe for only 40 $ per printed issue. All published volumes are available in full text free-of-charge online at www. tjh.com.tr.


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Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey Telephone: +90 312 490 98 97
 Fax: +90 312 490 98 68 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
 Web page: www.tjh.com.tr
 E-mail: info@tjh.com.tr

 Permissions
 Requests for permission to reproduce published material should be sent to the editorial office. Editor: Professor Dr. Reyhan Küçükkaya Adress: İlkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya, Ankara, Turkey
 Telephone: +90 312 490 98 97
 Fax: +90 312 490 98 68
 Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh
 Web page: www.tjh.com.tr
 E-mail: info@tjh.com.tr Publisher Galenos Yayınevi Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey Telephone : +90 212 621 99 25 Fax : +90 212 621 99 27 info@galenos.com.tr Instructions for Authors Instructions for authors are published in the journal and at www.tjh.com.tr Material Disclaimer Authors are responsible for the manuscripts they publish in The Turkish Journal of Hematology. The editor, editorial board, and publisher do not accept any responsibility for published manuscripts. If you use a table or figure (or some data in a table or figure) from another source, cite the source directly in the figure or table legend. The journal is printed on acid-free paper. Editorial Policy Following receipt of each manuscript, a checklist is completed by the Editorial Assistant. The Editorial Assistant checks that each manuscript contains all required components and adheres to the author guidelines, after which time it will be forwarded to the Editor in Chief. Following the Editor in Chief’s evaluation, each manuscript is forwarded to the Associate Editor, who in turn assigns reviewers. Generally, all manuscripts will be reviewed by at least three reviewers selected by the Associate Editor, based on their relevant expertise. Associate editor could be assigned as a reviewer along with the reviewers. After the reviewing process, all manuscripts are evaluated in the Editorial Board Meeting. Turkish Journal of Hematology’s editor and Editorial Board members are active researchers. It is possible that they would desire to submit their manuscript to the Turkish Journal of Hematology. This may be creating a conflict of interest. These manuscripts will not be evaluated by the submitting editor(s). The review process will be managed and decisions made by editor-in-chief who will act independently. In some situation, this process will be overseen by an outside independent expert in reviewing submissions from editors.


TURKISH JOURNAL OF HEMATOLOGY INSTRUCTIONS FOR AUTHORS The Turkish Journal of Hematology accepts invited review articles, research articles, brief reports, letters to the editor, and hematological images that are relevant to the scope of hematology, on the condition that they have not been previously published elsewhere. Basic science manuscripts, such as randomized, cohort, cross-sectional, and case-control studies, are given preference. All manuscripts are subject to editorial revision to ensure they conform to the style adopted by the journal. There is a double-blind reviewing system. Review articles are solicited by the Editorin-Chief. Authors wishing to submit an unsolicited review article should contact the Editor-in-Chief prior to submission in order to screen the proposed topic for relevance and priority. The Turkish Journal of Hematology does not charge any article submission or processing charges. Manuscripts should be prepared according to ICMJE guidelines (http:// www.icmje.org/). Original manuscripts require a structured abstract. Label each section of the structured abstract with the appropriate subheading (Objective, Materials and Methods, Results, and Conclusion). Letters to the editor do not require an abstract. Research or project support should be acknowledged as a footnote on the title page. Technical and other assistance should be provided on the title page. Original Manuscripts Title Page Title: The title should provide important information regarding the manuscript’s content. The title must specify that the study is a cohort study, cross-sectional study, case-control study, or randomized study (i.e. Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability of ferrous succinate tablet formulations without correction for baseline circadian changes in iron concentration in healthy Chinese male subjects: A single-dose, randomized, 2-period crossover study. Clin Ther 2011;33:2054-2059). The title page should include the authors’ names, degrees, and institutional/ professional affiliations and a short title, abbreviations, keywords, financial disclosure statement, and conflict of interest statement. If a manuscript includes authors from more than one institution, each author’s name should be followed by a superscript number that corresponds to their institution, which is listed separately. Please provide contact information for the corresponding author, including name, e-mail address, and telephone and fax numbers. Running Head: The running head should not be more than 40 characters, including spaces, and should be located at the bottom of the title page. Word Count: A word count for the manuscript, excluding abstract, acknowledgments, figure and table legends, and references, should be provided and should not exceed 2500 words. The word count for the abstract should not exceed 300 words.

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Conflict of Interest Statement: To prevent potential conflicts of interest from being overlooked, this statement must be included in each manuscript. In case there are conflicts of interest, every author should complete the ICMJE general declaration form, which can be obtained at http://www.icmje.org/downloads/coi_disclosure.zip Abstract and Keywords: The second page should include an abstract that does not exceed 300 words. For manuscripts sent by authors in Turkey, a title and abstract in Turkish are also required. As most readers read the abstract first, it is critically important. Moreover, as various electronic databases integrate only abstracts into their index, important findings should be presented in the abstract. Objective: The abstract should state the objective (the purpose of the study and hypothesis) and summarize the rationale for the study. Materials and Methods: Important methods should be written respectively. Results: Important findings and results should be provided here. Conclusion: The study’s new and important findings should be highlighted and interpreted. Other types of manuscripts, such as reviews, brief reports, and editorials, will be published according to uniform requirements. Provide 3-10 keywords below the abstract to assist indexers. Use terms from the Index Medicus Medical Subject Headings List (for randomized studies a CONSORT abstract should be provided: http:// www.consort-statement.org). Introduction: The introduction should include an overview of the relevant literature presented in summary form (one page), and whatever remains interesting, unique, problematic, relevant, or unknown about the topic must be specified. The introduction should conclude with the rationale for the study, its design, and its objective(s). Materials and Methods: Clearly describe the selection of observational or experimental participants, such as patients, laboratory animals, and controls, including inclusion and exclusion criteria and a description of the source population. Identify the methods and procedures in sufficient detail to allow other researchers to reproduce your results. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for using them and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. The section should include only information that was available at the time the plan or protocol for the study was devised (https://www.strobe-statement.org/ fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_combined. pdf). Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify


the reported results. Statistically important data should be given in the text, tables, and figures. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present your results in logical sequence in the text, tables, and figures. Do not present all the data provided in the tables and/or figures in the text; emphasize and/or summarize only important findings, results, and observations in the text. For clinical studies provide the number of samples, cases, and controls included in the study. Discrepancies between the planned number and obtained number of participants should be explained. Comparisons and statistically important values (i.e. p-value and confidence interval) should be provided. Discussion: This section should include a discussion of the data. New and important findings/results and the conclusions they lead to should be emphasized. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by the data. Do not repeat the findings/results in detail; important findings/ results should be compared with those of similar studies in the literature, along with a summarization. In other words, similarities or differences in the obtained findings/results with those previously reported should be discussed. Study Limitations: Limitations of the study should be detailed. In addition, an evaluation of the implications of the obtained findings/ results for future research should be outlined. Conclusion: The conclusion of the study should be highlighted. References Cite references in the text, tables, and figures with numbers in square brackets. Number references consecutively according to the order in which they first appear in the text. Journal titles should be abbreviated according to the style used in Index Medicus (consult List of Journals Indexed in Index Medicus). Include among the references any paper accepted, but not yet published, designating the journal followed by “in press”. Examples of References: 1. List all authors Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow transplantation from unrelated donors for patients with aplastic anemia after failure of immunosuppressive therapy. Blood 2006;108:1485-1491. 2. Organization as author Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of syngeneic bone marrow graft without preconditioning in post-hepatitis marrow aplasia. Lancet 1977;2:742-744.

4. Book Chapter Perutz MF. Molecular anatomy and physiology of hemoglobin. In: Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management. New York, Cambridge University Press, 2000. 5. Abstract Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract). 6. Letter to the Editor Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further definition of 20q deletion in myeloid leukemia using fluorescence in situ hybridization. Blood 1994;84:2821-2823. 7. Supplement Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant 2005;9(Suppl 7):81-86. Brief Reports Abstract length: Not to exceed 150 words. Article length: Not to exceed 1200 words. Introduction: State the purpose and summarize the rationale for the study. Materials and Methods: Clearly describe the selection of the observational or experimental participants. Identify the methods and procedures in sufficient detail. Provide references to established methods (including statistical methods), provide references to brief modified methods, and provide the rationale for their use and an evaluation of their limitations. Identify all drugs and chemicals used, including generic names, doses, and routes of administration. Statistics: Describe the statistical methods used in enough detail to enable a knowledgeable reader with access to the original data to verify the reported findings/results. Provide details about randomization, describe treatment complications, provide the number of observations, and specify all computer programs used. Results: Present the findings/results in a logical sequence in the text, tables, and figures. Do not repeat all the findings/results in the tables and figures in the text; emphasize and/or summarize only those that are most important. Discussion: Highlight the new and important findings/results of the study and the conclusions they lead to. Link the conclusions with the goals of the study, but avoid unqualified statements and conclusions not completely supported by your data. Invited Review Articles Abstract length: Not to exceed 300 words.

3. Book

Article length: Not to exceed 4000 words.

Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger, 1961.

Review articles should not include more than 100 references. Reviews should include a conclusion, in which a new hypothesis or study about the

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subject may be posited. Do not publish methods for literature search or level of evidence. Authors who will prepare review articles should already have published research articles on the relevant subject. The study’s new and important findings should be highlighted and interpreted in the Conclusion section. There should be a maximum of two authors for review articles. Images in Hematology Article length: Not to exceed 200 words. Authors can submit for consideration illustrations or photos that are interesting, instructive, and visually attractive, along with a few lines of explanatory text and references. Images in Hematology can include no more than 200 words of text, 5 references, and 3 figures or tables. No abstract, discussion, or conclusion is required, but please include a brief title. Letters to the Editor Article length: Not to exceed 500 words. Letters can include no more than 500 words of text, 5-10 references, and 1 figure or table. No abstract is required, but please include a brief title. Tables Supply each table in a separate file. Number tables according to the order in which they appear in the text, and supply a brief caption for each. Give each column a short or abbreviated heading. Write explanatory statistical measures of variation, such as standard deviation or standard error of mean. Be sure that each table is cited in the text. Figures Figures should be professionally drawn and/or photographed. Authors should number figures according to the order in which they appear in the text. Figures include graphs, charts, photographs, and illustrations. Each figure should be accompanied by a legend that does not exceed 50 words. Use abbreviations only if they have been introduced in the text. Authors are also required to provide the level of magnification for histological slides. Explain the internal scale and identify the staining method used. Figures should be submitted as separate files, not in the text file. Highresolution image files are not preferred for initial submission as the file sizes may be too large. The total file size of the PDF for peer review should not exceed 5 MB. Authorship Each author should have participated sufficiently in the work to assume public responsibility for the content. Any portion of a manuscript that is critical to its main conclusions must be the responsibility of at least one author. Contributor’s Statement All submissions should contain a contributor’s statement page. Each statement should contain substantial contributions to idea and design, acquisition of data, and analysis and interpretation of findings. All

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persons designated as an author should qualify for authorship, and all those that qualify should be listed. Each author should have participated sufficiently in the work to take responsibility for appropriate portions of the text. Acknowledgments Acknowledge support received from individuals, organizations, grants, corporations, and any other source. For work involving a biomedical product or potential product partially or wholly supported by corporate funding, a note stating, “This study was financially supported (in part) with funds provided by (company name) to (authors’ initials)”, must be included. Grant support, if received, needs to be stated and the specific granting institutions’ names and grant numbers provided when applicable. Authors are expected to disclose on the title page any commercial or other associations that might pose a conflict of interest in connection with the submitted manuscript. All funding sources that supported the work and the institutional and/or corporate affiliations of the authors should be acknowledged on the title page. Ethics When reporting experiments conducted with humans indicate that the procedures were in accordance with ethical standards set forth by the committee that oversees human subject research. Approval of research protocols by the relevant ethics committee, in accordance with international agreements (Helsinki Declaration of 1975, revised 2013 available at https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involving-humansubjects/), is required for all experimental, clinical, and drug studies. Patient names, initials, and hospital identification numbers should not be used. Manuscripts reporting the results of experimental investigations conducted with humans must state that the study protocol received institutional review board approval and that the participants provided informed consent. Non-compliance with scientific accuracy is not in accord with scientific ethics. Plagiarism: To re-publish, in whole or in part, the contents of another author’s publication as one’s own without providing a reference. Fabrication: To publish data and findings/results that do not exist. Duplication: Use of data from another publication, which includes republishing a manuscript in different languages. Salami slicing: To create more than one publication by dividing the results of a study unnecessarily. We disapprove of such unethical practices as plagiarism, fabrication, duplication, and salami slicing, as well as efforts to influence the review process with such practices as gifting authorship, inappropriate acknowledgments, and references. Additionally, authors must respect participants‘ right to privacy. On the other hand, short abstracts published in congress books that do not exceed 400 words and present data of preliminary research, and


those that are presented in an electronic environment, are not considered as previously published work. Authors in such a situation must declare this status on the first page of the manuscript and in the cover letter. (The COPE flowchart is available at http://publicationethics.org.) We use iThenticate to screen all submissions for plagiarism before publication. Conditions of Publication All authors are required to affirm the following statements before their manuscript is considered: 1. The manuscript is being submitted only to The Turkish Journal of Hematology; 2. The manuscript will not be submitted elsewhere while under consideration by The Turkish Journal of Hematology; 3. The manuscript has not been published elsewhere, and should it be published in The Turkish Journal of Hematology it will not be published elsewhere without the permission of the editors (these restrictions do not apply to abstracts or to press reports for presentations at scientific meetings); 4. All authors are responsible for the manuscript’s content; 5. All authors participated in the study concept and design, analysis and interpretation of the data, and drafting or revising of the manuscript and have approved the manuscript as submitted. In addition, all authors are required to disclose any professional affiliation, financial agreement, or other involvement with any company whose product figures prominently in the submitted manuscript. Authors of accepted manuscripts will receive electronic page proofs and are responsible for proofreading and checking the entire article within two days. Failure to return the proof in two days will delay publication. If the authors cannot be reached by email or telephone within two weeks, the manuscript will be rejected and will not be published in the journal.

Copyright At the time of submission all authors will receive instructions for submitting an online copyright form. No manuscript will be considered for review until all authors have completed their copyright form. Please note, it is our practice not to accept copyright forms via fax, e-mail, or postal service unless there is a problem with the online author accounts that cannot be resolved. Every effort should be made to use the online copyright system. Corresponding authors can log in to the submission system at any time to check the status of any co-author’s copyright form. All accepted manuscripts become the permanent property of The Turkish Journal of Hematology and may not be published elsewhere, in whole or in part, without written permission.

An extensive list of conversion factors can be found at https://www. nist.gov/sites/default/files/documents/pml/wmd/metric/SP1038.pdf. For more details, see http://www.amamanualofstyle.com/oso/public/jama/ si_conversion_table.html.

Abbreviations and Symbols Use only standard abbreviations. Avoid abbreviations in the title and abstract. The full term for an abbreviation should precede its first use in the text, unless it is a standard abbreviation. All acronyms used in the text should be expanded at first mention, followed by the abbreviation in parentheses; thereafter the acronym only should appear in the text. Acronyms may be used in the abstract if they occur 3 or more times therein, but must be reintroduced in the body of the text. Generally, abbreviations should be limited to those defined in the AMA Manual of Style, current edition. A list of each abbreviation (and the corresponding full term) used in the manuscript must be provided on the title page.

Online Manuscript Submission Process The Turkish Journal of Hematology uses submission software powered by ScholarOne Manuscripts. The website for submissions to The Turkish Journal of Hematology is http://mc.manuscriptcentral.com/tjh. This system is quick and convenient, both for authors and reviewers.

Setting Up an Account New users to the submission site will need to register and enter their account details before they can submit a manuscript. Log in, or click the “Create Account” button if you are a first-time user. To create a new account: After clicking the “Create Account” button, enter your name and e-mail address, and then click the “Next” button. Your e-mail address is very important. Enter your institution and address information, as appropriate, and then click the “Next” Button. Enter a user ID and password of your choice, select your area of expertise, and then click the “Finish” button. If you have an account, but have forgotten your log-in details, go to “Password Help” on the journal’s online submission system and enter your e-mail address. The system will send you an automatic user ID and a new temporary password.

Units of Measurement

Full instructions and support are available on the site, and a user ID and password can be obtained during your first visit. Full support for authors is provided. Each page has a “Get Help Now” icon that connects directly to the online support system. Contact the journal administrator with any questions about submitting your manuscript to the journal (info@tjh.com.tr). For ScholarOne Manuscripts customer support, click on the “Get Help Now” link on the top right-hand corner of every page on the site.

Measurements should be reported using the metric system, according to the International System of Units (SI). Consult the SI Unit Conversion Guide, New England Journal of Medicine Books, 1992.

Log in to your author center. Once you have logged in, click the “Submit a Manuscript” link in the menu bar. Enter the appropriate data and answer

Note: We cannot accept any copyright form that has been altered, revised, amended, or otherwise changed. Our original copyright form must be used as is.

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The Electronic Submission Process


the questions. You may copy and paste directly from your manuscript. Click the “Next” button on each screen to save your work and advance to the next screen.

Upload Files Click on the “Browse” button and locate the file on your computer. Select the appropriate designation for each file in the drop-down menu next to the “Browse” button. When you have selected all the files you want to upload, click the “Upload Files” button. Review your submission before sending to the journal. Click the “Submit” button when you are finished reviewing. You can use ScholarOne Manuscripts at any time to check the status of your submission. The journal’s editorial office will inform you by e-mail once a decision has been made. After your manuscript has been submitted, a checklist will then be completed by the Editorial Assistant. The Editorial Assistant will check that the manuscript contains all required components and adheres to the author guidelines. Once the Editorial Assistant is satisfied with the manuscript it will be forwarded to the Senior Editor, who will assign an editor and reviewers.

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CONTENTS Review 282 The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

Şadiye Mehtat Ünlü, Hayri Özsan, Sülen Sarıoğlu

Commentary 289 Time to Cure Hairy Cell Leukemia

Ilana Levy, Tamar Tadmor

291

Research Articles

Retrospective Evaluation of Hairy Cell Leukemia Patients Treated with Three Different First-Line Treatment Modalities in the Last Two Decades: A Single-Center Experience Şeniz Öngören, Ahmet Emre Eşkazan, Selin Berk, Tuğrul Elverdi, Ayşe Salihoğlu, Muhlis Cem Ar, Zafer Başlar, Yıldız Aydın, Nükhet Tüzüner, Teoman Soysal

300

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics Narges Rezaei, Nargess Arandi, Behnaz Valibeigi, Sezaneh Haghpanah, Mehdi Khansalar, Mani Ramzi

307

Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia Ahmet Menteşe, Nergiz Erkut, Selim Demir, Serap Özer Yaman, Ayşegül Sümer, Şeniz Doğramacı, Ahmet Alver, Mehmet Sönmez

314

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment Emine Begüm Gençer, Pınar Yurdakul, Klara Dalva, Meral Beksaç

321

Effectiveness of Visual Methods in Information Procedures for Stem Cell Recipients and Donors Çağla Sarıtürk, Çiğdem Gereklioğlu, Aslı Korur, Süheyl Asma, Mahmut Yeral, Soner Solmaz, Nurhilal Büyükkurt, Songül Tepebaşı, İlknur Kozanoğlu, Can Boğa, Hakan Özdoğu

328

Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma Carl Hsieh, Vani Rajashekharaiah

334

Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats Hong Qian, Tao Yuan, Jian Tong, Wen-shuang Sun, Jiajia Jin, Wen-xiang Chen, Jia Meng, Nirong Bao, Jianning Zhao

340

Brief Reports

High Infection-Related Mortality in Pediatric Acute Myeloid Leukemia without Preventive Antibiotics and Antifungals: Retrospective Cohort Study of a Single Center from a Middle-Income Country Emine Zengin, Nazan Sarper, Sema Aylan Gelen, Uğur Demirsoy, Meriban Karadoğan, Suar Çakı Kılıç, Selim Öncel, Emin Sami Arısoy, Devrim Dündar

345

Hematopoietic Stem Cell Transplantation in Primary Immunodeficiency Patients in the Black Sea Region of Turkey Alişan Yıldıran, Mehmet Halil Çeliksoy, Stephan Borte, Şükrü Nail Güner, Murat Elli, Tunç Fışgın, Emel Özyürek, Recep Sancak, Gönül Oğur

350

Images in Hematology

Bullous Pyoderma Gangrenosum in a Patient with Acute Myelogenous Leukemia as a Pathergic Reaction after Bone Marrow Biopsy Nur Efe İris, Reyhan Diz-Küçükkaya, Mutlu Arat, Zahide Eriş

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352

Giant Intracranial Solitary Plasmacytoma Osman Kara, Tayfur Toptaş, Işık Atagündüz, Süheyla Bozkurt, Önder Şirikçi, Tülin Fıratlı Tuğlular

354

Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia Yasushi Kubota, Yuka Hirakawa, Kazuo Wakayama, Shinya Kimura

356

Letters to the Editor

358

Chronic Active Parietal Osteomyelitis Due to Salmonella typhi in a Patient with Sickle Cell Anemia Ahmad Antar, George Karam, Maurice Kfoury, Nadim El-Majzoub

359

Acquired Leukocyte Inclusion Bodies Resembling Döhle Bodies During Acute Cholangitis Gökhan Özgür, Musa Barış Aykan, Murat Yıldırım, Selim Sayın, Ahmet Uygun, Cengiz Beyan

360

Three Novel Calreticulin Mutations in Two Turkish Patients Veysel Sabri Hançer, Hüseyin Tokgöz, Serkan Güvenç, Ümran Çalışkan, Murat Büyükdoğan

362

Imatinib-Induced Interstitial Pneumonitis Successfully Switched to Nilotinib in a Patient with Prior History of Mycobacterium tuberculosis Infection Zhuan-Bo Luo, Ning Xu, Xiao-Ping Huang, Guifang Ouyang

364

Prostate Involvement in a Patient with Follicular Lymphoma Seda Yılmaz, Sinan Demircioğlu, Özlen Bektaş, Özcan Çeneli, Sıdıka Fındık

366

Coexistence of EZH2, NOTCH1, IL7R, and PHF6 Mutations in Adult T-cell Acute Lymphoblastic Leukemia Xilian Zhou, Yan Gu, Qi Han, Mario Soliman, Chunhua Song, Zheng Ge

369

Circulating Tumor Cells in Neuroblastoma Mili Jain, Ashutosh Kumar, Sanjay Mishra, Nishant Verma, Madhu Mati Goel

370

Megakaryocytic Emperipolesis Associated with Thrombocytopenia: Causative or Coincidence? Manu Goyal, Sreeja Thandilath Thekkelakayil, Anurag Gupta

372

First Observation of Hemoglobin San Diego, a High Oxygen Affinity Hemoglobin Variant, in Turkey Ebru Yılmaz Keskin, Ali Fettah, Ana Catarina Oliveira, Şule Toprak, Andreia Lopes, Celeste Bento

374

A Case of Para-Bombay Phenotype Caused by Homozygous Mutation of the FUT1 Gene Jung-Kuang Yu, Yi-Hong Liu, Tze-Kiong Er

376

A Myopathy, Lactic Acidosis, Sideroblastic Anemia (MLASA) Case Due to a Novel PUS1 Mutation Çiğdem Seher Kasapkara, Leyla Tümer, Nadia Zanetti, Fatih Ezgü, Eleonora Lamantea, Massimo Zeviani

378

Frequency and Risk Factors for Secondary Malignancies in Patients with Mycosis Fungoides Fatma Pelin Cengiz, Nazan Emiroğlu, Nahide Onsun

380

Leishmaniasis: Bone Marrow Aspirate Smear and Rapid Antibody Test Beuy Joob, Viroj Wiwanitkit

381

Receiver Operating Characteristic Curve Analysis May be Helpful to Study the Prognostic Value of miR-155 in B-Cell Non-Hodgkin Lymphoma Long Su

Liver Transplantation in a Patient with Acquired Dysfibrinogenemia Who Presented with Subdural Hematoma: A Case Report Şencan Acar, Gökhan Güngör, Murat Dayangaç, Reyhan Diz-Küçükkaya, Yaman Tokat, Murat Akyıldız

34th Volume Index Author Index 2017

Subject Index 2017

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Advisory Board of This Issue (December 2017) Abdulahad Doğan, Turkey Ahmet Emre Eşkazan, Turkey Ahmet Muzaffer Demir, Turkey Akif Selim Yavuz, Turkey Anıl Tombak, Turkey Bülent Eser, Turkey Burhan Engin, Turkey Cem Ar, Turkey Chloe Anthlas, UK Dilber Talia İleri, Turkey Dushyant Kumar, India Düzgün Özatlı, Turkey Ebru Koca, Turkey Emre Tekgündüz, Turkey Engin Özçivici, Turkey Erdal Karaöz, Turkey Erdal Kurtoğlu, Turkey Eren Altun, Turkey Graeme Quest, Canada Hale Ören, Turkey

Handan Dinçaslan, Turkey Hüseyin Onay, Turkey James Yu, USA Jamile Shammo, USA Jansen Seheult, USA John M. Bennett, USA Kanjaksha Ghosh, India Mahmut Bayık, Turkey Majid Shahabi, Iran Maria Papaioannou, Greece Mehmet Sönmez, Turkey Melih Aktan, Turkey Meral Beksaç, Turkey Michael Grever, USA Müge Sayitoğlu, Turkey Mutlu Arat, Turkey Nader Cohan, Iran Nejat Akar, Turkey Oliver Karanfılski, Macedonia Poojan Agarwal, India

Rajat Kumar Agarwal, India Rejin Kebudi, Turkey Rosemary Sparrow, Australia Ruchi Gupta, India Rümeyza Kazancıoğlu, Turkey Sema Anak, Turkey Sevil Çatal, Turkey Sigbjørn Berentsen, Norway Sujata Mallick, India Tamar Tadmor, Israel Tiraje Celkan, Turkey Türkan Patıroğlu, Turkey Ujjwayini Ray, India Ümit Yavuz Malkan, Turkey Vildan Güngörer, Turkey Vildan Özkocaman, Turkey Volkan Hazar, Turkey Yahya Büyükaşık, Turkey Yüksel Aliyazıcıoğlu, Turkey


REVIEW DOI: 10.4274/tjh.2017.0197 Turk J Hematol 2017;34:282-288

The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance Monoclonal Gammopatilerin Her Klinik Evresinde Böbrek Etkilenim Paternleri Şadiye Mehtat Ünlü1, Hayri Özsan2, Sülen Sarıoğlu1 Dokuz Eylül University Faculty of Medicine, Department of Pathology, İzmir, Turkey Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey

1 2

Abstract

Öz

Multiple myeloma (MM) is one of the most important clonal malignant plasma cell disorders and renal involvement is associated with poor prognosis. Although there are several reasons for renal impairment in MM, the main cause is the toxic effects of monoclonal proteins. Although cast nephropathy is the best known and unchallenged diagnosis for hematologists and pathologists, the renal effects of monoclonal gammopathy can be various. Monoclonal gammopathy of renal significance was proposed by the International Kidney and Monoclonal Gammopathy Research Group for renal lesions in monoclonal gammopathy in recent years. Renal lesions in monoclonal gammopathy can be grouped as follows: light chain (cast) nephropathy, acute tubular injury/necrosis, tubulointerstitial nephritis, amyloidosis, monoclonal Ig deposition diseases, immunotactoid glomerulopathy, type I cryoglobulinemia, proliferative glomerulonephritis with monoclonal IgG deposits, C3 glomerulopathy with monoclonal gammopathy, and crystal-storing histiocytosis, considering the previous and new terminology. In this study, renal involvement of monoclonal gammopathies, in terms of previous and new terminology, was reviewed.

Multipl miyelom (MM) malign plazma hücre hastalıklarının en önemlilerinden biridir ve böbrek tutulumu kötü prognozla ilişkilidir. Multipl miyelomda böbrek fonksiyonunu etkileyecek çeşitli faktörler bulunmakla birlikte ana etken monoklonal proteinlerin toksik etkisidir. Monoklonal gammopatilerde böbrek tutulumları içinde hematolog ve patologlar tarafından en iyi bilinen ve tanısı sorunsuz olan “kast nefropatisi” olmakla birlikte çok farklı tutulum tipleri olabilir. Yakın zamanda “International Kidney and Monoclonal Gammopathy Research Group” tarafından, monoklonal gammopatilerdeki böbrek tutulumlarını bir çatı altında toplayan “renal öneme sahip monoklonal gammopati” terimi önerildi. Monoklonal gammopatilerdeki böbrek lezyonları, önceki ve yeni terminoloji uyarınca; hafif zincir kast nefropati, akut tübüler zedelenme/nekroz, tubulointerstisyel nefrit, amiloidoz, monoklonal Ig depo hastalıkları, immünotaktoid glomerülopati, tip I kriyoglobulinemi, monoklonal IgG depoziti ilişkili proliferatif glomerülonefrit, monoklonal gammopati ilişkili C3 glomerülopati ve kristal/histiyosit depo hastalığı olarak gruplanabilir. Bu makalede, monoklonal gammopatilerdeki böbrek tutulumu yeni ve önceki terminolojiler gözden geçirildi.

Keywords: Monoclonal gammopathy of renal significance, Plasma cell disorders, Multiple myeloma, Renal involvement, Kidney, Cast nephropathy

Anahtar Sözcükler: Renal öneme sahip monoklonal gammopati, Plazma hücre hastalıkları, Multipl miyelom, Böbrek tutulumu, Böbrek, Kast nefropati

Introduction Each specific immunoglobulin (Ig) molecule, which is synthesized by plasma cells, includes two identical heavy chains and two light chains (LCs). There are 5 types of heavy chains (ϒ/α/μ/δ/Є), and immunoglobulins get their names (IgG, IgA, IgM, IgD, IgE) according to these, while there are 2 types of LC (К/λ). Each heavy chain and LC has constant domains and variable domains. Variable domains include the antigen-binding region. Heavy chains and LCs are synthesized independently of each other and

finally unite in the endoplasmic reticulum, and the structure called immunoglobulin is unified here [1,2]. Normally, LCs are freely filtered through the glomerular basal membrane because they are low-molecular-weight proteins and are reabsorbed by the proximal tubules, endocytosed and catabolized in the lysosome. After the catabolization process, their amino acids return to the circulation. There are cubilinmegalin receptors on the brush-border of proximal tubular cells, which are very important for the control of LC endocytosis.

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Şadiye Mehtat ÜNLÜ, M.D., Dokuz Eylül University Faculty of Medicine, Department of Pathology, İzmir, Turkey Phone : +90 232 412 34 15 E-mail : mehtat.unlu@deu.edu.tr ORCID-ID: orcid.org/0000-0002-7170-7594

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Received/Geliş tarihi: May 18, 2017 Accepted/Kabul tarihi: August 22, 2017


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The heavy chains do not cross the glomerular filtration barrier [3,4,5]. In plasma cell dyscrasia, there is monoclonal plasma cell proliferation and a single type of whole Ig or a subunit or just a LC, which is synthesized by the clone, and the type of these protein fragments causes damage to the kidneys at a varying degree or the amount of the monoclonal LC in the filtrate exceeds the capacity of proximal tubular cells. Approximately 85% of all LCs with plasma cell dyscrasia are nephrotoxic. Most of them are tubulopathic (70%) and so they affect the tubulointerstitial compartment, and the rest of them are glomerulopathic and affect the glomerular compartment. Of course, some host factors are deterministic for this pattern and the grade of the damage, while the molecular and physicochemical characteristics of the LC affecting different compartments are unknown [6]. Monoclonal protein secretion is a typical feature of plasma cell disorders and may affect the kidneys in several ways. Multiple myeloma (MM) is one of the most important clonal malignant plasma cell disorders and renal involvement is associated with poor prognosis [7]. Clonal evaluation steps for MM include monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM [8,9]. While MGUS and smoldering MM patients do not require therapy except for clinical trial settings, if the patients have myeloma defining events such as  CRAB (hypercalcemia, renal impairment, anemia, and bone lesions), treatment becomes obligatory.  One of the CRAB finding is renal impairment and its incidence ranges from 20% to 50% according to how it is defined [7,9]. The International Myeloma Working Group consensus defines kidney impairment as an acute deterioration of kidney function that results in a serum creatinine level of more than 2.0 mg/dL, but kidney biopsy is currently not proposed. That means that the real incidence of nephropathy is not clear. Although the main cause for renal impairment is the toxic effects of monoclonal proteins, there are several other factors like hypercalcemia, dehydration, nephrotoxic drugs, and contrast agents that can aggravate the underlying disease [9]. Although MGUS and smoldering MM seem to be more benign disorders and do not require therapy, they may cause some renal conditions and those should be treated. Until recent time, in the writings on this subject, “MM, MGUS, or smoldering myeloma” has probably been the most important classification for hematologists while “glomerulopathic/ tubulopathic pattern” has been the most important for pathologists. Although these classifications still maintain their importance, they have become inadequate for the diagnosis, monitoring, and treatment of kidney lesions. Although MM and cast nephropathy are the most straightforward diagnoses for hematologists and pathologists, there are uncertain

areas in this wide clinical and morphologic spectrum for both hematologists and pathologists. According to the treatment guidelines, chemotherapy is indicated when the patient has symptoms related to the underlying plasmacytic or lymphocytic proliferation [10,11]. At this stage, due to the lack of end-stage organ involvement according to the definitions of smoldering MM (>3 g/dL of monoclonal protein and/or >10% bone marrow plasma cells)  and MGUS  (<3 g/dL of monoclonal protein and/ or <10% bone marrow plasma), they do not receive treatment. Although this group looks like benign disease to hematologists, the same situation is not true for the kidneys [11,12,13]. Some patients with proteinuria or acute renal failure consult with  nephrologists  before hematologists and they get a diagnosis from renal biopsy if the pathologist is experienced in nephropathology, because renal injury patterns other than cast nephropathy and amyloidosis can be very silent or can be confused with other renal diseases. Until recently, in cases of plasma cell dyscrasia, renal involvements have been grouped into the following categories: LC (cast) nephropathy, acute tubular injury/necrosis (ATD),  tubulointerstitial nephritis (TIN),  amyloidosis, and monoclonal Ig deposition diseases (MIDDs). Light chain (cast) nephropathy is characterized by acute renal deterioration or clear renal failure and an uncomplicated histopathological diagnosis.  Because the formation of the myeloma cast begins in the collecting ducts, the medulla has special importance in the biopsy. The casts generally contain the LCs  and Tamm-Horsfall protein and sometimes cellular debris and crystals and giant cells. While monoclonal LC staining is important in immunofluorescence, equally strong staining must also not be ignored (Figure 1) [6,14,15,16].

Figure 1. Light chain cast nephropathy: A) classical large myeloma casts show irregular shape and fracture planes (hematoxylin and eosin; 100x); B) giant cell surrounding the casts (hematoxylin and eosin; 200x); C) metachromatic staining of the myeloma casts (Masson’s trichrome; 200x). 283


Ünlü ŞM, et al: The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

ATD is especially characterized by proximal tubular damage because the LCs are metabolized in the lysosomes of these cells. ATD can be the only histological lesion in a biopsy or it may be combined with other findings with associated  monoclonal gammopathy. The clinical manifestation is a varying degree of renal failure according to the severity of the tubular injuries.  Light chain proximal tubulopathy  is  characterized  by periodic acid-Schiff-negative crystals or non-crystallized LCs in the cytoplasm of proximal tubular cells.  Pathologic LCs are almost always kappa in proximal tubulopathy with crystal formation and appear to be hypereosinophilic under a light microscope. In addition, round/rectangular free structures can be detected in the cytoplasm of proximal tubules by electron microscope. Due to the loss of crystals during standard frozen sectioning,  direct  immunofluorescence  (DIF) is not a proper method to demonstrate these structures [17,18]. In the non-crystallized cases, pathologic LCs can be kappa or lambda and can be detected in classical DIF. They appear to be hypereosinophilic inclusions in the cytoplasm of proximal tubules in hematoxylin-eosin, dark positive dots in periodic acid-methionine silver, and electron-dense large phagolysosomes by  electron microscope.  Since the proximal tubule epithelial cells are the physiologic catabolism area for LCs, the acute or chronic signs of tubular injury should be well demonstrated morphologically by the pathologist to differentiate the physiologic process. Fanconi syndrome, which is characterized by electrolyte wasting and aminoaciduria, is most frequently accompanied by LC proximal tubulopathy with crystals (Figure 2) [19,20].

Figure 2. Light chain proximal tubulopathy: A) dark positive dots are a clue for the accumulation of light chain protein in proximal tubules (PAMS; 400x); B) cytoplasmic granular staining with direct immunofluorescence for lambda light chain (fluorescein, 400x).

Turk J Hematol 2017;34:282-288

TIN is  quite rare in the plasma cell dyscrasia-associated disease group. Acute renal failure is the most common clinical manifestation. The histomorphological findings are identical to the other inflammatory TIN findings and the morphological diagnosis is very easy; however, it is difficult to establish the connection with plasma cell dyscrasia in terms of etiology and the rate of establishing the linkage between the two diseases is very low. Linear monotypic light staining throughout the tubular basement membranes sometimes can be helpful (Figure 3) [6,21,22,23]. Amyloidosis is a group of misfolded protein disorders and has the structure of fibrils. Amyloidosis with plasma cell dyscrasia is an important systemic problem.  Amyloid light-chain  (AL) amyloidosis stains with Congo red just like the amyloid A type and exhibits yellow/orange/green colors under polarized light [24]. Amyloid fibrils consist of generally light (most frequently λ) and sometimes heavy chain fragments.  Renal involvement is not infrequent (70%-80%) and it mostly affects the glomeruli and vessels. Although renal involvement is common, cardiac involvement is important for early mortality.  Amyloidogenic LCs are directly toxic to the myocardium and rapid suppression is crucial for mortality. It is important to  emphasize  that amyloidosis patients mostly have a low-grade plasma cell clone and only 20% of patients meet the criteria for MM (Figure 4) [25,26,27,28,29]. MIDDs  are systemic like amyloidosis and are observed in approximately 3%-5% of myeloma cases. Although immunoglobulin is stored in many organs, the kidneys are most commonly affected. Except for a few cases, usually the immunoglobulin depositions consist of LCs. Most light-chain deposition disease patients have proteinuria, with 50% of them

Figure 3. Light chain-related tubulointerstitial nephritis: A-B) interstitial lymphocyte-rich inflammation (hematoxylin and eosin; 100x and 200x); C) very few cellular cast formations may be the only clue for monoclonal gammopathy (hematoxylin and eosin; 400x).

Figure 4. AL amyloidosis. Acellular brick red mesangial deposits with Congo red in the glomerulus (A) and also a vessel wall (B) (400x). C) Yellow-orange birefringence in glomerulus and vessels under polarized light with Congo red (200x). D) Mesangial lambda light chain amyloid deposits with direct immunofluorescence (fluorescein, 400x). 284


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being in the nephrotic range and about 30% of cases having acute renal failure in the diagnosis. In more than half of the cases, clinical findings meet the criteria for MM. However, it should be kept in mind that a significant number of cases have normal bone marrow biopsy results. Most light microscopic findings are nodular glomerulopathy and monotypic LC deposition in the glomerular-tubular basement membrane and in vessels walls with immunofluorescence (Figure 5) [18,22,30,31,32,33].

diagnosis of MM or not, because they know very well that MM is characterized  by high tumor mass and high monoclonal Ig level.

A new term, monoclonal gammopathy of renal significance (MGRS), was recently proposed by the International Kidney and Monoclonal Gammopathy Research Group for renal lesions in monoclonal gammopathy. All renal lesions related to monoclonal gammopathy were reclassified under the title of MGRS and it was emphasized that the kidneys of patients whose clinical symptoms do not meet  the criteria for MM, Waldenström macroglobulinemia (WM),  chronic lymphocytic leukemia  (CLL), or other plasma/B-cell proliferative disorders [12] should be monitored (Schema 1). Cast nephropathy and WM (both of which are well recognized for therapy by nephrologists and hematologists) causing acute renal failure are excluded from this description, directly related to high tumor load. Furthermore, as our experience supports, different patterns with different levels can be seen in the same case. If the patient has acute renal failure due to cast nephropathy, hematologists should immediately determine whether the patient has a clinical

Renal dysfunction is a common complication (approximately 20%-25%) in active MM cases and the grade of renal dysfunction is probably correlated with myeloma cell load. Half of patients (50%) even show an improvement in the renal dysfunction with the treatment of myeloma, but the remaining ones have some degree of persistent chronic kidney disease and 2%-12% of these patients need renal transplantation. The high tumor burden and excessive monoclonal protein secretion are the main courses of the renal lesions in this group and the most common manifestation is acute renal failure due to cast nephropathy  [34,35].  In MGRS cases, however, tumor burden is not high and the determination of the latent renal injury is based on the physicochemical property of paraprotein [12,36]. As shown in Schema 1, MGRS-associated renal lesions mainly affect the glomerulus.

Figure 5. C3 glomerulopathy with monoclonal gammopathy: A) mesangial matrix expansion (hematoxylin and eosin; 400x); B) positivity of the mesangial complement C3c with direct immunofluorescence (fluorescein, 400x).

Schema 1. MGRS-associated renal lesions (modified from Bridoux et al. [36]). AH: Immunoglobulin heavy chain amyloidosis, AHL: immunoglobulin heavy and light chain amyloidosis, AL: immunoglobulin light chain amyloidosis, GN: glomerulonephritis, HCDD: heavy chain deposition disease, LCDD: light chain deposition disease, LHCDD: light and heavy chain deposition disease, MGRS: monoclonal gammopathy of renal significance.

However, in MGRS, the patient has a small clone of lymphoplasmacytic cells and the structural and biological features rather than the amounts of immunoglobulin are especially important for renal disease.

Fibrillary glomerulonephritis is characterized by fibril structures, resembling amyloidosis. However, these fibrils do not create the amyloid form and do not react with Congo red. Very few are cases associated with monoclonal gammopathy and most cases show  a membranoproliferative glomerulonephritis (MPGN) pattern under light microscope and IgG4, IgG1, and polyclonal LC positivity in immunofluorescence. The fibrils are non-branching and randomly oriented, similar to the amyloid fibrils ultrastructurally in electron microscopic assessment, but the fibril diameters are thicker (12-25 nm in diameter) than the amyloid fibrils (8-12 nm in diameter) [37,38,39]. Immunotactoid glomerulopathy  is  characterized by microtubule  organization  consisting of monoclonal Ig. More than half of such patients have CLL or a small lymphoma and rarely a low-grade plasma cell clone.  In renal biopsy, membranous nephropathy and the MPGN pattern are mostly seen by light microscope and generally monocytic IgG, C3, and LC positivity are seen in immunofluorescence. Similarly to other fibrillary lesions, electron microscopic assessment is very critical for the diagnosis [36,37,40,41]. Type I cryoglobulinemia arises from monoclonal immunoglobulin. In several diseases in which the entire Ig is secreted, such as MGUS, WM, or other B-cell lymphoid disorders, the cryoglobulins can be detected. Renal involvement is more common in the IgG type of cryoglobulinemia and about 30% of patients are affected. Despite episodes of acute renal failure or nephrotic syndrome, the essential process is a chronic glomerular injury. 285


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Endocapillary glomerulonephritis or MPGN patterns are the most common morphological findings with the hyaline-protein thrombi in the glomerulus. Monoclonal heavy chains and LCs (most frequently IgG-k) with complements (C3/C4/c1q) are detected in immunofluorescence [42,43,44]. Proliferative glomerulonephritis with monoclonal IgG deposits is a new entity that shows non-organized glomerular deposits most commonly consisting of IgG3-k. Membranoproliferative/ endocapillary proliferative and atypical membranous are the most commonly observed patterns by light microscope. Unlike in other deposit diseases (Randall-type heavy chain deposition disease/light and heavy chain deposition disease), monoclonal protein consists of the entire Ig and is detected only in glomeruli by immunofluorescence. Because the light and immunofluorescence microscopic appearances resemble immune complex glomerulonephritis, if immunoglobulin subgroups are not examined by immunofluorescence routinely, monoclonal accumulation may be missed by the pathologist. The possibility of detection of the circulating monoclonal IgG3 is low compared to IgG1/IgG2 and that is another challenge in diagnosis [33,34,35,45,46,47,48]. C3 glomerulopathy with monoclonal gammopathy  is a different entity among the other MGRS lesions. Although the manifestation of the disease is glomerulonephritis, there are not any Ig deposits in the kidneys. C3 glomerulopathy is  characterized  only by C3 deposits in the glomeruli by immunofluorescence and it can be divided into two main groups according to the ultrastructure of the deposit under an electron microscope. One of them is C3 glomerulonephritis and it is characterized by subendothelial/subepithelial or mesangial granular deposits upon electron microscopy. The other is dense deposit disease and it is characterized by “sausage-like” highdensity deposits in GBM [49,50]. The dysregulation of the alternative complement pathway is the main problem in both diseases. Some patients with C3 glomerulopathy have plasma cell dyscrasia and circulating monoclonal Ig. There are several hypotheses about the relationship between C3 glomerulopathy and monoclonal gammopathy [36]. This topic is not the subject of this article; however, it is important to emphasize that patients with the diagnosis of C3 glomerulopathy proven by biopsy should be investigated by the clinician in terms of monoclonal gammopathy (Figure 5). Crystal-storing histiocytosis is a very rare disease associated with MM or MGRS. Although several sites can be affected, including the kidneys, perirenal adipose tissue, and the lungs, bone marrow involvement is most typical.  Regardless of the site of involvement, the histiocytes with intralysosomal accumulation of Ig as crystals are determined by light microscope and most of the kappa LC cases are identified by DIF. We have no experience in the kidneys for crystal-storing histiocytosis [51,52]. 286

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Treatment Approach If the patient has a diagnosis of MM and has renal impairment, the treatment is more standard. The immediate start of antimyeloma therapy, dialysis (high cut-off hemodialysis), supportive care, high fluid intake, and avoidance of nephrotoxic agents are parts of standard  of care.  The value of plasma exchange is controversial; some studies showed encouraging results. These approaches may result in improvement of renal disease. Independence from dialysis is an important prognostic factor for survival. With the development of new agents such as thalidomide, bortezomib, and lenalidomide, the results are much better.  Proteasome  inhibitor (PI) (bortezomib)-based regimens are cornerstones in this setting. For eligible patients, autologous stem cell transplantation (ASCT) with 100-140 mg/ m2 melphalan is feasible [7]. In settings without overt myeloma patients, the decision about the necessity of anti-myeloma therapy may be difficult. However, MGRS is associated with high morbidity due to renal disease and sometimes systemic findings may occur based on the monoclonal immunoglobulin. MGRS regroups all renal disorders caused by monoclonal proteins secreted by a nonmalignant B-cell clone (AL amyloidosis, cryoglobulinemia, MIDDs, Fanconi syndrome). In AL amyloidosis, the decision for myeloma-like therapy can be easily made, because the disease is usually systemic and has clinical features like a malignant disease rather than benign disorders. On the other hand, there are sufficient studies and data in this area, because it is a more common disorder, and standard care includes high-dose dexamethasone + melphalan or bortezomib-based regimens and ASCT in eligible patients [7]. In MIDDs, hematologic responses are best achieved with ASCT or PI-based therapies and are associated with improved renal outcomes [53].  Renal transplantation is feasible for MGRS, but to avoid recurrence after transplantation, control of the responsible B-cell clone is important [7,54]. However, it is not clear that small B-cell clones are truly curable; thus, the risk of disease recurrence cannot be eliminated totally. Early diagnosis is important; if treatment is begun while renal functions are still preserved, long-term outcome results are better. MGRS is a heterogeneous and relatively rare entity and more collaborative studies and efforts of nephrologists and hematologists are required to improve its management.

Important Aspects - Paraprotein can cause injury to the kidneys independent of its concentration. - MGRS patients have a small B-cell clone and a low level of circulating paraprotein. Therefore, serum electrophoresis may not be sufficient and more detailed investigations may be


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Ünlü ŞM, et al: The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

required to detect monoclonal protein, like immunofixation and serum free LC assays, especially if the pathologist has any suspicion. - The suppression of nephrotoxic monoclonal proteins in MGRS is very important for renal and also patient survival. - Regular monitoring of the renal function, proteinuria, or hematuria can be very helpful for the early detection of the MGRS renal lesion with biopsy and the early effective treatment of cases with the diagnosis of monoclonal gammopathy with or without clinical importance for hematologists. Authorship Contributions Concept: Ş.M.Ü, H.Ö., S.S.; Design: Ş.M.Ü, H.Ö., S.S.; Data Collection or Processing: Ş.M.Ü, H.Ö., S.S.; Analysis or Interpretation: Ş.M.Ü, H.Ö., S.S.; Literature Search: Ş.M.Ü, H.Ö., S.S.; Writing: Ş.M.Ü, H.Ö., S.S. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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COMMENTARY DOI: 10.4274/tjh.2017.0296 Turk J Hematol 2017;34:289-290

Time to Cure Hairy Cell Leukemia Tüylü Hücreli Löseminin Kür Edilme Zamanı Ilana Levy1, Tamar Tadmor2,3 Bnai Zion Medical Center, Internal Medicine B Department, Haifa, Israel Bnai Zion Medical Center, Hematology Unit, Haifa, Israel 3 Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel 1 2

To the Editor, Major advances in the treatment of hairy cell leukemia (HCL) occurred during the 1980s when the purine analogs (PAs) cladribine [1] and pentostatin [2] were introduced. These agents dramatically altered the clinical course and outcome of this disease, achieving a 10-year overall survival (OS) rate of approximately 90%. However, in the last 30 years, first-line therapy for patients with HCL and even second-line therapy have not been substantially changed. In the current issue of this journal, Öngören et al. [3] report a retrospective analysis of 67 patients treated for classic HCL and compare 3 different first-line treatment modalities. Among them, 31 patients received cladribine therapy, 19 received interferonalpha (IFN-α), 16 underwent splenectomy, and 1 was treated with rituximab monotherapy. Patients treated with a PA as firstline therapy achieved the highest overall response rate (ORR) and significantly longer progression-free survival (PFS) with the lowest relapse rate, but had a similar OS rate when compared to other treatment modalities. However, in terms of therapy-related complications, there was a high rate of infections, which were mostly bacterial, with the highest rate reported in the cladribinetreated group. These results are in line with previous reports indicating that IFN-α and splenectomy are much less frequently used now. Pentostatin and cladribine are both equally recommended as first-line therapy, achieving equivalent efficacies. Both are associated with low rates of relapse or refractory disease (R/R) [4,5,6,7,8], indicating that HCL is potentially curable [5,7,8]. Nevertheless, HCL patients do relapse after PA therapy, and the rate or timing of relapse is associated with both complete

remission (CR) and minimal residual disease (MRD) status [6]. Indeed, patients with HCL and MRD positivity have shorter treatment-free intervals than those in CR after PA therapy [8]. Moreover, for each consequent relapse, the response rate to retreatment with PA decreases [5,7]. Taking all the above into consideration, in the future we should encourage the development of novel combinations or the use of consolidation therapies after the first response with PA is achieved, particularly in those patients who are MRD-positive, in an attempt to achieve more durable responses. Another disadvantage that should be carefully considered is the high rate of PA toxicity, which includes bone marrow suppression associated with neutropenia, lymphopenia, T-cell dysfunction, or stem cell toxicity with the development of hypoplasia and aplasia [5,6,9,10]. Similar complications were also described in the study by Öngören et al. [3]. Novel agents with a lower toxicity profile are currently being tested as alternative therapy or in combination with PA. Rituximab, a monoclonal antibody, has been studied as monotherapy or in combination with PA, both in the frontline setting or at relapse [11]. Results are encouraging, and rituximab seems to be well tolerated while side effects are quite rare. Other agents are also being used now for R/R HCL patients. These drugs are not used as frontline therapy but have a favorable toxicity profile. These include recombinant immunotoxins targeting CD22 (BL22, moxetumomab pasudox) [12] or the BRAF inhibitor (vemurafenib) [4] and have shown positive results in R/R disease [5]. Finally, the Bruton tyrosine kinase inhibitor ibrutinib appears to shorten the survival of hairy cells and block

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Tamar TADMOR M.D., Bnai Zion Medical Center, Hematology Unit, Haifa, Israel Phone : +972 483 594 07 E-mail : tamar.tadmor@b-zion.org.il ORCID-ID: orcid.org/0000-0002-3435-8612

Received/Geliş tarihi: August 07, 2017 Accepted/Kabul tarihi: August 25, 2017

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cell proliferation and intracellular signaling in vitro [13], and it is effective in some preliminary reports of therapy in refractory disease [14]. However, in this respect, due to the rarity of the disease, data are still incomplete and mostly based on small case series, retrospective studies, or phase I clinical trials [12,13,14,15]. Based on the recently published HCL consensus by Grever et al., [6] PAs remain the only first-line therapy for HCL, while vemurafenib is recommended in some cases with uncontrolled infection prior to therapy with PAs due to its ability to improve low blood counts. However, this agent should be replaced by a PA as soon as the infectious status is controlled. In regard to relapsed disease, both vemurafenib monotherapy and rituximab in combination with PA may be recommended, although repeated PA therapy is preferred in patients who achieve long first remissions of more than 60 months [6]. Other novel therapies such as ibrutinib or immunotoxin conjugates are still mostly used in clinical trials and are not included in current guidelines. In conclusion, as reported by Öngören et al. [3], PAs remain the most effective treatment for classic HCL in terms of PFS and ORR when compared to earlier therapies such as IFN-α or splenectomy, and this approach has not been changed over the past 30 years. Perhaps the time has arrived to challenge this approach and improve the outcome of HCL by using PAs in combination with some of the available biological agents, either as frontline or consolidation therapy for patients with classical HCL, in an attempt to cure this chronic neoplastic disorder. Keywords: Hairy cell leukemia, Purine analogs, Cladribine, Pentostatin Anahtar Sözcükler: Tüylü hücreli lösemi, Pürin analogları, Kladribin, Pentostatin Authorship Contributions Surgical and Medical Practices: I.L., T.T.; Concept: T.T.; Design: T.T. Data Collection or Processing: I.L., T.T.; Analysis or Interpretation I.L., T.T.; Literature Search I.L., T.T.; Writing: I.L., T.T. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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References 1. Piro LD, Carrera CJ, Carson DA, Beutler E. Lasting remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine. N Engl J Med 1990;322:1117-1121. 2. Saven A, Piro LD. Complete remissions in hairy cell leukemia with 2-chlorodeoxyadenosine after failure with 2-deoxycoformycin. Ann Intern Med 1993;119:278-283. 3. Öngören Ş, Eşkazan AE, Berk S, Elverdi T, Salihoğlu A, Ar MC, Başlar Z, Aydın Y, Tüzüner N, Soysal T. Retrospective evaluation of hairy cell leukemia patients treated with 3 different first-line treatment modalities in the last two decades: a single center experience. Turk J Hematol 2017;34:291-299. 4. Tiacci E, Park JH, De Carolis L, Chung SS, Broccoli A, Scott S, Zaja F, Devlin S, Pulsoni A, Chung YR, Cimminiello M, Kim E, Rossi D, Stone RM, Motta G, Saven A, Varettoni M, Altman JK, Anastasia A, Grever MR, Ambrosetti A, Rai KR, Fraticelli V, Lacouture ME, Carella AM, Levine RL, Leoni P, Rambaldi A, Falzetti F, Ascani S, Capponi M, Martelli MP, Park CY, Pileri SA, Rosen N, Foà R, Berger MF, Zinzani PL, Abdel-Wahab O, Falini B, Tallman MS. Targeting mutant BRAF in relapsed or refractory hairy-cell leukemia. N Engl J Med 2015;373:1733-1747. 5. Sarvaria A, Topp Z, Saven A. Current therapy and new directions in the treatment of hairy cell leukemia: a review. JAMA Oncol 2016;2:123-129. 6. Grever MR, Abdel-Wahab O, Andritsos LA, Banerji V, Barrientos J, Blachly JS, Call TG, Catovsky D, Dearden C, Demeter J, Else M, Forconi F, Gozzetti A, Ho AD, Johnston JB, Jones J, Juliusson G, Kraut E, Kreitman RJ, Larratt L, Lauria F, Lozanski G, Montserrat E, Parikh SA, Park JH, Polliack A, Quest GR, Rai KR, Ravandi F, Robak T, Saven A, Seymour JF, Tadmor T, Tallman MS, Tam C, Tiacci E, Troussard X, Zent CS, Zenz T, Zinzani PL, Falini B. Consensus guidelines for the diagnosis and management of patients with classic hairy cell leukemia. Blood 2017;129:553-560. 7. Else M, Ruchlemer R, Osuji N, Del Giudice I, Matutes E, Woodman A, Wotherspoon A, Swansbury J, Dearden C, Catovsky D. Long remissions in hairy cell leukemia with purine analogs: a report of 219 patients with a median follow-up of 12.5 years. Cancer 2005;104:2442-2448. 8. López Rubio M, Da Silva C, Loscertales J, Seri C, Baltasar P, Colado E, Pérez Fernández I, Osma M, Gomis F, González M, Jarque I, Vargas M, Monzó E, Monteagudo D, Orts MI, Pardal E, Carbonell F, Perez Calvo C, Garcia-Marco JA. Hairy cell leukemia treated initially with purine analogs: a retrospective study of 107 patients from the Spanish Cooperative Group on Chronic Lymphocytic Leukemia (GELLC). Leuk Lymphoma 2014;55:1007-1012. 9. Tadmor T. Purine analog toxicity in patients with hairy cell leukemia. Leuk Lymphoma 2011;52(Suppl 2):38-42. 10. Lad DP, Malhotra P, Khadwal A, Prakash G, Suri V, Kumari S, Jain S, Das R, Varma N, Varma S. Outcomes of splenectomy versus cladribine in hairy cell leukemia in resource limited settings. Leuk Lymphoma 2014;55:1428-1430. 11. Leclerc M, Suarez F, Noël MP, Vekhoff A, Troussard X, Claisse JF, Thieblemont C, Maloisel F, Beguin Y, Tamburini J, Barbe C, Delmer A. Rituximab therapy for hairy cell leukemia: a retrospective study of 41 cases. Ann Hematol 2015;94:89-95. 12. Kreitman RJ, Tallman MS, Robak T, Coutre S, Wilson WH, Stetler-Stevenson M, Fitzgerald DJ, Lechleider R, Pastan I. Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol 2012;30:1822-1828. 13. Sivina M, Kreitman RJ, Arons E, Ravandi F, Burger JA. The BTK inhibitor ibrutinib (PCI-32765) blocks hairy cell leukaemia survival, proliferation and BCR signalling: a new therapeutic approach. Br J Haematol 2014;166:177-188. 14. Bohn JP, Wanner D, Steurer M. Ibrutinib for relapsed refractory hairy cell leukemia variant. Leuk Lymphoma 2017;58:1224-1226. 15. Jones J, Andritsos L, Kreitman RJ, Ravandi F, Schiffer C, Call TG, Lozanski G, Harris P, Sexton J, Ruppert AS, Grever MR. Efficacy and safety of the Bruton tyrosine kinase inhibitor ibrutinib in patients with hairy cell leukemia: stage 1 results of a phase 2 study. Blood 2016;128:1215.


RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0443 Turk J Hematol 2017;34:291-299

Retrospective Evaluation of Hairy Cell Leukemia Patients Treated with Three Different First-Line Treatment Modalities in the Last Two Decades: A Single-Center Experience Geçmiş İki Dekatta Üç Farklı Birinci Basamak Tedavisi Alan Tüylü Hücreli Lösemi Hastalarının Geriye Dönük Değerlendirmesi: Tek Merkez Deneyimi Şeniz Öngören1, Ahmet Emre Eşkazan1, Selin Berk1, Tuğrul Elverdi1, Ayşe Salihoğlu1, Muhlis Cem Ar1, Zafer Başlar1, Yıldız Aydın1, Nükhet Tüzüner2, Teoman Soysal1 İstanbul University Cerrahpaşa Faculty of Medicine, Department of Hematology, İstanbul, Turkey İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pathology, İstanbul, Turkey

1 2

Abstract

Öz

Objective: In this study, we retrospectively analyzed the clinical outcome, treatment responses, infectious complications, and survival rates of 71 hairy cell leukemia (HCL) cases.

Amaç: Bu çalışmada, geriye dönük olarak 71 tüylü hücreli lösemi (SHL) olgusunu klinik sonuçlar, tedavi yanıtları, enfeksiyon komplikasyonları ve sağkalım oranları açısından analiz ettik.

Materials and Methods: Sixty-seven patients received a first-line treatment and 2-chlorodeoxyadenosine (cladribine-2-CdA) was administered in 31 cases, 19 patients received interferon-alpha (INF-α), splenectomy was performed in 16 cases, and rituximab was used in one.

Gereç ve Yöntemler: Altmış yedi hasta birinci basamak tedavi almıştı ve 2-klorodeoksiadenozin (kladribin-2-CdA) 31 olguya uygulanmış, 19 hasta interferon-alfa (INF-α) almış, splenektomi 16 olguda uygulanmış ve rituksimab ise bir hastada kullanılmıştı.

Results: Although the highest overall response rate (ORR) was observed in patients receiving 2-CdA upfront, ORRs were comparable in the 2-CdA, INF-α, and splenectomy subgroups. Relapse rates were significantly lower in patients who received first-line 2-CdA. The progression-free survival (PFS) rate with 2-CdA was significantly higher than in patients with INF-α and splenectomy, but we found similar overall survival rates with all three upfront treatment modalities. Infections including tuberculosis were a major problem. Conclusion: Although purine analogues have improved the ORRs and PFS, there is still much progress to make with regard to overall survival and relapsed/refractory disease in patients with HCL. Keywords: Cladribine, Hairy cell leukemia, Interferon, Splenectomy

Introduction Hairy cell leukemia (HCL) is a rare mature B-cell neoplasm characterized by the accumulation of atypical lymphocytes with prominent cytoplasmic projections in the bone marrow and spleen, resulting in pancytopenia and splenomegaly [1,2].

Bulgular: En yüksek toplam yanıt oranı (TYO) birinci basamak 2-CdA alan hastalarda görülmüş olsa da, TYO’lar 2-CdA, INF-α ve splenektomi alt gruplarında benzerdi. Nüks oranları birinci basamak 2-CdA alan hastalarda anlamlı olarak daha azdı. Progresyonsuz sağkalım (PS) oranı 2-CdA alanlarda INF-α ve splenektomi hastalarına göre anlamlı olarak daha yüksek olmakla birlikte, her üç birinci basamak tedavi yaklaşımı ile toplam sağkalım (TS) oranları benzer olarak bulundu. Tüberkülozun da dahil olduğu enfeksiyonlar önemli bir problemdi. Sonuç: Her ne kadar pürin analogları TYO ve PS’yi iyileştirmiş olsa da, SHL hastalarında TS ve nüks/dirençli hastalık açısından daha yapılması gereken çok şey vardır. Anahtar Sözcükler: Kladribin, Tüylü hücreli lösemi, İnterferon, Splenektomi

Most patients eventually require therapy owing to worsening cytopenias, frequent and life-threatening infections, and/ or symptomatic splenomegaly. Several treatment modalities, including splenectomy and immunotherapy with interferonalpha (INF-α), were used with various clinical and hematologic

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Şeniz ÖNGÖREN, M.D., İstanbul University Cerrahpaşa Faculty of Medicine, Department of Hematology, İstanbul, Turkey Phone : +90 532 334 16 64 E-mail : senizongoren@hotmail.com ORCID-ID: orcid.org/0000-0002-2809-5510

Received/Geliş tarihi: November 10, 2016 Accepted/Kabul tarihi: July 26, 2017

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responses until the introduction of the purine nucleoside analogues 2-chlorodeoxyadenosine (cladribine, 2-CdA) and 2′-deoxycoformycin (pentostatin) [3,4,5]. Purine analogues have resulted in higher complete response (CR) rates and durable remissions, and they have become the treatment of choice in most cases [6]. Monoclonal antibodies (i.e. rituximab) and immunotoxins are currently recommended for relapsed/ refractory (R/R) cases [7,8,9]. Also among the target-oriented therapeutic options, the BRAF inhibitor vemurafenib can be used in patients with R/R HCL [10,11]. In this study, we retrospectively analyzed the clinical outcome, treatment responses, infectious complications, and survival rates of HCL patients treated in our institution with three treatment modalities (splenectomy, INF-α, and 2-CdA) as first-line therapy between 1991 and 2014.

Materials and Methods Patients A total of 71 patients with HCL, who were diagnosed and followed in our clinic over the past 20 years, were included in this study. Diagnosis of HCL was established by morphological, flowcytometric, and immunohistochemical analysis of peripheral blood, bone marrow, and/or spleen specimens. Information on the patients’ characteristics, presenting signs and symptoms, treatment modalities and outcomes, and infections were retrospectively taken from the patients’ files. While defining the patient cohort, we excluded cases with variant HCL. Treatment Modalities Patients were divided into 3 subgroups according to the first-line treatments (i.e. splenectomy, INF-α, and 2-CdA) that they had received. Splenectomy was performed either laparoscopically or via open surgery. 2-CdA was given either by continuous intravenous infusion at a dose of 0.1 mg/kg/ day over 7 consecutive days, or by 2-h intravenous infusion at a dose of 0.1 mg/kg once a week for 7 consecutive weeks, depending on whether the patients received it as an inpatient or outpatient treatment, respectively. While on 2-CdA, the patients were given cotrimoxazole prophylaxis against Pneumocystis jirovecii pneumonia. INF-α was administered subcutaneously at a starting dose of 3 MU 3 times a week and maintained with subsequent toxicity-based dose adjustments. Rituximab was administered at the conventional dose of 375 mg/m2 weekly for 4 consecutive weeks, as suggested before [7].

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disappearance of hepatosplenomegaly and cytopenias. Normalization of peripheral blood counts together with an at least 50% reduction in the size of organomegaly and the volume of bone marrow HCs, plus <5% circulating HCs, was designated as partial response (PR). Presence of CR or PR was defined as overall response (OR), and any response other than a CR or PR was considered as no response. During response evaluation, a bone marrow biopsy was performed 3 months after finishing 2-CdA treatment. Furthermore, relapse after CR was defined as the reappearance of HCs in the peripheral blood or bone marrow, development of cytopenias, and/or splenomegaly on physical examination. Relapse after PR indicated a >50% increase of residual disease. Overall survival (OS) denotes the time from the first treatment until the time of death or last follow-up. The duration of progression-free survival (PFS) was calculated from the onset of any first-line treatment until the date of progression. Time to next treatment (TTNT) was calculated as the time from the end of the previous treatment to the institution of the next therapy. Statistical Analysis Student’s t-test was used for the comparison of the quantitative variables. Qualitative variables between groups were compared using the chi-square test. The Kaplan-Meier method was used for survival analysis [13]. Survival rates were compared by using the log-rank test. All tests were two-sided, and p<0.05 was considered as statistically significant. All analyses were performed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results Sixty-two patients (87%) were male and the median age was 49 years (range: 31-76 years). There were 53 patients (75%) with splenomegaly, and the numbers of patients with hepatomegaly and lymphadenopathy were 32 (45%) and 27 (38%), respectively. The demographic features of the entire cohort including median leukocyte and platelet counts and hemoglobin levels at diagnosis are displayed in Table 1. The median duration of follow-up was 57 months (range: 1-217 months). At diagnosis, all patients had bone marrow biopsy, and flow cytometric evaluation was performed for 47 patients (66%) from peripheral blood and/ or bone marrow. At diagnosis, hematoxylin and eosin staining, reticulin staining, tartrate-resistant acid phosphatase staining, and immunohistochemistry for CD20 and annexin A1 were performed from the bone marrow aspiration and trephine biopsy.

Definition of Response and Survival

Treatments and Outcomes

Response to treatment was assessed using the criteria described in the consensus resolution of 1987 [12]. Accordingly, CR was defined as the morphological absence of hairy cells (HCs) in the blood and the bone marrow in addition to complete

Among the patient cohort, there were 4 patients who did not receive treatment. Two of them were lost to follow-up, one patient died due to severe infection, and one had an acute myocardial infarction (AMI) and died before any treatment was

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initiated (Figure 1). First-Line Treatment Sixty-seven (94%) patients received a first-line treatment. 2-CdA was administered for 31 patients (46%), 19 patients (28%) received INF-α, splenectomy was performed in 16 cases (24%), and rituximab was used in one case (2%) (Figure 1). Although patients in the splenectomy arm were younger than those in the other 2 arms, the differences were not statistically significant (Table 2). The 3 treatment subgroups were equally balanced regarding sex distribution, median hemoglobin levels, and leukocyte and platelet counts (Table 2). There were 30, 16, and 13 patients who achieved OR after the first-line treatment with 2-CdA, INF-α, and splenectomy, respectively (Figure 1). The OR rates (ORRs) in the 2-CdA, INF-α, and splenectomy subgroups

were 97%, 84%, and 81%, respectively, and although the ORR of 2-CdA treatment was superior to those of the other 2 groups, the differences were not significant (Figure 2). During the firstline treatment, 2 patients (one on 2-CdA and one on INF-α) died due to infection (Figure 1). No postoperative complications were observed in patients with splenectomy. Five of the 31 patients who received 2-CdA as the first-line therapy required a second-line treatment after a median TTNT of 23 months (range: 3-58 months) (Table 2). There were 10 patients in the first-line INF-α subgroup who progressed and needed a second-line treatment following a median TTNT of 21 months (range: 1-96 months). Eleven of the 16 patients who underwent first-line splenectomy received a second-line therapy due to relapsed disease at a median TTNT of 5 months (range: 2-73 months). Relapse rates were significantly lower in patients who received first-line 2-CdA than those who were treated with INF-α or splenectomy (p=0.007 and p<0.0001, respectively). However, this was not significantly different when the INF-α and splenectomy subgroups were compared (p=0.339) (Table 2). Although the patients with first-line splenectomy had a shorter median TTNT than those with 2-CdA and INF-α, the difference did not reach statistical significance (Table 2). The only patient who received rituximab as frontline treatment remained refractory to that therapy, and she needed further treatment. Second-Line Treatment

Figure 1. The distribution of first-line treatment modalities and outcomes. 2-CdA: Cladribine, HCL: hairy cell leukemia, INF-α: interferon-alpha, RTX: rituximab, SPL: splenectomy, ORR: overall response rate, NRR: non-response rate. *See text for details.

Table 1. The baseline characteristics of the patients. Parameter

Entire cohort (n=71)

Sex - male/female, n (%)

62 (87)/9 (13)

Median age, years (min-max)

49 (31-76)

Median leukocyte count, x109/L (min-max)

3.0 (0.5-9.4)

Median hemoglobin level, g/dL (min-max)  

10 (4-17)

Median platelet count, x109/L (min-max)

51.5 (21-124)

Splenomegaly - yes/no, n (%) Mean spleen size, cm (min-max)*

53 (75)/18 (25) 11.4 (2-26)

Hepatomegaly - yes/no, n (%)

32 (45)/39 (55)

Lymphadenopathy - yes/no, n (%)

27 (38)/44 (62)

Median follow-up duration, months (min-max)

57 (1-217)

*Spleen size was measured by physical examination and stated as the distance from the left costal margin in cm.

With a median TTNT of 13 months (range: 1-96 months), 27 patients required a second-line treatment due to R/R disease. Twelve patients received INF-α, 11 patients were treated with 2-CdA, 3 patients underwent splenectomy, and one received fludarabine. Eight and 2 of the 12 patients who received second-line INF-α had been initially treated with splenectomy and 2-CdA,

Figure 2. Response rates according to the different first-line treatment modalities. *2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy. ORR: Overall response rate, NRR: non-response rate, 2-CdA: cladribine, INF-α: interferon-alpha.

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respectively. In the remaining 2 patients the previous treatment was also INF-α. The ORR was 100% in patients who received second-line INF-α, with only 3 patients requiring a third-line treatment due to relapsed disease. In patients who had 2-CdA as a second-line treatment, 7 had received INF-α as the first-line therapy, and 2 had splenectomy. The other 2 had been treated with rituximab and 2-CdA previously. All the patients responded to second-line 2-CdA (the ORR was 100%), and only one relapsed after a follow-up of 48 months. Splenectomy was performed in 3 patients as a second-line treatment. All of them initially responded to splenectomy; however, 2 needed further treatment due to relapsed disease. The patient who received fludarabine as a second-line therapy achieved PR but was then lost to follow-up after 9 months.

Third-Line Treatment There were 6 patients who needed third-line treatment. INF-α, 2-CdA, and rituximab were used for 3, 2, and one of them, respectively, following a median TTNT of 36 months (range: 8-63 months). Among patients receiving third-line INF-α, one achieved and maintained CR throughout the entire follow-up. The other 2 remained refractory to INF-α treatment; one died, and the other patient proceeded to fourth-line treatment. One of the 2 patients who received 2-CdA as a third-line treatment achieved and maintained CR and the other patient died due to refractory disease. The patient who received third-line weekly rituximab could not complete the 4th week of treatment due to an allergic reaction, which had happened following the 3rd dose of the drug, and had to proceed to fourth-line therapy.

Table 2. Patient characteristics and treatment outcomes in patients with 3 first-line treatment options. First-Line Treatment (n=67)† Parameter

2-CdA (n=31)

INF-α (n=19)

Splenectomy (n=16)

p value

Sex - male/female, n (%)

27 (87)/4 (13)

18 (95)/1 (5)

14 (88)/2 (12)

0.387* 0.969** 0.453***

Median age, years (min-max)

52 (33-75)

50 (33-76)

47.5 (31-64)

0.564* 0.094** 0.061***

Median leukocyte count, x109/L (min-max)

3.1 (1.6-7.2)

3.0 (0.5-9.4)

3.4 (0.5-9.1)

0.601* 0.510** 0.392***

Median hemoglobin level, g/dL (min-max)

11 (5-17)

10 (4-14)

9 (5-15)

0.098* 0.053** 0.721***

Median platelet count, x109/L (min-max)

62 (22-108)

42.5 (21-124)

56 (29-81)

0.624* 0.552** 0.888***

Patients needing further treatment(s) due to R/R disease, n (%)

5 (16)

10 (53)

11 (69)

0.007* <0.0001** 0.339***

Median TTNT, months (min-max)

23 (3-58)

21 (1-96)

5 (2-73)

0.902* 0.370** 0.190***

Median follow-up, months (min-max)

53 (5-213)

57 (2-154)

83 (15-217)

0.739* 0.120** 0.230***

One patient received rituximab as a first-line treatment, *2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy.

R/R: Relapsed/refractory, TTNT: time to next treatment, 2-CdA: cladribine.

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Fourth-Line Treatment There were only 2 patients who received a fourth-line treatment. One of them was treated with INF-α and the other with rituximab. The patient who received INF-α died due to refractory disease, and the patient who was given rituximab achieved CR. Infections In 47 patients, 76 infectious episodes were noted during the entire follow-up period (Table 3). Bacterial infections were the leading cause, and 64 bacterial infection episodes were observed. Of these 65 episodes, 14 were observed after diagnosis prior to the initiation of any anti-HCL treatment, whereas 50 episodes were noted during treatment (Table 3). The infections were diagnosed by means of cultures, radiological imaging techniques, and tissue biopsies as indicated. There were 18 patients who required hospitalization (mostly due to neutropenic fever), and in 17 cases infections occurred within 30 days after completion of treatment (all patients received 2-CdA). There were 7 infection episodes caused by Mycobacterium tuberculosis in 6 patients (5 pulmonary cases, 2 disseminated). In 2 patients tuberculosis was diagnosed synchronously with HCL, and 5 episodes occurred during/after anti-HCL treatment. In patients for whom tuberculosis was diagnosed prior to treatment initiation, one had pulmonary tuberculosis and he experienced disseminated disease 3 months after completing 2-CdA. The other patient had disseminated tuberculosis at diagnosis, which was diagnosed via splenectomy. That patient then received INF-α due to relapse without any recurrence of tuberculosis. None of the patients with tuberculosis had

drug-resistant disease, and none of them had any known comorbidities including diabetes. There were also 2 patients with invasive pulmonary aspergillosis (IPA). Six of the 10 episodes of viral infections were related to flu. One of these patients had influenza A virus subtype H1N1 infection (swine flu) and had to be followed in the intensive care unit. Overall, 3 patients died due to infection, and one of them died because of a febrile neutropenic episode prior to treatment initiation. In the remaining 2 cases, infections occurred during/ after treatment; IPA was the reason for death in one patient receiving first-line INF-α and the other patient died due to sepsis following frontline 2-CdA administration. The distribution of the infections and outcomes are shown in Table 3. Survival During the follow-up, 10 patients (12%) died (7 due to refractory disease and/or infections, 3 due to AMI and sudden cardiac death) and 2 patients were lost to follow-up. PFS and OS rates for the entire cohort after a median follow-up of 57 months were 62% (Figure 3A) and 83% (Figure 3B), respectively. With regard to the first-line treatment, the PFS rate was significantly higher for patients who received 2-CdA than those for patients who were treated with INF-α and splenectomy (p=0.01 and p<0.0001, respectively). However, PFS did not statistically differ between patients who were treated with INF-α and those who underwent splenectomy (p=0.213) (Figure 4A). There was no statistically significant OS difference between these 3 treatment modalities, although survival rates achieved with first-line 2-CdA and INF-α seemed to be superior to those achieved with splenectomy (Figure 4B).

Figure 3. The progression-free survival (A) and overall survival (B) of the entire cohort. 2-CdA: Cladribine, INF-α: interferon-alpha.

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Table 3. The distribution of infections among the patient cohort. Type of infection

Number of events, n

Timing of infection, n

Type of treatment*

Prior to treatment initiation

During/after treatment

Outcome of infection (resolved/ death), n

Viral (n=10) Flu Herpes labialis HBV

6 2 1

5 0 0

1 2 1

2-CdA (n=1) 2-CdA (n=1) IFN (n=1)

HCV

1

0

1

2-CdA (n=1) IFN (n=1) [SPL, n=1]

Bacterial (n=64) Pneumonia

13

7

6

2-CdA (n=4) [IFN, n=1] IFN (n=2) [SPL, n=2]

13/0

Tuberculosis

7

2

5

IFN (n=2) [SPL, n=1] 2-CdA (n=2) RTX (n=1)

7/0

Febrile neutropenia

21

3

18

2-CdA (n=15) [IFN, n=3; RTX, n=1] IFN (n=2) [SPL, n=1] RTX (n=1)

20/1

Soft tissue infection

6

1

5

2-CdA (n=1) [RTX, n=1] IFN (n=4) [SPL, n=2]

6/0

Dental infection

3

0

3

IFN (n=1) [SPL, n=1] 2-CdA (n=2) [IFN, n=1]

3/0

Paronychia

2

0

2

IFN (n=2) [SPL, n=1]

2/0

Sepsis

4

0

4

2-CdA (n=3) [IFN, n=1] IFN (n=1) [SPL, n=1]

3/1 (2-CdA)

Conjunctivitis

2

1

1

2-CdA (n=1)

2/0

Septic arthritis

1

0

1

IFN (n=1)

1/0

Meningitis

1

0

1

IFN (n=1)

1/0

Urinary tract infection

3

0

3

3/0

Splenic abscess

1

0

1

2-CdA (n=2) [RTX, n=1] RTX (n=1) IFN (n=1)

Fungal (n=2) Invasive pulmonary aspergillosis

2

0

2

IFN (n=1) 2-CdA (n=1)

2/1 (IFN)

10/0

1/0

*In this column, the number of patients who received the defined most recent therapies is shown in parentheses, whereas the number of patients with a previous anti-HCL therapy is displayed in square brackets. 2-CdA: Cladribine, HBV: hepatitis B virus, HCV: hepatitis C virus, INF-α: interferon-alpha, SPL: splenectomy, HCL: hairy cell leukemia.

Discussion In this study we retrospectively evaluated the demographic features of patients with HCL and assessed the efficacy and tolerability of the main first-line treatments together with the subsequent therapy options and outcomes. We also identified the infections associated with the course of the disease. HCL 296

occurs more frequently in males, and the median age of diagnosis is 52 years [14]. Our patient cohort showed characteristics that were similar to the previously reported literature with a median age of 49 years and a male/female ratio of approximately 7:1. In our cohort, we identified peripheral and/or visceral lymphadenopathy in 27 patients (38%). In the most recent


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consensus guidelines for the diagnosis/management of patients with classic HCL, performing imaging studies for the detection of, e.g., lymphadenopathy was optional [15]. It was recommended that these procedures be reserved for patients in a clinical trial or those with associated symptoms referable to these systems [15]. We did not routinely perform imaging studies for all of our patients, but rather only for patients with signs/symptoms for which ultrasonography and/or computerized tomography scanning were indicated. Furthermore, patients with HCL may develop infections (e.g., tuberculosis) or secondary primary tumors [16], where lymphadenopathy could be observed in the course of the disease. Most probably, in some of the patients, these enlarged lymph nodes are reactive or are due to other conditions, including infections. After the introduction of the purine analogues, the treatment algorithm of HCL evolved greatly [5]. Before that, splenectomy and IFN-α were the mainstays of the treatment [3,4]. The management of HCL in Turkey also changed from the first years of the 2000s onwards with the availability of 2-CdA in the country, and prior to that time, patients with HCL were receiving mainly INF-α and splenectomy upfront. Thereafter, 2-CdA became the standard choice of treatment for most patients with HCL. Earlier data indicate that patients who were treated with first-line splenectomy were usually younger than patients receiving IFN-α. Both splenectomy and IFN-α have been associated with favorable clinical and hematologic responses. However, the median survival with these treatment modalities was approximately 4 years [3,4]. These earlier findings were confirmed by our results indicating high but not durable ORRs with first-line splenectomy and IFN-α. We observed relapse rates as high as 70% and 50% in patients treated with splenectomy and IFN-α, respectively.

Öngören Ş, et al: First-Line Treatment in Patients with HCL

With the upfront-usage of 2-CdA more responses have been reported to be higher and durable. In line with the literature, we only noted 16% R/R disease and a median treatment-free period of 23 months in our patients treated upfront with 2-CdA. This was quite similar to what was reported by Saven et al. [17], demonstrating a relapse rate of 26% after a median follow-up of 29 months. However, with longer follow-up, relapse rates tended to rise to 40% among patients who received 2-CdA upfront [18]. Twenty-seven patients of our cohort (41%) had to be given a second-line treatment for R/R disease after a median TTNT of approximately 1 year. This was consistent with the findings of Zinzani et al. [19], who found that nearly 44% of patients relapsed after a median of 2.7 years. In our cohort the median TTNT was shorter than that observed in the cohort of Zinzani et al. [19], and most probably the reason for this was the higher percentage of upfront purine analogue usage (85/121, 70%) among their patients than ours (31/66, 47%). As expected, patients receiving first-line 2-CdA had significantly lower relapse rates than those treated with INF-α and splenectomy. Second-line 2-CdA treatment in our cohort of patients resulted in excellent response rates (ORR=100%). Relapse was observed in one patient only, who also received second-line 2-CdA. Retreating relapsed patients with an additional course of purine analogues is a reasonable option. Zinzani et al. [19] recommended repeating the same treatment regimen with purine analogues in relapse settings, although changing to a different purine analogue might yield a better result. Unfortunately, 2-CdA is the only approved and available purine analogue in Turkey, so we did not have the opportunity of using other purine analogues like pentostatin in patients who relapsed after 2-CdA.

Figure 4. The progression-free survival (A) and overall survival (B) when patients were divided into 3 groups according to the first-line treatment. *2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy. 2-CdA: Cladribine, INF-α: interferon-alpha.

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Although the PFS rate with 2-CdA was significantly higher than those with INF-α and splenectomy, we found similar OS rates with all three upfront treatment modalities. Most probably one of the reasons for this is the relatively short follow-up duration of our study. In addition to that, since most of the patients with HCL may relapse during follow-up, sequentially re-challenging the previous successful treatment(s) as well as the administration of alternative effective agents might have a positive impact on OS. In our cohort, switching to other potent therapies at relapse could be another reasonable explanation for the comparable OS rates between these 3 first-line treatment groups. A median follow-up duration of 57 months might not be enough to show OS benefit in patients with chronic leukemias such as HCL. After four lines of treatments with a median follow-up of approximately 6 years, 10 patients died and 2 were lost to follow-up, giving an OS rate of 83%, which was consistent with the OS rate of 87% that was reported in the article by Zinzani et al. [19]. One of the most important clinical problems in patients with HCL is the development of severe and sometimes lifethreatening infections [20]. Gram-positive and gram-negative organisms, Aspergillus, and other fungi are the most common pathogens [21], but tuberculosis [22] and herpes zoster [23] can be observed, as well. In our patient cohort, by far bacterial and fungal infections were the most common, and 3 patients died due to severe bacterial infection, sepsis, and IPA. We also had 6 patients with 7 episodes of tuberculosis infection. Two patients had tuberculosis and HCL at diagnosis, and 5 episodes occurred during/after anti-HCL treatment. Tuberculosis is an important issue since it can lead to the misdiagnosis of patients for HCL relapse [22]. Thus, when a patient with HCL presents with fever, tuberculosis should always be kept in mind, especially where tuberculosis is endemic. We had 2 patients with herpes labialis and 1 patient with influenza A virus subtype H1N1 infection, which has also been documented in earlier reports on patients with HCL [24].

Conclusion In conclusion, with the introduction of the purine analogues, the treatment of HCL has been greatly changed. Among our patient cohort, although ORRs were comparable for first-line 2-CdA, INF-α, and splenectomy, patients with frontline 2-CdA had superior ORRs with more durable responses with a higher PFS rate than splenectomy and INF-α cases. The OS rate of the entire cohort was consistent with the current literature. Infections including tuberculosis were a major problem, which caused morbidity and mortality. Although purine analogues improved the CR rates and PFS, there is still much progress to be made with regard to OS and R/R disease. In that sense, the addition of monoclonal antibodies 298

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to purine analogues or incorporation of new target-oriented therapeutic agents such as BRAF, Bruton’s tyrosine kinase, or phosphoinositide 3-kinase inhibitors into treatment regimens might help change the prognosis of the disease further, especially for younger patients and for those who would poorly tolerate the current therapy options. Ethics Ethics Committee Approval: Since the study has a retrospective design, Ethics Committee approval is not needed. Informed Consent: Not applicable. Authorship Contributions Surgical and Medical Practices: Ş.Ö., A.E.E., S.B., T.E., A.S., M.C.A., Z.B., Y.A., N.T., T.S.; Concept:  Ş.Ö., A.E.E.; Design:  Ş.Ö., A.E.E.; Data Collection or Processing:  Ş.Ö., A.E.E., S.B.; Analysis or Interpretation:  Ş.Ö., A.E.E.; Literature Search:  Ş.Ö., A.E.E.; Writing: Ş.Ö., A.E.E. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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Saven A, Varettoni M, Altman JK, Anastasia A, Grever MR, Ambrosetti A, Rai KR, Fraticelli V, Lacouture ME, Carella AM, Levine RL, Leoni P, Rambaldi A, Falzetti F, Ascani S, Capponi M, Martelli MP, Park CY, Pileri SA, Rosen N, Foà R, Berger MF, Zinzani PL, Abdel-Wahab O, Falini B, Tallman MS. Targeting mutant BRAF in relapsed or refractory hairy-cell leukemia. N Engl J Med 2015;373:1733-1747. 12. [No authors listed.] Consensus resolution: proposed criteria for evaluation of response to treatment in hairy cell leukemia. Leukemia 1987;1:405. 13. Kaplan E, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958;53:457-481. 14. Cannon T, Mobarek D, Wegge J, Tabbara IA. Hairy cell leukemia: current concepts. Cancer Invest 2008;26:860-865. 15. Grever MR, Abdel-Wahab O, Andritsos LA, Banerji V, Barrientos J, Blachly JS, Call TG, Catovsky D, Dearden C, Demeter J, Else M, Forconi F, Gozzetti A, Ho AD, Johnston JB, Jones J, Juliusson G, Kraut E, Kreitman RJ, Larratt L, Lauria F, Lozanski G, Montserrat E, Parikh SA, Park JH, Polliack A, Quest GR, Rai KR, Ravandi F, Robak T, Saven A, Seymour JF, Tadmor T, Tallman MS, Tam C, Tiacci E, Troussard X, Zent CS, Zenz T, Zinzani PL, Falini B. Consensus guidelines for the diagnosis and management of patients with classic hairy cell leukemia. Blood 2017;129:553-560. 16. Aydin SO, Eskazan AE, Aki H, Ozguroglu M, Baslar Z, Soysal T. Synchronous detection of hairy cell leukemia and HIV-negative Kaposi’s sarcoma of the

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lymph node: a diagnostic challenge and a rare coincidence. Case Rep Oncol 2011;4:439-444. 17. Saven A, Burian C, Koziol JA, Piro LD. Long-term follow-up of patients with hairy cell leukemia after cladribine treatment. Blood 1998;92:1918-1926. 18. Grever MR, Zinzani PL. Long-term follow-up studies in hairy cell leukemia. Leuk Lymphoma 2009;50(Suppl 1):23-26. 19. Zinzani PL, Pellegrini C, Stefoni V, Derenzini E, Gandolfi L, Broccoli A, Argnani L, Quirini F, Pileri S, Baccarani M. Hairy cell leukemia: evaluation of the long-term outcome in 121 patients. Cancer 2010;116:4788-4792. 20. Kraut E. Infectious complications in hairy cell leukemia. Leuk Lymphoma 2011;52(Suppl 2):50-52. 21. Golomb HM, Hadad LJ. Infectious complications in 127 patients with hairy cell leukemia. Am J Hematol 1984;16:393-401. 22. Gogia A, Raina V, Gupta R. Disseminated tuberculosis mimicking relapse in hairy cell leukemia. Indian J Cancer 2013;50:321. 23. Cheson BD, Sorensen JM, Vena DA, Montello MJ, Barrett JA, Damasio E, Tallman M, Annino L, Connors J, Coiffier B, Lauria F. Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine via the Group C protocol mechanism of the National Cancer Institute: a report of 979 patients. J Clin Oncol 1998;16:3007-3015. 24. Nicolini A, Perazzo A. Influenza A H1N1 pneumonia in a patient with hairycell leukemia. Monaldi Arch Chest Dis 2010;73:92-94.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0489 Turk J Hematol 2017;34:300-306

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics Normal Karyotipli İran’lı Erişkin Akut Miyeloid Lösemi Hastalarında FMS-Benzeri Tirozin Kinaz 3 (FLT3) ve Nükleofosmin 1 (NPM1): Mutasyon Durumu ile Klinik ve Laboratuvar Karakteristikleri Narges Rezaei1, Nargess Arandi1, Behnaz Valibeigi2, Sezaneh Haghpanah1, Mehdi Khansalar3, Mani Ramzi1 Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Department of Pathology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 3 Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran 1 2

Abstract

Öz Öz

Objective: In this study, we evaluated the frequency of FMS-like tyrosine kinase 3 (FLT3-ITD and FLT3-TKD) and nucleophosmin (NPM1) mutations in Iranian patients with cytogenetically normal acute myeloid leukemia (CN-AML). The clinical and laboratory characteristics were compared between wild-type and mutant cases.

Amaç: Bu çalışmada, İran’lı normal sitogenetikli akut miyeloid lösemi (NS-AML) hastalarında FMS-benzeri tirozin kinaz 3 (FLT3-ITD ve FLT3-TKD) ile nükleofosmin 1 (NPM1) mutasyonlarının sıklığını değerlendirdik. Mutant olmayan (yabanıl-wild-type) ve mutant olgular klinik ve laboratuvar özellikler açısından mukayese edildi.

Materials and Methods: Seventy newly diagnosed de novo AML patients were recruited at the time of diagnosis prior to chemotherapy; among them, 54 had CN-AML. For detecting mutations, the FLT3 and NPM1 genes were amplified by the polymerase chain reaction method, followed by direct sequencing.

Gereç ve Yöntemler: Yetmiş yeni tanı de novo AML hastası kemoterapi uygulanması öncesinde çalısmaya dahil edildi; bunların 54’ü NS-AML idi. Mutasyonları tespit etmek için, FLT3 ve NPM1 genleri polimeraz zincir reaksiyonu ile amplifiye edildi ve bu işlemi direkt dizileme takip etti.

Results: Our results showed that the frequencies of FLT3-ITD, FLT3TKD, and NPM1 mutations in CN-AML patients were 25.9%, 5.9%, and 20.8%, respectively. The most frequent NPM1 mutation type was the type A mutation. The FLT3-ITD mutation was seen more frequently in non-M3 patients compared with M3 patients. No mutation was observed in either the FLT3-TKD or the NPM1 gene in patients in the M3 French-American-British group. There was no significant association between the presence of FLT3-ITD and NPM1 mutations in CN-AML patients (p>0.05). The frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation was higher in CN-AML patients in comparison with AML patients with cytogenetic aberrations, although the differences were not statistically significant (p>0.05). There were no significant differences in mean white blood cell and platelet counts, serum hemoglobin levels, and bone marrow blast percentages between patients with wild-type and mutant FLT3-ITD and NPM1 genes

Bulgular: NS-AML hastalarında FLT3-ITD, FLT3-TKD ve NPM1 mutasyonlarının sıklıkları sırasıyla %25,9; %5,9 ve %20,8 olarak bulunmuştur. En sık gözlenen NPM1 mutasyon tipi, tip A mutasyonuydu. FLT3-ITD mutasyonu M3 hastalarına göre M3-dışı olgularda daha sık görülmekteydi. Fransız-Amerikan-İngiliz M3 grubundaki hastalarda FLT3-TKD veya NPM1 genine ait mutasyon tespit edilmedi. NS-AML hastalarında FLT3-ITD ve NPM1 mutasyonlarının varlığı açısından anlamlı ilişki yoktu (p>0,05). FLT3-ITD, FLT3-TKD ve NPM1 mutasyon sıklığı, her ne kadar istatistiksel olarak anlamlı farklılık saptanmasa da (p>0,05), NS-AML hastalarında sitogenetik aberasyonu olan AML olgularına göre daha fazlaydı. FLT3-ITD ve NPM1 genleri açısından mutant olan ve olmayan hastalarda ortalama lökosit ve trombosit sayıları, serum hemoglobin düzeyleri ve kemik iliği blast yüzdeleri arasında anlamlı farklılık yoktu (p>0,05). Yaş ve cinsiyete göre FLT3ITD veya NPM1 mutasyonlarının sıklıkları açısından farklılık tespit edilmedi (p>0,05).

Address for Correspondence/Yazışma Adresi: Nargess ARANDI, M.D., Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Phone : +98 713 612 22 63 E-mail : arandin@sums.ac.ir ORCID-ID: orcid.org/0000-0001-6489-0979

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Received/Geliş tarihi: December 20, 2016 Accepted/Kabul tarihi: March 10, 2017


Turk J Hematol 2017;34:300-306

(p>0.05). No difference was observed in the frequency of FLT3-ITD or NPM1 mutation regarding age or sex (p>0.05). Conclusion: Given the high stability of NPM1 during the disease course, it can be used in combination with FLT3 as well as other known genetic markers to monitor patients, especially for minimal residual disease detection.

Rezaei N, et al: FLT3 and NPM1 Mutation in Iranian CN-AML Patients

Sonuç: NPM1 hastalık sürecindeki yüksek kararlılığı nedeniyle, özellikle minimal kalıntı hastalık tespiti açısından FLT3 veya diğer bilinen genetik belirteçler ile kombine olarak hastaların izlenmesinde kullanılabilir. Anahtar Sözcükler: Akut miyeloid lösemi, Gen mutasyonu, FLT3, NPM1

Keywords: Acute myeloid leukemia, Gene mutation, FLT3, NPM1

Introduction Acute myeloid leukemia (AML) is the most common hematologic malignancy, characterized by uncontrolled proliferation of hematopoietic stem cells resulting in abnormal accumulation of myeloblasts [1]. Generally, based on the cytogenetic abnormalities, the prognosis of AML patients is categorized into three risk groups: good, intermediate, and poor [2]. However, about 50% of AML patients have the normal cytogenetic feature (CN-AML), which represents a diverse subset of patients who are usually classified into an intermediate risk group [3]. Recently, assessment of molecular abnormalities has proven to be a useful marker for risk stratification of these patients into good and poor risk subgroups [3,4,5,6]. In this regard, somatic mutations of the FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and Wilms’ tumor 1 (WT1) genes have been well studied [3,7,8,9]. FLT3 is a member of the class III receptor tyrosine kinase (RTK) family, normally expressed in early bone marrow precursors and playing an important role in the regulation of hematopoietic cell proliferation and differentiation [10]. Binding of the FLT3 ligand to its receptor recruits and activates several signaling molecules affecting cell proliferation, differentiation, and survival [11]. The FLT3 receptor consists of five extracellular immunoglobulin-like domains (Ig1-Ig5), a transmembrane domain, a juxtamembrane domain (JM), and the two intracellular tyrosine kinase domains (TK1 and TK2) [12,13,14]. FLT3 is one of the most frequently mutated genes as approximately 30% of all AML patients have a mutated form of it [15]. Two types of activating mutations have been identified in the FLT3 gene: internal tandem duplication (FLT3-ITD) of the region between exon 11 and 12 in the JM domain (occurring in 20%-25% of AML patients), and a point mutation at codon 835 of exon 17 in the TK domain (FLT3-TKD, also known as D835Y, and occurring in 5%-7% of AML patients) [8,16]. Both mutations contribute to constitutive activation of the FLT3 receptor [8]. It has been shown that the FLT3-ITD mutation has an inverse correlation with patient survival and can be used as an important poor prognostic factor to predict clinical outcomes in AML patients, especially those with normal karyotypes. However, data on the correlation between FLT3-TKD and AML disease outcome are highly limited [3,4,7,17]. The nucleophosmin gene encodes the NPM1 protein, which functions as a chaperone that shuttles between the nucleus

and cytoplasm [3,5,7,8]. NPM1 regulates different intracellular processes such as transport of preribosomal particles, responses to stress stimuli, DNA repair, centromere duplications, and the activity and stability of tumor suppressor genes like p53 [3]. Mutation within exon 12 of the NPM1 gene, which is the most frequent mutation in AML patients (about 35% in all adult AML patients and 50%-60% of CN-AML cases), results in abnormal expression and localization of the protein within the cytoplasm [3]. The most common NPM1 mutation (type A mutation, occurring in 75%-80% of cases) is the insertion of the TCTG tetranucleotide at position 956-959 in exon 12, but other less common mutations in exon 12 have also been described [18,19]. There are various reports describing that NPM1 mutation is mostly associated with FLT3-ITD mutation and it has been shown that NPM1 can be considered a favorable prognostic marker in the absence of FLT3-ITD mutation [3,4,7,17]. Accordingly, in this study, FLT3 and NPM1 mutations were evaluated in adult Iranian patients with de novo CN-AML and its correlations with clinical and laboratory parameters were also assessed.

Materials and Methods Patient Selection This study included 70 newly diagnosed adult patients with de novo AML who were referred to the Shiraz Namazi Hospital, affiliated to Shiraz University of Medical Sciences, from November 2014 to May 2016. All patients were recruited at the time of diagnosis prior to chemotherapy. AML was diagnosed using morphology, cytochemistry, and immunophenotyping. Clinical and laboratory data, including French-American-British (FAB) subclass, complete blood count, blast percentage, and hemoglobin (Hb) level, were also collected. All patients received standard induction chemotherapy, which consisted of daunorubicin at 45 mg/m2 on days 1 to 3 and cytarabine at 100-200 mg/m2 on days 1 to 7, followed by high doses of a cytarabine-based consolidation phase (cytarabine at mg/m2 3 every 12 h for 3 days, repeated for 2 to 3 cycles). This study was approved by the Ethics Committee of Shiraz University of Medical Sciences and written informed consent was obtained from all the participants. 301


Rezaei N, et al: FLT3 and NPM1 Mutation in Iranian CN-AML Patients

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Cytogenetic Analysis

Detection of the NPM1 Mutation

Karyotypes were analyzed by standard G-banding technique [20]. Chromosomal abnormalities were tested by reverse transcriptase polymerase chain reaction (PCR) for AML1-ETO and CBFB-MYH11. Among the 70 AML patients, 16 had abnormal karyotypes: one patient had inv (16) translocation, one had both t (8;21) and inv (16), 12 had t (15;17), and the remaining two patients had other translocations. The 54 patients who were negative for these chromosomal abnormalities were considered as having CN-AML.

Exon 12 of the NPM1 gene was amplified using specific primer NPM1-F 5’-TTAACTCTCTGGTGGTAGAATGAA-3’ and NPM1-R 5’-CAAGACTATTTGCCATTCCTAAC-3’. The PCR reaction was performed in a similar volume as was used for the FLT3-ITD mutation. PCR conditions included initial denaturation at 95 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 57 °C for 60 s, and 72 °C for 75 s with final extension at 72 °C for 5 min. The PCR products were purified and directly sequenced with reverse primer NPM1-R2 5’-GGCATTTTGGACAACACA-3’ using the ABI Prism 3730XL DNA sequencing analyzer and analyzed by Chromas software (version 2.4.3).

Sample Collection Five milliliters of fresh peripheral blood and/or bone marrow samples was collected in ethylenediaminetetraacetic acidcontaining tubes. DNA was extracted with a DNA extraction kit (GeNet Bio, Korea) and stored at -80 °C. Detection of the FLT3-ITD Mutation For detection of the FLT3-ITD mutation, the JM domain between exons 11 and 12 was amplified using specific forward primer FLT.11F 5’-GCAATTTAGGTATGAAAGCCAGC 3’ and reverse primer FLT.12R 5’-CTTTCAGCATTTTGACGGCAACC-3’. The PCR reaction was performed in a total volume of 50 µL containing 200 ng of genomic DNA, 10X PCR buffer (100 mM Tris-HCl, pH 8.8, 500 mM KCl), 2 mM MgCl2, 200 µM dNTPs, 10 pM of each primer, and 1 U of Taq DNA polymerase. PCR conditions included initial denaturation at 95 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s with a final extension at 72 °C for 5 min. PCR reaction was conducted in a PCR T100 thermocycler (Applied Biosystems, USA). The 329-bp PCR products were run on 3% agarose gel stained with DNA SafeStain Dye and visualized under UV light. Samples with additional longer PCR products were identified as FLT3-ITD+. All mutant samples were verified by direct sequencing using the ABI Prism 3730XL DNA sequencing analyzer. The sequencing results were analyzed by Chromas software (version 2.4.3). Detecting of the FLT3-TKD Mutation For detection of the FLT3-TKD mutation, the specific forward primer FLT.17F 5’-CCGCCAGGAACGTGCTTG-3’ and reverse primer FLT.17R 5’-GCAGCCTCACATTGCCCC-3’ were used. The PCR reaction was performed in a total volume of 15 µL with similar reagents as used for the FLT3-ITD mutation, except for the primers. PCR conditions were also the same, except for the annealing temperature, which was 65 °C for 30 s. The amplification reaction was conducted in a PCR T100 thermocycler (Applied Biosystems). The 119-bp PCR products were then digested with 2 U of EcoRV at 37 °C for 17 h, run on 3% agarose gel stained with DNA SafeStain Dye, and visualized under UV light. The presence of an undigested PCR product was an indication of a mutant sample. 302

Statistical Analysis The statistical analysis of data was done using SPSS 18 (SPSS Inc., USA). For comparison of qualitative data between wildtype and mutant patients, chi-square and Fisher exact tests were performed. Independent sample t-tests and Mann-Whitney U tests were used to compare quantitative data between wildtype and mutant patients. A p-value of less than 0.05 was considered statistically significant.

Results This study included 70 newly diagnosed adult patients with de novo AML (49 males and 21 females, mean age: 47.73±18.64 years, minimum - maximum: 17-87 years). The demographic and laboratory data of all the patients are shown in Table 1. Screening for the Mutation of the FLT3 and NPM1 Genes in CN-AML The chromatograms of FLT3-ITD and NPM1 sequencing are shown in Figure 1. Of all 54 CN-AML patients, 14 (25.9%) had the FLT3-ITD mutation, while 40 (74.1%) had the normal FLT3 gene. In Table 1. Demographic and laboratory data of acute myeloid leukemia patients. Variables

Values

Sex, number Male (%) Female (%)

49 (70%) 21 (30%)

Age, years, mean ± SD

47.73±18.64

Laboratory data, median (minimum - maximum) WBC count (x103/mL)

9 (0.3-164.6)

Platelet count (x103/mL)

49 (7-300)

Serum Hb (g/dL)

8.6 (4.4-13.4)

Blast count (%)

90 (50-99)

WBC: White blood cells, Hb: hemoglobin, SD: standard deviation.


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addition, of the 52 patients genotyped for FLT3-TKD mutation status, 3 (5.9%) were mutant and 48 (94.1%) were normal. One patient had both FLT3-ITD and FLT3-TKD mutations. Of the 53 CN-AML patients genotyped for the NPM1 gene, 11 (20.8%) had NPM1 mutation and 42 (79.2%) had wild-type NPM1. From the 11 patients with mutant NPM1, 8 (72.8%) had type A, 1 (9.1%) had type C, and 1 (9.1%) had type D mutation. One patient (AML-20) had a unique mutation pattern that did not belong to a typical NPM1 mutation type. Of 11 patients with mutated NPM1, 5 (45.5%) were also positive for FLT3-

ITD, while none had FLT3-TKD mutation. Thirty-three patients had the wild-type form of both the FLT3-ITD and NPM1 genes. There was no significant correlation between the presence of the FLT3-ITD mutation and NPM1 mutation in CN-AML patients (p>0.05).

FLT3 and NPM1 Mutations and Different Clinical and Laboratory Parameters in CN-AML The mean white blood cell (WBC) and platelet counts, serum Hb level, and percentage of blasts in the bone marrow were compared between mutant and wild-type groups of CN-AML patients (Table 2). As shown in Table 2, there were no significant differences in mean WBC and platelet counts, serum Hb level, or percentage of blasts in the bone marrow between patients with wild-type and mutant FLT3-ITD and NPM1 genes. Moreover, the mean age of AML patients did not differ between wild-type and mutant patients for the FLT3-ITD and NPM1 mutations (p=0.287 and p=0.387, respectively). No significant differences were observed between male and female patients in cases of FLT3-ITD and NPM1 mutation frequency (p=0.450 and p=0.545, respectively).

FLT3 and NPM1 Mutation in AML Patients with Different FAB Groups and Cytogenetic Aberrations Of 70 de novo AML patients, 17 had FLT3-ITD, 3 had FLT3-TKD, and 12 had NPM1 mutations. The frequencies of these mutations in patients with different cytogenetic abnormalities are shown in Table 3. Although the frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation was higher in CN-AML patients in comparison with AML patients with cytogenetic aberrations, the differences were not statistically significant (p>0.05, data not shown).

Figure 1. Sequencing results for FLT3-ITD and NPM1 mutation: A and B are representative of patients with wild-type and mutant FLT3-ITD gene, respectively. C and D are representative of patients with wild-type and mutant NPM1 gene, respectively. The arrows show the mutation site.

Since the AML subtypes of some patients were not defined, AML patients were divided into M3 and non-M3 groups according to the FAB classification. As a result, 12 (17.1%) were M3 and 58 (82.9%) were non-M3. The FLT3 and NPM1 mutation status was analyzed in AML patients according to FAB groups. The results showed that there were no differences between the mutation status of the FLT3-ITD, FLT3-TKD, and NPM1 genes in the M3 and non-M3 FAB subtypes (Table 4). No mutation was observed in either FLT3-TKD or NPM1 genes in patients of the

Table 2. Comparison of baseline characteristics between wild-type and mutant groups. FLT3-ITD

p-value

NPM1

p-value

Clinical characteristics

(-)

(+)

(-)

(+)

WBC count (x10 /mL)

23.74±36.21

33.19±45.4

0.538

21.44±34.65

42.45±48.07

0.116

Platelets (x103/mL)

80.14±68.3

65.5±46

0.911

76.81±66.54

73±49.28

0.861

Serum Hb (g/dL)

8.69±2.02

8.92±2.02

0.719

8.6±1.75

9.31±2.74

0.429

Blast count (%)

83.17±9.36

82.64±13.22

0.912

81.59±11.9

86.67±8.16

0.347

3

WBC: White blood cells, Hb: hemoglobin.

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Turk J Hematol 2017;34:300-306

Table 3. The frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutations in acute myeloid leukemia patients with different cytogenetic statuses. Mutation type

FLT3-ITD (+) (n=17)

FLT3-TKD (+) (n=3)

NPM1 (+) (n=12)

Normal cytogenetics

14

3

11

t(8;21)

-

-

-

t(15;17)

3

-

-

inv(16)

-

-

-

t(8;21) + inv(16)

-

-

1

Other mutation

-

-

-

Table 4. FLT3 and NPM1 mutation status in different French-American-British groups.

FLT3-ITD

p-value

FLT3-TKD

p-value

NPM1

p-value

FAB subtypes

(-) (%)

(+) (%)

(-) (%)

(+) (%)

(-) (%)

(+) (%)

Non-M3

44 (83%)

14 (82.4%)

0.604

52 (81.3%)

3 (100%)

0.548

45 (79%)

12 (100%)

0.080

M3

9 (17%)

3 (17.6%)

12 (18.7%)

0 (0%)

12 (21%)

0 (0%)

FAB: French-American-British.

M3 FAB group. The FLT3-ITD mutation was more frequent in non-M3 patients compared to M3 patients (82.4% vs. 17.6%, respectively; Table 4).

Discussion Genetic abnormalities are one of the most common features observed in AML patients, of which genetic variations of the FLT3, NPM1, DNMT3A, IDH1/2, and WT1 genes have been given more attention [3,7]. In the current study, we analyzed the frequency of FLT3 and NPM1 mutation in 54 adult de novo AML patients with normal karyotypes (CN-AML). The results showed that the frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutations was 25.9%, 5.9%, and 20.8%, respectively. The most frequent NPM1 mutant type was the type A mutation. Our results are consistent with previous studies that described the FLT3-ITD mutation in 25%-35%, FLT3-TKD mutation in 7%-10%, and NPM1 in 50%-60% of CNAML cases [7,21]. In a study of 39 CN-AML patients by Aly et al. [22], the frequency of FLT3-ITD was reported to be 15.4%, while Fröhling et al. [23] and Kainz et al. [24] found that the frequency of FLT3-ITD was 32% and 30% in CN-AML patients, respectively. In addition, Falini et al. [18] showed that the frequency of NPM1 mutation was 61.7%, while different mutation rates were reported by Zhang et al. [25] (14.3%), Döhner et al. [26] (48.3%), and Boissel et al. [27] (47%). The discrepancy in the frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation between our study and others may be due to different population groups as well as the number of cases in the abovementioned studies. Consistent with previous reports, our results also demonstrated that the frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation was higher in CN-AML patients in comparison with AML patients with cytogenetic aberrations [3,7,28]. 304

No mutation was detected in the FLT3-TKD or NPM1 gene in patients in the M3 FAB group. FLT3-ITD mutation was more frequent in non-M3 patients compared to M3 ones. Consistent with our results, Falini et al. [18], Thiede et al. [19], and Suzuki et al. [29] reported no NPM1 mutation in the M3 subtype. In addition, Verhaak et al. [30] reported a lower frequency of NPM1 mutation in M3 and M0 in comparison with other subgroups. Therefore, it seems that both FLT3 and NPM1 mutations are generally mostly seen in AML patients with normal cytogenetics. Evaluation of the clinical characteristics of the patients revealed that there were no significant differences in mean WBC and platelet counts, serum Hb level, or bone marrow blast percentage between patients with wild-type and mutant FLT3-ITD and NPM1 genes. No difference was observed in the frequency of FLT3-ITD or NPM1 mutation regarding age or sex. Consistent with our findings, Dehbi et al. [31] reported no significant association between FLT3-ITD mutation and WBC and platelet counts or blast percentage. Bao et al. [32] also did not observe any differences in FLT3-ITD mutation frequency according to age or sex. However, higher WBC counts and increased blast percentages in FLT3-ITD-positive patients were reported by Fröhling et al. [23]. Moreover, Haferlach et al. [33] showed a strong association of bone marrow blast percentage with NPM1 and FLT3-ITD mutations. Gale et al. [28] and Döhner et al. [26] reported that a significant correlation existed between the presence of the FLT3-ITD mutation and the NPM1 mutation. However, there was no significant correlation between the concomitant mutation of both the FLT3-ITD and the NPM1 gene in our study, which might be due to the different sample sizes and also the type of AML (CN-AML in our study and unselected AML patients in the study by Gale et al. [28]). It has been demonstrated that the FLT3-ITD mutation promotes constitutive activation of the FLT3 receptor, leading to ligand-


Turk J Hematol 2017;34:300-306

Rezaei N, et al: FLT3 and NPM1 Mutation in Iranian CN-AML Patients

independent cell stimulation and subsequent uncontrolled proliferation of leukemic blasts [3,8]. Mutation in exon 12 of NPM1 leads to aberrant cytoplasmic accumulation of the NPM1, which might contribute to leukemogenesis [21]. Association of the mutation in both of these genes with clinical outcome has been shown in various studies; NPM1 has been shown to be associated with good prognosis, especially in the absence of the FLT3-ITD mutation, while FLT3-ITD has been independently considered as a worse prognostic factor that significantly reduces patients’ survival [22,26,28,30,34,35].

of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

According to our findings, the higher incidence of both the FLT3 and the NPM1 mutation in CN-AML patients underscores that both FLT3 and NPM1 can be used as candidate genetic markers for predicting the prognosis of CN-AML patients. In line with these genes, other known prognostic genetic markers like the DNMT3A and IDH genes should be considered, which are under further investigation by our group. Due to time limitations, it was not possible to follow our patients for a longer period of time in order to conduct survival analysis. However, further screening of patients for FLT3 and NPM1 mutations could be useful to verify the clinical significance of these genes for AML population prognosis, and especially for assessment of the presence of the remaining clones as minimal residual disease. In this regard, the value of increasing the number of patients in the studied population should be taken into account.

2. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. Blood 1998;92:2322-2333.

Conclusion

9. Toogeh G, Ramzi M, Faranoush M, Amirizadeh N, Haghpanah S, Moghadam M, Cohan N. Prevalence and prognostic impact of Wilms’ tumor 1 (WT1) gene, including SNP rs16754 in cytogenetically normal acute myeloblastic leukemia (CN-AML): an Iranian experience. Clin Lymphoma Myeloma Leuk 2016;16:21-26.

In conclusion, given the high stability of NPM1 during the disease course, it can be used in combination with FLT3 as well as other known genetic markers to monitor Iranian CN-AML patients, especially for minimal residual disease detection. Acknowledgments The authors wish to thank the Research Consultation Center for its editorial assistance. This study was financially supported with funds provided by Shiraz University of Medical Sciences, Grant Number 93-01-32-8647. Ethics Ethics Committee Approval: This study was approved by the Ethics Committee of Shiraz University of Medical Sciences. Informed Consent: Written informed consent was obtained from all the participants. Authorship Contributions Surgical and Medical Practices: M.R.; Concept: N.A.; Design: N.A.; Data Collection or Processing:  N.R., B.V., M.K.; Analysis or Interpretation: S.H.; Literature Search: N.R.; Writing: N.A. Conflict of Interest: The authors of this paper have no conflicts

Financial Disclosure: This study was financially supported with funds provided by Shiraz University of Medical Sciences, Grant Number 93-01-32-8647.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0341 Turk J Hematol 2017;34:307-313

Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia Akut Miyeloid Lösemi Hastalarında Karbonik Anhidraz I ve II Otoantikorları Ahmet Menteşe1, Nergiz Erkut2, Selim Demir3, Serap Özer Yaman4, Ayşegül Sümer5, Şeniz Doğramacı4, Ahmet Alver4,6, Mehmet Sönmez2 Karadeniz Technical University Vocational School of Health Sciences, Program of Medical Laboratory Techniques, Trabzon, Turkey Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey 3 Karadeniz Technical University Faculty of Health Sciences, Department of Nutrition and Dietetics, Trabzon, Turkey 4 Karadeniz Technical University Faculty of Medicine, Department of Medical Biochemistry, Trabzon, Turkey 5 Recep Tayyip Erdoğan University Faculty of Health Services, Department of Nursing, Rize, Turkey 6 Recep Tayyip Erdoğan University Faculty of Medicine, Department of Medical Biochemistry, Rize, Turkey 1 2

Abstract

Öz

Objective: Cancer, one of the principal causes of death, is a global social health problem. Autoantibodies developed against the organism’s self-antigens are detected in the sera of subjects with cancer. In recent years carbonic anhydrase (CA) I and II autoantibodies have been shown in some autoimmune diseases and carcinomas, but the mechanisms underlying this immune response have not yet been explained. The aim of this study was to evaluate CA I and II autoantibodies in patients with acute myeloid leukemia (AML) and to provide a novel perspective regarding the autoimmune basis of the disease.

Amaç: Kanser, dünyadaki başlıca ölüm nedenlerinden birisi olup, küresel bir toplum sağlığı sorunudur. Organizmanın kendi antijenlerine karşı gelişen otoantikorlar pek çok kanser hastasının serumunda tespit edilmiştir. Son yıllarda karbonik anhidraz (KA) I ve II otoantikorlarının varlığı bazı otoimmün hastalıklarda ve kanser türlerinde gösterilmiştir, ancak bu immün yanıtın altında yatan mekanizmalar henüz açıklanabilmiş değildir. Bu çalışmanın amacı, akut miyeloid lösemili (AML) kişilerde, KA I ve II otoantikorlarının varlığını değerlendirmek ve hastalığın otoimmün temeline dair yeni bir bakış açısı sağlamaktır.

Materials and Methods: Anti-CA I and II antibody levels were investigated using ELISA in serum samples from 30 patients with AML and 30 healthy peers.

Gereç ve Yöntemler: Otuz hasta ve 30 sağlıklı kontrolden elde edilen serum örneklerinde anti-KA I ve II antikor düzeyleri ELISA yöntemiyle belirlendi.

Results: Anti-CA I and II antibody titers in the AML group were significantly higher compared with the control group (p=0.0001 and 0.018, respectively). A strong positive correlation was also determined between titers of anti-CA I and II antibodies (r=0.613, p=0.0001).

Bulgular: AML grubundaki anti-KA I ve II antikor düzeyleri kontrol grubu (p= sırasıyla 0,0001 ve 0,018) ile karşılaştırıldığında anlamlı derecede yüksek bulundu. Ayrıca KA I ve II otoantikor seviyeleri arasında güçlü bir pozitif korelasyon saptandı (r=0,613; p=0,0001).

Conclusion: Our results suggest that these autoantibodies may be involved in the pathogenesis of AML. More extensive studies are now needed to reveal the entire mechanism.

Sonuç: Elde edilen sonuçlar bu otoantikorların AML patogenezinde rolü olabileceğini düşündürmektedir. Kesin mekanizmayı ortaya çıkarabilmek için daha kapsamlı çalışmalar gereklidir.

Keywords: Acute myeloid leukemia, Autoantibody, Cancer, Carbonic anhydrase

Anahtar Sözcükler: Akut miyeloid lösemi, Otoantikor, Kanser, Karbonik anhidraz

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Ahmet MENTEŞE, PhD, Karadeniz Technical University Vocational School of Health Sciences, Program of Medical Laboratory Techniques, Trabzon, Turkey Phone : + 90 462 377 78 76 E-mail : amentese028@gmail.com ORCID-ID: orcid.org/0000-0003-2036-5317

Received/Geliş tarihi: August 22, 2016 Accepted/Kabul tarihi: February 28, 2017

307


Menteşe A, et al: Serum Anti-Carbonic Anhydrase Antibodies in AML

Introduction Cancer is the second most important cause of mortality and a major public health problem worldwide [1]. Acute myeloid leukemia (AML) is a complex and particularly heterogeneous clonal disease involving arrest of differentiation in the myeloid lineage along with deposition of immature progenitors in bone marrow, thus concluding in hematopoietic failure [2].  The pathogenesis of AML involves various disorders, such as mutations in transcription factors or epigenetic modifiers, aberrant signaling pathways, excessive expression of the gene involved in multidrug resistance, abnormal immune function, and abnormalities in the bone marrow microenvironment [3].  Malignant diseases progress with the stimulation of autoimmunity, characterized by the formation of antibodies against their own antigens. Autoantibodies can be observed in the sera of patients with solid tumors and  hematological malignancies [4,5]. These autoantibodies are regarded as early biomarkers for some types of cancer [6,7,8]. Carbonic anhydrases (CAs) are vitally important enzymes responsible for the regulation of acid-base homeostasis in both healthy and pathological conditions. Members of the CA family contain  16 isoenzymes that differ from one another in terms of tissue distribution, cell localization, catalytic activity, and resistance to inhibitors.  They perform several  functions, such as transport of carbon dioxide, pH regulation, ion transport, formation of stomach acidity, bone resorption, calcification, and tumorigenesis  during cancer cell development and invasion [9,10]. CA I and II are both cytosolic enzymes present in significant numbers in erythrocytes. CA I is the second most plentiful protein in erythrocytes after hemoglobin. CA II is a highly active isoenzyme involved in much total CA activity in a number of tissues.  CA I and/or II autoantibodies have recently been demonstrated in various pathological conditions, such as autoimmune diseases (systemic lupus erythematosus, primary biliary cirrhosis, rheumatoid arthritis, and Sjögren’s syndrome) and carcinomas (lung, colon, and prostate). However, the mechanisms underlying this immune response have not yet been explained [11,12,13,14]. The purpose of this study was to investigate CA I and II autoantibodies in patients with AML and to provide a novel perspective regarding the autoimmune basis of the disease.

Materials and Methods Study Group Informed consent was obtained from all patients and controls. Approval for the study was granted by the local ethics committee. Thirty  patients newly diagnosed with AML were included as the study group and 30 healthy peers as the control group. Diagnosis of AML was made and verified by a panel of 308

Turk J Hematol 2017;34:307-313

hematologists who also classified each case according to the French-American-British (FAB) classification [15]. The subtypes of AML according to FAB were as follows: M0: 1 (3.3%); M1: 1 (3.3%); M2: 13 (43.3%); M3: 3 (10%); M4: 9 (30%); M5: 2 (6.6%); M6: 1 (3.3%). Patients were selected from individuals presenting to the hematology clinic and referred from other practitioners. The study group consisted of 17 women and 13 men with a mean age of 52.8±6.3 years, and the control group of 17 women and 13 men with a mean age of 51.9±14.1. Patients with renal, coronary, or liver failure and chronic inflammatory diseases or anemia, and subjects receiving chemotherapy or using oral contraceptives and anticoagulants, were excluded from the study. Blood samples of 5 mL from each individual were placed into vacutainer tubes without anticoagulant. These were then centrifuged at 1800xg for 10 min. Serum samples were stored at -80 °C until being used for measurements. Platelet (PLT), hemoglobin (Hb), hematocrit (Hct), and white blood cell (WBC) levels were determined with a Beckman Coulter autoanalyzer. Determination of Serum Autoantibody to CA I and II ELISA is frequently used to detect autoantibodies in blood samples since it is economical, simple, and quick to perform [16,17]. It has also been widely used for the evaluation of CA I and II autoantibodies in different pathological conditions in previous reports [12,13,18,19,20,21]. Serum CA I and II autoantibodies were therefore determined using the ELISA method as previously described elsewhere [18]. Briefly, flat-bottomed plates were coated with CA I or II (10 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) in carbonate buffer (pH 9.6). These were then incubated for 18 h at 4 °C. In the next stage, the wells were washed four times with phosphate buffer (PBS) (pH 7) before being blocked with 3% skim milk in PBS at room temperature for 2 h. The wells were next washed again four times with PBS containing 0.05% Tween-20 before incubation with 100 µL of 1:200 diluted serum for 2 h. Following these washing procedures, each individual well was incubated for 2 h with 100 µL of a 1:2000 solution of peroxidase-conjugated anti-human IgG anti-serum (SigmaAldrich, St. Louis, MO, USA) in 3% skim milk in PBS. A further five washes were performed with PBS containing 0.05% Tween-20, and the wells were then incubated with 100 µL of substrate solution for 20 min. Reactions were halted by adding 100 µL of 2 M sulfuric acid to each well. The resulting absorbance was measured at 480 nm (Molecular Devices, Sunnyvale, CA, USA). Control wells containing no CA I or II were also employed for ELISA investigation of each serum studied. All assays were performed in duplicate. The specific binding of serum antibody to CA II was calculated as the mean absorbance of the antigencoated wells minus the mean absorbance of the control wells. The results were expressed as absorbance units.


Menteşe A, et al: Serum Anti-Carbonic Anhydrase Antibodies in AML

Turk J Hematol 2017;34:307-313

Statistical Analysis Data are shown as mean ± standard deviation for normally distributed and median (interquartile range) for non-normally distributed variables. Statistical analysis was performed with SPSS 13.0 (Chicago, IL, USA) and MedCalc (Version 12.3, Mariakerke, Belgium) statistical software. Compatibility with normal distribution was determined using the KolmogorovSmirnov test. Differences between the two groups were analyzed using Student’s t-test for normally distributed data. Correlation analysis was calculated using Pearson’s correlation coefficient and the nonparametric equivalent Spearman’s rank correlation coefficient at a 95% confidence interval. Receiver operating characteristic (ROC) curves were used to detect the discriminatory dominance of CA I and II autoantibodies for the identification of AML. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were determined from ROC graphs for autoantibodies of CA I and II. p<0.05 was regarded as significant.

value of anti-CA I antibody for healthy subjects was 0.092±0.018, and the absorbance was higher than 0.146. Positive results were obtained in 23 of the 30 cases of AML (Figure 1A). The mean absorbance value of the  AML  group was significantly higher (p=0.018) than that of the healthy controls (Table 1). The mean absorbance +3SD of healthy subjects was also positive. The mean absorbance value of anti-CA II antibody for the healthy subjects was 0.079±0.024, and the absorbance was higher than 0.151. Positive results were obtained in 7 of the 30 cases of AML (Figure 1B).  We also observed a strong positive correlation between titers of anti-CA I and II antibodies (r=0.613, p=0.0001). ROC curve analysis was also used to quantify serum Hb, Hct, PLT, WBC, and anti-CA I and II levels. Values for cut-off points, area under the curve, sensitivity, specificity, PPV, and NPV for individual parameters are shown in Table 2 and Figure 2.

Results Thirty AML patients and 30 healthy subjects were included in this study. There was no significant difference in terms of mean age between the study and control groups. Levels of anti-CA I and II antibodies in patients with AML and control subjects are shown in Figures 1A and 1B, respectively. The mean absorbance value of the AML group was significantly higher (p=0.0001) than that of the healthy subjects (Table 1). The mean absorbance +3 standard deviations (SD) of healthy subjects was determined as positive. The mean absorbance

Figure 1. Anti-CA I (A) and anti-CA II (B) antibodies in sera of patients with acute myeloid leukemia and healthy controls. The dotted line indicates plus 3 SD of health control sera (A480=0.146 ABSU for anti-CA I antibody, A480=0.151 for anti-CA II antibody). ABSU: Absorbance units, SD: standard deviation, CA: carbonic anhydrase.

Table 1. Clinical characteristics of the two groups. AML group (n=30)

Control group (n=30)

p-values

Anti-CA I Ab (ABSU)

0.195 (0.148-0.311) 0.269±0.211

0.091 (0.074-0.112) 0.092±0.018

0.0001*

Anti-CA II Ab (ABSU)

0.105 (0.069-0.146) 0.127±0.092

0.077 (0.059-0.096) 0.079±0.024

0.018*

Hemoglobin (g/dL)

9.90±2.96

14.8±0.680

0.0001

Hematocrit (%)

28.4±8.42

44.0±1.33

0.0001

Leukocytes (cells/µL)

62907±52501

7436±1336

0.0001

Platelets (cells/µL)

47000 (16750-78000) 64567±57865

253000 (226000-277500) 256233±42242

0.0001*

Data were expressed as mean±SD, median (interquartile range for 25-75%). p shows differences between the control and AML groups using Student’s t test, *p shows differences between the control and AML groups using the Mann-Whitney U test. ABSU: Absorbance units, AML: acute myeloid leukemia, CA: carbonic anhydrase.

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Table 2. Receiver operator characteristic curve analysis of hemoglobin, hematocrit, platelet, white blood cell, and anti-carbonic anhydrase I and II antibody levels and their sensitivity, specificity, positive predictive value, and negative predictive value. AUC

Cut-off Point

Sensitivity

Specificity

PPV

NPV

Anti-CA I

0.919 (0.819-0.973)

>0.123

80 (61-92)

100 (88-100)

100 (86-100)

83 (67-94)

Anti-CA II

0.658 (0.524-0.775)

>0.097

60 (41-77)

76 (58-90)

72 (51-88)

66 (48-81)

Hb

0.906 (0.803-0.966)

<12

83 (65-94)

100 (88-100)

100 (86-100)

86 (68-95)

Hct

0.923 (0.824-0.976)

<40

90 (73-98)

100 (88-100)

100 (87-100)

91 (76-98)

PLT

0.998 (0.937-1.000)

<198000

100 (88-100)

97 (83-99)

97 (83-100)

100 (88-100)

WBC

0.909 (0.806-0.968)

>8890

87 (69-96)

97 (83-99)

96 (81-99)

88 (72-97)

Sensitivity, specificity, PPV, and NPV values were expressed as % within a 95% confidence interval. ROC: Receiver operator characteristic, PPV: positive predictive value, NPV: negative predictive value, AUC: under the curve, CA: carbonic anhydrase, Hb: hemoglobin, Hct: hematocrit, PLT: platelet, WBC: white blood cell.

Figure 2. ROC curve analysis for all parameters in patients with acute myeloid leukemia. CA: Carbonic anhydrase, Hct: hematocrit, PLT: platelet, WBC: white blood cell.

Discussion Cancer is the second most important cause of mortality, and millions of people either have or have had the disease. An estimated 1.68 million new cancer cases and 595690 deaths from cancer are predicted to have occurred in the United States in 2016. Leukemia is one of the most common forms of cancer [1]. AML is a heterogeneous disease with marked malignancy of hematopoietic progenitor cells committed to the myeloid lineage. This phenomenon is most common in subjects aged over 70, and AML constitutes approximately 30% of all cases of leukemia [1,22]. Several mutated or overexpressed proteins seem to be processed and presented to the immune system as tumor antigens, leading to humoral and/or cellular responses [23]. 310

Autoimmunity is well known to be potentially associated with cancer, and one of the forms of its expression is the development of autoantibodies and eventually autoimmune disease. Detection of autoantibodies may therefore be the first sign of cancer [24]. The ideal tumor biomarker would make it possible to detect cancer with a simple blood test. The serum biomarkers available today are based on the measurement of cancer antigens, such as prostate-specific antigen, carcinoembryonic antigen, the cancer antigens (CA15-3, CA19-9, and CA125), extracellular protein kinase A, anti-oncoprotein (HER-2/neu), anti-tumor suppression antigen (p53), anti-proliferation associated antigens (cyclin A, cyclin B1, and CDKs), anti-onconeural antigens (Hu and Yo), and anti-cancer/testis antigens (NY-ESO-1 and MAGE-1) [4,25]. There has therefore been considerable research in recent years into the identification of new biochemical diagnostic markers for the early detection of AMLs [3,22,26,27]. Analysis of serum autoantibodies may become a useful tool for clinicians in screening for cancer and diagnosis of AML. However, these markers exhibit limited specificity and sensitivity, and their levels can also rise even under benign conditions or during gestation. There is therefore an urgent need for novel biomarkers capable of adoption into routine clinical use in the diagnosis of AML and other cancer types [25]. Autoantibodies are common in cancer patients. The autoantibody response in AML patients has been considered in previous studies, such as Wilms tumor gene product [28], single-stranded DNA [29], anti-cardiolipin antibodies [30], the M-phase phosphoprotein 11 (MPP11) [31], receptor for hyaluronan acid-mediated motility (RHAMM) [32], and RHAMM-like protein [33]. This study is the first report to show an increased autoimmune response to both CA I and II in the sera of AML patients. The prevalence of CA I and II autoantibodies in patients with AML in this study was 76.6% and 23.3%, respectively. The presence of autoantibodies against CA I and II has been observed in many pathological conditions,


Turk J Hematol 2017;34:307-313

such as metabolic syndrome, recurrent pregnancy loss, acute anterior uveitis, gastric cancer, Graves’ disease, preeclampsia, and rheumatoid arthritis. The prevalence of CA I autoantibody is reported in the range of 9.6%-20%, and that of autoantibodies against CA II in the range of 4.6%-72.5% [13,19,20,34,35]. From this perspective, our results were consistent with the literature.  Protection of the acid-base balance is of considerable importance in tumorigenesis. Extracellular hydrogen ion concentrations in solid tumors are reported to be higher than those in normal neighboring tissues [11,36]. Tumor cells express ion transport protein, such as vacuolar-type H+-ATPase, Cl-/HCO3-, and Na+/ H+  exchangers between the inner and outer regions of the cell, thus creating a pH gradient. Many tumor cells synthesize CAs that catalyze the production of H+  and HCO3-  ions [11]. CAs are currently the subject of significant research into carcinogenesis and tumor invasion. Studies have recently shown an incremental  expression  of specific  cytosolic CA I and II in some carcinomas, including leukemia, and  in the blast cells of AML [11,36,37]. CA I and II interact with various molecules due to their cellular localizations, functions, and wide tissue distribution. These proteins are therefore becoming target molecules in the body. Autoantibodies against CA I and II have recently been demonstrated in many pathological conditions, such as cancer, and autoimmune and idiopathic diseases.  Although the mechanisms involved  have not been identified  exactly, oxidative stress has been reported to be potentially significant in the formation of these autoantibodies [13,14,19,21,38,39]. Oxidative stress results from acceleration of the rate of free radical formation and/or a decrease in the rate at which these are eliminated. In either condition a severe imbalance occurs between free radical formation and the antioxidant defense mechanism [40]. Increased reactive oxygen or nitrogen species (ROS) lead to tissue injury and compromise numerous biomolecules, including proteins, nucleic acids, structural carbohydrates, and lipids. The reaction of ROS with lipids causes these molecules to undergo oxidative breakdown. Malondialdehyde (MDA) is a one-end product of lipid peroxidation capable of being covalently bound to proteins, and especially to the Ɛ-amino groups of lysine residues. These oxidative disturbances may influence the immune system, resulting in the development of specific autoimmune processes [41]. The lipid peroxidation end-products 4-hydroxy-2-nonenal (HNE) and MDA are known to alter proteins and to modify their antigenic properties [42]. One study of erythrocytes proved that CA II is the first target of HNE [43]. Numerous anti-MDA-modified proteins have been detected in systemic diseases, such as systemic lupus erythematosus, periarteritis nodosa, scleroderma, atherosclerosis, and rheumatoid arthritis in previous studies. It has also been suggested that these autoantibodies may be of predictive value for systemic diseases [41,44,45,46,47]. Studies in the literature have shown that the levels of such oxidative

Menteşe A, et al: Serum Anti-Carbonic Anhydrase Antibodies in AML

stress parameters as MDA, advanced oxidation protein products, 8-hydroxydeoxyguanosine, and protein carbonyl increase in the sera of patients with AML, while the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and monoamine oxidase decrease [48].  In light of these data, we anticipate that oxidative byproducts, including MDA, might generate the spread of neoantigens and confirm a potential association between autoimmunity and oxidative stress. Study Limitations The major limitation of this study is the relatively small sample size of the patient and control groups.

Conclusion In conclusion, CA I and II autoantibody titers were significantly higher in subjects with AML compared to the controls. More extensive studies are now needed to reveal the entire mechanism involved. Ethics Ethics Committee Approval: Approval for the study was granted by the Karadeniz Technical University Faculty of Medicine Ethics Council under reference no 2016/31. Informed Consent: Informed consent was obtained from all individual participants included in the study. Authorship Contributions Surgical and Medical Practices:  A.M., N.E., A.A., M.S.; Concept: A.M., N.E., A.A., M.S.; Design: A.M., N.E., S.D., A.S.; Data Collection or Processing: A.M., N.E., S.D., S.Ö.Y., Ş.D.; Analysis or Interpretation: A.M., N.E., S.D., A.S.; Literature Search: A.M., N.E., S.Ö.Y., Ş.D.; Writing: A.M., N.E., A.A., M.S., S.D. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. 

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0214 Turk J Hematol 2017;34:314-320

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment İnsan Göbek Kordon Kanı Hematopoetik Kök Hücre Değerlendirmesinde Hızlı ve Etkin Bir Değerlendirme Yöntemi: Akım Sitometrik Aldehit Dehidrogenaz Testi Emine Begüm Gençer1,2 , Pınar Yurdakul1,3 , Klara Dalva4, Meral Beksaç5 Ankara University Faculty of Medicine, Cord Blood Bank, Ankara, Turkey Ankara University Faculty of Medicine, Biotechnology Institute, Ankara, Turkey 3 TOBB Economics Technology and University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey 4 Ankara University Faculty of Medicine, Stem Cell Research Institute, Ankara, Turkey 5 Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey 1 2

Abstract

Öz

Objective: Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for  prediction of  engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method. 

Amaç: Granülositer makrofaj koloni oluşturma (CFU-GM) testi kordon kanı (KK) hematopoietik kök hücre engrafman potensiyelini ölçmek için kullanılan bir yöntemdir. Aldehit dehidrogenaz (ALDH) enzimi ölçüm yöntemide hematopoetik kök hücre (HKH) kalitesini belirlemek amacıyla kullanılan daha yeni bir metottur. Çalışmamızda fenotipik ve fonksiyonel olarak korelasyon analizi yapılarak HKH ölçümünde en etkili metodu bulmayı amaçladık.

Materials and Methods: In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units. 

Gereç ve Yöntemler: Bu çalışmada taze ve donma çözme sonrası KK ünitelerinde CD34+  ve ALDH+  hücrelerle CFU-GM kapasiteleri araştırılmıştır. 

Results: Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 postthaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92) x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/ granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant. 

Bulgular: Otuz taze KK ünitesinde her KK için ortalama değerler: Toplam çekirdekli hücre sayısı (TNC): 93,8±30,1x107, CD34+: 3,85±2,55x106, ALDH+: 3,14±2,55 x106, CFU-GM: 2,64±1,96x105. On dokuz KK ünitesinde donma çözme sonrası hücre değerleri: TNC: 32,79±17,27x107, CD34+: 2,18±3,17x106, ALDH+: 2,01±2,81x106, CFUGM: 0,74±0,92x105’dir. Bulgularımız; taze KK’da TNC, CD34 ve ALDH; CFU-GM, CFU-GEMM ve BFU-E ile korelasyon gösterirken (TNC, r=0,47, r=0,35, r=0,41; CD34+, r=0,44, r=0,54 r=0,41; ve ALDH, r=0,63 r=0,45 r=0,6) donma çözme sonrası KK’da korelasyon sırasıyla CFU-GM, CFU-GEMM, ve BFU-E için, TNC r=0,59, r=0,46, r=0,56, CD34+ r=0,67, r=0,48, r=0,61 ve ALDH r=0,61, r=0,67, r=0,67 olarak saptanmıştır. Bütün bulgularımız istatistiksel olarak anlamlı çıkmıştır.  Sonuç:  Çalışmamız, ALDH aktivitesi tayin metodu HKH tayininde geleneksel yöntemlerle özellikle donma çözme sonrası örnekler açısından korelasyon göstermiştir. Böylelikle hızlı, ucuz bir metod

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Meral BEKSAÇ, M.D., Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey Phone : +90 0312 595 79 88 E-mail : meral.beksac@medicine.ankara.edu.tr ORCID-ID: orcid.org/0000-0003-1797-8657

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Received/Geliş tarihi: June 10, 2016 Accepted/Kabul tarihi: December 01, 2016


Turk J Hematol 2017;34:314-320

Conclusion: In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

olarak ALDH diğer HKH belirlemede kullanılan yöntemlere üstün olabilecek kapasitededir. Anahtar Sözcükler: Göbek kordon kanı, Aldehit dehidrogenaz, Koloni oluşturan birim granülositer/makrofaj

Keywords: Cord blood, Aldehyde dehydrogenase, Colony-forming unit-granulocyte/macrophage

Introduction Recent scientific evidence demonstrates that different subtypes of CD34+ cells in the cord blood (CB) hematopoietic stem cell (HSC) niche have different engraftment potentials [1,2]. It is of crucial importance to determine the quality of the CB particularly following freeze/thaw cycles. Two different approaches can be used to assess the functionality and population-forming capacities of CB HSCs along with the gold standard method of the International Society of Hematotherapy and Graft Engineering (ISHAGE) [3]. Ex vivo colony-forming unit (CFU) assays are the most widely used tests for determining HSC functions, but they possess serious drawbacks such as difficulty in routine application, lack of standardization, labor-intensive nature, and long turnaround time [4]. One of the likely reasons for this is probably the fact that while being predictive of shortterm re-populating cells, CFU assays could not determine longterm populating cells effectively. Long-term populating cells have been shown to provide long-term immune reconstitution after CB transplantation (CBT); thus, it is of crucial importance to assess their numbers. The measurement of aldehyde dehydrogenase (ALDH) activity can therefore be much more accurate due to the intracellular presence of this enzyme [5]. It was reported that ALDH enzyme expression is high in early HSCs in the bone marrow and CB [6,7]. A few published studied correlated high ALDH activity with better permanent engraftment following HSC transplantation [5,7,8,9,10,11]. In the first such study by Lioznov et al. [12], it was reported that ALDH expression is a practical marker to assess HSC activity for both stem and progenitor cells before bone marrow and peripheral blood transplantation. There are hardly any data for CB investigating the phenotypic and functional properties of CB HSCs and the correlation of ALDH activity with CFU potential in pre- and post-thaw CB HSCs [5,7,11,13,14]. In this study, we aimed to correlate phenotypic assays with functional assays to find the most predictive method for fresh and post-thaw CB.

Materials and Methods CB Unit Selection and Processing A total of 50 CB units from consenting maternal donors collected at the Ankara University Faculty of Medicine’s Cord Blood Bank were included in this study. Thirty CB units that met volume and total number of nucleated cell (TNC) eligibility criteria (>70 mL and

100x107/U, respectively) were processed and used immediately for the fresh group and 20 non-conforming CB units that had been reserved for research purposes were included as the post-thaw group (1 unit was discarded due to CFU culture contamination). CB units were processed automatically with a Sepax 2 device (Biosafe) and TNC counts, post-processing CD34+ cell enumeration, and cell viabilities were assessed for every CB unit. Post-Thaw Washing Nineteen non-conforming CB units were thawed in a 37 °C water bath and samples of 10 mL were taken into conical tubes. In order to remove DMSO, CB units were washed using a washing solution (10% dextran 40 (BioFleks), 20% human serum albumin (Centurion Pharma), and PBS (Lonza) at 4:1:3/8 (v/v), respectively). Upon thawing of the CB units they were washed twice with 1:1 (v/v) washing solution After discarding the supernatant, the pellet was re-suspended in washing solution by gentle mixing. Determination of TNC/CD34 Viability and Counts The number of TNCs for all units (30 fresh and 19 post-thaw units) was assessed by complete blood counting with an automated cell counter (Beckman Coulter, LH780). CB unit CD34+ cell enumeration and detection of cell viability by 7-aminoactinomycin dye was performed using a Stem-Kit upon the acquisition of the data with an FC 500 instrument (Beckman Coulter). The analysis was performed using an ISHAGE single test platform. ALDH Analysis The ALDEFLUOR assay (StemCell Technologies) was used for the detection of ALDH expression in fresh and post-thaw CB HSCs. ALDH activity was measured by the protocol recommended by the manufacturer. Briefly, cell suspensions were adjusted to 106 cells/mL with 1500 µL of ALDEFLUOR assay buffer after red blood cell depletion. ALDEFLUOR reagent (10 µL) was added to each tube, followed by 5 min of centrifugation at 300×g. Supernatants were stained with FITC-ALDH, APC A-750-CD38, phycocyanin (PC) 7-CD34, chrome orange-CD45 (Beckman Coulter), PE-CD73, and PC 5-CD90 (Becton Dickinson) antibodies and analyzed by flow cytometry (Beckman Coulter FC500). Diethylaminobenzaldehyde reagent was used to suppress ALDH activity in control tubes. Using the ALDH activity assay, CB HSCs were categorized as ALDH+ and ALDH-. 315


Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

Turk J Hematol 2017;34:314-320

from CB, both after processing and after thawing. We analyzed different cellular fractions, namely TNCs; CD34+, ALDH+, CD34+ ALDH+, ALDH+ CD34+, and ALDH+ CD34+ CD90+ CD38- cells; and colony-forming units of granulocytes/macrophages (CFU-GM), CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E), for both fresh and post-thawed units. Table 1 demonstrates the mean, median, and minimum-maximum values of the aforementioned parameters for fresh and post-thawed samples.

CFU Assays CFU assays were implemented according to the manufacturer’s recommendations (StemCell Technologies CFU Manual, MA28404) and modified from Lee et al. [19]. First, 100 µL of CB sample was removed from all CB units, and after the addition of 80 µL of HetaSep and 300 µL of Iscove’s modified Dulbecco medium containing 2% fetal bovine serum (StemCell Technologies), the mixture was incubated at 37 °C in 5% CO2 for 20 min (Sanyo CO2 Incubator). Cells (5x105 cells/mL) were transferred to 3 mL of MethoCult Express medium. After 14 days, colonies were counted and different morphologies as well as numbers of CFUs were recorded using an inverted light microscope (Olympus/IX51). The number of colonies was calculated as the mean value for two dishes.

Table 2 provides the correlation values for ALDH positivity and TNC, CD34+, and CD34+ CD90+ CD38- cell numbers as well as CFU-GM, CFU-GEMM, and BFU-E colony counts among all CB samples. When fresh samples were analyzed, ALDH activity correlated well with all the cell populations investigated; TNC, ALDH+, and CD34+ fractions were found to be highly correlated both with CFU-GM, CFU-GEMM, and BFU-E and with each other. The correlation coefficients remained significant for fresh and post-thawed samples, and when post-thaw data were analyzed, TNCs, CD34+, and ALDH were also found to be statistically correlated with CFU-GM, CFU-GEMM, and BFU-E (Table 2).

Statistical Analysis Pearson correlation coefficient tests (if data distribution was normal) and Spearman rank correlation coefficient tests (if data distribution was not in the normal range) were used to assess the correlations. All statistical analyses were performed with SPSS 15.0.

Among all parameters compared, the most striking correlation was detected for CFU-GM numbers and ALDH positivity for fresh CB units (r=0.629, p<0.001); post-thaw analyses also revealed a correlation for CFU-GM and ALDH+ cells when the same parameters were investigated (r=0.608, p=0.006; Table 2). When CFU-GM numbers were tested against all parameters for

Results In this study we aimed to compare three different methods in terms of efficiency to assess different re-populating HSCs Table 1. Characteristics of all fresh and post-thaw CB units tested. Variables (fresh CB units, n=30; post-thaw CB units, n=19)

Mean ± SD

Median

Minimum - maximum

TNC (x107/U)

Fresh

93.8±30.1

85.5

46.8-168.8

Post-thaw

32.79±17.27

31.05

9.2-47.6

CD34+ (x106/U)

Fresh

3.85±2.55

3.6

0.94-11.64

Post-thaw

2.18±3.17

1.18

0.5-14.5

ALDH (x106/U)

Fresh

3.14±2.55

2.6

0.12-8.48

Post-thaw

2.01±2.81

1.38

0.20-11.3

ALDH /CD34 (x106/U)

Fresh

2.97±2.02

2.4

0.11-8.12

Post-thaw

1.82±2.63

1.28

0.17-1.69

CD34+/ALDH+ (x106/U)

Fresh

3.72±2.28

2.9

0.34-8.94

Post-thaw

3.01±4.33

1.65

0.29-15.31

ALDH /CD90 /CD34 /CD38(x106/U)

Fresh

0.19±0.19

0.1

0.005-0.795

Post-thaw

0.40±0.55

0.2

0.01-1.92

CFU-GM (x105/U)

Fresh

2.64±1.96

2.14

0.25-7.67

Post-thaw

0.74±0.92

0.41

0.02-2.92

CFU-GEMM (x105/U)

Fresh

3.86±2.73

3.6

0.66-11.49

Post-thaw

0.70±0.98

0.36

0-4.27

BFU-E (x105/U)

Fresh

5.30±3.53

5.6

0.12-16.22

Post-thaw

0.44±0.74

0.19

0-2.9

+

+

+

+

+

+

SD: Standard deviation, CB: cord blood, TNC: total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU-GEMM: colony-forming units - granulocytes-macrophages/ megakaryocytic cells, BFU-E: burst forming units of erythroids, GM: granulocytes/macrophages.

316


+

+

Post-thaw

Post-thaw

Fresh

Post-thaw

Fresh

Post-thaw

Fresh

Fresh

Post-thaw

r=0.541*

r=0.516**

r=0.935**

r=0.799**

r=0.507*

r=0.671**

ALDH+ (x106/U)

r=0.988**

r=0.999**

r=0.931**

r=0.799**

r=0.931**

r=0.786**

r=0.935**

r=0.786**

r=0.971**

r=0.967**

r=0.474*

r=0.432 NS

r=0.556**

CD34+ ALDH+ (x106/U)

r=0.674**

ALDH+ CD34+ (x106/U)

r=0.867**

r=0.431*

r=0.877**

r=0.616**

r=0.868**

r=0.615**

r=0.889**

r=0.446*

r=0.414 NS

r=0.624**

6

(x10 /U)

ALDH+ CD90+  CD 34+ CD38-

r=0.594**

r=0.559**

r=0.670**

r=0.426*

r=0.611**

r=0.623**

r=0.608**

r=0.629**

r=0.665**

r=0.444*

r=0.591**

r=0.476**

CFU-GM (x105/U)

r=0.561*

r=0.567**

r=0.500*

r=0.393*

r=0.594**

r=0.558**

r=0.673**

r=0.435*

r=0.672**

r=0.451*

r=0.476*

r=0.543**

r=0.461*

r=0.354NS

CFU-GEMM (x105/U)

r=0.554*

r=0.398NS

r=0.510*

r=0.683**

r=0.644**

r=0.678**

r=0.610**

r=0.375*

r=0.634**

r=0.586**

r=0.668**

r=0.596**

r=0.610**

r=0.414*

r=0.556*

r=0.580**

(x105/U)

BFU-E

TNC: Total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU: colony-forming units - granulocytes-macrophages/megakaryocytic cells, BFU-E: burst forming units of erythroids, GM: granulocytes/macrophages.

*p<0.05,**p<0.01, NS: p>0.05.

BFU-E (x105/U)

CFU-GEMM (x105/U)

CFU-GM (x105/U)

+

ALDH CD90 CD34 CD38- (x106/U)

CD34+ ALDH+(x106/U)

+

Post-thaw

+

Fresh

Fresh

ALDH CD34 (x10 /U)

Post-thaw

ALDH+ (x106/U)

6

Post-thaw

Fresh

Fresh

(x10 /U)

(x10 /U) 6

CD34+ 7

TNC

CD34+ (x106/U)

TNCs (x107/U)

Variables

Table 2. Correlations between different groups of cells demonstrating hematopoietic activity in fresh and post-thaw cord blood units.

Turk J Hematol 2017;34:314-320 Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

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Turk J Hematol 2017;34:314-320

Figure 1. Correlation values of TNCs, ALDH, and CD34+ cells with CFU-GM and ALDH+ CD34+ cells. Graphs A-D denote correlations for fresh CB units: TNCs and CFU-GM (A); CD34+ cells and CFU-GM (B); ALDH+ cells and CFU-GM (C); CD34+ cells and ALDH+ cells (D). Graphs E-H denote correlations for post-thaw samples: TNCs and CFU-GM (E); CD34+ and CFU-GM (F); ALDH+ and CFU-GM (G); CD34+ cells and ALDH+ cells (H). CB: Cord blood, TNC: total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU-GM: colony-forming units granulocytes/macrophages.

post-thawed samples, an even higher correlation was detected in CD34+ cells, which were also positive for ALDH (r=0.670, p=0.002). On the other hand, there was no significant correlation between TNCs and ALDH+ cells, which were also positive for CD34 (r=0.432, p=0.065). These correlations are shown in Figure 1. Table 2 shows r values for all parameters investigated.

Discussion

Shoulars et al. [11] developed an ALDH-based method to estimate the post-thaw quality of CB units. The results of their study, similar to ours, demonstrated that ALDH activity is highly correlated with CFU counts and can be integrated into routine CB unit release procedures prior to transplantation. Thus, our findings, which show the highest correlation between in vitro CFU counts and ALDH activity compared to TNCs or CD34, are confirmed by this very recent publication.

One of the recent methods described for the rapid and accurate detection of functional CB cell fractions is ALDH activity measurement in CB HSCs. To date, five studies have looked at ALDH levels in CB units [5,7,11,13,14]. Our study is unique in terms of having a detailed post-thaw analysis and to our knowledge it is also the first to determine the capacity of ALDH+ CD90+ CD34+ CD38- cells, a group of cells that possess high engraftment capacity. Most of the papers in the literature have focused on HSCs with high ALDH activity with conflicting results related to their role in engraftment following HSC transplantation [11,14,15,16]. An experiment carried out by Pearce et al. [17] showed that ALDH and CD34 double-positive cells constitute 63% of lineage-negative cells for TNCs and only ALDH+ cells improved engraftment. Storms et al. [18] classified HSCs as being CD34+/-, ALDH+, and ALDH- and demonstrated that only CD34+ ALDH+ cells were efficient in terms of longterm and short-term re-population capacities.

We aimed to compare three different approaches to assess different re-populating HSCs from CB, both fresh and after thawing. In our study, CD34+ cells were found to constitute 0.49±0.26% of all TNCs and 0.35±0.21% of ALDH+ cells. Additionally, 86.98±13% of all CD34+ cells were found to be ALDH+, and within all ALDH+ cells, 94.54±5.3% were CD34+. Among all CB units tested, the rate of ALDH+ CD34- cells was found to be 5.46% and 13.02% were ALDH- but CD34+. Different research groups sought to identify different cellular populations by ALDH staining intensities, but only two of them compared the ALDH activity of different CB sub-populations [5,19]. The clinical significance of those populations remains to be determined. In a study by Lee et al., [7] CD34 cells were found to constitute 0.14±0.10% of all TNCs. In comparison, CD34 positivity was seen in 0.49±0.26% of all TNCs in our study. Unlike our results, Lee et al. [7] detected less ALDH positivity among CD34+ cells (74.5±13.8%), and of the entire ALDH+

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Turk J Hematol 2017;34:314-320

population, 69.9±15.5% of cells were shown to express CD34. Another similar investigation by Storms et al. [18] demonstrated even higher HSC rates in fresh CB samples: 0.9±0.5% of TNCs were CD34+, but 47.9±14.3% of those cells were ALDH+. ALDH+ cells constituted 0.96±0.5% of TNCs and 50.9±18.3% of ALDH+ cells were CD34+ [18]. In Gentry et al.’s [10] study CD34+ cell count was found to be 0.15±0.08% of all TNCs, and 0.05±0.02% of TNCs expressed high ALDH. Attia et al. [14] reported that ALDH activity detection is not only quick and easy to perform but also it does not affect the cell viability or re-populating capacity of CB cells, which may be a serious drawback of some CD34 detection systems [20]. Ikeda et al. [13] suggested that prior to CBT the ALDH assessment method could be an alternative approach to the selection of CB units for unrelated donors [14]. All of the results from the papers mentioned here are in favor of our findings indicating the utility of an ALDH-based approach for CBT settings. Characterization of the sub-populations of CB is crucial because high cell doses with adequate viability predict the outcome after CBT. Engraftment is generally ensured when highly CD34+ cells are used, but occasionally a graft with partially dysfunctional cells due to freeze/thaw processes can affect the cells’ shortterm and long-term re-populating capacities [20]. In vitro manipulations have been shown to interfere with membrane CD34 expression without hampering HSC functionality [21,22]. With the ALDH analysis approach, HSCs with relatively high engraftment capacity but with limited or no growth in CFU tests can easily be detected. As we sought to determine the correlation levels for TNCs and ALDH+ and CD34+ cells with CFU capacities for both fresh and post-thaw samples, we demonstrated that ALDH positivity correlated highly with CFU-GM capacity (r=0.629, p<0.001) in fresh samples. On the other hand, the highest correlation was detected for CFU-GM numbers and CD34+ cells for the post-thaw group of CB units (r=0.655, p<0.001) (Table 2). With a similar approach, Lee et al. [19] analyzed ALDH+, CD34+, ALDH+ within CD34+, and CD34+ within ALDH+ cell populations and CFU-GM and CFU-GEMM capacities in 245 CB units, both fresh and after thawing. Unlike our results, CFU-GM count was not found to be correlated with TNCs in their study. In addition to Lee et al.’s [19] approach, ALDH+ CD90+ CD34+ CD38- cell populations and BFU-E capacities were also analyzed in our study. Lee et al. [19] did not provide any data related to post-thaw samples in terms of TNC/ALDH and TNC/CD34+ ratios, but we demonstrated in our study that 0.66±0.4% of TNCs were CD34+ cells and 0.37±0.27% of TNCs were ALDH+ cells. In a phase 1 study by Gentry et al. [10], CD34+ cell counts were claimed to be the sole post-thaw CB quality predictor, indicating a lower transplant-related mortality, but post-thaw comparisons of CFU-GM counts versus ALDH activity were not

Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

conducted. In our study, ALDH+ cells correlated well with CFUGM for post-thaw samples (Table 2). Similar to our results, a positive correlation was detected between ALDH+ cells and CFUGM positivity (r=0.40, p=0.03) in Frandberg et al.’s [5] study, but their work did not reveal any correlation of CD34+ cells with total CFU count (r=0.36, p=0.051). In addition to ALDH positivity, determination of CD90+ CD34+ CD38- cells may also be a good predictor of engraftment both for fresh and post-thaw CB units. The ALDH+ CD90+ CD34+ CD38group of progenitors is unique with their high engraftment and re-populating capacities [23,24,25]. To our knowledge, our study is the first to examine the ALDH capacity of these particular cells. When we analyzed ALDH positivity in CD90+ CD34+ CD38cells, CFU-GM, CFU-GEMM, and BFU-E counts were found to be well correlated for both fresh and post-thaw CB units (Table 2). Putman et al. [9] performed CFU tests for ALDHhi and ALDHlo cellular populations of CB BFU-E, CFU-GM, and CFU-GEMM. In our study the highest correlation of ALDH+ cells was found with CFU-GM and BFU-E for fresh samples. Putman et al. [9] found that the CB ALDHhi population was significantly enriched for human hematopoietic progenitor function. Owing to the nature of ALDH as an intracellular enzyme, it may be less affected by centrifuge force and thus may reflect the actual HSC population in a more realistic manner. By using ALDH as a marker of functionality, the disadvantage of a likely false negativity caused by CD34+ cell counting could also be overcome. Similar to our results, Shoulars et al. [11] from Duke University recently demonstrated that ALDH activity measured from post-thaw segments highly correlated with CFUs. They suggested that measurement of ALDHbr CD34+ cells might indicate CFU potency and thus engraftment capacity.

Conclusion In light of our results and other recently published studies, we propose that ALDH activity determination can substitute for CFU-GM tests. This fast and inexpensive method has the potential to overcome the weaknesses of other techniques, such as the limitations of CD34 counting due to the internalization of membrane CD34 expression or lack of standardization and long turnaround time of CFU assays. We are aware of the limitations in engraftment prediction by phenotype-based analysis. ALDH measurement, as confirmed by us, has the highest correlation with in vitro functional assays. Currently CD34 and CFUGM assays, accepted as golden standards, are expected to be replaced by ALDH measurement, which is a fast, reproducible, and accurate assessment tool. Acknowledgments This study was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, 114S829). We are grateful to Handan Karakaya for her kind assistance in the CB 319


Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

processing lab and to Sema Meriç for her assistance in flow cytometry analysis. We would like to thank Dr. Sinan Beksaç and Dr. Doruk C. Katlan for their assistance with CB collection. This study was presented as a poster at the 8th National Bone Marrow Transplantation and Stem Cell Therapy Congress as “Phenotypical Analysis of Ex Vivo Granulocyte Colony-Forming Human Umbilical Cord Blood Cells”. Ethics Ethics Committee Approval: Ankara University Faculty of Medicine, Clinical Research Ethics Committee (04-17313/11.03.2013). Informed Consent: This study was based on blood bank data. Authorship Contributions Surgical and Medical Practices: M.B.; Concept: M.B., P.Y.; Design: M.B.; Data Collection or Processing: E.B.G.; Analysis or Interpretation:  E.B.G., K.D.; Literature Search:  E.B.G., P.Y.; Writing: E.B.G. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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9. Putman DM, Liu KY, Broughton HC, Bell GI, Hess DA. Umbilical cord bloodderived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury. Stem Cells 2012;30:2248-2260. 10. Gentry T, Deibert E, Foster SJ, Haley R, Kurtzberg J, Balber AE. Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood. Cytotherapy 2007;9:569-576. 11. Shoulars K, Noldner P, Troy JD, Cheatham L, Parrish A, Page K, Gentry T, Balber AE, Kurtzberg J. Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay. Blood 2016;127:2346-2354. 12. Lioznov MV, Freiberger P, Kroger N, Zander AR, Fehse B. Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants. Bone Marrow Transplant 2005;35:909-914. 13. Ikeda H, Toyama D, Matsuno R, Fujimoto Y, Isoyama K. Aldehyde dehydrogenase activity as a marker of quality in cryopreserved cord blood. Showa University Journal of Medical Sciences 2013;25:297-306. 14. Attia FM, El Baz AA, Naeim MA, Hassan AM, Mohamed AAM, El Barbary MA. Flowcytometric analysis of aldehyde dehydrogenase activity in mononuclear cells from umbilical cord blood. International Journal of Medicine and Medical Sciences 2014;6:151-157. 15. Allan DS, Keeney M, Howson-Jan K, Popma J, Weir K, Bhatia M, Sutherland DR, Chin-Yee IH. Number of viable CD34+ cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant 2002;29:967-972. 16. Emminger W, Emminger-Schmidmeier W, Höcker P, Hinterberger W, Gadner H. Myeloid progenitor cells (CFU-GM) predict engraftment kinetics in autologous transplantation in children. Bone Marrow Transplant 1989;4:415-420. 17. Pearce DJ, Taussig D, Simpson C, Allen K, Rohatiner AZ, Lister TA, Bonnet D. Characterization of cells with a high aldehyde dehydrogenase activity from cord blood and acute myeloid leukemia samples. Stem Cells 2005;23:752760. 18. Storms RW, Green PD, Safford KM, Niedzwiecki D, Cogle CR, Colvin OM, Chao NJ, Rice HE, Smith CA. Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34. Blood 2005;106:95-102. 19. Lee HR, Song EY, Shin S, Roh EY, Yoon JH, Kim BJ. Quality of cord blood cryopreserved for up to 5 years. Blood Res 2014;49:54-60. 20. Duggleby RC, Querol S, Davy RC, Fry LJ, Gibson DA, Horton RB, Mahmood SN, Gomez SG, Madrigal JA. Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment. Transfusion 2012;52:549-559. 21. Mehrishi JN. Current and historical perspectives on methodological flaws in processing umbilical cord blood. Transfusion 2013;53:2667-2674. 22. Mehrishi JN, Bakacs T. A novel method of CD34+ cell separation from umbilical cord blood. Transfusion 2013;53:2675-2680. 23. Notta F, Doulatov S, Dick JE. Engraftment of human hematopoietic stem cells is more efficient in female NOD/SCID/IL-2Rgc-null recipients. Blood 2010;115:3704-3707. 24. Sumikuma T, Shimazaki C, Inaba T, Ochiai N, Okano A, Hatsuse M, Ashihara E, Nakagawa M. CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation. Br J Haematol 2002;117:238-244. 25. Raynaud CM, Butler JM, Halabi NM, Ahmad FS, Ahmed B, Rafii S, Rafii A. Endothelial cells provide a niche for placental hematopoietic stem/ progenitor cell expansion through broad transcriptomic modification. Stem Cell Res 2013;11:1074-1090.


RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0118 Turk J Hematol 2017;34:321-327

Effectiveness of Visual Methods in Information Procedures for Stem Cell Recipients and Donors Kök Hücre Alıcıları ve Donör Bilgilendirme İşleminde Görsel Yöntemlerin Etkinliği Çağla Sarıtürk1, Çiğdem Gereklioğlu2, Aslı Korur2, Süheyl Asma2, Mahmut Yeral3, Soner Solmaz3, Nurhilal Büyükkurt3, Songül Tepebaşı3, İlknur Kozanoğlu3, Can Boğa3, Hakan Özdoğu3 Başkent University Adana Application and Research Center, Department of Biostatistics, Adana, Turkey Başkent University Faculty of Medicine, Department of Family Medicine, Adana, Turkey 3 Başkent University Faculty of Medicine, Adana Adult Bone Marrow Transplantation Center, Adana, Turkey 1 2

Abstract

Öz

Objective: Obtaining informed consent from hematopoietic stem cell recipients and donors is a critical step in the transplantation process. Anxiety may affect their understanding of the provided information. However, use of audiovisual methods may facilitate understanding. In this prospective randomized study, we investigated the effectiveness of using an audiovisual method of providing information to patients and donors in combination with the standard model.

Amaç: Hematopoietik kök hücre alıcıları ve donörlerden bilgilendirilmiş onam alınması, nakil sürecinin en önemli basamağıdır. Görsel yöntemlerden yararlanılması anlamayı kolaylaştırabilir. Bu prospektif randomize çalışmada sözel ve yazılı bilgilendirmeye ilave olarak audiovizüel yöntem kullanılmasının standart yönteme göre etkinliğinin araştırılması amaçlanmıştır.

Materials and Methods: A 10-min informational animation was prepared for this purpose. In total, 82 participants were randomly assigned to two groups: group 1 received the additional audiovisual information and group 2 received standard information. A 20-item questionnaire was administered to participants at the end of the informational session.

Gereç ve Yöntemler: On dakikalık kısa bir bilgilendirme animasyonu hazırlatıldı. Toplam 82 katılımcı rastgele 2 gruba ayrıldı: Grup 1 (görsel bilgilendirme yönteminin ilave edildiği grup) ve grup 2 (standart yöntem uygulanan grup). Bilgilendirme işlemi sonunda katılımcılara 20 soruluk bir anket uygulandı. Ayrıca, yeni yöntemin sonuçlarının kişiler arası farklılıktan etkilenip etkilenmediği test edildi.

Results: A reliability test and factor analysis showed that the questionnaire was reliable and valid. For all participants, the mean overall satisfaction score was 184.8±19.8 (maximum possible score of 200). However, for satisfaction with information about written informed consent, group 1 scored significantly higher than group 2 (p=0.039). Satisfaction level was not affected by age, education level, or differences between the physicians conducting the informative session.

Bulgular: Güvenirlik testi ve faktör analizi anketin güvenilir ve geçerli olduğunu gösterdi. Tüm olgular için genel memnuniyet ortalama değeri 200 üzerinden 184,8±19,8 olarak bulundu. Yazılı bilgilendirilmiş onam form memnuniyeti ortalama puanları yönünden grup 1’deki olguların memnuniyetleri grup 2’ye oranla anlamlı olarak daha yüksek olduğu saptandı (p=0,039). Sözel olarak bilgi veren doktor ve bilgilendirme animasyonu memnuniyeti bakımından gruplar arasında fark saptanmadı. Olguların memnuniyet seviyesi yaş, eğitim durumu ve bilgilendiren kişiler arası farklılıktan etkilenmedi.

Conclusion: This study shows that using audiovisual tools may contribute to a better understanding of the informed consent procedure and potential risks of stem cell transplantation.

Sonuç: Bu çalışma, görsel yöntemlerin kullanılmasının işlem ve muhtemel risklerinin daha iyi anlaşılmasına katkı sağlayabileceğini göstermektedir.

Keywords: Hematopoietic stem cell, Donor, Informed consent, Audiovisual method, Bone marrow transplantation

Anahtar Sözcükler: Hematopoietik kök hücre, Donör, Bilgilendirilmiş onam, Audovizuel yöntem, Kemik iliği nakli

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Can BOĞA, M.D., Başkent University Adana Application and Research Center, Department of Biostatistics, Adana, Turkey Phone : +90 322 327 27 27-2162 E-mail : drcanboga@hotmail.com ORCID-ID: orcid.org/0000-0002-9680-1958

Received/Geliş tarihi: March 23, 2016 Accepted/Kabul tarihi: July 20, 2016

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Introduction Stem cell transplantation (SCT) is a procedure with severe morbidity and mortality, but it also has the potential for long-term survival and recovery [1]. An informed discussion with the patient and his or her relatives and a comprehensive examination of the patient and the donor that includes psychosocial aspects are central to pre-transplant preparation [2]. SCT cannot be performed without collaboration with the patients and their relatives, as treatment may result in shortand long-term changes that affect the patient’s life. Therefore, patient contribution is essential for a detailed educational discussion and provision of informed consent [3,4,5,6,7]. The rationale, procedure, and potential outcomes for SCT can be difficult to understand [1,3]. As the patient may have severe anxiety due to an often difficult diagnosis and potentially fatal outcome, it is unrealistic to expect the patient to easily understand this information. Therefore, to overcome this difficulty, transplant doctors have developed their own communication methods based on personal experiences. The reason for SCT is often not clear to donors and recipients, and the benefits and drawbacks of transplantation may need to be discussed in detail. The correct timing for transplantation is another issue. In many situations, transplantation may be postponed until other therapeutic methods are attempted [3]. Once transplantation becomes feasible, both short- and longterm adverse events are discussed. Patients are informed clearly and objectively about potential side effects. The possibility of procedure-related death and other severe conditions (e.g., admission to the intensive care unit or life support) are also discussed [7]. Requirements for interventional procedures to evaluate potential side effects are also covered. Nonfatal side effects (e.g., chronic graft-versus-host disease) are mentioned as possible long-term effects. Informed consent is only obtained after this information is clearly communicated in accordance with laws, regulations, and standards [7]. The goal of patient and donor education is to help them understand and accurately evaluate the information and risks. Therefore, there needs to be verification of patient and donor understanding throughout the educational procedure for SCT [7]. However, there are a limited number of reports describing the effectiveness of visual methods in patient/donor education about SCT. On this study, we investigated the effectiveness of an informational animation for transplant patients and donors in pre-transplant education.

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scheduled to undergo autologous or allogeneic peripheral SCT at the Adana Bone Marrow Transplantation Unit of Başkent University Faculty of Medicine and donors from whom peripheral stem cell collection for allogeneic transplantation was planned. The standard operating procedure (SOP: KITKU 005) was applied to patients and donors after the council decision had been obtained for transplantation and cell collection, in accordance with JACIE standards. The transplant coordinator invited donors and patients to participate in the study. The clinical medical director, transplant doctor, and transplant coordinator also participated in the informational meeting, and a transplant nurse participated when necessary. Donors were asked to attend the session alone in accordance with the donor privacy principle. However, patients could request that first-degree relatives attend the session with them. Participants were randomly selected and divided into two groups. Group 1 was the study group, exposed to audiovisual information in addition to standard verbal and written information. Group 2 was the control group and received only standard verbal and written information. The transplant coordinator obtained feedback from participants and completed a questionnaire that measured the quality of the information session. To eliminate ethical problems, the audiovisual information was provided for group 2 (control group) after their initial feedback on the information session. The verbal information in the sessions was delivered by two separate transplant doctors to test whether the audiovisual method was affected by interpersonal differences. The results were evaluated in accordance with the rules stated in the Clinical Trials section of the JACIE standards by the Study Board of the Başkent University Adana Bone Marrow Transplantation Unit. Approval was obtained from the Başkent University Scientific Research Board. Verbal and Written Information

Materials and Methods

Both groups received standard verbal and written information. This covered disease status, purpose of the treatment, treatment principles, stem cell collection procedure, pretreatment assessment, the drugs used and their side effects, infusion of stem cells, benefits expected from the treatment, treatment risks and side effects, other treatment options, and disposal of the cellular product. These topics were prepared locally in accordance with international standards (FACT-JACIE standards) to meet donor and recipient information requirements. Patients and donors were able to ask questions after the information session had been completed [4,5,6,7]. The information session lasted up to 30 min.

Study Design

Information Animation

This study was conducted between June 2013 and July 2014 using a prospective, randomized, cross-sectional singlecenter design. The sample comprised adult patients who were

The information animation was based on the flow of the topics discussed in the verbal and written information session. Some topics were covered in movie format and others were shown as

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graphics and images. A Three D Studio Max program used to prepare the program and the technical support was provided by a technical company (Teknik Medya, Adana, Turkey). The visual animation lasted 10 min, and there were Turkish, Arabic, and English language and caption options.

distribution, and the Mann-Whitney U test was used for variables not showing parametric distribution. A p-value of less than 0.05 was considered statistically significant.

Obtaining Patient and Donor Feedback

In total, 92 subjects who were scheduled to undergo autologous or allogeneic hematopoietic peripheral SCT and peripheral stem cell donors for allogeneic transplantation were invited to participate in the study. Of these, 82 (89%) agreed to participate, and 41 participants were assigned to each group. The ten individuals who did not agree to participate were allogeneic SCT recipients. The mean age of participants was 47±14 years (range: 15-67). Mean age of both group 1 and group 2 was 47±14 years (p=0.886). Nine participants (11%) were sibling donors, all in group 1. Participants’ demographic characteristics are shown in Table 1. No statistically significant differences were found between groups in terms of sex, marital status, educational status, center where their diagnosis was made, and patient diagnoses (p>0.05, for all). Before starting the reliability and validity analysis, the present researchers reviewed the questionnaires to determine the participants who had repeated the same answer. This was not found to be a significant problem, and analysis continued with 82 participants. The questionnaire used to determine patient satisfaction had a Cronbach’s alpha coefficient of 0.94 (95% confidence interval [CI] 0.92-0.96), indicating that it was highly reliable. We used factor analysis to measure the validity of the scale. The 20-item satisfaction scale comprised three parts. Consistency of data in factor analysis was measured with KMO sample sufficiency and Bartlett’s test of sphericity. The KMO value was 0.769 and the Bartlett’s test results were statistically significant (χ2=2216.4, p=0.0001). The results of both tests showed that factor analysis of satisfaction scale data was appropriate. In factor analysis of the 20-item satisfaction scale, questions with sample adequacy below 0.50 were investigated. No questions were eliminated because no statement showed a factor load below 0.50. Factor analysis detected three factors, all of which had eigenvalues of ≥1, and there were no overlapping expressions. Factor 1 comprised seven items and explained 25.6% of the total variance, factor 2 explained 15.4% of the variance, and factor 3 explained 25.7%. The total variance explained by three factors was 66.7% (Table 2). The Cronbach’s alphas for the satisfaction scale were 0.95 for written consent, 0.91 for the informing doctor, and 0.90 for the informational animation. This indicates that all sections were highly reliable. The first physician conducted information sessions for 41 participants (21 in group 1, 20 in group 2). The second physician conducted the information sessions for the remaining 41 participants (19 in group 1, 22 in group 2). Both physicians had 10 years of experience as transplant physicians.

A 20-item questionnaire was used to collect feedback including demographic data (age, sex, education status, job, and the institute where the patient was first diagnosed). In addition, a 20-item scale was prepared to measure participants’ satisfaction. Seven items assessed satisfaction with the written informed consent form, seven items were about the information provided by the doctor, and six items focused on the audiovisual information. After an interactive interview, participants scored each question from 1 to 10 based on their satisfaction level. The questions were pre-tested with 10 randomly selected healthy subjects before the study to confirm intelligibility. The scores of the 20 questions were summed to give the overall satisfaction level. The seven questions concerning the written informed consent tested satisfaction with the information on the informed consent form regarding issues such as side effects, stages of treatment, and treatment method. Questions about the doctor who provided the information evaluated the same issues. The questions measuring satisfaction with the information animation also evaluated how the patient understood the stages of the disease and the treatment process. Statistical Analysis Statistical analysis was performed with SPSS 17.0. Categorical measurements were summarized as number and percentage and continuous measurements as mean and standard deviation (median and minimum - maximum where needed). Chi-square or Fisher’s exact tests were used for comparison of categorical variables. The inter-rater agreement was analyzed with kappa statistics. The consistency between questions was evaluated using Cronbach’s alpha coefficient. The value of Cronbach’s alpha coefficient reflects the reliability and internal consistency of the scale (<0.40 indicates that a scale is not reliable, 0.40 to <0.50 indicates very low reliability, 0.50-0.60 low reliability, 0.60-0.70 sufficient reliability, 0.70-0.90 high reliability, and ≥0.90 very high reliability). The reliability of the scale was tested with factor analysis. The appropriateness of the data structure for factor analysis was evaluated with the KaiserMeyer-Olkin (KMO) test, where <0.50 indicates that factor analysis could not be continued, 0.50 and 0.60 are interpreted as poor, 0.60-0.70 as weak, 0.70-0.80 as moderate, 0.800.90 as good, and above 0.90 as excellent. Bartlett’s test of sphericity was used to test the association between statements. Distributions were controlled for inter-group comparisons. Student’s t-test was used for variables showing parametric

Results

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Table 1. Characteristics of responder patients at the time of the information.

Total

Group 2

Group 1

n

%

n

%

n

%

p-value

Female

36

43.9

15

35.7

21

52.5

0.182

Male

46

56.1

27

64.3

19

47.5

Married

67

81.7

35

83.3

32

80.0

Single

12

14.6

6

14.3

6

15.0

Divorced-Widow

3

3.7

1

2.4

2

5.0

Primary School

47

57.3

23

54.8

24

60.0

Middle School

9

11.0

4

9.5

5

12.5

High School

13

15.9

9

21.4

4

10.0

Academic

13

15.8

6

14.3

7

17.5

Başkent University

41

50.0

20

47.6

21

52.5

Other

41

50.0

22

52.4

19

47.5

NHL

18

22.0

11

26.2

7

17.5

ALL

9

11.0

5

11.9

4

10.0

Multiple myeloma

26

31.7

12

28.6

14

35.0

CML

1

1.2

1

2.4

0

0

Acute Leukemia (de novo)

2

2.4

1

2.4

1

2.5

Hodgkin’s Lymphoma

2

2.4

2

4.8

0

0

Sickle Cell Disease

2

2.4

1

2.4

1

2.5

MDS

3

3.7

1

2.4

2

5,0

tAML

2

2.4

1

2.4

1

2.5

Sex

Marital Status 0.811

Educational Status 0.555

Origin of First Diagnosis 0.825

Diagnosis 0.149

NHL: Non-Hodgkin’s lymphoma, ALL: acute lymphoblastic leukemia, AML: acute myeloblastic leukemia, CML: chronic myelocytic leukemia, MDS: myelodysplastic syndrome, tAML: transformed acute myeloblastic leukemia.

The Cronbach’s alpha for participants’ satisfaction with the patient/donor information session was 0.94 (95% CI 0.92-0.96). Table 3 shows participants’ satisfaction with the information session. Satisfaction with the written informed consent, the informing doctor, and the informational animation was measured with an overall satisfaction level that was high (184.8±19.8) compared with the maximum value of 200. There was no significant difference between groups with regard to overall satisfaction. However, a statistically significant difference was found between groups for satisfaction with the written informed consent form. Patient satisfaction was greater in group 1 (p=0.039) (Table 3; Figure 1). There were no significant differences between groups for satisfaction with the doctor who provided the information and the informational animation. The level of satisfaction was not affected by sex or educational status in either group (p>0.05 for all). 324

Figure 1. Patient satisfaction with the written consent. CI: Confidence interval.


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Table 2. Factor analysis. Factor loadings

Factors explaining the variance

Satisfaction with the written consent Information in the informed consent is understandable enough

0.851

The treatment plan is understandable in the informed consent

0.700

The benefits of the treatment are understandable enough in the informed consent

0.805

The risks and side effects of the treatment are understandable enough in the informed consent

0.829

The procedure of stem cell transplantation is understandable enough in the informed consent

0.865

The method of stem cell collection from the blood has been described clearly in the informed consent form

0.693

The medications that will be used during the treatment and their side effects are understandable enough in the informed consent

0.638

25.384

Satisfaction with the physician My doctor has provided me enough information about my disease

0.664

My doctor has provided me information about the stages of my treatment

0.601

My doctor has provided me detailed information about the risks and the side effects of the recommended treatment

0.597

My doctor has provided me enough information about the treatment options other than this treatment method

0.770

My doctor has provided me information about the management of the side effects

0.731

My doctor has answered my questions in detail

0.729

I know that I can always reach my doctor when I have some questions in my mind

0.783

15.374

Satisfaction with the informational animation I could find more clear answers to my questions following the animation

0.829

I could better understand the stages of my treatment through the animation

0.521

I could better understand the benefits of the treatment through the animation

0.747

I could better understand the risks and the side effects of the treatment through the animation

0.799

I could better understand how the bone marrow transplantation would be performed through the animation

0.830

I could better understand the stem cell collection procedure through the animation

0.787

25.478

Table 3. Participant satisfaction.

Total Group 1 Group 2

Overall satisfaction

Satisfaction with the written informed consent

Satisfaction with the informing physician

Satisfaction with the informational animation

n

82

82

82

82

Mean ± SD

184.8±19.8

62.3±8.5

65.8±7.4

56.7±6.3

n

42

42

42

42

Mean ± SD

185.7±22.2

64.2±8.5

64.7±8.6

56.8±6.6

n

40

40

40

40

Mean ± SD

183.8±17.2

60.3±8.2

67.0±5.8

56.5±6.2

p

0.671

0.039

0.161

0.827

SD: Standard deviation.

Discussion This study investigated the effectiveness of audiovisual information in providing essential information during the informed consent process for patients and donors. There were differences in patient/donor satisfaction between the session that included

audiovisual information and that comprising standard verbal/ written explanations. To our knowledge, this is the first study to investigate the effects of audiovisual materials in providing information to hematopoietic stem cell recipients and donors. In general, the use of audiovisual materials facilitates learning and reduces learning time [8,9]. This observation has been supported 325


Sarıtürk Ç, et al: Visual Information for Stem Cell Donation

physiologically [10,11] and is reminiscent of the Chinese saying “I forget if I hear, I remember if I see, I learn if I do”. In this context, our study aimed to develop an information technique using audiovisual methods and demonstrate its efficacy with a verification study in the context of an important issue such as bone marrow transplantation and stem cell donation. Hematopoietic SCT is an effective treatment that is performed for many life-threatening diseases; however, it may result in significant morbidity and mortality [3]. International standards and national laws and regulations require that informed consent be obtained from bone marrow recipients and bone marrow and stem cell donors [7]. These individuals are informed about the rationale of the procedure, expectations, application technique, potential difficulties, and other options if the procedure is not approved. Verbal and written information is provided. The main goal of this information is to enable the subject to make accurate risk assessments and provide informed consent. This is related to correct understanding of the information [12]. A standard operating procedure was produced for the information methods used in this study. In this procedure, we determined the information field, the individuals responsible for patient/donor information, the individuals who would attend the formal meeting, national laws and regulations, and the information required for FACT-JACIE standards [7]. The duration of the informational session and the materials used in the session and consent process were standardized. Approval was obtained from parents or custodians for subjects aged <18 years and those who were not able to give consent (e.g., disabled subjects). A special arrangement was made for pediatric patients or donors (i.e. a psychiatrist joining the interview). We found no statistically significant difference between groups for overall satisfaction. However, satisfaction with the written informed consent form was greater in the study group compared with the control group. Participants in the study group answered questions about treatment stages after sequentially being given written, verbal, and video explanations, and they were satisfied with the information given about their disease. However, the control group provided initial feedback after the written and verbal information session and additional feedback after they viewed the animation. Our results indicated that the video helped patients obtain a more accurate understanding of their disease. Current FACT-JACIE standards (version 6.0) do not require visual materials in addition to verbal and written information for hematopoietic stem cell recipients and donors [7]; verbal and written information is considered sufficient. However, the results of our study support the theory that there are benefits to providing information using audiovisual materials, such as a decrease in perception difficulty arising from language and cultural differences, intellectual differences, and aging. In the present study, participants in both groups evaluated their experiences of the physician who conveyed information 326

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as similar, which may be regarded as a limitation of the study. However, as bone marrow transplantation is a critical issue, it is likely that doctors use similar statements to convey essential information.

Conclusion In conclusion, using audiovisual materials and standard methods for providing information to bone marrow recipients and donors may positively affect patient/donor perception and overcome unnecessary anxiety. Although we obtained data about patient/ donor satisfaction at the time of providing informed consent, further studies may reveal whether better understanding of the transplant/donation procedure would result in better transplant/ donation experience and outcomes. Ethics Ethics Committee Approval: Başkent University Application and Research Center (KA13/165).

Adana

Informed Consent: It was taken. Authorship Contributions Surgical and Medical Practices: Ç.G., A.K., S.A., M.Y., S.S., N.B., C.B., H.Ö.; Concept: Ç.S., İ.K., C.B., H.Ö.; Design: Ç.S., İ.K., C.B., H.Ö.; Data Collection or Processing: S.T., İ.K., C.B., H.Ö.; Analysis or Interpretation: Ç.S., C.B., H.Ö.; Literature Search: Ç.S., Ç.G., A.K., S.A., M.Y., S.S., N.B., S.T., İ.K., C.B., H.Ö.; Writing: Ç.S., Ç.G., C.B. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0343 Turk J Hematol 2017;34:328-333

Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma L-Karnitinin Depolanmış Sıçan Kanı Üzerine Etkisi: Plazmada Yapılan Bir Çalışma Carl Hsieh, Vani Rajashekharaiah Jain University, Center for Post Graduate Studies, Department of Biotechnology, Bangalore, India

Abstract

Öz

Objective: Plasma acts as a good indicator of oxidative stress in blood. L-Carnitine is an antioxidant that reduces metabolic stress in cells, thereby providing a protective effect against oxidative stress (OS). L-Carnitine as an additive in storage has not been explored. Thus, this study attempts to analyze the role of L-carnitine in blood storage solution, citrate phosphate dextrose adenine (CPDA)-1, through OS markers including antioxidant enzymes, lipid peroxidation, and protein oxidation.

Amaç: Plazma kanda oksidatif stresin iyi bir göstergesi olarak görev yapar. L-karnitin hücrelerde metabolik stresi azaltan böylece oksidatif strese (OS) karşı koruyucu etki sağlayan bir antioksidandır. L-Karnitinin depolamada katkı maddesi olarak kullanımı araştırılmamıştır. Bu nedenle, bu çalışmada kan depolama solüsyonu olan sitrat fosfat dekstroz adenin (CPDA)-1 ‘e eklenen L-karnitinin rolü antioksidan enzimler, lipid peroksidasyonu ve protein oksidasyonunu içeren OS göstergeleri aracılığı ile analiz edilmektedir.

Materials and Methods: Blood was collected from male Wistar rats and stored in CPDA-1 solution with L-carnitine (10 mM, 30 mM, and 60 mM: groups LC 10, LC 30, and LC 60, respectively) and without L-carnitine (control group). Plasma was isolated every 5th day and the OS markers were analyzed.

Gereç ve Yöntemler: Wistar sıçanlarından kan örneği alındı ve CPDA1 solüsyonunda L-karnitin ile (10 mM, 30 mM, ve 60 mM: Sırasıyla LC 10, LC 30 ve LC 60 grupları) ve L-karnitinsiz (kontrol grup) olarak depolandı. Plazma her 5. günde izole edildi ve OS göstergeleri analiz edildi.

Results: Superoxide dismutase (SOD) and sulfhydryl (SH) increased over storage in controls, LC 30, and LC 60. Catalase increased in LC 30 and LC 60 during storage. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PrC) levels in all groups increased initially and reduced towards the end of storage. SOD and SH levels were maintained while TBARS and PrC levels increased in LC 10.

Bulgular: Süperoksit dismutaz (SOD) ve sülfidril (SH) kontroller de (LC30 ve LC60) depolama boyunca arttı. Katalaz LC30 ve LC60 da depolama sırasında arttı. Tiyobarbitürik asit reaktif maddesi (TBARS) ve protein karbonil (PrC) düzeyleri tüm gruplarda başlangıçta arttı ve depolama sonuna doğru azaldı. SOD ve SH düzeyleri LC 10’da korunurken TBARS ve PrC düzeyleri arttı.

Conclusion: L-Carnitine was beneficial in terms of increased antioxidant capacity and SH and decreased lipid peroxidation. This forms the basis for further studies on L-carnitine as a constituent in storage solutions.

Sonuç: L-Karnitin antioksidan kapasite ve SH artışı ile lipid peroksidasyonu azalması açısından faydalı idi. Bu durum, L-karnitinin depolama solüsyonlarında bileşen olarak ileri çalışmaları yapılması için bir temel oluşturmaktadır.

Keywords: L-Carnitine, Plasma, peroxidation, Protein oxidation

Anahtar Sözcükler: L-Karnitin, Plazma, Antioksidan enzimler, Lipid peroksidasyonu, Protein oksidasyonu

Antioxidant

enzymes,

Lipid

Introduction L-Carnitine (L-3 hydroxy-4-N-N-N-trimethylaminobutyrate) is an essential nutrient that the body uses to convert fat into energy. It is required for the transport of fatty acids from the cytosol into the mitochondria during breakdown of lipids via β-oxidation. It acts as an antioxidant that reduces metabolic stress in cells, thereby providing a protective effect against

lipid peroxidation and oxidative stress (OS) in the phospholipid membrane and the myocardial and endothelial cells [1]. Disturbances in the redox state can cause OS, which is an imbalance between the production of reactive oxygen species (ROS) and the biological system’s natural ability to detoxify these intermediates or repair the resulting damage caused by them [2]. OS is also induced during storage of blood. The

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Vani RAJASHEKHARAIAH, PhD, Jain University, Center for Post Graduate Studies, Department of Biotechnology, Bangalore, India Phone : +91 988 6178 584 E-mail : tiwari.vani@gmail.com ORCID-ID: orcid.org/0000-0002-4155-0960

328

Received/Geliş tarihi: August 24, 2016 Accepted/Kabul tarihi: December 28, 2016


Turk J Hematol 2017;34:328-333

constituents of plasma change during storage due to cell metabolism and the activation of proteolytic activity [3]. These changes can be attributed to the depletion of glucose levels in plasma, cationic pump failure, hemoconcentration, and leakage of cell constituents and metabolites in erythrocytes [4]. Plasma reflects the overall OS environment in whole blood as it holds all the cellular components in suspension. Thus, plasma serves as a good candidate for assessing the changes occurring during whole blood storage. The study of d’Almeida et al. [5] reported that 1 week of rat blood storage is equivalent to 4 weeks of human blood storage. Thus, rat blood was used for studying storage lesions. The ability of L-carnitine to combat OS was studied by Li et al. [1], where it was found that L-carnitine could protect hepatocytes through its antioxidant effect. L-Carnitine could reduce free radical-induced oxidative damage of intermittent hypoxia exposure, thus delaying muscle fatigue [6,7]. Various studies have reported on the effects of L-carnitine on other blood components (erythrocytes and platelets) [8,9,10,11]. Although there are reports on plasma storage [12,13,14,15], very few have focused on plasma isolated from stored blood. However, the influence of Carnitine on stored blood is still unclear. Thus, this study attempts to analyze the role of L-carnitine as a constituent in blood storage solution through OS markers (antioxidant enzymes, lipid peroxidation, and protein oxidation) in plasma.

Materials and Methods Animal care and maintenance was in accordance with the Ethical Committee regulations (841/b/04/CPCSEA). Chemicals Epinephrine, thiobarbituric acid (TBA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). All other chemicals used were of reagent grade and organic solvents were of spectral grade. Blood Sampling Animals were lightly anesthetized with ether and restrained in dorsal recumbency as described earlier [16]. In brief, the syringe needle was inserted just below the xiphoid cartilage and slightly to the left of the midline. Blood was carefully aspirated from the heart into polypropylene collection tubes with citrate phosphate dextrose adenine (CPDA)-1. Experimental Design Blood was drawn from 4-month-old male Wistar rats and stored over a period of 20 days at 4 °C in CPDA-1. The samples from 20 animals were divided into 4 groups of 5 animals each: i) controls,

Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood

ii) LC 10 (samples with L-carnitine at a concentration of 10 mM), iii) LC 30 (samples with L-carnitine at a concentration of 30 mM), and iv) LC 60 (samples with L-carnitine at a concentration of 60 mM). Plasma was isolated from whole blood at regular intervals of 5 days and biomarkers, i.e. antioxidant enzymes, lipid peroxidation, and protein oxidation products, were assessed. Plasma Separation Plasma was isolated in microcentrifuge tubes by centrifuging for 20 min at 1000 × g. The plasma was removed and stored in isotonic phosphate buffer at -20 °C for further assays [17]. Antioxidant Enzymes Superoxide Dismutase [EC 1.15.1.1]: Superoxide dismutase (SOD) was measured by the method of Mishra and Fridovich [18]. Plasma was added to carbonate buffer (0.05 M). Epinephrine was added to the mixture and absorbance was measured at 480 nm. SOD activity was expressed as the amount of enzyme that inhibited oxidation of epinephrine by 50%. Catalase [EC 1.11.1.6]: Catalase (CAT) was determined by the method of Aebi [19]. Briefly, plasma with absolute alcohol was incubated at 0 °C. An aliquot was taken up with 6.6 mM H2O2 and the decrease in absorbance was measured at 240 nm. An extinction coefficient of 43.6 M cm-1 was used to determine enzyme activity. Lipid Peroxidation: Thiobarbituric Acid Reactive Substances Thiobarbituric acid reactive substances (TBARS) content was determined by the method of Bar-Or et al. [20]. Plasma with 0.9% sodium chloride was incubated at 37 °C for 20 min, and then 0.8 M HCl containing 12.5% trichloroacetic acid (TCA) and 1% TBA was added and samples were kept in a boiling water bath for 20 min and cooled at 4 °C. Centrifugation was carried out at 1500 × g and absorbance was measured at 532 nm. TBARS content was calculated by using the extinction coefficient of 1.56x105 M-1 cm-1. Protein Oxidation Protein carbonyls: Protein carbonyl (PrC) content was determined by the method of Reznick and Packer [21]. PrC content was measured by forming a labeled protein hydrazone derivative using 2,4-dinitrophenyl hydrazine (DNPH), which was then quantified spectrophotometrically. Briefly, after precipitation of protein with an equal volume of 1% TCA, the pellet was resuspended in 10 mM DNPH. Samples were kept in the dark for 1 h. An equal volume of 20% TCA was added and left on ice for 10 min and then centrifuged at 3000 × g, and the pellet was washed with an ethanol-ethyl acetate mixture (1:1) to remove the free DNPH and lipid contaminants. The final pellet was dissolved in 6 M guanidine HCl in 133 mM Tris and absorbance was measured 329


Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood

at 370 nm. PrC content was calculated by using the extinction coefficient of 20,000 M-1 cm-1. Protein Sulfhydryls The protein sulfhydryl (P-SH) concentration in the proteins was measured as described by Habeeb [22]. In brief, 0.08 mol/L sodium phosphate buffer containing 0.5 mg/mL Na2ethylenediaminetetraacetic acid and 2% SDS was added to the sample in each assay tube, and then 0.1 mL of 5,5’-DTNB was added and the solution was vortexed. Color was allowed to develop at room temperature and absorbance was measured at 412 nm. P-SH was calculated from the net absorbance and molar absorptivity, 13,600 M-1 L-1 cm-1. Protein Protein was determined in the plasma by the method of Lowry et al. [23] using BSA as the standard.

Turk J Hematol 2017;34:328-333

Catalase Changes in CAT were significant with storage in all groups. Control and LC 10 CAT levels were maintained over storage. LC 30 and LC 60 levels were significantly increased at days 15 and 20 (Figure 2). Lipid Peroxidation - Thiobarbituric Acid Reactive Substances Changes in TBARS were significant in controls during storage. TBARS peaked on day 15 in controls. An increase in TBARS was observed in LC 10 over storage. TBARS content was maximum on day 10 in LC 30 and LC 60 samples (Figure 3). Protein Oxidation Protein carbonyls: Changes in PrC were significant in all groups with storage. PrC levels were increased at days 15 and 20 in

Statistical Analysis Results are represented as mean ± standard error. The Kolmogorov-Smirnov test was performed for suitability of the data. Values between the groups (storage period) and subgroups (antioxidants) were analyzed by two-way ANOVA and differences were considered significant at p<0.05. The Bonferroni post-test was performed using GraphPad Prism 6 software.

Results Superoxide Dismutase Significant changes in SOD were observed in all groups with storage. SOD in controls increased during storage. SOD was maintained in LC 10 throughout the storage period. LC 30 and LC 60 samples showed increments in SOD over storage (Figure 1).

Figure 2. Catalase activity in plasma isolated from stored blood. LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM. Values are mean ± SE of five animals/group. Two-way ANOVA was performed between the groups and subgroups to analyze catalase activity followed by the Bonferroni post-test, using GraphPad Prism 6 software. Changes between the groups are represented in upper case. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.

Figure 1. Superoxide dismutase activity in plasma isolated from stored blood.

Figure 3. Thiobarbituric acid reactive substances in plasma isolated from stored blood.

LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM.

LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM. Values are mean ± SE of five animals/group. Two-way ANOVA was performed between the groups and subgroups to analyze thiobarbituric acid reactive substances followed by the Bonferroni post-test, using GraphPad Prism 6 software. Changes between the groups are represented in upper case. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.

Values are mean ± SE of five animals/group. Two-way ANOVA was performed between the groups and subgroups to analyze superoxide dismutase activity followed by the Bonferroni post-test, using GraphPad Prism 6 software. Changes between the groups are represented in upper case. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.

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controls. PrC was significantly higher than in the other groups throughout the storage period in LC 10. PrC peaked on day 15 in LC 30 and LC 60 samples (Figure 4). Protein sulfhydryls: P-SH levels varied significantly over storage. P-SH increased gradually throughout the storage period in controls. P-SH was maintained in LC 10 samples over the storage period. P-SH was significantly higher in LC 30 and LC 60 than controls and LC 10 (Figure 5).

Discussion This study assessed whole blood through plasma, as it holds all the blood components in suspension. It gives an overall view of the OS microenvironment during storage. SODs are a group of enzymes that catalyze the conversion of superoxide into H2O2 and O2. Increase in the activity of SOD is

Figure 4. Protein carbonyls in plasma isolated from stored blood. LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM. Values are mean ± SE of five animals/group. Two-way ANOVA was performed between the groups and subgroups to analyze protein carbonyl followed by Bonferroni post-test, using GraphPad Prism 6 software. Changes between the groups are represented in upper case. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.

Figure 5. Protein sulfhydryls in plasma isolated from stored blood. LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM. Values are mean ± SE of five animals/group. Two-way ANOVA was performed between the groups and subgroups to analyze protein sulfhydryl followed by Bonferroni post-test, using GraphPad Prism 6 software. Changes between the groups are represented in upper case. Changes within the groups are represented in lower case. Those not sharing the same letters are significantly different.

Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood

generally a sign of increased formation of superoxide radicals and thus elevated OS [24]. This was evident in our results of increased SOD activity during storage. L-Carnitine is a scavenger of free radicals and protects the cells from OS [25]. Similar results were observed in our study, where L-carnitine upregulated SOD [1,26]. SOD increased on day 10 and peaked on day 15 in controls, due to maximum ROS [27]. The decrease on day 20 in controls can be attributed to ROS overwhelming the antioxidant enzyme capacity and thus inactivating the enzyme. A similar trend was observed in LC 10 from day 15. SOD varied from day 10 onwards in LC 30, which can be attributed to the modulation of the enzyme activity by L-carnitine in proportion to superoxides. CAT degrades H2O2 to H2O and O2. H2O2 can also be scavenged by glutathione peroxidase (GPX) [28]. CAT activity was low initially and increased over storage. This may be due to the activity of GPX scavenging H2O2 at lower concentrations, while CAT decomposes H2O2 only at high concentrations [29,30]. CAT levels were highest on day 15 in controls, similar to SOD, due to maximum ROS being produced on that day [23]. CAT expression in LC 10 was in accordance with the SOD levels, where the levels dropped on day 15. Li et al. [1] and Cao et al. [26] showed that L-carnitine increased CAT expression, which was also observed in our results. The increase in CAT in LC 30 and LC 60 can be attributed to L-carnitine’s ability to upregulate antioxidant enzyme activity. TBARS, a biomarker of lipid peroxidation, is a reasonable reflection of a nonlipophilic peroxidation product, malondialdehyde [26]. The peak on day 15 in controls may be attributed to greater amounts of ROS generated [27]. L-Carnitine at concentrations of 30 mM and 60 mM reduced TBARS over storage. This may be due to L-carnitine’s ability to scavenge ROS and upregulate antioxidant enzymes at higher concentrations. L-Carnitine also has the property of preventing the accumulation of lipid peroxidation end products, hence causing the decrease in TBARS [31]. This was evident in our results of lipid peroxidation. Oxidative cleavage of the protein backbone, oxidation of amino acids, or binding of aldehydes produced from lipid peroxidation produces PrC. It is formed early and circulates in the blood for longer periods as it is more stable than lipid peroxidation products [32]. Protein oxidation products are effective biomarkers of OS due to their long half-lives [33]. PrC increased in controls over storage, indicative of oxidative insult and protein damage. Addition of L-carnitine at 30 mM and 60 mM did not alter the PrC levels significantly, suggesting that L-carnitine could not alter the formation of carbonyls. P-SH gets oxidized to disulfides, which is a reversible reaction. It is mainly present in the cysteine components of proteins and generally at lower concentrations in glutathione [34]. 331


Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood

P-SH increased over storage in controls, which indicates that the endogenous antioxidant system could combat OS during storage. The increase in P-SH with L-carnitine indicates that it could protect sulfhydryl groups against oxidation or was effective in catalyzing the reversible change of disulfides to sulfhydryls [26]. Arduini et al. [11] reported L-carnitine to be beneficial at 5 mM in terms of increased ATP concentrations and reduced hemolysis over storage. However, our study showed that L-carnitine at 10 mM could not prevent protein oxidation and lipid peroxidation, but LC 30 and LC 60 had reduced oxidative damage through reduced TBARS and elevated P-SH and antioxidant enzymes (SOD and CAT).

Conclusion In conclusion, antioxidant enzymes in plasma could combat the ROS generated during storage. Our study showed that L-carnitine at higher concentrations can be further explored as a constituent of storage solutions as it significantly upregulated the antioxidant capacity of plasma and reduced oxidative damage during storage. Therefore, L-carnitine is a promising constituent in blood storage solutions. Acknowledgments The authors acknowledge Dr. Leela Iyengar, Ms. Soumya Ravikumar, Mrs. Manasa K, and Jain University for their support. Ethics Ethics Committee Approval: The Committee for the Purpose of Control and Supervision of Experiments on Animals (841/b/04/ CPCSEA). Informed Consent: N/A. Authorship Contributions Surgical and Medical Practices (Sample collection): C.H.; Concept: V.R.; Design: V.R.; Data Collection or Processing: C.H.; Analysis or Interpretation: C.H.; Literature Search: C.H.; Writing: C.H., V.R. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

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RESEARCH ARTICLE DOI: 10.4274/tjh.2016.0469 Turk J Hematol 2017;34:334-339

Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats Antioksidanlar Sıçanlarda Oksidatif Stres ile Oluşan Gizli Kan Kaybını Zayıflatır Hong Qian1, Tao Yuan2, Jian Tong3, Wen-shuang Sun1, Jiajia Jin4, Wen-xiang Chen5, Jia Meng2, Nirong Bao2, Jianning Zhao2 Southeast University Nanjing General Hospital of Nanjing Military Command, Clinic of Orthopedics, Nanjing, China Nanjing University Faculty of Medicine, Jinling Hospital, Clinic of Orthopedics, Nanjing, China 3 Nanjing University Faculty of Medicine, Nanjing General Hospital of Nanjing Military Command, Clinic of Orthopedics, Nanjing, China 4 Southeast University Nanjing General Hospital of Nanjing Military Command, Department of Respiratory Medicine, Nanjing, China 5 Southern Medical University Faculty of Medicine, Department of Orthopedics, Nanjing, China 1 2

Abstract

Öz

Objective: Hidden blood loss (HBL), commonly seen after total knee or hip arthroplasty, causes postoperative anemia even after reinfusion or blood transfusion based on the visible blood loss volume. Recent studies demonstrated that oxidative stress might be involved in HBL. However, whether the antioxidants proanthocyanidin (PA) or hydrogen water (HW) can ameliorate HBL remains poorly understood. The aim of this study was to evaluate the effects of PA and HW on HBL.

Amaç: Total diz veya kalça artroplastisinden sonra yaygın olarak görülen gizli kan kaybı (GKK), görülebilir kan hacmi kaybına dayanan reinfüzyon veya kan nakli sonrasında bile postoperatif anemiye neden olur. Son yıllarda yapılan çalışmalar oksidatif stresin GKK’yla ilişkili olabileceğini göstermiştir. Bununla birlikte, proantosiyanidin (PA) veya hidrojenli su (HS) antioksidanlarının GKK’yi iyileştirip iyileştirmediği anlaşılamamıştır. Bu çalışmanın amacı PA ve HS’nin GKK üzerindeki etkilerini değerlendirmektir.

Materials and Methods: A rat HBL model was established through administration of linoleic acid with or without treatment with PA or HW. The levels of hemoglobin (Hb), red blood cell (RBC) count, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, malondialdehyde (MDA), and ferryl Hb were measured. Results: RBC and Hb values as well as the activity of SOD and GSHPX were reduced after administration of linoleic acid, which was ameliorated by treatment with PA or HW. In addition, the quantity of MDA was significantly decreased with the administration of PA or HW. Conclusion: PA and HW could ameliorate HBL in a rat model by reducing oxidative stress, suggesting that they might be used as a novel therapeutic approach in the prophylaxis or treatment of HBL in clinics. Keywords: Hidden blood loss, Antioxidants, Proanthocyanidin, Hydrogen water

Gereç ve Yöntemler: PA veya HS ile muamele edilmiş veya edilmemiş olan linoleik asit uygulaması ile bir sıçan GKK modeli oluşturulmuştur. Hemoglobin (Hb), kırmızı kan hücresi sayısı (RBC), süperoksit dismutaz (SOD) aktivitesi, glutatyon peroksidaz (GSH-PX) aktivitesi, malondialdehit (MDA) ve ferril Hb düzeyleri ölçüldü. Bulgular: PA veya HS verilen linoleik asit uygulaması ile RBC ve Hb değerlerinin yanı sıra SOD ve GSH-PX aktiviteleri azaltıldı. Buna ek olarak, PA veya HS uygulaması ile MDA miktarı önemli ölçüde azaldı. Sonuç: PA ve HS bir sıçan modelinde oksidatif stresi azaltarak GKK’yi düzeltebildiğinden, kliniklerdeki GKK profilaksisi veya tedavisinde yeni bir terapötik yaklaşım olarak kullanılabileceklerini düşündürmektedir. Anahtar Sözcükler: Gizli kan kaybı, Antioksidanlar, Proantosiyanidin, Hidrojenli su

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Jianning ZHAO, M.D., Nanjing University Faculty of Medicine, Jinling Hospital, Clinic of Orthopedics, Nanjing, China Phone : +90 532 325 10 65 E-mail : zhaojianning.0207@163.com ORCID-ID: orcid.org/0000-0001-7169-8117

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Received/Geliş tarihi: December 04, 2016 Accepted/Kabul tarihi: March 07, 2017


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Introduction Artificial joint replacements are widely employed to alleviate pain and improve the quality of patients’ lives [1]. The  rates of primary and total hip arthroplasty (THA)  and  total knee arthroplasty (TKA) are estimated to increase by 174%-673% by 2030 as the population ages [2]. However, hidden blood loss (HBL) predominantly occurs after artificial joint replacement, such as in cases of TKA and THA [3]. The consequential acute anemia and transfusions are major concerns for joint surgeons. The pathogenesis of HBL is very complicated, involving several factors. A recent study demonstrated that free fatty acids (FFAs) generated from fatty emboli in the blood circulation are responsible for HBL through peroxidation injury of membrane molecules of red blood cells (RBCs) and hemoglobin (Hb) [4]. In addition, antioxidants administered intra- or postoperatively are predicted to play a protective role in erythrocyte oxidation and potentially reduce the volume of HBL after arthroplasty, suggesting that oxidation might be involved in the pathogenesis of HBL. Consistent with this, our previous study also demonstrated that FFAs can induce RBC and Hb damage via reactive oxygen species (ROS) toxicity in vivo [5]. As a natural antioxidant extract from grape seeds, proanthocyanidin (PA) possesses a wide range of bioavailability [6]. PA exhibits higher protective effects against DNA damage and lipid peroxidation induced by ROS compared with β-carotene, vitamin C, and vitamin E [7]. PA is a safe and effective bioavailable antioxidant and ROS scavenger, which is used for the treatment of ischemia/reperfusion injuries of multiple organs, malignant tumor progression, carcinogenesis, gastrointestinal disorders, and Parkinson and Alzheimer  disease [6]. As a new antioxidant, hydrogen water (HW) has also been applied to prevent and treat oxidative stress-associated illnesses using the establishment of animal models [8,9,10]. HW has been proven to selectively remove strong oxidants including peroxynitrite and hydroxyl radicals. Alternatively, ROS play a physiological role in preventing cells from experiencing oxidative stress [11]. Considering the role of oxidative stress in the pathogenesis of HBL, whether PA and/or HW as antioxidants ameliorate HBL remains poorly understood. The objective of this study was to evaluate the effect of PA and HW on HBL as well as to compare their protective effects by measuring the levels of Hb, RBC count, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), and ferryl Hb.

Materials and Methods Animals Forty 10-week-old male Sprague-Dawley rats weighing 250±20 g were obtained from the Nanjing University Model Animal Research Center. All animals were fed daily with rat feed and potable water or HW under appropriate laboratory conditions at

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24 °C with a 12-h light/dark cycle. The animals were randomly assigned into four groups (n=10 per group). Experimental procedures were performed strictly according to the Guide for the Care and Use of Laboratory Animals proposed by the National Research Council in 1996. All animals were properly monitored. Animal ethics approval was obtained for this research. All experimental procedures conducted complied with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Institutional Care and Use Committee of Nanjing University. Preoperatively, all animals were anesthetized via ether inhalation. Instruments and Reagents Instruments used included a hematology analyzer (SYSMEX XE5000, Kobe, Japan), centrifuge (Hermle Universal Centrifuge Z323, Gosheim, Germany), microplate reader (Bio-Rad 680, Hercules, CA, USA), polarizing microscope (Nikon Eclipse 50I, Tokyo, Japan), spectrophotometer (Hewlett Packard 8453 UVvisible diode array spectrophotometer, Palo Alto, CA, USA), HWgenerating apparatus (Bio Coke Laboratory, Tokyo, Japan), and hydrogen sensor (DHS-001, ABLE, Tokyo, Japan).  The concentration of MDA and the activities of SOD and GSH-PX were measured with commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Linoleic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). PA was purchased from Shanghai Aladdin Bio-Chem Technology Institute (Shanghai, China). HW was prepared by dissolving H2 gas in drinking water under high pressure of 0.4 MPa using the HW-generating apparatus. Rats were supplied with HW (0.7 mM) through a closed glass vessel (300 mL) equipped with an outlet line containing 2 ball bearings to prevent water degassing. The H2 concentration of HW was detected with a hydrogen sensor (Unisense, Aarhus, Denmark). Experimental Protocol and Drugs The procedures below were performed on the rats in all four groups and the dose used was selected as previously described. The control group (CON) rats were given potable water and injected with ethanol alone (0.5 mL, 20%) via intravenous administration into the tail vein after 2 weeks of feeding. The linoleic acid (LIN) group animals (receiving LIN) received potable water  and were injected with 0.5 mL of 60 mmol/L linoleic acid  diluted in 20% ethanol  by intravenous administration into the tail vein  after 2 weeks of feeding. The LIN+PA group received a 100 mg/kg dose of PA diluted with potable water  daily [12,13] and was injected with  0.5 mL of 60 mmol/L linoleic acid diluted in 20% ethanol by intravenous administration into the tail vein  after 2 weeks of feeding [5]. The LIN+HW group received HW daily and was injected with 0.5 mL of 60 mmol/L linoleic acid diluted in 20% ethanol by intravenous administration into the tail vein following 2 weeks of feeding [5]. 335


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During all treatments, rats were monitored daily and were weighed one to six times per day until the end of the experiment. None of the rats had any notable discomfort throughout the experiment. Routine and Biochemical Analysis of Blood Blood samples were taken from the caudal vein under anesthesia (0.5 mL each time) at the beginning of the injection and 24, 48, and 72 h following administration. RBC, hematocrit, and Hb levels were detected with a hematology analyzer immediately after sampling collection. Morphological changes of blood cells were observed following Wright’s staining under a polarizing microscope. The remaining blood samples were centrifuged and stored at 80 °C for subsequent biochemical analysis. MDA, T-SOD, and GSH-PX activities were measured by spectrophotometer. The absorbance values were detected at 532 nm, 550 nm, and 412 nm wavelengths [12]. Spectral changes of Hb in the LIN and LIN+PA groups were quantitatively measured by spectrophotometer. Hb at a concentration of 10 mM was mixed with 0.1 M sodium phosphate buffer containing 100 mM DTPA. All experimental procedures were conducted at 25 °C [14].

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Hb values were reduced by (0.66±0.34)×1012/L and 16.3±8.25 g/L, and in the LIN+PA group those values were decreased by (0.35±0.1)×1012/L and 9.1±4.01 g/L, respectively. A significant difference was noted in the changes between the LIN and LIN+PA groups. After 48 h of administration, the changes of RBC and Hb levels of the LIN group and the LIN+PA group were still significantly different. In the LIN+HW group, we found the RBC and Hb values decreased by (0.45±0.22)×1012/L and 10.7±3.56 g/L after 24 h, respectively, with a tendency of alleviation of the reduction of RBC and Hb levels. After 48 h, the decreases of RBC and Hb (respectively (0.72±0.23)×1012/L and 18.2±5.85 g/L) in the LIN+HW group were significantly different compared to those of the LIN group (respectively (1.15±0.48)×1012/L and 25.7±8.38 g/L). Oxidative Stress Markers The activities of SOD and GSH-PX in the LIN group significantly declined after 24 h of administration, reached the lowest levels after 48 h, and had mild increases after 72 h. Both the LIN+PA and the LIN+HW group showed a similar variation tendency in these two markers. However, the SOD and GSH-PX activities

Statistical Analysis Statistical analysis was performed using SPSS 19.0. All data were expressed as mean ± standard deviation. The KolmogorovSmirnov test was performed and we concluded that the observed data were from a population specified by normal distribution. One-way analysis of variance (ANOVA) was performed followed by the Tukey test. p<0.05 was considered statistically significant.

Results Daily consumption of water and body weight among all groups were monitored. Rats in the CON group consumed 20.0±3.5 mL of potable water daily, while the LIN group consumed 21.0±2.7 mL of potable water daily. In the LIN+PA group, daily consumption of PA solution was 22.0±2.4 mL, while the LIN+HW group consumed 24.0±3.4 mL of HW daily. Water consumption and body weight did not significantly differ among the four groups. Routine Blood Tests Before linoleic acid administration, no significant differences were observed in RBC and Hb levels among the four groups. After administration of a dose of 0.5 mL of 60 mmol/L linoleic acid, RBC and Hb levels significantly changed compared with the control group (Figure 1), which showed that an in vivo HBL model had been established successfully. We further analyzed the RBC and Hb levels of the LIN+PA and LIN+HW groups compared to those of the LIN group. After 24 h of administration, the Hb and RBC levels had decreased to different extents in the three experimental groups. In the LIN group, the RBC and 336

Figure 1. Changes of hemoglobin and red blood cell levels with time between control (sham) group and experimental groups. Values are presented as the mean ± standard deviation, n=10 for all groups. *Compared with the control group, p<0.05, #Compared with the linoleic acid group, p<0.05. LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water, RBC: red blood cell, Hb: hemoglobin.


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in groups LIN+PA and LIN+HW were both obviously higher than those of the LIN group at each time point (Figure 2). The MDA concentration in the LIN group reached a peak after 24 h and then started to decrease slowly. The LIN+PA and LIN+HW groups also displayed a similar changing pattern in MDA level. However, both SOD and GSH-PX activities in groups LIN+PA and LIN+HW were consistently lower than those of the LIN group (Figure 2). Ferryl Hb was present and formed by reacting with H2O2, which was confirmed by the characteristic absorbance band around 620 nm via the reaction with sulfide ions [15]. The effect of linoleic acid upon the hemolysis of RBCs, either by itself or in conjunction with ROS, can be utilized to assess the severity of oxidative injury of erythrocytes [16]. Blood samples were

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collected from these three groups before administration and every 24 h thereafter. Absorbance peak values were detected at a wavelength of approximately 425 nm, consistent with the Soret peak of ferryl Hb (Figure 3). Histologic Investigations In the LIN group, a number of shrunken, deformed, and ruptured blood cells were seen compared with the control group and the morphological changes were the most distinct after 24 h of administration. In contrast, we could also identify some shrunken and deformed RBC in groups LIN+PA and LIN+HW, but ruptured blood cells could hardly be found in those two groups (Figure 4).

Figure 3. Changes in absorbance at 425 nm among the linoleic acid, linoleic acid+proanthocyanidin, and linoleic acid+hydrogen water groups. Values are presented as the mean Âą standard deviation, n=10 for all groups. *Compared with the control group, p<0.05. LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water.

Figure 2. Changes in T-superoxide dismutase, glutathione peroxidase, and malondialdehyde among the linoleic acid, linoleic acid+proanthocyanidin, and linoleic acid+hydrogen water groups. Values are presented as the mean Âą standard deviation, n=10 for all groups. *Compared with the control group, p<0.05. LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water, SOD: superoxide dismutase, GSH-PX: glutathione peroxidase, MDA: malondialdehyde.

Figure 4. Protective effects of proanthocyanidin and hydrogen water on red blood cells. Blood samples were collected before administration and then every 24 h thereafter. Stains were added to the blood smears to observe erythrocyte morphological changes. Images are magnified at 400x. After 24 h, cell morphology was obviously changed in the linoleic acid group (A-D) with a large number of red blood cells shrunken (black arrows), deformed (blue arrows), and even ruptured (red arrows); in contrast, in the linoleic acid+proanthocyanidin group (E-H) and LIN+HW group (I-L), ruptured cells could hardly be identified. LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water.

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Discussion Many recent studies have investigated the pathophysiological mechanisms and therapeutic strategies for HBL [17,18,19], but to our knowledge, our team provided the first evidence that oxidative stress can lead to HBL [10] and that antioxidant treatment with PA or HW ameliorated HBL, suggesting they may represent a possible therapeutic choice for HBL in clinical practice. HBL is a severe complication after TKA and THA [20]. Although several theories concerning the mechanisms of HBL have been proposed, no theory is convincing enough to explain the pathological mechanism. Pattison et al. [21]  proposed that hemolysis may partly contribute to postoperative loss, but they did not provide a pathological mechanism. Faris et al. [22] demonstrated that hemolysis was detected after reinfusion with an average volume of 1.3 L of blood, but hemoglobinuria did not occur due to the activity of Hb. In contrast, Shen et al. [23]  showed that no statistical significance was observed in HBL between the reinfusion and non-reinfused groups. Li et al. [24]  reported that administration of a tourniquet could significantly increase HBL in their study, but as much as 600 mL of HBL can be detected without using a tourniquet. Moreover, the theory of the “third compartment” was proposed to explain the mechanism underlying HBL. Erskine et al. [25] reported that unexplained blood loss was completely due to perioperative bleeding, probably into the tissue compartments. However, it is not reasonable that the bleeding would be “pressing” into tissue compartments because of commonly used techniques, such as negative pressure drainage and pressure dressing. Therefore, subsequent investigation is required to fully unravel the mechanisms underlying HBL. The increased intramedullary pressure in TKA and THA plays a vital role in the pathogenesis of fatty metabolism [26,27]. In addition to the clinical association between fatty emboli and cardiopulmonary function, the metabolites of fatty emboli, FFAs, can stimulate ROS production in neutrophils [28] and exert a negative biological effect on erythrocytes. After ROS were stimulated and the oxidants accumulated, osmotic fragility of RBCs increased through oxidizing polyunsaturated fatty acids derived from the RBC membranes [29] and cytosolic ferrous Hb [30]. Given the critical role of ROS in the damage or injury of RBCs, this study investigated the antioxidant effect of PA and HW on linoleic acid-induced oxidative stress by measuring GSHPx, SOD, and MDA. Our results showed that SOD and GSHPx activities were increased in the experimental groups with the use of PA or HW, and the SOD and GSH-Px activities of each experimental group were significantly decreased after 24 h of LIN administration, indicating that linoleic acid plays a vital role 338

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in promoting oxidation responses in the body and reducing SOD and GSH-Px activity. In the LIN+PA and LIN+HW groups, SOD and GSH-Px showed significantly elevated activities compared with the LIN group. These findings can be interpreted as PA and HW exhibiting a positive effect on SOD and GSH-Px activity. The amount of MDA was significantly decreased due to the effect of linoleic acid, suggesting the presence of oxidative stress in the culture medium. In this study, the quantity of MDA was significantly decreased with the administration of PA or HW [9,12], consistent with previous studies  showing  that the elevation of MDA level induced by lipid peroxidation was counteracted by the administration of PA or HW. Although the present study indicates that PA possesses higher anti-HBL effects compared with HW, no significant variation occurred in our study considering the dosage and duration of PA administration. Oxidative injury changes the structure and function of Hb, leading to Hb denaturation and precipitation. The resultant product is known as methemoglobin [19]. Hydrophilic hydrogen peroxide is capable of directly penetrating the RBC membrane and oxidizing Hb into ferryl Hb [20]. The heme proteins oxidized into the ferryl species by peroxides are widely regarded as the initiators of a variety of lipid peroxidation and lipid pseudo-peroxidase responses [21]. Hypochlorous acid can oxidize glutathione and membrane protein-SH groups and elevates the osmotic fragility. In addition, it also induces cell membrane deformation via lipid oxidation [3]. Multiple investigations have indicated that ferrous Hb can be oxidized into ferryl Hb by H2O2 and hypochlorous acid. Ferryl Hb loses the capacity of carrying oxygen. Nevertheless, GSH-Px is able to decrease the formation of methemoglobin by 93% when Hb is oxidized by H2O2 [22], highlighting the pivotal role of linoleic acid in mediating Hb oxidation and subsequent cross-linking of the oxidation-reduction responses. Several limitations have to be acknowledged in this study. First, our studies suggest that FFAs could cause HBL, which could be ameliorated through treatment with antioxidant drugs, but we cannot draw the conclusion that oxidative stress produced by FFAs leading to the toxicity of RBCs is the only pathophysiological mechanism underlying postoperative blood loss. Second, the appropriate therapeutic dose and timing of PA and HW administration and the combination therapy of these two drugs need further investigation. The significance of the current experiment is that HBL induced by ROS increase can be counteracted by antioxidant therapy. 

Conclusion In conclusion, PA and HW could ameliorate HBL in a rat model by reducing oxidative stress, suggesting they might be used as novel therapeutic approaches in the prophylaxis or treatment of HBL in clinical practice.


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Ethics Ethics Committee Approval: Animal studies were approved by the Ethic Committeee of Jinling Hospital and were strictly performed following the Institutional Animal Care and User guidelines. Informed Consent: N/A. Authorship Contributions Surgical and Medical Practices: H.Q., T.Y.; Concept:  J.Z.; Design: N.B.; Data Collection or Processing: J.T., W.C.; Analysis or Interpretation: W.S:, J.J.; Literature Search:  Q.H., J.M.; Writing: Q.H. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included. Financial Disclosure: This study was supported by the Clinical Science and Technology Foundation of Jiangsu Province (BL2012002), the Natural Science Foundation of Jiangsu Province (BK2012776), and the National Natural Science Foundation of China (Grant No. 81000814).

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9. Miyazaki N, Yamaguchi O, Nomiya M, Aikawa K, Kimura J. Preventive effect of hydrogen water on the development of detrusor overactivity in a rat model of bladder outlet obstruction. J Urol 2016;195:780-787. 10. Hasegawa S, Ito M, Fukami M, Hashimoto M, Hirayama M, Ohno K. Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice. Redox Rep 2017;22:26-34. 11. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007;13:688-694. 12. Bakar E, Ulucam E, Cerkezkayabekir A. Investigation of the protective effects of proanthocyanidin and vitamin E against the toxic effect caused by formaldehyde on the liver tissue. Environ Toxicol 2015;30:1406-1415. 13. Lee YA, Kim YJ, Cho EJ, Yokozawa T. Ameliorative effects of proanthocyanidin on oxidative stress and inflammation in streptozotocin-induced diabetic rats. J Agric Food Chem 2007;55:9395-9400. 14. Winterbourn CC. Oxidative reactions of hemoglobin. Methods Enzymol 1990;186:265-272. 15. Potor L, Bányai E, Becs G, Soares MP, Balla G, Balla J, Jeney V. Atherogenesis may involve the prooxidant and proinflammatory effects of ferryl hemoglobin. Oxid Med Cell Longev 2013;2013:676425. 16. Rao YP, Lokesh BR. Modulatory effects of α-linolenic acid on generation of reactive oxygen species in elaidic acid enriched peritoneal macrophages in rats. Indian J Exp Biol 2014;52:860-869. 17. Xie J, Ma J, Yao H, Yue C, Pei F. Multiple boluses of intravenous tranexamic acid to reduce hidden blood loss after primary total knee arthroplasty without tourniquet: a randomized clinical trial. J Arthroplasty 2016;31:2458-2464. 18. Wang K, Ni S, Li Z, Zhong Q, Li R, Li H, Ke Y, Lin J. The effects of tourniquet use in total knee arthroplasty: a randomized, controlled trial. Knee Surg Sports Traumatol Arthrosc 2017;25:2849-2857. 19. Huang GP, Jia XF, Xiang Z, Ji Y, Wu GY, Tang Y, Li J, Zhang J. Tranexamic acid reduces hidden blood loss in patients undergoing total knee arthroplasty: a comparative study and meta-analysis. Med Sci Monit 2016;22:797-802. 20. Sehat KR, Evans RL, Newman JH. Hidden blood loss following hip and knee arthroplasty. Correct management of blood loss should take hidden loss into account. J Bone Joint Surg Br 2004;86:561-565. 21. Pattison E, Protheroe K, Pringle RM, Kennedy AC, Dick WC. Reduction in haemoglobin after knee joint surgery. Ann Rheum Dis 1973;32:582-584. 22. Faris PM, Ritter MA, Keating EM, Valeri CR. Unwashed filtered shed blood collected after knee and hip arthroplasties. A source of autologous red blood cells. J Bone Joint Surg Am 1991;73:1169-1178. 23. Shen HL, Li Z, Feng ML, Cao GL. Analysis on hidden blood loss of total knee arthroplasty in treating knee osteoarthritis. Chin Med J (Engl) 2011;124:1653-1656. 24. Li B, Wen Y, Wu H, Qian Q, Lin X, Zhao H. The effect of tourniquet use on hidden blood loss in total knee arthroplasty. Int Orthop 2009;33:1263-1268. 25. Erskine JG, Fraser C, Simpson R, Protheroe K, Walker ID. Blood loss with knee joint replacement. J R Coll Surg Edinb 1981;26:295-297. 26. Esmaeilnejad B, Tavassoli M, Asri-Rezaei S, Dalir-Naghadeh B. Evaluation of antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis. Vet Parasitol 2012;185:124-130. 27. Kato N, Nakanishi K, Yoshino S, Ogawa R. Abnormal echogenic findings detected by transesophageal echocardiography and cardiorespiratory impairment during total knee arthroplasty with tourniquet. Anesthesiology 2002;97:1123-1128. 28. Qian M, Eaton JW. Free fatty acids enhance hypochlorous acid production by activated neutrophils. J Lab Clin Med 1994;124:86-95. 29. Roy A, Sil PC. Tertiary butyl hydroperoxide induced oxidative damage in mice erythrocytes: protection by taurine. Pathophysiology 2012;19:137-148. 30. van den Berg JJ, Op den Kamp JA, Lubin BH, Roelofsen B, Kuypers FA. Kinetics and site specificity of hydroperoxide-induced oxidative damage in red blood cells. Free Rad Biol Med 1992;12:487-498.

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BRIEF REPORT DOI: 10.4274/tjh.2017.0052 Turk J Hematol 2017;34:340-344

High Infection-Related Mortality in Pediatric Acute Myeloid Leukemia without Preventive Antibiotics and Antifungals: Retrospective Cohort Study of a Single Center from a Middle-Income Country Çocukluk Çağı Akut Miyeloid Lösemilerinde Koruyucu Antibiyotik ve Antifungal Kullanılmadığında Yüksek Enfeksiyon İlişkili Mortalite: Orta Gelir Grubundaki Bir Ülkeden Tek Merkezin Retrospektif Kohort Çalışması Emine Zengin1, Nazan Sarper1, Sema Aylan Gelen1, Uğur Demirsoy1, Meriban Karadoğan1, Suar Çakı Kılıç1, Selim Öncel2, Emin Sami Arısoy2, Devrim Dündar3 Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Infectious Diseases, Kocaeli, Turkey 3 Kocaeli University Faculty of Medicine, Department of Microbiology, Kocaeli, Turkey 1 2

Abstract

Öz

Objective: This study aimed to evaluate infection-related mortality in patients with acute myeloid leukemia (AML) treated without preventive antibiotics and antifungals in a middle-income country.

Amaç: Orta gelir düzeyindeki bir ülkede koruyucu antibiyotik ve antifungal kullanılmadan tedavi edilen akut miyeloid lösemi (AML) tanılı hastalarda enfeksiyona bağlı ölümlerin değerlendirilmesidir.

Materials and Methods: Infection-related mortality was evaluated retrospectively in 49 pediatric patients.

Gereç ve Yöntemler: Kırk dokuz AML tanılı çocuk hastada enfeksiyona bağlı ölümler retrospektif olarak değerlendirildi.

Results: A total of 173 chemotherapy courses were administered as first-line chemotherapy. Four patients died during induction: one patient due to intracranial bleeding, two patients due to typhlitis, and one patient due to invasive fungal infection with pulmonary vascular invasion and massive bleeding. Another two patients died with resistant disease. During consolidation there were four infectionrelated deaths and one death due to cardiotoxicity. In first-line chemotherapy mortality was 22% (11/49); infection-related mortality was 14% (7/49). Event-free survival and overall survival at 6 years were 42.9% and 61.2% (95% CI: 44-76 and 66-99 months), respectively.

Bulgular: İlk basamak tedavi olarak toplam 173 kemoterapi kürü uygulandı. İndüksiyon sırasında bir hasta beyin kanaması, iki hasta tiflit ve bir hasta invazif mantar enfeksiyonunun akciğer damar duvarını hasarlaması sonucu gelişen yoğun kanamayla olmak üzere toplam dört hasta kaybedildi. İki hasta da dirençli hastalık ile kaybedildi. Konsolidasyon sırasında beş ölüm vardı; dördü bakteriyemi ve sepsis, biri kardiyotoksisite ilişkiliydi. İlk basamak tedavide ölüm oranı %22 (11/49) olup, bunların %14’ü (7/49) enfeksiyonla ilişkiliydi. Olaysız sağkalım ve genel sağkalım sırasıyla 6 yılda %42,9 ve %61,2 (%95 güven aralığı: 44-76 ve 66-99 ay) bulundu.

Conclusion: Due to considerable infection-related deaths, antibacterial and mold-active antifungal prophylaxis may be tried during neutropenic periods in pediatric AML.

Sonuç: Dikkate değer sayıda enfeksiyon ölümleri nedeniyle antibakteriyel ve küf mantarlarına etkili koruyucu tedaviler, ortagelir düzeyindeki ülkelerde, AML tanılı çocuk hastaların nötropenik dönemlerinde kullanılabilir.

Keywords: Acute myeloid leukemia, Pediatric leukemia, Febrile neutropenia, Infection

Anahtar Sözcükler: Akut miyeloid lösemi, Çocukluk çağı lösemileri, Nötropenik ateş, Enfeksiyon

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Nazan SARPER, M.D., Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey Phone : +90 262 303 72 16 E-mail : nazan_sarper@hotmail.com ORCID-ID: orcid.org/0000-0003-1599-774X

340

Received/Geliş tarihi: February 12, 2017 Accepted/Kabul tarihi: March 28, 2017


Zengin E, et al: Infection-Related Mortality in Pediatric AML

Turk J Hematol 2017;34:340-344

Introduction

Statistical Analysis

Children and adolescents  with acute myeloid leukemia (AML) are at risk of severe infectious complications as a result of prolonged neutropenia. It was reported that the main causes of death during chemotherapy are infections [1]. Studies show that  prophylaxis with antibiotics and antifungals reduced infections,  hospitalization days, and mortality [2,3,4], but the emergence of resistance, particularly  in vancomycin-resistant enterococci (VRE) and gram-negative bacteria, is another dilemma [5]. 

Data were analyzed using SPSS 13. Descriptive statistics were employed and are reported as absolute frequencies or percentages for qualitative data and as medians and range or means and standard deviations for quantitative data. Comparisons of frequency distribution were analyzed with the nonparametric statistics of the chi-square test or the KruskalWallis test. For survival, Kaplan-Meier analysis was performed. All tests were two-tailed and p<0.05 was considered statistically significant.

The aim of this study was to document infection-related mortality (IRM) of patients with de novo  AML and myelodysplastic syndrome (MDS)/AML during first-line chemotherapy  courses and compare the results with the literature data.

Results

Materials and Methods This retrospective study was performed in a university hospital’s pediatric hematology unit. The hospital records of all de novo AML/MDS patients aged <18 years diagnosed from June 2005 through February 2016 were reviewed by two experienced hematologists of the unit. Patients with Down syndrome were also included. Before starting chemotherapy, all the parents/ legal guardians gave informed consent for the treatment and for the usage of patient data in the research. The ethics committee of the institution also approved the study. The United Kingdom Medical Research Council (MRC) AML-10 chemotherapy protocol was used as first-line chemotherapy with some modifications and no randomizations [1]. A modification was the substitution of amsacrine with idarubicine 10 mg/m2 on  days 0 and 1 in some patients due to unavailability of the drug. Patients with Down syndrome were treated with reducedintensity chemotherapy. Patients stayed in two-bed rooms with a bathroom and there was no high-efficiency particulate air (HEPA) filtration. Co-trimoxazole prophylaxis was administered. Granulocyte colony stimulating factor (G-CSF) was only used in consolidation phases if there was severe infection with hemodynamic instability. Patients generally were not discharged until remission, but in the subsequent chemotherapy courses they were followed as outpatients if there were no infections. In the first years of the study, ceftazidime and then piperacillin/ tazobactam or cefepime were used as empirical monotherapy. Carbapenems and teicoplanin were administered as initial empirical therapy in hemodynamically unstable patients. If there were any respiratory symptoms at initial presentation of febrile neutropenia or if fever persisted longer than 96 h, serum galactomannan monitoring was started and chest computerized tomography and abdominal ultrasound imaging were performed. Empirical mold-active antifungals were introduced after 96 h. Patients were referred to some other centers when hematopoietic stem cell transplantation (HSCT) was indicated.

Forty-nine patients (32 boys, 17 girls) with AML were diagnosed and 173 chemotherapy courses were administered as first-line chemotherapy. Ten of the patients had acute promyelocytic leukemia, 4 patients had Down syndrome, and 3 patients had MDS/AML (one had myelofibrosis). The overall remission rate with first-line chemotherapy was 85.7%. During induction and consolidation there were 11 deaths. In first-line chemotherapy mortality was 22% (11/49); IRM was 14% (7/49) (Figure 1). In 682 sterile-site cultures, 47 cases of pathogen growth were observed. The isolated pathogens of the three infectionrelated deaths were  Candida guilliermondii (septicemia and pneumonia), extended spectrum β-lactamase-positive Klebsiella pneumoniae  (septicemia),  and  Enterobacter cloacae (septicemia). Except for the patient with Candida septicemia who had sudden pulmonary hemorrhage, all the patients required intensive care. Gram-negative bacteria isolation was more frequent than gram-positive (67.2% versus 32.7%).  Escherichia  coli  and  K. pneumoniae  were the most predominant isolates. Viridans streptococci were rarely isolated; there were three  Streptococcus mitis  isolations from this

Figure 1. Outcome of pediatric patients with AML/MDS. CR: Complete remission, CT: chemotherapy, IFI: invasive fungal infection, ICH: intracranial hemorrhage, HSCT: hematopoietic stem cell transplantation.

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Zengin E, et al: Infection-Related Mortality in Pediatric AML

Turk J Hematol 2017;34:340-344

Table 1. Characteristics of the infectious episodes in pediatric patients with acute myeloid leukemia.  

ADE I

ADE II

MACE**

MIDAC

# of chemotherapy courses 

49

45

41

38

# of febrile episodes

62

41

30

43

# of febrile episodes per chemotherapy course

1.3

0.9

0.7

1.1

# of episodes with fever of unknown origin (%)

12 (19.4)

13 (31.7)

6 (20)

12 (27.9)

*# of clinically documented infections (%)

38 (61.3)

15 (36.6)

14 (46.7)

16 (37.2)

# of microbiologically documented infections (%)

12 (19.4)

13 (31.7)

10 (33.3)

15 (34.9)

# of discharged patients after chemotherapy courses

5

36

36

32

Mean outpatient days of discharged patients after chemotherapy courses

4.3±7.5

12.1±9

16.2±10

9.5±7.3

Mean period off antimicrobials and antifungals during chemotherapy courses (days)

9.2±9.9

20.2±9.4

21.9±9.8

14.2±7.8

*Clinically documented infections also include possible fungal infections, **MACE or modified MACE.

or vancomycin with oral ciprofloxacin reduced bacterial sepsis and days of hospitalization. Prophylactic oral voriconazole was also used in that study but it did not reduce fungal infections. IRM was 2.5% [2].

Figure 2. Kaplan-Meier analysis of event-free and overall survival. group. Characteristics of the infectious episodes and identified pathogens are demonstrated in Tables 1 and 2, respectively. In the first remission, 4 matched related donor (MRD) and 1 matched unrelated donor (MUD) transplants were performed. Eight patients had allogeneic HSCT in the second remission (4 MRD, 2 MUD, and 2 haploidentical transplants), and two of these patients had second transplants. Of the 11 transplanted patients, 8 were alive in a median of 54 (range: 6-84) months (Figure 1). Event-free survival and overall survival at 6 years were 42.9% and 61.2% (95% CI: 44-76 and 66-99 months), respectively (Figure 2). 

Discussion Sixty-one percent of the patients were alive during the retrospective study period. During the first-line treatment there were 11 deaths (22%), 7 of which (14%) were infection-related. In the present study, with less intensive chemotherapy, out of 4 patients with Down syndrome there was only one infectionrelated death during induction. In a multicenter study of children also treated with the MRCAML-10 protocol, IRM was 9.1% [1]. Single-occupancy rooms with HEPA filtration and more training about hand washing may reduce infections. It was reported that prophylactic oral cephalosporins did not significantly reduce bacterial sepsis, but intravenous cefepime 342

In a multicenter trial some centers used antibacterial (penicillin or vancomycin and others), antifungal, and G-CSF prophylaxis. All the centers used fluconazole prophylaxis. The authors concluded that antibacterial prophylaxis reduced sterilesite infections. Prophylactic G-CSF reduced bacterial and Clostridium difficile infections. Mandatory hospitalization did not reduce bacterial/fungal infection and nonrelapse mortality but did increase C. difficile infections [6]. We hospitalized patients until remission but in the subsequent courses we discharged them. Mandatory hospitalization might prevent some infection-related deaths. One patient died at home with infection and another’s admission was delayed. In another study, vancomycin and ceftazidime or cefepime prophylaxis was administered in addition to fluconazole, voriconazole, or micafungin. There was only one infection-related death due to candidal pneumonia. When induction I was excluded, preventive antibiotics reduced infections compared to no prophylaxis. The hospital rooms were all single-occupancies and most of them had HEPA filtration. However, after emergence of VRE and  C. difficile  infections, vancomycin was removed from the preventive therapy and cefepime mono therapy was started [7]. In the multicenter trial AML-Berlin-Frankfurt-Münster (BFM) 2004, excluding patients with Down syndrome, the infectionrelated morbidity rate was 3.3 per patient and IRM was only 1.5%. There was a reduction in IRM compared to the previous AML-BFM 93 study. This was explained by the administration of anti-mold active prophylaxis (amphotericin B, voriconazole, or posaconazole) in more than 70% of the chemotherapy cycles and prompt institution of empiric therapy that should include a glycopeptide in severely ill patients and regular training courses in the education of the pediatric hematologists [8].


Zengin E, et al: Infection-Related Mortality in Pediatric AML

Turk J Hematol 2017;34:340-344

Table 2. Isolated pathogens during chemotherapy courses of pediatric patients with acute myeloblastic leukemia.

ADE I

ADE II

MACE*

MIDAC

# of eligible courses

49

45

41

38

Staphylococcus epidermidis

3

1

-

5

Micrococcus luteus

1

-

2

-

Streptococcus mitis

-

2

-

1

Enterococcus faecalis

1

1

-

-

Kocuria kristinae

1

-

-

-

Escherichia coli

4

2

2

10

Klebsiella pneumoniae

-

3

1

5

Pseudomonas aeruginosa

-

1

1

1

Enterobacter cloacae

1

2

-

1

Acinetobacter spp.

1

-

-

Proteus mirabilis

-

1

-

-

Elizabethkingia meningoseptica

1

-

-

-

# of fungal isolates

Candida albicans

1

-

1

-

Candida parapsilosis

1

-

-

-

Candida guilliermondii

2

-

-

-

Aspergillus fumigatus

-

-

-

1

Aspergillus niger

-

-

1

-

# of gram-positive isolates

# of gram-negative isolates

*MACE or modified MACE.

Conclusion In a multicenter study of pediatric AML from Turkey, similar to our study, overall survival was 58.8% and IRM was 16.6%. The authors also suggested that better supportive care may improve outcomes [9].    In light of the above literature we think that, to reduce IRM, we should try prophylaxis in patients with pediatric AML. Prophylactic piperacillin/tazobactam or cefepime and voriconazole can be administered in the induction. After induction oral  ciprofloxacin  and voriconazole can be tried in preventive therapy when the absolute neutrophil count (ANC) falls below 0.5x109/L and discontinued once the ANC recovers to >0.1x109/L. Prophylaxis may justify the potential risk from emerging resistant bacteria. Ethics Ethics Committee Approval: Kocaeli University Noninterventional Clinical Research Ethics Board (approval number: 2016/288 - 2016/18.1).

Informed Consent: Informed consent was obtained from parents for publication of the patient data at the start of chemotherapy. Authorship Contribution Surgical and Medical Practices: E.Z., N.S., M.K., S.A.G., U.D., S.Ç.K., D.D., S.Ö., E.S.A.; Concept: N.S.; Design: N.S.; Data Collection or Processing: E.Z., N.S.; Analysis or Interpretation: E.Z.; Literature Search: N.S.; Writing: N.S. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Riley LC, Hann IM, Wheatley K, Stevens RF. Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party. Br J Haematol 1999;106:436-444. 2. Kurt B, Flynn P, Shenep JL, Pounds S, Lensing S, Ribeiro RC, Pui CH, Razzouk BI, Rubnitz JE. Prophylactic antibiotics reduce morbidity due to septicemia during intensive treatment for pediatric acute myeloid leukemia. Cancer 2008;113:376-382.

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Zengin E, et al: Infection-Related Mortality in Pediatric AML

3. Yeh TC, Liu HC, Hou JY, Chen KH, Huang TH, Chang CY, Liang DC. Severe infections in children with acute leukemia undergoing intensive chemotherapy can successfully be prevented by ciprofloxacin, voriconazole, or micafungin prophylaxis. Cancer 2014;120:1255-1262. 4. Feng X, Ruan Y, He Y, Zhang Y, Wu X, Liu H, Liu X, He L, Li C. Prophylactic first-line antibiotics reduce infectious fever and shorten hospital stay during chemotherapy-induced agranulocytosis in childhood acute myeloid leukemia. Acta Haematol 2014;132:112-117. 5. Inaba H, Gaur AH, Cao X, Flynn PM, Pounds SB, Avutu V, Marszal LN, Howard SC, Pui CH, Ribeiro RC, Hayden RT, Rubnitz JE. Feasibility, efficacy, and adverse effects of outpatient antibacterial prophylaxis in children with acute myeloid leukemia. Cancer 2014;120:1985-1992. 6. Sung L, Aplenc R, Alonzo TA, Gerbing RB, Lehrnbecher T, Gamis AS. Effectiveness of supportive care measures to reduce infections in pediatric AML: a report from the Children’s Oncology Group. Blood 2013;121:3573-3577.

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7. Nolt D, Lindemulder S, Meyrowitz J, Chang BH, Malempati S, Thomas G, Stork L. Preventive antibiotics in pediatric patients with acute myeloid leukemia (AML). Pediatr Blood Cancer 2015;62:1149-1154. 8. Bochennek K, Hassler A, Perner C, Gilfert J, Schöning S, Klingebiel T, Reinhardt D, Creutzig U, Lehrnbecher T. Infectious complications in children with acute myeloid leukemia: decreased mortality in multicenter trial AMLBFM 2004. Blood Cancer J 2016;6:e382. 9. Ozyurek E, Vergin C, Büyükavcı M, Kılınç Y, Timur V, Özbek N, Celkan T, Erduran E, Olcay L, Çetingül N, Patıroğlu T, Atabay B, Bör Ö, Ünal S, Güler E, Koç A, Gülen H, Kürekçi E, Söker M, Balkan C, Polat A, Tunç B, Ören H. The outcomes of Turkish children with acute myeloid leukemia treated on AMLBerlin-Munster-Frankfurt (AML-BFM) protocol: Turkish AML-BFM Study Group. In: 4th International Congress on Leukemia Lymphoma Myeloma (Abstracts); 22-25 May; İstanbul, Turkey, 2013.


BRIEF REPORT DOI: 10.4274/tjh.2016.0477 Turk J Hematol 2017;34:345-349

Hematopoietic Stem Cell Transplantation in Primary Immunodeficiency Patients in the Black Sea Region of Turkey Primer İmmün Yetmezlikli Hastalarda Hematopoetik Kök Hücre Transplantasyonu; Türkiye’de Karadeniz Bölgesi Deneyimi Alişan Yıldıran1, Mehmet Halil Çeliksoy1, Stephan Borte2, Şükrü Nail Güner1, Murat Elli3, Tunç Fışgın3, Emel Özyürek3, Recep Sancak1, Gönül Oğur4 Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey Leipzig University, Translational Centre for Regenerative Medicine, Leipzig, Germany 3 Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Samsun, Turkey 4 Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Genetic, Samsun, Turkey 1 2

Abstract

Öz

Hematopoietic stem cell transplantation is a promising curative therapy for many combined primary immunodeficiencies and phagocytic disorders. We retrospectively reviewed pediatric cases of patients diagnosed with primary immunodeficiencies and scheduled for hematopoietic stem cell transplantation. We identified 22 patients (median age, 6 months; age range, 1 month to 10 years) with various diagnoses who received hematopoietic stem cell transplantation. The patient diagnoses included severe combined immunodeficiency (n=11), Chediak-Higashi syndrome (n=2), leukocyte adhesion deficiency (n=2), MHC class 2 deficiency (n=2), chronic granulomatous syndrome (n=2), hemophagocytic lymphohistiocytosis (n=1), Wiskott-Aldrich syndrome (n=1), and Omenn syndrome (n=1). Of the 22 patients, 7 received human leukocyte antigen-matched related hematopoietic stem cell transplantation, 12 received haploidentical hematopoietic stem cell transplantation, and 2 received matched unrelated hematopoietic stem cell transplantation. The results showed that 5 patients had graft failure. Fourteen patients survived, yielding an overall survival rate of 67%. Screening newborn infants for primary immunodeficiency diseases may result in timely administration of hematopoietic stem cell transplantation.

Birçok kombine primer immün yetmezlik ve fagositer bozukluk için hematopoetik kök hücre nakli küratif bir tedavidir. Bu çalışmada, primer immün yetmezlik tanısı alan ve hematopoetik kök hücre nakli yapılan hastaları retrospektif olarak inceledik. Yirmi iki hasta belirlendi. Hastaların hematopoetik kök hücre nakli sırasındaki ortanca yaşları 6 ay (minimum-maksimum: 1 ay-10 yaş) idi. Hastaların tanıları ağır kombine immün yetmezlik (n=11), Cheidak Higashi sendromu (n=2), lökosit adezyon defekti (n=2), MHC sınıf-2 eksikliği (n=2), kronik granülomatoz hastalık (n=2), hemofagositik lenfohistiyositoz (n=1), Wiskott-Aldrich sendromu (n=1) ve Omenn’s sendromu (n=1) idi. Yedi hastaya tam insan lökosit antijen uyumlu, 12 hastaya yarı uyumlu ve 2 hastaya insan lökosit antijen uyumsuz vericiden hematopoetik kök hücre nakli yapıldı. Beş hasta da graft başarısız oldu. On dört hasta hayatta kaldı ve ortalama sağkalım %67 idi. Bu hastalık için yenidoğan taramaları yapılması ile hematopoetik kök hücre transplantasyonları zamanında yapılabilir. Anahtar Sözcükler: Hematopoetik kök hücre, Transplantasyon, Çocuk, İmmün yetmezlik

Keywords: Hematopoietic stem cell, Transplantation, Children, Immunodeficiency

Introduction Primary immunodeficiency (PID) disorders are a group of heterogeneous diseases, many of which are caused by monogenic defects, resulting in susceptibility to life-threatening

infections, uncontrolled inflammation, or autoimmunity. In 1968, successful transplantation was performed in two patients, one with severe combined immunodeficiency (SCID)  and one with Wiskott-Aldrich syndrome (WAS). These cases represented the first successful  hematopoietic stem cell transplantation

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Mehmet Halil ÇELİKSOY, M.D., Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey Phone : +90 362 312 19 19 E-mail : drmhc@hotmail.com ORCID-ID: orcid.org/0000-0002-4164-4668

Received/Geliş tarihi: December 09, 2016 Accepted/Kabul tarihi: April 11, 2017

345


Yıldıran A, et al: HSC Transplantation in Patients with PID

(HSCT) procedures, ushering in a new era of curative therapies for treating PID disorders [1,2,3]. To date, only one report has described HSCT therapies for PID disorders in Turkey [4]. The aim of this study was to retrospectively document all pediatric cases of patients diagnosed with PID disorders and considered for HSCT therapy at our pediatric transplantation center.

Materials and Methods In total, 22 infants were diagnosed with PID; 19 of these patients underwent HSCT at the Ondokuz Mayıs University Faculty of Medicine, Department of Pediatrics, Pediatric Transplantation Unit, between June 2010 and December 2013. One patient died shortly after diagnosis. Of the 22 patients, 11 were diagnosed with SCID, 2 with MHC class 2 deficiency, 2 with leukocyte adhesion deficiency (LAD), 2 with chronic granulomatous disease (CGD), 2 (siblings) with ChediakHigashi syndrome (CHS), 1 with WAS, 1 with hemophagocytic lymphohistiocytosis, and 1 with Omenn syndrome (Tables 1 and 2). All patients met the European Society for Immunodeficiencies - Pan-American Group for Immunodeficiency diagnostic criteria for PID disease [5]. In terms of the phenotypic profiles of patients with SCID, seven displayed T-B-NK+ and four showed T-B+NK+ profiles. The molecular defects of two patients with SCID could not be determined. In total, eight patients with SCID and three without SCID underwent haploidentical CD34+ stem cell transplantation. Additionally, one patient with CGD and one patient with WAS underwent HSCT from matched unrelated donors at another center.

Results Patient Characteristics The patients’ ages at diagnosis of SCID ranged from 2 to 8 months (median: 3 months). Parental consanguinity was determined in seven (64%) patients with SCID. Pneumonia and diarrhea were common complaints in patients with SCID. Parental consanguinity was determined in nine (82%) nonSCID patients. Two patients with SCID were referred by another center for HSCT 3 months after diagnosis. Only one SCID patient was found to be positive for cytomegalovirus antigenemia at the time of diagnosis; therefore, a conditioning regimen was not administered. No engrafted maternal T cells were detected in patients with SCID at the time of diagnosis. All patients with SCID were lymphopenic and had few T cells (CD3+ cells <30%). The age of the non-SCID patients at the time of HSCT ranged from 3 to 120 months (median: 13 months). Failure to thrive was the most common complaint in non-SCID patients. Although parental consanguinity was determined in 64% of SCID and 82% of non-SCID patients, only six patients (27%) had matched related donors. The characteristics and transplantation data from SCID and non-SCID cases are shown in Tables 1 and 2. 346

Turk J Hematol 2017;34:345-349

Complications A common complication in our patients was graft failure (40%) that required repeated transplantations. Grade I-II acute graftversus-host disease (GVHD) was observed in four patients (three SCID and one non-SCID) after HSCT. Outcomes and complications in the SCID and non-SCID cases are shown in Tables 3 and 4.

Discussion Established in 2009, our bone morrow transplantation center was the first of its kind in the Black Sea Region of Turkey. We reviewed all pediatric patients diagnosed with PID who were scheduled to receive HSCT at Ondokuz Mayıs University between June 2010 and December 2013 (n=22). A similar recent study in two Balkan countries reported only 15 SCID cases during a 24year period [6]. Cipe et al. [4] reported haploidentical HSCT in 18 patients in the capital city of Turkey during a 10-year period; however, many of these patients were from other regions of the country. Although four of our patients came from other regions (two with SCID, one with Omenn syndrome, and one with LAD), our patient numbers suggested that the prevalence of PID disorders should have been higher in the Black Sea Region of Turkey because of the high rate of consanguinity. Yorulmaz et al. [7] reported that the parental consanguinity rate was 37.5% in patients with PID; in another region of the country, these rates were 84%, 75%, and 73% in patients with SCID, phagocytic system defects, and common variable immune disease, respectively. As our prenatal consanguinity rates were lower than expected, we suggest that newborn screenings for PID disorders should be mandatory, at least in our region. In the near future, we plan to apply the screening method developed by Borte et al. [8], which includes a robust triplex polymerase chain reaction method for quantitation of T-cell  receptor excision circles and κ-deleting recombination excision circles using single-punch Guthrie cards. We expect to identify patients with SCID, X-linked agammaglobulinemia, ataxia telangiectasia, Nijmegen breakage syndrome, and other severe immunodeficiency syndromes characterized by the absence of T or B cells with this method. Cipe et al. [4] concluded that human leukocyte antigen (HLA)haploidentical transplantation from parental donors represents a readily available treatment option, especially for patients with SCID, offering a high probability of cure. In our study, 12 patients received haploidentical HSCT, 7 received HLA-matched HSCT from related donors, and 2 received HLA-matched HSCT from unrelated donors. There are several concerns regarding the safety of haploidentical HSCT, as it may cause a delay in the successful outcome in patients with PID disorders. Our study and others [4,9] showed that T cell-depleted haploidentical HSCT is a life-saving treatment in patients with PID disorders.


M

M

M

M

F

F

M

F

F

F

F

1 FD*

2 MK

3 MO

4 IY**

5 PA

6 YFK**

7 BY

8 ET

9 FZS

10 EK

11 GS

-

-

+

-

+

+

+

-

+

+

+

C#

Pneumonia/diarrhea

FTT/oral thrush

Diarrhea

Pneumonia

Pneumonia/diarrhea

Pneumonia/diarrhea

Pneumonia/diarrhea/ oral thrush

Pneumonia

Diarrhea

Pneumonia/diarrhea

Pneumonia/FTT/oral thrush

at presentation

Symptoms

T-B+NK+

T-B-NK+

T-B+NK-

T-B+NK+

T-B-NK+

T-B-NK+

T-B-NK+

T-B-NK+

T-B+NK+

T-B-NK+

T-B-NK+

Diagnosis

6

2

2

8

3

3

5

2

3

5

6

(months)

8

3

2

8

4

7

6

6

5

7

7

(months)

Age at diagnosis Age at HSCT

Haplo

Haplo

ND

Haplo

MRD

Haplo

Haplo

Haplo

Haplo

Haplo

MRD

Type of HSCT

Mother/PBSCT

Mother/PBSCT

ND

Mother/PBSCT

Sister/BM

Father/PBSCT

Mother/Father/ PBSCT

Father/PBSCT

Mother/PBSCT

Father/PBSCT

Brother/BM

Donor/source

Yes, 4 trials

No

Died

Died

No

Yes, 3 trials

Yes, 3 trials

No

No

No

No

Graft failure

FLU/BU

FLU/BU

ND

None

FLU/BU

None

None

None

FLU/BU

FLU/BU/ATG

None

Conditioning regimen

CsA

CsA

ND

CsA

CsA

CsA

CsA

CsA

CsA

CsA

CsA

GvHD prophylaxis

JAK3

ND

ND

JAK3

RAG1

JAK3

RAG2

RAG1

IL2RG

RAG1

RAG2

Mutation

M

F

F

F

M

M

F

F

F

M

M

1 SA*

2 EDT

3 PGC

4 SGE***

5 MK

6 TTD***

7 IB

8 MNA**

9 MA**

10 HCÇ##

11 YEK##

+

+

+

+

+

+

-

-

+

+

+

C#

Petechiae

Aspergillosis/FTT

HSM

HSM

Diarrhea/FTT

Omphalitis/ diarrhea

Aspergillosis/FTT

Erythroderma/FTT

Otitis

Pneumonia/FTT

Pneumonia/FTT

at presentation

Symptoms

WAS

CGD

CHS

CHS

MHC class 2

LAD

CGD

Omenn

LAD

MHC class 2

HLH

Diagnosis

48

75

3

4

21

4

120

3

13

12

57

(months)

Age at HSCT

MUD

MUD

1 Ag MM

MRD

MRD

MRD

Haplo

Haplo

MRD

MRD

Haplo

Type of HSCT

-

-

Father/PBSCT

Mother/BM

Brother/BM

Sister/BM

Father/PBSCT

Mother/PBSCT

MRD/BM

MRD/BM

Father/PBSCT

Donor/source

No

No

No

No

No

No

No

Yes 2 trials

No

Yes

No

Graft failure

BU/FLU/Alem

BU/FLU/Alem

BU/FLU/ATG

BU/FLU/ATG

BU/FLU

FLU/BU/ATG

Thio/BU/FLU/Alem

FLU/BU

FLU/BU

FLU/BU

None

CsA

CsA

CsA

CsA

CsA

CsA

CsA

CsA

CsA

CsA

CsA

Conditioning regimen GvHD prophylaxis

*Exome sequencing showed ATM frame-shift mutation after he died, **Siblings, ***Referred from another region of Turkey, ## HSCT performed at Akdeniz University. CHS: Chediak-Higashi syndrome, BM: bone marrow, Thio: thiotepa, BU: busulfan, CsA: cyclosporine, F: female, FTT: failure to thrive, FLU: fludarabine, GvHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation, M: male, MRD: matched related donor, MM: mismatched, ATG: antithymocyte globulin, Alem: alemtuzumab, NK: natural killer cell, PBSCT: peripheral blood stem cell transplantation, SCID: severe combined immunodeficiency, T: T cell, HSM: hepatosplenomegaly, ND: not done, C#: consanguinity.

Sex

Patient

Table 2. Characteristics and transplantation data of non-severe combined immunodeficiency patients.

*Conley et al. [5], **Referred from another region of Turkey. B: B cell, BM: bone marrow, BU: busulfan, CsA: cyclosporine, F: female, FTT: failure to thrive, FLU: fludarabine, GvHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation, M: male, MRD: matched related donor, ATG: antithymocyte globulin, NK: natural killer cell, PBSCT: peripheral blood stem cell transplantation, SCID: severe combined immunodeficiency, T: T cell, ND: not done, C#: consanguinity.

Sex

Patient

Table 1. Characteristics and transplantation data of severe combined immunodeficiency patients. Turk J Hematol 2017;34:345-349 Yıldıran A, et al: HSC Transplantation in Patients with PID

347


348

Yes

Yes

No

No

No

No

Yes

No

No

No

Yes

1 FD

2 MK

3 MO

4 IY

5 PA

6 YFK*

7 BY

8 ET**

9 FZS***

10 EK

11 GS

No

No

No

No

No

Yes

No

No

No

No

No

reactivation

CMV

No

No

Yes/staph

Yes/staph

No

No

No

No

Catheter/ Acinetobacter

No

No

Bacterial infections/cause

No

No

Yes/unknown

No

No

CMV

No

No

No

No

No

Viral infections/cause

Skin

No

ND

No

No

No

No

No

Skin

No

Skin

aGvHD/site

No

Yes/pneumothorax

No

No

No

No

No

No

Hemophagocytosis

PICU admission/cause

No

No

Autoimmune anemia No

ND

No

No

No

Skin

No

No

No

No

cGvHD/site

98%/no

80%/yes

ND

ND

35%/no

0%/yes

17%/yes

87%/yes

100%/no

95%/yes

90%/yes

Last chimerism/ need for IVIG

Alive

Alive

Dead

Dead

Alive

Dead

Alive

Alive

Alive

Alive

Dead

Patient status

No

No

No

No

No

No

No

No

No

No

No

1 SA

2 EDT

3 PGC

4 SGE

5 MK

6 TTD

7 IB

8 MNA

9 MA

10 HCC

11 YEK

No

No

No

No

No

No

No

No

No

No

No

reactivation

CMV

No Cryptosporidium No No No Unknown

No

S. haemolyticus E. faecium P. aeruginosa No

CMV

No

No

No

CMV

CMV

No

No

No

No

Viral infections/ cause

No

Bacterial infections/ cause

No

Skin

No

No

No

Skin

No

No

No

No

No

aGvHD/ site

No

No

No

No

No

Skin

No

No

No

No

No

cGvHD/ site

No

No

Yes/hemophagocytosis

No

No

No

Yes/ineffective ventilation

No

No

Yes/sepsis

Yes/hemophagocytosis

PICU admission/cause

ND: Not done, aGvHD: acute graft-versus-host disease, cGvHD: chronic graft-versus-host disease, CMV: cytomegalovirus, PICU: pediatric intensive care unit.

BCG activation

Patient

Table 4. Outcomes and complications of non-SCID patients.

ND: Not done, aGvHD: acute graft-versus-host disease, cGvHD: chronic graft-versus-host disease, CMV: cytomegalovirus, BCG: bacillus Calmette-Guerin, PICU: pediatric intensive care unit.

100%

ND

ND

100%

100%

90%

ND

5%

100%

ND

ND

Alive

Alive

Alive

Alive

Alive

Alive

Dead

Dead

Alive

Dead

Dead

Last chimerism Patient status

*She was treated for CMV infection at another center before being sent to our clinic, **She had bilateral pneumothorax and Staphylococcus septicemia before being sent to our clinic, *** She was in sepsis before being referred to our clinic and died on the same day.

BCG activation

Patient

Table 3. Outcomes and complications of severe combined immunodeficiency patients.

Yıldıran A, et al: HSC Transplantation in Patients with PID Turk J Hematol 2017;34:345-349


Yıldıran A, et al: HSC Transplantation in Patients with PID

Turk J Hematol 2017;34:345-349

In recent report from Jordan, a country that resembles Turkey socially, Amayiri et al. [10] concluded that delayed diagnosis (or referral) and reactivation of bacillus Calmette-Guerin (BCG) are unique challenges for patients with PID disorders. Similarly, delayed diagnosis is an important problem in our region because of the insufficient number of immunologists and lack of physician awareness about PID disorders [11]. BCG vaccine reactivation has an important effect on the prognosis of combined immunodeficiencies, but this vaccine also helps to identify interferon gamma/interleukin 2 axis defects with BCGitis [12] and to determine T-lymphocyte function with a positive tuberculin (purified protein derivative) test. Epidemiological studies in various countries have shown that X-linked common gamma-chain deficiency is the most common type of SCID, affecting almost half of all patients. In our patients with SCID, 55% had RAG1 and RAG2, 33% had JAK3, and 11% had IL2RG mutations. The present study showed that RAG mutations are more prevalent in SCID cases in Turkey than in Europe and the United States [13]. However, gamma-chain deficiencies are rare in the Greek population [14]. The higher incidence of RAG mutations in our region could be related to high parental consanguinity. We used the CliniMACS method for efficient T-cell depletion prior to transplantation. After applying this method, we observed acute (18%-28%) and chronic (9%-18%) GVHD in the SCID and non-SCID cases. Our patients displayed low infection rates and BCG activation and less need for treatment in the pediatric intensive care unit (PICU), which could have been due to the use of prophylactic antituberculosis treatment. A previous study addressing the outcomes of and mortalityrelated risk factors for pediatric patients with PID requiring PICU admission reported respiratory problems as the leading cause for hospital admission [15]. In our study, six patients required PICU admission, mostly due to severe infection and respiratory problems.

Conclusion This study showed that PID disorders are common and that the delayed diagnosis of such disorders is an important problem in the Black Sea Region of Turkey. Routine screening for these diseases should be performed in newborn infants. Acknowledgments We would like to thank Professor Dr. Aydan İkincioğulları and Professor Dr. Fikret Arpacı for their material and moral support. Ethics Ethics Committee Approval: Retrospective study. Informed Consent: Retrospective study.

Authorship Contributions Surgical and Medical Practices: M.H.Ç., A.Y., S.B., Ş.N.G., M.E., T.F., E.Ö., R.S., G.O.; Concept: A.Y.; Design: A.Y.; Data Collection or Processing:  A.Y.; Analysis or Interpretation:  A.Y.; Literature Search: M.H.Ç., A.Y.; Writing: M.H.Ç., A.Y. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Hagin D, Burroughs L, Torgerson TR. Hematopoietic stem cell transplant for immune deficiency and immune dysregulation disorders. Immunol Allergy Clin North Am 2015;35:695-711. 2. Gatti R, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 1968;2:1366-1369. 3. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet 1968;2:1364-1366. 4. Cipe FE, Dogu F, Aytekin C, Yuksek M, Kendirli T, Yildiran A, Bozdogan G, Karatas D, Reisli I, Dalva K, Arpacı F, Ikinciogullari A. HLA-haploidentical transplantations for primary immunodeficiencies: a single-center experience. Pediatr Transplant 2012;16:451-457. 5. Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary immunodeficiencies. Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol 1999;93:190-197. 6. Pasic S, Vujic D, Veljković D, Slavkovic B, Mostarica-Stojkovic M, Minic P, Minic A, Ristic G, Giliani S, Villa A, Sobacchi C, Lilić D, Abinun M. Severe combined immunodeficiency in Serbia and Montenegro between years 1986 and 2010: a single-center experience. J Clin Immunol 2014;34:304-308. 7. Yorulmaz A, Artaç H, Kara R, Keleş S, Reisli İ. Primer immün yetmezlikli 1054 olgunun retrospektif değerlendirmesi. Astım Alerji İmmünoloji 2008;6:127134 (in Turkish). 8. Borte S, von Döbeln U, Fasth A, Wang N, Janzi M, Winiarski J, Sack U, PanHammarström Q, Borte M, Hammarström L. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex realtime PCR. Blood 2012;119:2552-2555. 9. Arpacı F, Tezcan İ, Kuzhan O, Yalman N, Uçkan D, Kürekçi AE, İkincioğulları A, Özet A, Tanyeli A. G-CSF-mobilized haploidentical peripheral blood stem cell transplantation in children with poor prognostic nonmalignant disorders. Am J Hematol 2008;83:133-136. 10. Amayiri N, Al-Zaben A, Ghatasheh L, Frangoul H, Hussein AA. Hematopoietic stem cell transplantation for children with primary immunodeficiency diseases: single center experience in Jordan. Pediatr Transplant 2013;17:394-402. 11. Yüksek M, İkincioğulları A, Doğu F, Elhan A, Yüksek N, Reisli I, Babacan E. Primary immune deficiency disease awareness among a group of Turkish physicians. Turk J Pediatr 2010;52:372-377. 12. Yıldıran A, Ak E, Akyol Ş, Sancak R, Picard C, Dogu F, Ikinciogullari A. Yaygın BCG enfeksiyonu olan IL12R defekti olgusu. Deneysel ve Klinik Tıp Dergisi 2010;27:85-87 (in Turkish). 13. Buckley RH. Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution. Annu Rev Immunol 2004;22:625655. 14. Michos A, Tzanoudaki M, Villa A, Giliani S, Chrousos G, Kanariou M. Severe combined immunodeficiency in Greek children over a 20-year period: rarity of γc-chain deficiency (X-linked) type. J Clin Immunol 2011;31:778-783. 15. Odek C, Kendirli T, Doğu F, Yaman A, Vatansever G, Çipe F, Haskoloğlu Ş, Ateş C, İnce E, İkincioğulları A. Patients with primary immunodeficiencies in pediatric intensive care unit: outcomes and mortality-related risk factors. J Clin Immunol 2014;34:309-315.

349


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2016.0096 Turk J Hematol 2017;34:350-351

Bullous Pyoderma Gangrenosum in a Patient with Acute Myelogenous Leukemia as a Pathergic Reaction after Bone Marrow Biopsy Akut Miyelojenik Lösemili Olguda Kemik İliği Biyopsisi Sonrası Paterjik Reaksiyon Şeklinde Büllöz Piyoderma Gangrenosum Gelişmesi Nur Efe İris1,2, Reyhan Diz-Küçükkaya3, Mutlu Arat3, Zahide Eriş4 İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey Avrupa Florence Nightingale Hospital, Clinic of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey 3 İstanbul Bilim University Faculty of Medicine, Department of Internal Medicine Division of Hematology, İstanbul, Turkey 4 İstanbul Bilim University Faculty of Medicine, Department of Dermatology, İstanbul, Turkey 1 2

Figure 1. Bone marrow biopsy puncture area with 6x4 cm cribriform ulceration with expanding bullous margin.

A 59-year-old male patient presented with a wound over the sacral region on a bone marrow biopsy puncture that had been present for 3 weeks (Figure 1). There was an ulceration of 6x4 cm with a bullous margin. Bullous pyoderma gangrenosum (PG) was diagnosed by the dermatology consultant. Histopathologic examination of the biopsy specimen from the ulcer showed necrosis with an underlying mixed inflammatory cell infiltration

within the dermis extending to the subcutis. Cultures of skin biopsies were negative for bacteria, fungi, and atypical mycobacteria. A bone marrow biopsy showed acute myelogenous leukemia (AML) transformed from myelodysplastic syndrome. PG is an uncommon neutrophilic ulcerative skin disease. In contrast to its name, PG is neither an infectious nor a gangrenous condition. Pathergy is commonly observed, especially after

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Nur EFE İRİS, M.D., İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey Phone : +90 212 361 88 00 E-mail : nurefeiris@yahoo.coml ORCID-ID: orcid.org/0000-0002-4859-0009

350

Received/Geliş tarihi: March 10, 2016 Accepted/Kabul tarihi: June 02, 2016


Turk J Hematol 2017;34:350-351

Efe İris N, et al: Bullous PG in a Patient with AML as a Pathergic Reaction after Bone Marrow Biopsy

debridement of a lesion [1,2]. In PG there is an excessive inflammatory reaction to trauma of the skin by a needle. In this case there was a pathergic reaction after bone marrow biopsy. Definitive diagnosis requires both clinical recognition and exclusion of infectious or neoplastic disorders [3]. PG is usually associated with an underlying systemic disease [1,4]. Based on clinical morphology, PG is classified into four variants: ulcerative, pustular, bullous, and vegetative [5]. Bullous PG is commonly associated with myeloproliferative diseases [5]. Association with leukemia signifies a poor prognosis [5]. Our patient was in remission for AML, he underwent allogeneic hematopoietic stem cell transplantation, and the PG resolved completely. Keywords: Acute myelogenous leukemia, Bullous pyoderma gangrenosum, Pathergy Anahtar Sözcükler: Akut myeloid lösemi, Büllöz piyoderma gangrenosum, Paterji

Informed Consent: It was received. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Fox PL, Geyer AS, Husain S, Grossman ME. Bullous pyoderma gangrenosum as the presenting sign of fatal acute myelogenous leukemia. Leuk Lymphoma 2006;47:147-150. 2. Bennett ML, Jackson JM, Jorizzo JL, Fleischer AB Jr, White WL, Callen JP. Pyoderma gangrenosum. A comparison of typical and atypical forms with an emphasis on time to remission. Case review of 86 patients from 2 institutions. Medicine (Baltimore) 2000;79:37-46. 3. Callen JP, Dubin HV, Gehrke CF. Recurrent pyoderma gangrenosum and agnogenic myeloid metaplasia. Arch Dermatol 1977;113:1585-1586. 4. Srivastata M, Rencic A, Nousari HC. A rapidly expanding ulcer. Myelodysplastic syndrome-associated (paraneoplastic) pyoderma gangrenosum. Arch Dermatol 2003;139:531-536. 5. Powell FC, Su WP, Perry HO. Pyoderma gangrenosum: classification and management. J Am Acad Dermatol 1996;34:395-409.

351


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2016.0199 Turk J Hematol 2017;34:352-353

Giant Intracranial Solitary Plasmacytoma İntrakraniyal Dev Plazmositom Osman Kara1, Tayfur Toptaş1, Işık Atagündüz1, Süheyla Bozkurt2, Önder Şirikçi3, Tülin Fıratlı Tuğlular1 Marmara University Faculty of Medicine, Department of Hematology, İstanbul, Turkey Marmara University Hospital Faculty of Medicine, Department of Pathology, İstanbul, Turkey 3 Marmara University Hospital Faculty of Medicine, Department of Biochemistry, İstanbul, Turkey 1 3

Figure 1. The mass occupied the left frontoparietal region, displacing the left lateral ventricle and causing a shift of the midverge of the brain.

A 41-year-old man presented with complaints of severe headache and vomiting during the last 5 days. Neurological and systemic examination revealed no abnormality. A mass of 112x49 mm, which occupied the left frontoparietal parenchymal region, was evident on the T1 sequence of magnetic resonance imaging (Figure 1). This homogeneous contrasted mass displaced the left lateral ventricle and caused a shift of the mid-verge of the brain. The mass was totally removed (Figure 2). Pathological examination revealed a plasma cell dyscrasia with lambda monoclonality.

Figure 2. Postoperative cranial magnetic resonance imaging.

Bone marrow biopsy was consistent with a clonal plasma cell accumulation of 5%. Two tiny M-protein peaks were detected on serum protein electrophoresis, which was compatible with immunoglobulin (Ig) G-and IgA-lambda monoclonal bands on serum immunofixation electrophoresis (Figure 3). There were no other plasmacytomas or lytic lesions detected with positron emission tomography-computed tomography imaging. The patient was diagnosed with a solitary plasmacytoma and treated with radiotherapy only. He had no complaints at the sixth month after diagnosis.

©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Osman KARA, M.D., Marmara University Faculty of Medicine, Department of Hematology, İstanbul, Turkey Phone : +90 505 492 69 25 E-mail : dr_osmankara@hotmail.com ORCID-ID: orcid.org/0000-0001-9531-8123

352

Received/Geliş tarihi: May 31, 2016 Accepted/Kabul tarihi: August 15, 2016


Turk J Hematol 2017;34:352-353

Kara O, et al: Giant Intracranial Solitary Plasmacytoma

Keywords: Plasmacytoma, Myeloma, Intracranial Anahtar Sözcükler: Plazmasitom, Miyelom, İntrakraniyal Informed Consent: Was obtained from the patient. Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Figure 3. M-protein peaks on serum protein electrophoresis and monoclonal bands on immunofixation electrophoresis are depicted. Ig: Immunoglobulin.

353


IMAGES IN HEMATOLOGY DOI: 10.4274/tjh.2017.0203 Turk J Hematol 2017;34:354-355

Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia Mycoplasma pneumonia Pnömonisinde Alışılmamış Soğuk-Aracılı Lökoaglütinasyon Yasushi Kubota1,2, Yuka Hirakawa3, Kazuo Wakayama4, Shinya Kimura1 Saga University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Saga, Japan Saga University Faculty of Medicine, Department of Transfusion Medicine, Saga, Japan 3 Saga University Faculty of Medicine, Department of General Medicine, Saga, Japan 4 Saga University Faculty of Medicine, Department of Clinical Laboratory Medicine, Saga, Japan 1 2

Figure 1. A peripheral blood smear showed not only RBC agglutination (A) but also neutrophil aggregates, eosinophil aggregates, and monocyte aggregates (A-D). ©Copyright 2017 by Turkish Society of Hematology Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Yasushi KUBOTA, M.D. Saga University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Saga, Japan Phone : +81-952-34-23 66 E-mail : kubotay@cc.saga-u.ac.jp ORCID-ID: orcid.org/0000-0001-7785-1362

354

Received/Geliş tarihi: May 20, 2017 Accepted/Kabul tarihi: June 12, 2017


Turk J Hematol 2017;34:354-355

Kubota Y. et al. Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia

An 18-year-old woman was diagnosed with atypical pneumonia and treated with oral levofloxacin. Skin eruptions also appeared. On day 6 after admission, laboratory tests revealed the following: red blood cells (RBCs), 1.76x109/L; hemoglobin, 128 g/L; white blood cells (WBCs), 7x109/L with 56% neutrophils, 27% lymphocytes, 6% monocytes, 10.5% eosinophils, and 1% basophils. A peripheral blood smear showed not only RBC agglutination but also neutrophil aggregates, eosinophil aggregates, and monocyte aggregates (Figure 1). After warming to 37 °C, the agglutination disappeared. The RBC and WBC counts returned to 4.44x109/L and 9x109/L with 55% neutrophils, 26% lymphocytes, 6% monocytes, 12% eosinophils, and 1% basophils. Blood chemistry analysis showed total bilirubin of 0.4 mg/dL and lactate dehydrogenase of 510 U/L. A direct antiglobulin test showed 1+ anti-C3d and 1+ anti-C3b3d. A passive agglutination test in paired serum samples revealed seroconversion of M. pneumoniae antibodies (1:80 to 1:20,480). Cold agglutinin was detected to a titer of 1:8192.

leukoagglutination [4]. A previous series of four pediatric cases of M. pneumoniae infection, all of which showed leukoagglutination, reported that eruption, eosinophilia, a high titer of cold agglutinin, and a high titer of M. pneumoniae antibodies were observed [5]. When leukocytopenia occurs in patients with these symptoms, pseudoleukopenia induced by leukoagglutination should be recognized as one potential cause.

2.

Gertz MA. Management of cold haemolytic syndrome. Br J Haematol 2007;138:422-429.

Cold-induced erythrocyte agglutination is frequently observed in cases of M. pneumoniae infection, but leukoagglutination is rare [1,2]. Though the pathomechanism of leukoagglutination is still uncertain [3], it has been postulated that immunoglobulin M cold agglutinin directed against I antigens of the leukocyte membranes is responsible for transient cold-induced

3.

Glasser L. Pseudo-neutropenia secondary to leukoagglutination. Am J Hematol 2005;80:147.

Keywords: Leukoagglutination, Cold agglutinin, Mycoplasma pneumoniae, Eosinophilia, Pseudoleukopenia Anahtar Sözcükler: Lökoaglütinasyon, Soğuk aglütinin, Mycoplasma pneumoniae, Eozinofili, Psödolökopeni

References 1. Berentsen S, Randen U, Tjønnfjord GE. Cold agglutinin-mediated autoimmune hemolytic anemia. Hematol Oncol Clin North Am 2015;29:455471.

4. Pruzanski W, Faird N, Keystone E, Armstrong M. The influence of homogeneous cold agglutinins on polymorphonuclear and mononuclear phagocytes. Clin Immunol Immunopathol 1975;4:277-285. 5. Takiguchi M, Iizuka A, Nagao T. White blood cell aggregation in Mycoplasma pneumoniae infection. Syonika 1981;22:643-648 (in Japanese).

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LETTERS TO THE EDITOR Turk J Hematol 2017;34:356-381

Liver Transplantation in a Patient with Acquired Dysfibrinogenemia Who Presented with Subdural Hematoma: A Case Report Subdural Hematom ile Prezente Olan Edinsel Disfibrinojenemi Nedenli Karaciğer Nakli Uygulanan Hasta: Olgu Sunumu Şencan Acar1, Gökhan Güngör2, Murat Dayangaç3, Reyhan Diz-Küçükkaya4, Yaman Tokat3, Murat Akyıldız1 İstanbul Memorial Ataşehir Hospital, Liver Transplantation Unit, İstanbul, Turkey Konya Training and Research Hospital, Department of Internal Diseases, Konya, Turkey 3 İstanbul Bilim University Faculty of Medicine, Department of General Surgery, İstanbul, Turkey 4 İstanbul Bilim University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey 1 2

To the Editor, Fibrinogen is one of the most abundant proteins in the blood; normal levels range from 200 to 400 mg/dL. Fibrinogen is synthesized in the liver and is essential for the clotting of blood. It also binds to platelets, supports aggregation, and plays an important role in wound healing. Fibrinogen deficiencies can be caused by decreased levels (hypo- or afibrinogenemia) or defective function (dysfibrinogenemia). Dysfibrinogenemia may either be autosomal dominantly inherited or acquired and it can manifest as bleeding or thrombotic events, or in some cases both simultaneously. Situations causing acquired dysfibrinogenemia include chronic liver disease, malignancies, and autoimmune diseases. Herein, we report a liver transplant recipient with dysfibrinogenemia who presented with subdural hematoma due to liver cirrhosis.

The neurosurgery specialist suggested conservative treatment because of the high risk of surgery for the patient who had decompensated liver cirrhosis. The patient then consulted with the hematology department. Both the patient and his family had a negative history of bleeding. The peripheral blood smear was not consistent with disseminated intravascular coagulopathy (DIC). Thrombin time (TT) was 26 s (normal range: 16-20 s), fibrinogen activity was 106 (180-350), PT was 18.7 s (9.8-12.7 s), and activated partial thromboplastin time (aPTT) was 41.7 s (27-38.8 s). Results for the mixing test (the patient’s plasma was mixed with normal pool plasma), corrected PT, aPTT, TT, and factors X, V, VIII, and IX were within normal limits. Acquired dysfibrinogenemia because of liver cirrhosis was diagnosed based on thromboelastographic findings.

A 41-year-old male presented to the emergency department with headache and sicchasia. Cranial computerized tomography (CT) imaging showed subdural hematoma and surgical drainage was planned. Since he had thrombocytopenia, prolonged prothrombin time (PT), and hyperbilirubinemia, he was evaluated by a gastroenterology specialist and diagnosed with decompensated liver cirrhosis. Laboratory findings are shown in Table 1. Abdominal CT imaging showed liver cirrhosis, ascites, and splenomegaly.

Cryoprecipitate and fresh frozen plasma were administered until coagulation test results returned to normal, but the patient’s consciousness deteriorated. Subdural hematoma drainage was then performed and consciousness dramatically improved. The patient was placed on the waiting list for cadaveric liver transplantation due to decompensated liver cirrhosis. Cadaveric liver transplantation was performed 3 months later. Posttransplant follow-up coagulation tests were dramatically improved (Table 2).

Table 1. The patient’s laboratory findings. Hb: 9.9 g dL-1 WBC: 3560 µL

aPTT: 41.7 s (27-38.8 s)

GGT: 69 U L-1

Fibrinogen activity: 106 (180-350)

TT: 26 (16-20 s)

T.Bil: 3.2 mg dL

D-dimer: 1.43 (<0.7)

PLT: 74,000 µL-1

AST: 43 U L-1

D.Bil: 1.77 mg dL-1

Creatinine: 0.7 mg dL-1

INR: 1.7

ALT: 27 U L-1

Albumin: 4 g dL-1

Ammonia: 134 µg dL-1

PT: 18.7 s (9.8-12.7 s)

ALP: 26 U L-1

Globulin: 2.5 g dL-1

-1

-1

Hb: Hemoglobin, aPTT: activated partial thromboplastin time, GGT: gamma-glutamyltransferase, WBC: white blood cell, PLT: platelet, INR: international normalized ratio, PT: prothrombin time.

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Table 2. The patient’s hematological laboratory findings  upon liver transplantation follow-up. PT: 10.3 s aPTT: 31.5 s Fibrinogen activity: 218 D-dimer: 0.47 PT: Prothrombin time, aPTT: activated partial thromboplastin.

Dysfibrinogenemia can be caused by posttranslational sialylation of fibrinogen, as is seen in patients with chronic liver disease, and it can present with bleeding complications. Dysfibrinogenemia can also cause thrombotic complications if defective fibrinogen molecules are resistant to plasmin cleavage. In the presented case, dysfibrinogenemia secondary to liver cirrhosis resulted in serious bleeding that was cured following liver transplantation. In cases of portal hypertension and liver cirrhosis, defective hemostasis can occur due to a decrease in procoagulant and anticoagulant protein synthesis, a decrease in the destruction of activated coagulant factors, functionally abnormal fibrinogen synthesis, thrombocytopenia, and abnormal platelet function [1,2,3]. Abnormal fibrinogen is found in patients with diseases characterized by increased sialic acid content. However, dysfibrinogenemia may also occur in other systemic diseases such as multiple myeloma, autoimmune disorders, and malignancy, and with the usage of some medications (glucocorticoid, isotretinoin, and antileukemic agents) [4,5]. When dysfibrinogenemia is suspected, factor deficiencies and DIC should be excluded. Normal or high antigen levels and low functional activity are commonly seen in patients with dysfibrinogenemia [6].

LETTERS TO THE EDITOR

There is no specific treatment for dysfibrinogenemia and treatment should be personalized. Fresh frozen plasma or cryoprecipitate should be given for bleeding [7]. However, antithrombotic treatment (warfarin) should be offered in cases of thrombotic complications. Acquired dysfibrinogenemia may lead to fatal bleeding in cirrhotic patients and liver transplantation is the only curative treatment in cases of that rare complication. Keywords: Dysfibrinogenemia, Liver transplantation, Subdural hematoma Anahtar Sözcükler: Disfibrinojenemi, Karaciğer nakli, Subdural hematom Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Sallah S, Kato G. Evaluation of bleeding disorders. A detailed history and laboratory tests provide clues. Postgrad Med 1998;103:209-218. 2. Senzolo M, Burra P, Cholongitas E, Burroughs AK. New insights into the coagulopathy of liver disease and liver transplantation. World J Gastroenterol 2006;12:7725-7736. 3. Fuse I. Disorders of platelet function. Crit Rev Oncol Hematol 1996;22:1-25. 4. Undas A, Zabczyk M, Iwaniec T. Dysfibrinogenemia: from bleeding tendency to thromboembolic disorders. Boletim da SPHM 2011;26:5-17. 5. Undas A. Acquired dysfibrinogenemia in atherosclerotic vascular disease. Pol Arch Med Wewn 2011;121:310-318. 6. Beyan C. Diagnostic management in patients with thrombophilia. Turkiye Klinikleri J Int Med Sci 2005;1:71-81 (in Turkish with an abstract in English). 7. Soransen B, Bevan D. A critical evaluation of cryoprecipitate for replacement of fibrinogen. Br J Haematol 2010;149:834-843.

Address for Correspondence/Yazışma Adresi: Şencan ACAR, M.D., İstanbul Memorial Ataşehir Hospital, Liver Transplantation Unit, İstanbul, Turkey Phone : +90 533 769 71 96 E-mail : sencanacar@yahoo.com ORCID-ID: orcid.org/0000-0001-8086-0956

Received/Geliş tarihi: March 08, 2017 Accepted/Kabul tarihi: May 31, 2017 DOI: 10.4274/tjh.2017.0045

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Chronic Active Parietal Osteomyelitis Due to Salmonella typhi in a Patient with Sickle Cell Anemia Orak Hücreli Anemi Hastasında Salmonella typhi Kaynaklı Kronik Aktif Parietal Osteomiyelit Ahmad Antar1, George Karam2, Maurice Kfoury3, Nadim El-Majzoub4 Almoosa Specialist Hospital, Department of Internal Medicine, Division of Hematology-Oncology, Al-Ahsa, Saudi Arabia Almoosa Specialist Hospital, Department of Neurosurgery, Al-Ahsa, Saudi Arabia 3 Almoosa Specialist Hospital, Department of Diagnostic Radiology, Al-Ahsa, Saudi Arabia 4 American University of Beirut Medical Center, Department of Pathology and Laboratory Medicine, Beirut, Lebanon 1 2

To the Editor, Sickle cell disease (SCD) is a genetic disorder characterized by marked heterogeneity in clinical and hematologic severity, with musculoskeletal system manifestations being a major cause of morbidity and disability [1]. The increased susceptibility of SCD patients to infections, including osteomyelitis, has long been recognized with several mechanisms postulated including impaired splenic function, defects in complement activation, genetic factors, deficiencies in micronutrients, and the presence of infarcted or necrotic bone [2]. Salmonella is the most common cause of osteomyelitis in SCD, followed by Staphylococcus aureus and gram-negative enteric bacilli; this prevalence could be related to the fact that intravascular sickling of the bowel leads to patchy ischemic infarction [3,4]. The most common sites of osteomyelitis are the femur, tibia, or humerus. Patients usually present with acute onset of pain, swelling, and tenderness over the affected area in association with fever and elevated inflammatory markers. However, in some cases, osteomyelitis has atypical presentations with a more indolent course and often with abscess formation [5]. Here we present a 50-year-old female patient with sickle cell anemia (SCA) who developed parietal osteomyelitis with abscess formation and involvement of the dura due to Salmonella typhi, who was treated successfully by surgery followed by antibiotics. A 50-year-old Saudi female patient living in the Eastern Province of Saudi Arabia, diagnosed with SCA (HgS: 78%) with occasional vaso-occlusive crisis and no sickle cell-related complications, presented to us with a 1-month history of a painless right parietal subgaleal collection increasing in size over time with no history of trauma and no fever or neurological manifestations. Laboratory testing revealed an elevated white blood cell count and a high estimated sedimentation rate level (125 mm/h). Magnetic resonance imaging of the brain revealed an osteolytic defect centered on the right parietal bone and sizable subgaleal complex collection (Figure 1). The patient underwent right 358

Figure 1. Magnetic resonance imaging of the brain demonstrating peripheral enhancement of the scalp abscess alongside noticeable enhancement of the adjacent soft tissues, pathological bone, and underlying pachymeningeal layer enhanced on a coronal T1W slice (left) with a clear intra-osseous edematous edema in keeping with osteomyelitic changes isolating a central bone sequestrum, communicating with a subperiosteal/subgaleal fluid collection on a fluid attenuated inversion recovery weighted slice (right). parietal craniectomy with cranioplasty (removal of the right parietal subgaleal collection and the corresponding bone in addition to the invaded dura). Pathology of the specimen revealed a right parietal subgaleal abscess and right parietal bone chronic active osteomyelitis. Culture of the specimen grew Salmonella typhi. The morbidity of chronic osteomyelitis combined with other complications of SCD decreases patients’ quality of life. Patients with SCD are more prone to osteomyelitis. The most common causative organism is Salmonella. The usual manifestations of osteomyelitis are pain, swelling, tenderness, and fever. However, like in our case, sometimes osteomyelitis presents late, as a more indolent process often with abscess formation and in unusual and more critical sites. Our case highlights the atypical presentation of osteomyelitis in a patient with SCD, which could cause devastating complications if not treated properly, early, and by a multidisciplinary team approach.


LETTERS TO THE EDITOR

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Keywords: Sickle cell anemia, Osteomyelitis, Salmonella typhi

References

Anahtar

1. Almeida A, Roberts I. Bone involvement in sickle cell disease. Br J Haematol 2005;129:482-490.

Sözcükler:

Orak

hücreli

anemi,

Osteomiyelit,

Salmonella typhi

2. Booth C, Inusa B, Obaro SK. Infection in sickle cell disease: a review. Int J Infect Dis 2010;14:e2-e12.

Conflict of Interest: The authors of this paper have no conflicts

3. Atkins BL, Price EH, Tillyer L, Novelli V, Evans J. Salmonella osteomyelitis in sickle cell disease children in the East End of London. J Infect 1997;34:133-138.

of interest, including specific financial interests, relationships,

4. Burnett MW, Bass JW, Cook BA. Etiology of osteomyelitis complicating sickle cell disease. Pediatrics 1998;101:296-297.

and/or affiliations relevant to the subject matter or materials included.

5. Barrett-Connor E. Bacterial infection and sickle cell anemia. An analysis of 250 infections in 166 patients and a review of the literature. Medicine (Baltimore) 1971;50:97-112.

Address for Correspondence/Yazışma Adresi: Ahmad ANTAR, M.D., Almoosa Specialist Hospital, Department of Internal Medicine, Division of Hematology-Oncology, Al-Ahsa, Saudi Arabia Phone : +013 530 70 00 E-mail : a.antar@almoosahospital.com.sa ORCID-ID: orcid.org/0000-0003-1829-197X

Received/Geliş tarihi: March 06, 2017 Accepted/Kabul tarihi: June 30, 2017 DOI: 10.4274/tjh.2017.0094

Acquired Leukocyte Inclusion Bodies Resembling Döhle Bodies During Acute Cholangitis Akut Kolanjit Seyrinde Lökositlerde Döhle Benzeri Edinsel İnklüzyonlar Gökhan Özgür1, Musa Barış Aykan2, Murat Yıldırım1, Selim Sayın1, Ahmet Uygun3, Cengiz Beyan4 Gülhane Training and Research Hospital, Department of Hematology, Ankara, Turkey Health Sciences University, Gülhane Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey 3 Health Sciences University, Gülhane Faculty of Medicine, Department of Gastroenterology, Ankara, Turkey 4 TOBB University of Economics and Technology Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey 1 2

To the Editor, A 66-year-old woman was admitted to the gastroenterology department with epigastric pain, nausea, and subicterus. Her complaints had begun 6 h earlier. Her abdomen was soft and flat, with localized tenderness on palpation in the right subcostal area. Laboratory studies revealed a white cell count of 17.9x109/L, hemoglobin concentration of 14.4 g/dL, and platelet count of 48x109/L, and they were notable for elevated serum cholestatic enzymes. The abdominal ultrasound was remarkable for cholangitis. The patient received broad-spectrum antibiotics. A peripheral blood smear examination, performed to evaluate thrombocytopenia, revealed the presence of blue intracytoplasmic inclusions in neutrophils (Figures 1A-C). On the 11th day of treatment, her blood smear was examined once again and the Döhle body-like inclusions were resolved (Figure 1D). May-Hegglin anomaly is an uncommon autosomal dominant abnormality characterized by large, basophilic inclusion bodies (resembling Döhle bodies) in neutrophils [1,2]. Döhle bodies

Figure 1. A) Inclusion bodies in neutrophils and macrothrombocyte; B), C) inclusion bodies in neutrophils; D) peripheral blood smear after treatment. can be seen in bacterial infections. Hematologic findings of systemic diseases may be confused with hematological diseases such as May-Hegglin anomaly. We thought that the granules were Döhle bodies due to cholangitis. The disappearance of the inclusion bodies upon treatment is important in differential diagnosis.

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Keywords: Cholangitis, Döhle bodies, May-Hegglin anomaly

References

Anahtar Sözcükler: Döhle cisimciği, Kolanjit, May-Hegglin anomalisi

1. Saito H, Kunishima S. Historical hematology: May-Hegglin anomaly. Am J

Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

2. Gülen H, Erbay A, Kazancı E, Vergin C. A rare familial thrombocytopenia: May-

Hematol 2008;83:304-306.

Hegglin anomaly report of two cases and review of the literature. Turk J Haematol 2006;23:111-114.

Address for Correspondence/Yazışma Adresi: Gökhan Özgür, M.D., Gülhane Training and Research Hospital, Department of Hematology, Ankara, Turkey Phone : +90 312 304 4107 E-mail : gokhanozgur2010@hotmail.com ORCID-ID: orcid.org/0000-0003-0357-0503

Received/Geliş tarihi: March 21, 2017 Accepted/Kabul tarihi: July 26, 2017 DOI: 10.4274/tjh.2017.0121

Three Novel Calreticulin Mutations in Two Turkish Patients İki Türk Hastada Üç Yeni Kalretikulin Mutasyonu Veysel Sabri Hançer1, Hüseyin Tokgöz2, Serkan Güvenç3, Ümran Çalışkan4, Murat Büyükdoğan1 İstinye University Faculty of Medicine, Department of Medical Genetics, İstanbul, Turkey Selçuk University Meram Faculty of Medicine, Department of Pediatric Hematology, Konya, Turkey 3 Batman District State Hospital, Clinic of Hematology Batman, Turkey 4 Necmettin Erbakan University Meram Faculty of Medicine, Department of Pediatrics, Konya, Turkey 1 2

To the Editor, Calreticulin (CALR) mutations were first identified exclusively in JAK2-MPL-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF) at a rate of 60%-88%, accounting for 1/4 to 1/3 of all patients with ET and PMF [1,2,3]. As of today, more than 55 different types of mutations have been reported. The two most common mutations accounting for 85% of mutated cases are either a 52-bp deletion (type 1; c.1099_1150del; L367fs*46; 44%53% of cases) or a 5-bp insertion (type 2; c.1154_1155insTTGTC; K385fs*47; 32%-42% of cases). The remaining 15% include various other infrequent mutations that are often unique or found in only a few patients [4,5]. Here we present three CALR mutations in two patients with PMF and ET that have not been reported before as shown in Figure 1. Known CALR mutations and BCR-ABL, JAK-2 V617F, and MPL 515L/K test results were found to be negative. Patient 1: The patient was a 46-year-old man with low back pain. Magnetic resonance imaging scanning of the lumbosacral region revealed sacroiliitis on the left side and he was referred to a rheumatologist for further investigations. Anemia (Hb: 10.8 g/dL) and thrombocytosis (700x109/L) with a high lactate dehydrogenase level (351 U/L) were found in initial tests. The other tests for a possible rheumatologic disease, including

360

Figure 1. A) Electropherogram result of the primary myelofibrosis patient, B) electropherogram result of the essential thrombocythemia patient. human leukocyte antigen-B27, were all negative when the patient was seen. Physical examination was almost normal with no sign of organomegaly. Spleen size was also normal in the abdominal ultrasound. The peripheral blood smear showed dacrocytes, occasional myelocytes (1%), and metamyelocytes (1%). The bone marrow biopsy showed diffuse grade 3-4 reticulin fibrosis with atypical proliferation of megakaryocytes and increased cellularity consistent with PMF. Patient 2: A 9-year-old pediatric patient with thrombocytosis (2800x109/L) was identified in a routine check-up. Physical


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examination was normal except for mild splenomegaly. Complete blood count revealed increased platelet count (2800x109/L) with normal hemoglobin and leukocyte count. Platelets were very abundant and clustered in the peripheral blood smear. Bone marrow aspiration and biopsy examinations showed tri-lineage hematopoiesis with an increased number and clusters of megakaryocytes without fibrosis, which is consistent with ET. She had persistently elevated platelet counts ranging between 2000x109/L and 2800x109/L without any evidence of reactive/secondary thrombocytosis such as infections, medicine, autoimmune disorders, neoplasms, trauma, surgery, or hematological disorders such as iron deficient anemia, chronic hemolytic situations, and acute hemorrhages. Genomic DNA was extracted from whole blood, exon 9 of the CALR gene was amplified by polymerase chain reaction, and then the amplified fragments were sequenced. All nucleotide numbers refer to the wild-type cDNA sequence of CALR (NM_004343) as reported in Ensembl. Here we report three new CALR mutations [1-bp deletion; c.1116delA (D373fs*57) and c.1120 A>C] in the same patient with PMF and c.1108 G>T in a patient with ET. We performed germline testing from the cheek epithelium and both patient samples were confirmed as wild-type CALR. These novel mutations occurred and changed the amino acid sequence of the C domain amino acid residues, which will interfere with the calcium-binding capacity of the molecule. It is important to determine the type of mutation. Type 2-like CALR mutations are mainly associated with an ET phenotype, low risk of thrombosis, and indolent clinical course, while type 1-like mutations are mainly associated with a myelofibrosis phenotype and a high risk of progression from ET to myelofibrosis. The identification

LETTERS TO THE EDITOR

of new CALR mutations will improve our understanding of the pathophysiology of myeloproliferative neoplasms. Keywords: Essential thrombocythemia, Primary myelofibrosis, Calreticulin Anahtar Sözcükler: Esansiyel trombositemi, Primer miyelofibroz, Kalretikulin Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumie E, Milosevic JD, Them NC, Berg T, Gisslinger B, Pietra D, Chen D, Vladimer GI, Bagienski K, Milanesi C, Casetti IC, Sant’Antonio E, Ferretti V, Elena C, Schischlik F, Cleary C, Six M, Schalling M, Schönegger A, Bock C, Malcovati L, Pascutto C, Superti-Furga G, Cazzola M, Kralovics R. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med 2013;369:2379-2390. 2. Fu R, Zhang L, Yang R. Paediatric essential thrombocythaemia: clinical and molecular features, diagnosis and treatment. Br J Haematol 2013;163:295302. 3. Guglielmelli P, Nangalia J, Green AR, Vanucchi A.M. CALR mutations in myeloproliferative neoplasms: hidden behind the reticulum. Am J Hematol 2014;89:453-456. 4. Tefferi A, Wassie EA, Guglielmelli P, Nangat N, Belachew AA, Lasho TL, Finke C, Ketterling RP, Hanson CA, Pardanani A, Wolanskyj AP, Maffioli M, Casalone R, Pacilli A, Vannucchi AM, Passamonti F. Type 1 versus Type 2 calreticulin mutations in essential thrombocythemia: a collaborative study of 1027 patients. Am J Hematol 2014;89:121-124. 5. Giona F, Teofili L, Capodimonti S, Laurino M, Martini M, Marzella D, Palumbo G, Diverio D, Foà R, Larocca LM. CALR mutations in patients with essential thrombocythemia diagnosed in childhood and adolescence. Blood 2014;12:3677-3679.

Address for Correspondence/Yazışma Adresi: Veysel Sabri HANÇER, M.D., İstinye University Faculty of Medicine, Department of Medical Genetics, İstanbul, Turkey Phone : +90 533 634 30 14 E-mail : vshancer@yahoo.com ORCID-ID: orcid.org/0000-0003-2994-1077

Received/Geliş tarihi: April 05, 2017 Accepted/Kabul tarihi: July 26, 2017 DOI: 10.4274/tjh.2017.0146

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Imatinib-Induced Interstitial Pneumonitis Successfully Switched to Nilotinib in a Patient with Prior History of Mycobacterium tuberculosis Infection Mycobacterium tuberculosis Enfeksiyonu Öyküsü Olan Hastada Başarılı Bir Şekilde Nilotinibe Geçilen İmatinib ile Uyarılmış İnterstisyel Pnömonitis Zhuan-Bo Luo1, Ning Xu1, Xiao-Ping Huang1, Gui-fang Ouyang2 Department of Respiratory Diseases, Ningbo First Hospital, Affiliated Medical School of Ningbo University, Ningbo, China Department of Hematology, Ningbo First Hospital, Affiliated Medical School of Ningbo University, Ningbo, China

1 2

To the Editor, Imatinib mesylate (IM) has been proven to be an effective treatment of chronic myeloid leukemia (CML) and this drug is well tolerated [1]. Interstitial lung disease (ILD) associated with imatinib therapy is rare. We report the case of a patient who had a prior treatment history of Mycobacterium tuberculosis infection and developed interstitial pneumonia after 10 months of imatinib for CML and who has not relapsed since the introduction of the recent tyrosine kinase inhibitor nilotinib. A 48-year-old Chinese man was diagnosed with chronic-phase Philadelphia chromosome-positive CML in January 2015. His medical history was unremarkable, but he had a history of previous treatment for pulmonary tuberculosis 25 years ago. He was initially treated with IM at a dose of 400 mg daily, which was well tolerated. Complete hematological response was rapidly achieved after 2 months. Following the administration of imatinib, the patient gradually developed a dry cough and dyspnea on exertion. In November 2015, he visited the clinic because of progressing nonproductive cough. He had been treated with imatinib at a dose of 400 mg/day for 10 months. On examination, fine crackles were audible, predominantly in both posterior lower lung fields. No elevations of the acutephase reactants were detected, and the immunoglobulin E blood level was within the normal limits. Rheumatoid factor was negative, and antinuclear antibodies were positive at 1/100 with homogeneous staining. Sputum culture was negative and no acid-fast bacilli were observed. Lung function estimation demonstrated mild impairment of gas exchange with diffusing capacity [carbon monoxide diffusion in the lung (DLCO)] of 5.61 mmol/min per kPa (51.9% predicted) and mild restrictive impairment with forced vital capacity of 3.30 L (69.1% predicted). A chest radiograph showed fibrotic scar lesions in the left upper lung field associated with the previous pulmonary tuberculosis. A computed tomography scan (Figure 1A) showed significant 362

extension of the interstitial lung abnormalities, predominantly in the lower lobes. Bronchoscopy revealed normal airways, and histopathological analysis of the transbronchial lung biopsy demonstrated nonspecific interstitial pneumonitis, showing thickened alveolar septa with modest infiltration of chronic inflammatory cells and slight interstitial fibrosis (Figures 2A and 2B). As these findings were highly suggestive of imatinib-induced interstitial pneumonitis, this agent was discontinued and was replaced by nilotinib. At the same time, prednisone at 30 mg/ day was given during the initial days, and it was slowly tapered to 10 mg/day over 2 months. Because no signs of recurrence of pulmonary tuberculosis were detected and the patient was afraid of the side effects of anti-tuberculosis drugs, we did not give anti-tuberculosis prophylaxis, but we maintained close follow-up. This resulted in a gradual improvement in his clinical condition. Partial radiological resolution was observed after 4 months and further improved at 8 months (Figure 1B). DLCO improved to 6.78 mmol/min per kPa (62.8% predicted) and forced vital capacity was 4.50 L (83.3% predicted). The switch to nilotinib at 800 mg daily was well tolerated and followed by complete cytogenetic and major molecular response sustained for 8 months. IM is a targeted therapy that is highly active in patients with CML. It acts by inhibition of tyrosine kinase of the BCR-ABL fusion oncoprotein specific to CML. ILD is a rare adverse event associated with IM therapy. Few case series have been reported [2,3,4,5]. In the present case, the diagnosis of IM-induced ILD was made based on history, clinical symptoms, radiological findings, and pathological results. Furthermore, other etiologic factors for ILD were excluded via microbiologic and clinical studies. Until recently, there has been a lack of data for specific risk factors for the development of IM-induced ILD. However, the incidence of the disease seems higher in patients with preexisting


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LETTERS TO THE EDITOR

Figure 1. A) Chest computed tomography (CT) shows bilateral diffuse subpleural nodules, interlobular septal thickening, reticulation, and peribronchial ground glass opacities in both lungs. B) Chest CT scan 8 months after the switch to prednisone and nilotinib: lung abnormalities were decreased. pulmonary diseases. The largest case series study from Japan, which analyzed 27 patients with IM-induced ILD [6], revealed that preexisting lung disease was present in more than 40% of patients with IM-induced ILD. In this case, we had found fibrotic change and pleural thickening of the left upper lung associated with prior infection of Mycobacterium tuberculosis. Thus, the case raises the possibility of the association between IM-induced ILD and airway injury related to prior infection of Mycobacterium tuberculosis. Physicians caring for a patient presenting with respiratory symptoms while on imatinib therapy should consider interstitial pneumonitis, especially in patients with previous lung or airway injuries resulting from prior infection of Mycobacterium tuberculosis. Among the other tyrosine kinase inhibitors, similar pulmonary complications were reported with dasatinib [7] and gefitinib [8] in Japan, but not with nilotinib. In this case, 8 months after the introduction of nilotinib, interstitial pneumonitis had not recurred. Although the mechanistic basis for the absence of cross-intolerance is not fully understood, second-generation nilotinib appears to be an option in cases of ILD induced by other tyrosine kinase inhibitors.

Figure 2. A and B) Histopathological appearance of transbronchial lung biopsy specimens. Thickened alveolar septa with modest infiltration of chronic inflammatory cells and slight interstitial fibrosis are observed. Hematoxylin and eosin stain (H&E), 100x (A); H&E stain, 400x (B). Keywords: Imatinib mesylate, Interstitial pneumonitis, Chronic myeloid leukemia, Nilotinib, Tuberculosis Anahtar Sözcükler: İmatinib mesilat, İnterstisyel pnömonitis, Kronik miyeloid lösemi, Nilotinib, Tüberküloz Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031-1037. 2. Delomas T, Darne C, Besson C. Lack of recurrence of imatinib-induced interstitial lung disease with nilotinib. Leuk Lymphoma 2012;53:332-333.

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3. Dao K, Vedy D, Lopez J, Staneczek O, Buclin T, Livio F. Imatinib-induced dosedependent interstitial lung disease successfully switched to nilotinib: a case report with concentration exposure data. Int J Hematol 2013;97:299-300. 4. Lee NR, Jang JW, Kim HS, Yhim HY. Imatinib mesylate-induced interstitial lung disease in a patient with prior history of Mycobacterium tuberculosis infection. Korean J Intern Med 2015;30:550-553. 5. Eşkazan AE, Salihoğlu A, Erturan S, Soysal T. Interstitial pneumonitis in a patient with chronic myeloid leukemia. Turk J Hematol 2013;30:435-436. 6. Ohnishi K, Sakai F, Kudoh S, Ohno R. Twenty-seven cases of drug-induced

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interstitial lung disease associated with imatinib mesylate. Leukemia 2006;20:1162-1164. 7. Bergeron A, Rea D, Levy V, Picard C, Meignin V, Tamburini J, BruzzoniGiovanelli H, Calvo F, Tazi A, Rousselot P. Lung abnormalities after dasatinib treatment for chronic myeloid leukemia: a case series. Am J Respir Crit Care Med 2007;176:814-818. 8. Ando M, Okamoto I, Yamamoto N, Takeda K, Tamura K, Seto T, Ariyoshi Y, Fukuoka M. Predictive factors for interstitial lung disease, antitumor response, and survival in non-small-cell lung cancer patients treated with gefitinib. J Clin Oncol 2006;24:2549-2556.

Address for Correspondence/Yazışma Adresi: Zhuan-Bo LUO, M.D., Department of Respiratory Diseases, Ningbo First Hospital, Affiliated Medical School of Ningbo University, Ningbo, China E-mail : luozhuanbo2929@163.com ORCID-ID: orcid.org/0000-0003-0684-8363

Received/Geliş tarihi: April 12, 2017 Accepted/Kabul tarihi: July 28, 2017 DOI: 10.4274/tjh.2017.0155

Prostate Involvement in a Patient with Follicular Lymphoma Foliküler Lenfomalı Hastada Prostat Tutulumu Seda Yılmaz1, Sinan Demircioğlu1, Özlen Bektaş1, Özcan Çeneli1, Sıdıka Fındık2 Necmettin Erbakan University Meram Medicine Faculty, Department of Hematology, Konya, Turkey Necmettin Erbakan University Meram Medicine Faculty, Department of Pathology, Konya, Turkey

1 2

To the Editor, While extranodal involvement is observed in 50% of cases of non-Hodgkin’s lymphoma, prostatic involvement is rare. Prostatic lymphoma accounts for 0.09% of all prostate neoplasms and 0.1% of all non-Hodgkin’s lymphomas [1]. Our patient was monitored for 4 years and had stage 4BS follicular lymphoma (bone marrow involvement; mesenteric lymph nodes in the abdomen, the largest of which was measured as 9x4 cm; cervical and mediastinal lymph nodes; and splenomegaly and B symptoms) at the time of diagnosis. He received CVP (cyclophosphamide, vincristine, prednisolone), CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone), and gemcitabine therapy, respectively, and had lower urinary tract symptoms during follow-up. A hypertrophic prostate was palpated during the physical examination. The prostate-specific antigen (PSA) level was measured to be 8.3 (normal range: 0-4) ng/mL. Urinary analysis showed microscopic hematuria. Ultrasound examination detected a prostate volume of 60 mL. Transurethral resection of the prostate (TUR-P) pathology results showed a diffuse lymphocytic infiltration and positive staining for CD20, CD10, CD5, and BCL-2 (Figure 1). The symptoms of the patient regressed after treatment with rituximab plus bendamustine. Prostate cancer is the most common cancer among men worldwide. There were 1,618,000 cases with 366,000 deaths in 2015 [2]. Prostatic lymphoma is a rare condition that accounts for 0.09% of all prostate neoplasms. While extranodal involvement 364

Figure 1. Diffuse lymphocytic infiltration. is observed in about 50% of cases of non-Hodgkin’s lymphoma, prostatic involvement is rare. The usual clinical manifestations of prostatic involvement in lymphomas are lower urinary tract symptoms and acute urinary retention. High serum PSA levels are not typical for prostatic lymphoma. Our patient presented with high PSA levels. A study that investigated prostate materials from 4831 subjects determined lymphoma in 29 subjects (0.6%). Eleven (0.23%) subjects had a history of concurrent lymphoma [3]. In patients with prostate cancer, the incidence of non-Hodgkin’s lymphoma of the prostate was observed to be 0.2% in a series of 4319 radical prostatectomy cases [4] and 1.19% in another series of 1092 cases [5].


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In conclusion, prostatic lymphoma is clinically difficult to distinguish from benign prostatic hyperplasia and prostatic carcinoma as it occurs in the same age group and presents with similar symptoms; thus, the histopathological and immunohistochemical findings in TUR-P material are important. Early and appropriate treatment improves the patient’s quality and length of life. Keywords: Follicular involvement

lymphoma,

Extranodal,

Prostatic

Anahtar Sözcükler: Foliküler lenfoma, Ekstranodal, Prostat Tutulumu Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Sarris A, Dimopoulos M, Pugh W, Cabanillas F. Primary lymphoma of the prostate: good outcome with doxorubicin-based combination chemotherapy. J Urol 1995;153:1852-1854. 2. Global Burden of Disease Cancer Collaboration, Fitzmaurice C, Allen C, Barber RM, Barregard L, Bhutta ZA, Brenner H, Dicker DJ, ChimedOrchir O, Dandona R, Dandona L, Fleming T, Forouzanfar MH, Hancock J, Hay RJ, Hunter-Merrill R, Huynh C, Hosgood HD, Johnson CO, Jonas JB, Khubchandani J, Kumar GA, Kutz M, Lan Q, Larson HJ, Liang X, Lim SS, Lopez AD, MacIntyre MF, Marczak L, Marquez N, Mokdad AH, Pinho C, Pourmalek F, Salomon JA, Sanabria JR, Sandar L, Sartorius B, Schwartz SM, Shackelford

KA, Shibuya K, Stanaway J, Steiner C, Sun J, Takahashi K, Vollset SE, Vos T, Wagner JA, Wang H, Westerman R, Zeeb H, Zoeckler L, Abd-Allah F, Ahmed MB, Alabed S, Alam NK, Aldhahri SF, Alem G, Alemayohu MA, Ali R, AlRaddadi R, Amare A, Amoako Y, Artaman A, Asayesh H, Atnafu N, Awasthi A, Saleem HB, Barac A, Bedi N, Bensenor I, Berhane A, Bernabé E, Betsu B, Binagwaho A, Boneya D, Campos-Nonato , Castañeda-Orjuela C, CataláLópez F, Chiang P, Chibueze C, Chitheer A, Choi JY, Cowie B, Damtew S, das Neves J, Dey S, Dharmaratne S, Dhillon P, Ding E, Driscoll T, Ekwueme D, Endries AY, Farvid M, Farzadfar F, Fernandes J, Fischer F, G/Hiwot TT, Gebru A, Gopalani S, Hailu A, Horino M, Horita N, Husseini A, Huybrechts I, Inoue M, Islami F, Jakovljevic M, James S, Javanbakht M, Jee SH, Kasaeian A, Kedir MS, Khader YS, Khang YH, Kim D, Leigh J, Linn S, Lunevicius R, El Razek HMA, Malekzadeh R, Malta DC, Marcenes W, Markos D, Melaku YA, Meles KG, Mendoza W, Mengiste DT, Meretoja TJ, Miller TR, Mohammad KA, Mohammadi A, Mohammed S, Moradi-Lakeh M, Nagel G, Nand D, Le Nguyen Q, Nolte S, Ogbo FA, Oladimeji KE, Oren E, Pa M, Park EK, Pereira DM, Plass D, Qorbani M, Radfar A, Rafay A, Rahman M, Rana SM, Søreide K, Satpathy M, Sawhney M, Sepanlou SG, Shaikh MA, She J, Shiue I, Shore HR, Shrime MG, So S, Soneji S, Stathopoulou V, Stroumpoulis K, Sufiyan MB, Sykes BL, Tabarés-Seisdedos R, Tadese F, Tedla BA, Tessema GA, Thakur JS, Tran BX, Ukwaja KN, Uzochukwu BSC, Vlassov VV, Weiderpass E, Wubshet Terefe M, Yebyo HG, Yimam HH, Yonemoto N, Younis MZ, Yu C, Zaidi Z, Zaki MES, Zenebe ZM, Murray CJL, Naghavi M. Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the Global Burden of Disease Study. JAMA Oncol 2017;3:524-548. 3. Chu PG, Huang Q, Weiss LM. Incidental and concurrent malignant lymphomas discovered at the time of prostatectomy and prostate biopsy: a study of 29 cases. Am J Surg Pathol 2005;29:693-699. 4. Eisenberger CF, Walsh PC, Eisenberger MA, Chow NH, Partin AW, Mostwin JL, Marshall FF, Epstein JI, Schoenberg M. Incidental non-Hodgkin’s lymphoma in patients with localized prostate cancer. Urology 1999;53:175-179. 5. Terris MK, Hausdorff J, Freiha FS. Hematolymphoid malignancies diagnosed at the time of radical prostatectomy. J Urol 1997;158:1457-1459.

Address for Correspondence/Yazışma Adresi: Sinan DEMİRCİOĞLU, M.D., Necmettin Erbakan University Meram Medicine Faculty, Department of Hematology, Konya, Turkey Phone : +90 555 432 44 74 E-mail : sinandemircioglumd@gmail.com ORCID-ID: orcid.org/0000-0003-1277-5105

Received/Geliş tarihi: May 02, 2017 Accepted/Kabul tarihi: June 30, 2017 DOI: 10.4274/tjh.2017.0181

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Turk J Hematol 2017;34:356-381

Coexistence of EZH2, NOTCH1, IL7R, and PHF6 Mutations in Adult T-cell Acute Lymphoblastic Leukemia Erişkin T-hücre Akut Lenfoblastik Lösemi’sinde EZH2, NOTCH1, IL7R ve PHF6 Mutasyonlarının Birlikteliği Xilian Zhou1, Yan Gu1, Qi Han1, Mario Soliman2, Chunhua Song2, Zheng Ge1 Zhongda Hospital, Medical School of Southeast University Department of Hematology Nanjing, China Pennsylvania State University, Department of Pediatrics, Pennsylvania, USA

1 2

To the Editor, Enhancer of zestehomolog 2 (EZH2) mutations are reported in solid tumors [1,2,3] as well as leukemia, and they are most commonly detected in early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) [4,5,6,7,8], which is an extraordinarily aggressive malignancy of enigmatic genetic basis [9]. We screened EZH2 mutations in 146 Chinese adult ALL patients, among which 24.7% (36/146) cases were T-cell acute lymphoblastic leukemia (T-ALL) and 12.9% (4/31) T-ALL cases were identified as ETP-ALL. We found three EZH2 mutations in two patients with T-ALL. One patient had Mu#1:D730fs*1, a truncation mutation that was previously reported in acute myeloid leukemia, and the another patient had two new EZH2 mutations, Mu#2:K466T and Mu#3:T467fs*>3 (Figure 1). We also screened the mutations in other genes (Table 1). Strikingly, the EZH2 mutations coexisted with mutations of NOTCH1, IL7R, and PHF6 in the two patients and they responded poorly to chemotherapy and experienced difficult clinical histories and inferior outcomes (Table 1). Patient 1 was diagnosed with T-ALL with myeloid expression based on his bone marrow (BM) smear and immunophenotypes (Table 1). With the first inductive therapy (Table 1), the patient achieved complete remission (CR) with 0.1% blasts in the peripheral blood (PB) and 0.8% in BM. One year later, the patient relapsed with 90.4% lymphoblasts in the BM and 1.0% in the PB, and CR was achieved after the first chemotherapy. During the following treatment, he underwent an intramedullary and an extramedullary relapse infiltrating his left tonsil and then endured three more relapses. On the fifth relapse, the BM blast rate was 50.4%. Although the patient was treated with nelarabine, no CR was achieved in the subsequent treatments. Even though the BM blast rate was 5.2%, the patient died of infection during the BM suppression period after he received the last chemotherapy. We examined the EZH2 mutational status in the BM samples of the 1st relapse, 5th relapse, and 6 weeks after his 5th relapse; the EZH2 and NOTCH1 mutation status remained the same as in the first diagnosis even after the nelarabine treatment (Figure 1D). Patient 2 presented with 80.0% lymphoblasts in the PB and 78.0% blasts in the BM (Table 1). Two somatic mutations, K466T and T467fs*>3 in 366

Figure 1. Location and sequencing data of the EZH2 mutations. A) Mutation 1 (Mu#1:D730fs*1), located in exon 19, is a frame shift-creating insertion; on the protein level, it leads to a truncated protein with a length of 731 amino acids, which is located in the conserved catalytic SET domain(amino acids 618731). This domain is critical for the methyltransferase activity of EZH2. The other two mutations (Mu#2:K466T; Mu#3:T467fs*>3) located within exon 11 are anon-synonymous single-nucleotide substitution and a frame shift-creating deletion, respectively; on the protein level, they result in the substitution of EZH2 lysine466 to tyrosine and a truncation of the EZH2 protein, respectively. Mu#2 and Mu#3 are novel EZH2 mutations; both of them are located in the SANT domain of the EZH2 protein (amino acids 435-485), which is known to be incharge of the DNA binding. Mu#1 was detected in patient 1 and the other two in patient 2. Blue, pink, and yellow bars correspond to exons encoding the SANT domains, the cysteine-rich CXC domain, and the SET domain, respectively. The red arrows show EZH2 mutations. B-F) The direct sequencing data of EZH2 mutations (B, D, F) and wildtype (C, E). B: c.2187_2188insT p.D730fs*1; C: EZH2 exon 19 wildtype; D: Mu#1 after nelarabine treatment; E: EZH2 exon 11 wildtype; F: c.1397A>C; 1399delA p.K466T; T467fs*>3.


Turk J Hematol 2017;34:356-381

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Table 1. Clinical features of patients with EZH2 mutations.  

Patient 1

Patient 2

Age (years)/Sex

49/Male

41/Female

WBC, x109/L

9.7

64.9

Hemoglobin (g/L)

128

71

Platelets (x10 /L)

96

38

BM/PB blasts (%)

50.4/0

78.0/80.0

Patient 1 (nucleotide/mutant ID)

Patient 2 (nucleotide/mutant ID)

9

Mutation screening Gene

ALL (%) (n=146)

T-ALL (%) (n=36)

Exon

EZH2

1.4

5.6

Exon 19

2187_2188insT/COSM52999

Exon 11

1397A>C / (new) 1399delA / (new)

NOTCH1

75.0

74.4

Exon 34

7541_7542delCT/COSM13065

7329_7330delinsCCCA / COSM5752017

Exon 27

5033T>A / COSM21907

PHF6

33.3

33.3

Exon 2

134delG+insCC/(new)

IL7R

3.4

9.7

Exon 2

197T>C/COSM149813

197T>C / COSM149813

Exon 4

254G>A/COSM149814

254G>A / COSM149814

Exon 6

755_756ins9 / (new)

Exon 8

1066A>G/rs3194051

FBXW7

4.6

16.7

Exons 5-12

Negative

Negative

PTEN

12.1

12.5

Exons 1-9

Negative

Negative

CRLF2

27.7

17.2

Exons 1-6

Negative

33C>G / (new)

SH2B3

21.2

16.0

Exons 1-7

Negative

Negative

DNM2

14.7

15.2

Exon 6

789G>A/rs199976453

Exon 20

2139T>C/rs2229920

2139T>C / rs2229920

TP53

6.9

11.1

Exons 4-9

Negative

Negative

JAK1

7.0

14.8

Exons 13,14,16-19

Negative

Negative

Immunophenotype (%)

Patient 1

Patient2

CD34

64.3%

91%

CD13

28%

98%

CD33

97%

90%

CD3

32.5%

-

CD5

99.4%

38%

CD7

99.5%

63%

Hepatomegaly

Negative

Negative

Splenomegaly

Negative

Positive

Lymphadenopathy

Negative

Positive

IKZF1 deletion

Negative

Negative

BCR/ABL1

Negative

Negative

Complex karyotype

Negative

Negative

Treatment

1xHyperCVAD+2xIDA+FLAG+1xFLAG +1xBFM2002-HR-1+1xMOAP+4xCA G+Methylprednisolone+1xICE+3xNel arabine+1xDecitabine+0.5xCAG

1xHyper-CVAD+1xMA; no nelarabine treatment

1st CR time

42 days to achieve CR

Unknown

Relapse time after CR1

21 months

Unknown

Total relapse time

5

Lost to follow-up

Outcome

Death

Lost to follow-up

WBC: White blood cell, BM: bone marrow, PB: peripheral blood, CR: complete remission.

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Turk J Hematol 2017;34:356-381

EZH2 exon 11, were detected in her BM sample (Figure 1). No CR was achieved with the first induction therapy. Finally, the patient was administered methotrexate and cytarabine and endured a long period of BM suppression. Unfortunately, the patient was lost to follow-up. Our data indicated the oncogenic and poor prognostic effect of EZH2 mutations on T-ALL. The coexistence of EZH2 mutations with mutations in the NOTCH1, PHF6, and IL7R genes suggested a new mechanism underlying the tumorigenesis of EZH2 mutations in T-ALL. T-ALL and particularly ETP-ALL still have largely negative outcomes. In the past years, the effect of the use of nelarabine for relapsed and refractory T-ALL seemed to be negligible [10]. In our cohort, the first patient’s relapse, even after nelarabine treatment, revealed the insensitivity of patients with multiple mutations to such treatment. Moreover, our case report suggested that the gene mutations may be the cause of the failure of the drug treatment and emphasized the importance of developing more effective therapies as well as more active and tailored treatments for aggressive T-ALL.

References

Acknowledgment

7. Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, Paul JE, Boyle M, Woolcock BW, Kuchenbauer F, Yap D, Humphries RK, Griffith OL, Shah S, Zhu H, Kimbara M, Shashkin P, Charlot JF, Tcherpakov M, Corbett R, Tam A, Varhol R, Smailus D, Moksa M, Zhao Y, Delaney A, Qian H, Birol I, Schein J, Moore R, Holt R, Horsman DE, Connors JM, Jones S, Aparicio S, Hirst M, Gascoyne RD, Marra MA. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 2010;42:181-185.

This work was supported in part by the National Natural Science Foundation of China (81270613,30973376); Jiangsu Province Key Medical Talents (RC2011077); the Scientific Research Foundation for the Returned Overseas Chinese Scholars; State Education Ministry (39th); China Postdoctoral Science Foundation (20090461134); special grade of financial support from the China Postdoctoral Science Foundation (201003598); the Six Great Talent Peak Plan of Jiangsu (2010-WS-024); the Nanjing Municipal Bureau of Personnel (2009); the Fundamental Research Funds for the Central Universities (2242017K40271, 2242016K40143) (ZG); and the Milstein Medical Asian American Partnership Foundation Research Project Award in Hematology (2017) (ZG and CS). Keywords: EZH2, Adult, T-cell, Acute lymphoblastic leukemia Anahtar Sözcükler: EZH2, Erişkin, T-hücre, Akut lenfoblastik lösemi Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

1. Wassef M, Michaud A, Margueron R. Association between EZH2 expression, silencing of tumor suppressors and disease outcome in solid tumors. Cell Cycle 2016;15:2256-2262. 2. Gonzalez ME, Moore HM, Li X, Toy KA, Huang W, Sabel MS, Kidwell KM, Kleer CG. EZH2 expands breast stem cells through activation of NOTCH1 signaling. Proc Natl Acad Sci U S A 2014;111:3098-3103. 3. Katoh M. Mutation spectra of histone methyltransferases with canonical SET domains and EZH2-targeted therapy. Epigenomics-UK 2016;8:285-305. 4. Ernst T, Pflug A, Rinke J, Ernst J, Bierbach U, Beck JF, Hochhaus A, Gruhn B. A somatic EZH2 mutation in childhood acute myeloid leukemia. Leukemia 2012;26:1701-1703. 5. Guglielmelli P, Biamonte F, Score J, Hidalgo-Curtis C, Cervantes F, Maffioli M, Fanelli T, Ernst T, Winkelman N, Jones AV, Zoi K, Reiter A, Duncombe A, Villani L, Bosi A, Barosi G, Cross NC, Vannucchi AM. EZH2 mutational status predicts poor survival in myelofibrosis. Blood 2011;118:5227-5234. 6. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tönnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T, van der Reijden BA, Jansen JH. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010;42:665-667.

8. Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, Easton J, Chen X, Wang J, Rusch M, Lu C, Chen SC, Wei L, Collins-Underwood JR, Ma J, Roberts KG, Pounds SB, Ulyanov A, Becksfort J, Gupta P, Huether R, Kriwacki RW, Parker M, McGoldrick DJ, Zhao D, Alford D, Espy S, Bobba KC, Song G, Pei D, Cheng C, Roberts S, Barbato MI, Campana D, Coustan-Smith E, Shurtleff SA, Raimondi SC, Kleppe M, Cools J, Shimano KA, Hermiston ML, Doulatov S, Eppert K, Laurenti E, Notta F, Dick JE, Basso G, Hunger SP, Loh ML, Devidas M, Wood B, Winter S, Dunsmore KP, Fulton RS, Fulton LL, Hong X, Harris CC, Dooling DJ, Ochoa K, Johnson KJ, Obenauer JC, Evans WE, Pui CH, Naeve CW, Ley TJ, Mardis ER, Wilson RK, Downing JR, Mullighan CG. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012;481:157-163. 9. Schäfer V, Ernst J, Rinke J, Winkelmann N, Beck JF, Hochhaus A, Gruhn B, Ernst T. EZH2 mutations and promoter hypermethylation in childhood acute lymphoblastic leukemia. J Cancer Res Clin Oncol 2016;142:1641-1650. 10. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood 2015;126:833-841.

Address for Correspondence/Yazışma Adresi: Zheng GE, M.D., Zhongda Hospital, Medical School of Southeast University Department of Hematology Nanjing, China Phone : 86-25-83262468 E-mail : janege879@hotmail.com ORCID-ID: orcid.org/0000-0001-8028-1612

368

Received/Geliş tarihi: May 16, 2017 Accepted/Kabul tarihi: July 26, 2017 DOI: 10.4274/tjh.2017.0194


Turk J Hematol 2017;34:356-381

LETTERS TO THE EDITOR

Circulating Tumor Cells in Neuroblastoma Nöroblastomada Dolaşan Tümör Hücreleri Mili Jain1, Ashutosh Kumar1, Sanjay Mishra1, Nishant Verma2, Madhu Mati Goel1 King George’s Medical University, Department of Pathology, Uttar Pradesh, India King George’s Medical University, Department of Pediatrics, Uttar Pradesh, India

1 2

To the Editor, A 2-year-old girl presented with fever, hepatomegaly, and progressively increasing proptosis of the right eye for 1 month. Abdominal ultrasound revealed a well-defined multi-lobulated predominantly hyperechoic mass lesion of 10.9x2.5x6.1 cm with a few foci of coarse calcification and small cystic areas arising from the right suprarenal region. The lesion was inferiorly compressing the renal parenchyma; however, the interface was well maintained. Medially it was crossing the midline and encasing the aorta and its branches. The features were of neuroblastoma. The diagnosis was confirmed by TruCut biopsy from the suprarenal mass showing small round blue cells with salt and pepper chromatin. Immunohistochemistry was positive for synaptophysin. Non-contrast computerized tomography scanning of the head and orbit revealed a right retro-orbital mass with specks of calcification. The peripheral blood smears showed a few clusters and rosettes of circulating neuroblastoma cells. The bone marrow aspirate smears showed extensive infiltration by neuroblastoma cells dispersed singly, in clusters as well as in rosettes with central neuropils (Figure 1). The patient was categorized as stage IV as per the International Neuroblastoma Staging System. Neuroblastoma is the most common extracranial solid tumor in children. The mean age of presentation is 18 months and the majority of patients (90%) are diagnosed by 5 years of age [1]. The adrenal gland is the most common primary site. Approximately 75% of children have metastases to regional lymph nodes, bone marrow, cortical bones, the orbit, the liver, and skin at the time of diagnosis. The histology shows primitive neuroblasts with variable degrees of differentiation to Schwann cells and ganglion cells. Homer-Wright rosettes, i.e. neuroblasts surrounding a tangle of neuropils, are seen. Infants may present with blue-red cutaneous masses, referred to as blueberry muffin baby. The differential diagnosis includes alveolar rhabdomyosarcoma (ARMS), Ewing’s sarcoma/primitive neuroectodermal tumor (PNET), and lymphoma. ARMS cells are

Figure 1. Leishman staining. A, B) Peripheral blood smear (630x) showing neuroblastoma tumor cell cluster with nuclear molding and rosette-like arrangement. C) Bone marrow smear (400x) showing Homer-Wright rosette with neuroblasts surrounding central neuropil. more pleomorphic with abundant cytoplasm and are positive for myogenic markers (desmin, myogenin). Ewing’s sarcoma/ PNET patients are usually older; cells show finely stippled chromatin- and glycogen-filled cytoplasm, with expression of CD99. Neuroblastoma may clinically mimic acute leukemia [2]. These cells can be differentiated from blasts by expression of synaptophysin or neuron-specific enolase. The blasts are positive for leukocyte common antigen (LCA/CD45). Catecholamine metabolites homovanillic acid and vanillylmandelic acid are elevated in serum and urine in approximately 95% of patients. Metastatic disease can be evaluated using iodine123-metaiodobenzylguanidine (123-I-MIBG) scanning. N-MYC amplification is associated with poor prognosis. Morphologically identifiable circulating tumor cells (CTCs) are a rare finding [3]. Positive CTCs are associated with poor prognosis [4]. Keywords: Neuroblastoma, Circulating tumor cells, Metastasis Anahtar Sözcükler: Nöroblastoma, Dolaşan tümör hücreleri, Metastaz

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Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Brodeur G, Hogarty M, Bagatell R, Mosse YP, Maris JM Neuroblastoma. In: Pizzo P, Poplack D, (eds). Principles and Practice of Pediatric Oncology. Philadelphia, Lippincott Williams & Wilkins, 2016.

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2. Gökçe M, Aytaç S, Ünal Ş, Altan İ, Gümrük F, Çetin M. Acute megakaryoblastic leukemia with t(1;22) mimicking neuroblastoma in an infant. Turk J Hematol 2015;32:64-67. 3. Moodley V, Pool R. Circulating neuroblastoma cells in peripheral blood. Br J Haematol 2003;123:2. 4. Kuroda T, Morikawa N, Matsuoka K, Fujino A, Honna T, Nakagawa A, Kumagai M, Masaki H, Saeki M. Prognostic significance of circulating tumor cells and bone marrow micrometastasis in advanced neuroblastoma. J Pediatr Surg 2008;43:2182-2185.

Address for Correspondence/Yazışma Adresi: Mili JAIN, M.D., King George’s Medical University, Department of Pathology, Uttar Pradesh, India Phone : +91 979 354 60 90 E-mail : milijain786@gmail.com ORCID-ID: orcid.org/0000-0002-2598-1939

Received/Geliş tarihi: May 18, 2017 Accepted/Kabul tarihi: August 25, 2017 DOI: 10.4274/tjh.2017.0199

Megakaryocytic Emperipolesis Associated with Thrombocytopenia: Causative or Coincidence? Trombositopeni ile Birlikte Megakaryositik Emperipolez: Nedensel veya Tesadüf? Manu Goyal1, Sreeja Thandilath Thekkelakayil2, Anurag Gupta2,3 AmPath Hyderabad Hospital, Clinics of Hematopathology and Molecular Hematopathology, Telangana, India AmPath Hyderabad Hospital, Clinic of Hematopathology, Telangana, India 3 AmPath Hyderabad Hospital, Clinic of Cytogenetics, Telangana, India 1 2

To the Editor, Phagocytosis, emperipolesis, and entosis are physiological and pathological phenomena characterized by the engulfment of one cell into another cell [1]. Emperipolesis is defined as active penetration of one cell by another, which remains intact [2]. Emperipolesis differs from phagocytosis in that an engulfed cell exists temporarily within another cell and with an intact normal structure, while in phagocytosis, the engulfed cell is destroyed by the proteolytic action of lysosomal enzymes [1,2]. Entosis is a non-apoptotic cell death mechanism that occurs in cell populations deprived of matrix attachment [3,4]. A 31-year-old male presented with severe headache to the emergency department. He was afebrile without any organomegaly or neurological deficit. An urgent computed tomography scan of the brain showed subarachnoid hemorrhage. Complete blood counts revealed hemoglobin of 80 g/L, leukocyte count of 4.9x109/L, platelet count of 5x109/L, and a few giant platelets on peripheral smear. Prothrombin time, activated partial thromboplastin time, and fibrinogen were within the normal ranges. Bone marrow evaluation performed to assess the cause of severe thrombocytopenia showed normal erythropoiesis and myelopoiesis with increased megakaryocytes. These megakaryocytes showed neutrophils 370

Figure 1. Photomicrograph of the trephine biopsy shows megakaryocytic emperipolesis containing neutrophils (hematoxylin and eosin stain, original magnification 630x). with marked emperipolesis (Figure 1). There was no evidence of malignancy or infiltrate. A working diagnosis of immunemediated thrombocytopenia was issued and the patient was treated with steroids and intravenous immunoglobulins. In view of the marked thrombocytopenia and hemorrhagic complications, the patient was transfused with multiple units of single-donor platelets. Despite aggressive medical management,


Turk J Hematol 2017;34:356-381

his platelet counts did not improve. He was discharged against medical advice and lost to follow-up. Emperipolesis is a hallmark of Rosai-Dorfman disease (RDD); however, it can also be seen in both malignant hematolymphoid disorders (like Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemias, myeloproliferative disorders or myelodysplastic syndrome) and non-hematological malignancies (neuroblastoma, rhabdomyosarcoma) [1,5]. Emperipolesis can be either megakaryocytic or histiocytic. The former engulfs erythroblasts, myeloid cells, or neutrophils and is seen in hematolymphoid disorders, while the latter engulfs inflammatory cells (lymphocytes and plasma cells) as seen in RDD [1]. The exact mechanism for megakaryocytic emperipolesis is unknown. Centurione et al. [6] in their mice model suggested that abnormality in GATA1 transcription factor (either due to mutation or deletion) results in thrombocytopenia, megakaryocytic emperipolesis, and resultant myelofibrosis. Increased expression of P-selectin is known to mediate neutrophil sequestration on the outer surface of megakaryocytes, promoting increased neutrophil-megakaryocyte interactions [6,7]. A few studies indicated that the release of alpha-granular proteins, growth factors, and cytokines produced by megakaryocytes as well as neutrophil protease in the microenvironment induce emperipolesis [5,8]. The fate could be the cannibalism of the invading cell, host cell death, transcytosis, or division of both the invading and recipient cells [4,7]. Further research at the molecular level is needed to elucidate the underlying specific mechanisms. With regards to platelet counts, there have been few case reports of megakaryocytic emperipolesis associated with thrombocytosis, rarely in thrombocytopenia associated with myelodysplasia and none associated with immune-mediated thrombocytopenia [9]. In the present case, whether megakaryocytic emperipolesis was responsible for the thrombocytopenia or simply a coincidence is

LETTERS TO THE EDITOR

difficult to establish. We present this rare phenomenon so that similar observations cumulatively would help in resolving this complex issue. Keywords: Thrombocytopenia, Megakaryocytic emperipolesis, GATA1 Anahtar Sözcükler: emperipolez, GATA1

Trombositopeni,

Megakaryositik

Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Rastogi V, Sharma R, Misra SR, Yadav L, Sharma V. Emperipolesis - a review. J Clin Diagn Res 2014;8:ZM01-2. 2. Humble JG, Jayne WH, Pulvertaft RJ. Biological interaction between lymphocytes and other cells. Br J Haematol 1956;2:283-294. 3. Overholtzer M, Mailleux AA, Mouneimne G, Normand G, Schnitt SJ, King RW, Cibas ES, Brugge JS. A nonapoptotic cell death process, entosis, that occurs by cell-in-cell invasion. Cell 2007;131:966-979. 4. Xia P, Wang S, Guo Z, Yao X. Emperipolesis, entosis and beyond: dance with fate. Cell Res 2008;18:705-707. 5. Sable MN, Sehgal K, Gadage VS, Subramanian PG, Gujral S. Megakaryocytic emperipolesis: A histological finding in myelodysplastic syndrome. Indian J Pathol Microbiol 2009;52:599-600. 6. Centurione L, Di Baldassarre A, Zingariello M, Bosco D, Gatta V, Rana RA, Langella V, Di Virgilio A, Vannucchi AM, Migliaccio AR. Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1 low mice. Blood 2004;104:3573-3580. 7. Gupta N, Jadhav K, Shah V. Emperipolesis, entosis and cell cannibalism: demystifying the cloud. J Oral Maxillofac Pathol 2017;21:92-98. 8. Schmitt A, Jouault H, Guichard J, Wendling F, Drouin A, Cramer EM. Pathological interaction between megakaryocytes and polymorphonuclear leukocyte in myelofibrosis. Blood 2000;96:1342-1347. 9. Bobik R, Podolak-Dawidziak M, Kiełbiński M, Jeleń M, Wróbel T. Emperipolesis in megakaryocytes in patients with thrombocytosis in the course of myeloproliferative disorders. Acta Haematol Pol 1995;26:179183.

Address for Correspondence/Yazışma Adresi: Manu GOYAL, M.D., AmPath Hyderabad Hospital, Clinics of Hematopathology and Molecular Hematopathology, Telangana, India Phone : +09 866 969 997 E-mail : dr_manu31@yahoo.com ORCID-ID: orcid.org/0000-0003-1970-4270

Received/Geliş tarihi: May 25, 2017 Accepted/Kabul tarihi: August 22, 2017 DOI: 10.4274/tjh.2017.0211

371


LETTERS TO THE EDITOR

Turk J Hematol 2017;34:356-381

First Observation of Hemoglobin San Diego, a High Oxygen Affinity Hemoglobin Variant, in Turkey Türkiye’de Gözlenen İlk Hemoglobin San Diego (Oksijene İlgisi Yüksek Bir Hemoglobin Varyantı) Olgusu Ebru Yılmaz Keskin1, Ali Fettah1, Ana Catarina Oliveira2, Şule Toprak1, Andreia Lopes2, Celeste Bento2,3 Süleyman Demirel University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Isparta, Turkey Coimbra University, Centro Hospital, Clinic of Hematology, Coimbra, Portugal 3 CIAS, Coimbra University, Department of Life Sciences, Coimbra, Portugal 1 2

To the Editor, Congenital erythrocytosis (CE) or congenital polycythemia represents a rare clinical entity. High oxygen affinity hemoglobin (Hb) variants are a very rare cause of secondary CE. In 1966, Charache et al. [1] published the first case of a Hb variant associated with erythrocytosis, Hb Chesapeake. Since then, more than 220 variants with high oxygen affinity have been discovered and the autosomal dominant inheritance has been confirmed [2]. Many Hb variants have been reported so far from Turkey [3,4,5]. We report herein the first observation of Hb San Diego, a high oxygen affinity Hb variant, from Turkey in a case of CE. Case: A 15-year-old female patient residing in Kastamonu, Turkey, and examined due to the complaints of occasional headache, fatigue, dizziness, nausea, and chest pain was found to have an elevated Hb level. Erythrocytosis was also present in other family members, including her father and paternal grandmother (Figure 1). Both the father and grandmother had a history of several phlebotomies. Laboratory data are presented in Table 1. Serum biochemistry, abdominal ultrasonography, and echocardiographic examinations were all unremarkable. In addition to her family history consistent with a disorder transmitted autosomal dominantly, the finding of reduced P50 suggested the presence of a high oxygen affinity Hb. Hb electrophoresis performed with the high-performance liquid chromatography (HPLC) method with the device ZIVAK using the Hb Variant Whole Blood HPLC Analysis Kit yielded no abnormal Hb variant. The examination was repeated with Trinity Biotech’s Premier Hb9210 resolution method and displayed the presence of a Hb variant in both the patient and her father (Figure 1). Sanger sequencing analysis confirmed the associated mutation in the β-globin gene [Hb San Diego; β109(G11)Val→Met] (Figure 2). 372

Figure 1. A) Pedigree of the family with erythrocytosis and hemoglobin (Hb) San Diego, illustrating dominant mode of inheritance of erythrocytosis. The propositus is indicated with an arrow; B) high-performance liquid chromatography (premier Hb9210 resolution) showing the presence of Hb San Diego.

Figure 2. Identification of hemoglobin San Diego in β-globin gene by Sanger sequencing analysis in the index case. HbVar: Hemoglobin variant.


Turk J Hematol 2017;34:356-381

LETTERS TO THE EDITOR

Table 1. Laboratory findings of the patient at the time of admission.

Keywords: Abnormal hemoglobins, Hemoglobin San Diego, Hemoglobin variant

Hemoglobin (g/L)

169 (NR: 120-160)

Hematocrit (%)

50.6 (NR: 36-46)

Anahtar Sözcükler: Anormal hemoglobinler, Hemoglobin San Diego, Hemoglobin varyantı

Red blood cells (106/µL)

5.72 (NR: 4.1-5.1)

Reticulocyte ratio (%)

1.2 (NR: 0.3-1.5)

MCV (fL)

88.5 (NR: 70.6-95.6)

Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

White blood cells (10 /µL)

4.4 (NR: 4.5-13.0)

Platelets (10 /µL)

258 (NR: 150-400)

References

Ferritin (ng/mL)

12.4 (NR: 12-150)

1. Charache S, Weatherall DJ, Clegg JB. Polycythemia associated with a hemoglobinopathy. J Clin Invest 1966;45:813-822.

Vitamin B12 (pg/mL)

421 (NR: 200-820)

Folic acid (ng/mL)

6.3 (NR: 3.0-7.2)

2. Mangin O. High oxygen affinity hemoglobins. Rev Med Interne 2017;38:106112.

Homocysteine (µmol/L)

5.8 (NR: 5.0-13.0)

Erythropoietin (mIU/mL)

12.4 (NR: 4.3-20.0)

P50* (mmHg)

15.5 (NR: 22.6-29.4)

3

3

*P50 is the oxygen tension at which the hemoglobin molecule is one-half saturated. NR: Normal range, MCV: mean corpuscular volume.

Erythrocytosis may be the clinical manifestation of the presence of a high oxygen affinity Hb. Hb San Diego was first reported in 1974 in a Filipino family [6]. Thereafter, it has been described in subjects of different origins [7,8,9,10,11,12]. Our case represents the first one of Hb San Diego in Turkey. Although Hb San Diego was described as electrophoretically silent [6], it could be clearly identified using the new Trinity Biotech Premier Hb9210 resolution technology. In their study evaluating 70 patients with CE, Bento et al. [11] sequenced all the genes described as associated with CE and erythrocytosis molecular etiology was identified in only 25 (36%) subjects, a high oxygen affinity Hb being the cause in 14 (56%) of these 25 subjects. Determination of the P50 value, calculated easily from fresh venous blood gas samples, is a practical and useful test, and a decreased value may direct clinicians to order examinations regarding a Hb variant [12]. Some high oxygen affinity Hbs are electrophoretically silent but their identification can be rapidly done by direct sequencing of the globin genes (HBB and HBA). Management of CE caused by a high oxygen affinity Hb should be personalized, and it should primarily depend on smoking cessation and physical activity. Phlebotomy and platelet aggregation inhibitors’ prescription should be evaluated carefully, and blood donation is not advised [2].

3. Altay Ç. Abnormal hemoglobins in Turkey. Turk J Haematol 2002;19:63-74. 4. Akar E, Akar N. A review of abnormal hemoglobins in Turkey. Turk J Haematol 2007;24:143-145. 5. Akar N. An updated review of abnormal hemoglobins in the Turkish population. Turk J Haematol 2014;31:97-98. 6. Nute PE, Stamatoyannopoulos G, Hermodson MA, Roth D. Hemoglobinopathic erythrocytosis due to a new electrophoretically silent variant, Hemoglobin San Diego (β109 (G11) Val→ Met). J Clin Invest 1974;53:320-328. 7. González Fernández FA, Villegas A, Ropero P, Carreño MD, Anguita E, Polo M, Pascual A, Henández A. Haemoglobinopathies with high oxygen affinity. Experience of Erythropathology Cooperative Spanish Group. Ann Hematol 2009;88:235-238. 8. Loukopoulos D, Poyart C, Delanoe-Garin J, Matsis C, Arous N, Kister J, Loutradi-Anagnostou A, Blouquit Y, Fessas P, Thillet J, Rosa F, Galacteros F. Hemoglobin San Diego/beta zero thalassemia in a Greek adult. Hemoglobin 1986;10:143-159. 9. Coleman MB, Adams LG 3rd, Walker AM, Plonczynski MW, Harrell AH, Kark JA, Schechter GP. Hb San Diego [beta 109 (G11) Val-->Met] in an Iranian: further evidence for a mutational hot spot at position 109 of the betaglobin gene. Hemoglobin 1993;17:543-545. 10. Miniero R, Pullano MN, Oliverio AC, Benevento C, Madonna G, Altomare F, Giancotti L. Two Calabrian children with Hemoglobin San Diego. Child 2012;1:11. 11. Bento C, Percy MJ, Gardie B, Maia TM, van Wijk R, Perrotta S, Della Ragione F, Almeida H, Rossi C, Girodon F, Aström M, Neumann D, Schnittger S, Landin B, Minkov M, Randi ML, Richard S, Casadevall N, Vainchenker W, Rives S, Hermouet S, Ribeiro ML, McMullin MF, Cario H; ECE-Consortium, Chauveau A, Gimenez-Roqueplo AP, Bressac-de-Paillerets B, Altindirek D, Lorenzo F, Lambert F, Dan H, Gad-Lapiteau S, Catarina Oliveira A, Rossi C, Fraga C, Taradin G, Martin-Nuñez G, Vitória H, Diaz Aguado H, Palmblad J, Vidán J, Relvas L, Ribeiro ML, Luigi Larocca M, Luigia Randi M, Pedro Silveira M, Percy M, Gross M, Marques da Costa R, Beshara S, Ben-Ami T, Ugo V; ECEConsortium. Genetic basis of congenital erythrocytosis: mutation update and online databases. Hum Mutat 2014;35:15-26. 12. Bento C, Almeida H, Maia TM, Relvas L, Oliveira AC, Rossi C, Girodon F, Fernandez-Lago C, Aguado-Diaz A, Fraga C, Costa RM, Araujo AL, Silva J, Vitoria H, Miguel N, Silveira MP, Martin-Nuñez G, Ribeiro ML. Molecular study of congenital erythrocytosis in 70 unrelated patients revealed a potential causal mutation in less than half of the cases (Where is/are the missing gene(s)?). Eur J Haematol 2013;91:361-368.

Address for Correspondence/Yazışma Adresi: Ebru YILMAZ KESKİN, M.D., Received/Geliş tarihi: May 25, 2017 Süleyman Demirel University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Isparta, Turkey Accepted/Kabul tarihi: August 22, 2017 Phone : +90 505 558 36 11 E-mail : ebruyilmaz81@hotmail.com ORCID-ID: orcid.org/0000-0002-1462-9876 DOI: 10.4274/tjh.2017.0213

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Turk J Hematol 2017;34:356-381

A Case of Para-Bombay Phenotype Caused by Homozygous Mutation of the FUT1 Gene FUT1 Genindeki Homozigot Mutasyondan Kaynaklanan Bir Para-Bombay Fenotipi Olgusu Jung-Kuang Yu1, Yi-Hong Liu2, Tze-Kiong Er2,3 Asia University Hospital, Department of Orthopedics, Taichung, Taiwan Asia University Hospital, Division of Laboratory Medicine, Taichung, Taiwan 3 Asia University Faculty of Medicine, Department of Health and Nutrition Biotechnology, Taichung, Taiwan 1 2

To the Editor, A 79-year-old female patient presented at the hospital with osteoarthritis. Examination of the patient revealed hemoglobin level of 10.8 g/dL, RBC count of 3.45x106/µL, WBC count of 10.1x103/µL, and platelet count of 122x103/µL. Plasma levels of blood urea nitrogen, creatinine, sodium, potassium, and alanine aminotransferase were all within the normal ranges, while aspartate aminotransferase was slightly higher than normal. A blood sample obtained from the patient was submitted to our division for blood typing and cross-matching, with a request to receive 2 units of packed red blood cells. ABO typing was performed using standard serological techniques after an immediate spin. Testing the patient’s red blood cells revealed no detectable ABO antigens upon forward/cell grouping (group O blood type). On the other hand, reverse/serum grouping showed the presence of A antibodies in the serum (group B blood type). To resolve the discrepancy between cell and serum grouping we performed an agglutination examination of anti-H serum; the red blood cells from the sample did not exhibit an agglutination reaction. Additionally, secretor status was determined in order to assess the presence of soluble blood group substances. Our results showed the presence of B and H antigens in the saliva. Based on these results, the patient in the present case was diagnosed as having a para-Bombay B phenotype (Table 1, Figure 1). Genotyping of the ABO and FUT1 genes was also performed. Direct DNA sequencing of the patient’s ABO gene indicated the B/O1 genotype. To examine potential mutations in the FUT1 gene, we amplified and sequenced the full coding region of the gene. FUT1 gene sequence analysis revealed that the

patient harbored the homozygous mutation c.881_882delTT (p.Phe294Cysfs*40). A heterozygous mutation in FUT1 (880delTT) has been previously reported as the cause of the para-Bombay phenotype [1,2]. However, the homozygous mutation c.881_882delTT (p.Phe294Cysfs*40) only rarely causes the para-Bombay phenotype. Previously, a study indicated that homozygous mutations are a cause of the para-Bombay phenotype [3,4]. In patients with the para-Bombay blood group, ABH antigens are present in saliva but not expressed in red blood cells. The para-Bombay phenotype results either from an inactive FUT1 gene present together with a normal FUT2 gene or from a mutated FUT1 gene present with or without an active FUT2 gene [1]. H deficiency is slightly more common in Taiwan, affecting 1 of 8000 people [2]. More than 56 silencing or weakening FUT1 mutations have been reported in the dbRBC database (https:// www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.cgi?cmd=bgmut/ systems_info&system=hh).

Figure 1. Sequencing results confirm the wild-type (A) and the presence of the FUT1 homozygous mutation c. 881_882delTT (p. Phe294Cysfs*40) (B).

Table 1. Serologic and saliva test results of the patient: ABO group discrepancy.

Cell grouping Anti-A Anti-B 0

0

Anti-D

Serum grouping A1 cells B cells

4+

4+

0

O cells

Test for H antigen Anti-H

Saliva secretor status A1 cells B cells

0

0

2+

0

0= No agglutination, 1+= multiple small agglutinates with hazy supernatant, 2+= multiple large agglutinates with clear supernatant, 3+= 2-3 large agglutinates with clear supernatant, 4+= single large agglutinates.

374


Turk J Hematol 2017;34:356-381

In conclusion, identification of this phenotype is very important because this particular patient subgroup may be clinically mislabeled as group O. If patients with anti-H in their circulation receive transfusions of blood with the H antigen, it may cause a transfusion reaction such as an acute hemolytic reaction. Here we have reported a rare case of the para-Bombay phenotype caused by the homozygous mutation c.881_882delTT (p.Phe294Cysfs*40). Acknowledgments Our gratitude goes to Michael Burton, Asia University. This study was supported by grants from Asia University and China Medical University Hospital (ASIA-105-CMUH-07). Keywords: Para-Bombay, Phenotype, FUT1 gene, Blood transfusion

LETTERS TO THE EDITOR

Anahtar Sözcükler: Para-Bombay, Fenotip, FUT1 geni, Kan transfüzyonu Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Cai XH, Jin X, Fan LF, Lu Q, Xiang D. Molecular genetic analysis for the paraBombay blood group revealing two novel alleles in the FUT1 gene. Blood Transfus 2011;9:466-468. 2. Chen DP, Tseng CP, Wang WT, Peng CT, Tsao KC, Wu TL, Lin KT, Sun CF. Two prevalent h allele in para-Bombay haplotypes among 250,000 Taiwanese. Ann Clin Lab Sci 2004;34:314-318. 3. Zhang A, Chi Q, Ren B. Genomic analysis of para-Bombay individuals in south-eastern China: the possibility of linkage and disequilibrium between FUT1 and FUT2. Blood Tranfus 2015;13:472-477. 4. Lin-Chu M, Broadberry RE. Blood transfusion in the para-Bombay phenotype. Br J Haematol 1990;75:568-572.

Address for Correspondence/Yazışma Adresi: Tze-Kiong ER, M.D., Asia University Hospital, Division of Laboratory Medicine, Taichung, Taiwan E-mail : tzekiong92@gmail.com ORCID-ID: orcid.org/0000-0002-7068-1652

Received/Geliş tarihi: May 29, 2017 Accepted/Kabul tarihi: August 04, 2017 DOI: 10.4274/tjh.2017.0220

375


LETTERS TO THE EDITOR

Turk J Hematol 2017;34:356-381

A Myopathy, Lactic Acidosis, Sideroblastic Anemia (MLASA) Case Due to a Novel PUS1 Mutation PUS1 Geninde Yeni Mutasyon Saptanan Miyopati, Laktik Asidoz, Sideroblastik Anemi (MLASA) Olgusu Çiğdem Seher Kasapkara1, Leyla Tümer1, Nadia Zanetti2, Fatih Ezgü1, Eleonora Lamantea2, Massimo Zeviani2,3 Gazi University Faculty of Medicine, Division of Metabolism, Ankara, Turkey Fondazione IRCCS Carlo Besta, Molecular Neurogenetics Unit, Milan, Italy 3 Medical Research Council, Mitochondrial Biology Unit, Cambridge, United Kingdom 1 2

To the Editor, The patient, the first child of Turkish first-cousins, was born at term after an uncomplicated pregnancy. Birth parameters were normal. The family history was negative for hematological or neurological diseases. The newborn period was characterized by hypoglycemia, lactic acidemia (6.1 mmol/L; normal values: up to 1.9 mmol/L), and lactic, pyruvic, and dicarboxylic aciduria. At 10 months of age, hematological examination revealed marked sideroblastic anemia. He started to receive transfusions every 3-4 weeks until 14 months of age, when the blood parameters spontaneously normalized. He had exercise intolerance and delayed motor milestones (walking at 3.5 years of age). At 14 years of age, pallor, progressive muscle weakness, and lethargy occurred and sideroblastic anemia reappeared. The boy had mild mental insufficiency, profound generalized hypotrophy and weakness, and hyperlordosis of the trunk. He became transfusion-dependent, requiring packed cell transfusions every 2-3 weeks. The muscle biopsy showed subsarcolemmal abnormal mitochondrial aggregates and diffuse negative staining for cytochrome c oxidase. Due to the paucity of tissue, the biochemical evaluation of respiratory chain complexes was not performed. The clinical features oriented us towards a mitochondrial pathology; CoQ10 was given (400 mg/day) and dramatic improvement of muscle strength was observed with reduction of the frequency of blood transfusions. Unfortunately, the boy died when he was 18 years old due to severe respiratory failure. Myopathy, lactic acidosis, and sideroblastic anemia (MLASA) is a rare mitochondrial disease [1]: MLASA1 (MIM #600462) results from mutations in the PUS1 gene, encoding for pseudouridylate synthase 1, and the enzyme is located in both the nucleus and mitochondria, which is involved in posttranscriptional modification of cytoplasmic and mitochondrial tRNAs [2]; MLASA2 (MIM #613561) is caused by mutations in the YARS2 gene that encodes for the mitochondrial tyrosyltRNA synthetase [3]; and MLASA3 (MIM #500011) is caused by heteroplasmic mutation m.8969G>A in the mitochondrialDNA-encoded ATP6 gene (MTATP6) [4]. 376

Figure 1. Sequence analysis of the exon 2-intron 2 junction of the PUS1 gene in a control (A), in the patient (B), and in the patient’s mother (C). The yellow highlighted nucleotides belong to the consensus sequence of the splice donor site. In the red circle the mutated nucleotide is in homozygous (B) or heterozygous (C) form. Our patient had typical features of MLASA, so we analyzed the nuclear-encoded genes YARS2 and PUS1. YARS2 was normal, but we identified the novel homozygous mutation c.302A>G in exon 2 of the PUS1 gene, causing the substitution p.Gln101Arg in the protein (Figure 1). This variant is reported as a singleton in the ExAC browser, accounting for 0.001% of allele frequency, and absent in dbSNP and EVS databases. The p.Gln101Arg change was scored very high for the likelihood to be deleterious by different bioinformatics tools for predicting pathogenic variants, and furthermore the c.302A>G transition is predicted to modify the consensus sequence of the splice donor site of exon 2, probably affecting splicing (Table 1). The unavailability of the patient’s cells did not allow us to confirm this hypothesis.


Turk J Hematol 2017;34:356-381

LETTERS TO THE EDITOR

Table 1. Results of the in silico analysis obtained by different bioinformatics tools for the prediction of the impact of mutation on mRNA and protein. c.302A>G in splice donor site of exon 2 in the PUS1 gene Tool

Prediction

Site

BDGP

Donor site lost

http://www.fruitfly.org/seq_tools/splice.html

Human SpliceFinder 3.0

Alteration of the WT donor site, most probably affecting splicing

http://www.umd.be/HSF/

MutationTaster

Alteration within used splice site, likely to disturb normal splicing; donor lost

http://www.mutationtaster.org/

MutPredSplice

Splice affecting variant (0.78); loss of natural 5’ SS

http://www.mutdb.org/mutpredsplice

p.Gln101Arg in pseudouridylate synthase 1 Tool

Prediction

Site

MutationTaster

Disease (0.999)

http://www.mutationtaster.org/

MutPred2

Probably pathogenic (0.897)

http://mutpred2.mutdb.org/

Panther

Probably damaging (0.95)

http://www.pantherdb.org

Pmut (beta)

Disease (0.82)

http://mmb.pcb.ub.es/PMut/

PolyPhen-2

Probably damaging (0.931)

http://genetics.bwh.harvard.edu/pph2/

SIFT

Affected protein function

http://siftdna.org/

BDGP: Berkeley Drosophila Genome Project, SIFT: Sorting Intolerant From Tolerant.

To date, eleven PUS1-mutated patients from six families have been described [2,5,6,7,8] and five mutations are reported. Ours is the first alteration allegedly causing a splicing aberration according to prediction by in silico analysis. In our patient a high dose of CoQ10 improved the clinical condition for a while, although it did not reverse the course of the disease. To date, there is no effective therapy for MLASA, although many studies are in progress to address novel treatment options for mitochondrial diseases [9]. Our report expands the genetic spectrum of the MLASA syndrome, which must be considered in patients with congenital sideroblastic anemia associated with myopathy. Acknowledgments This work was supported by Fondazione Pierfranco e Luisa Mariani - CM23 (N.Z., E.L., M.Z.) and Institut de France - Grant NRJ (M.Z.). Keywords: Myopathy, Lactic acidosis, Sideroblastic anemia Anahtar Sözcükler: Miyopati, Laktik asidoz, Sideroblastik anemi Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Inbal A, Avissar N, Shaklai M, Kuritzky A, Schejter A, Ben-David E, Shanske S, Garty BZ. Myopathy, lactic acidosis, and sideroblastic anemia: a new syndrome. Am J Med Genet 1995;55:372-378. 2. Bykhovskaya Y, Casas K, Mengesha E, Inbal A, Fischel-Ghodsian N. Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA). Am J Hum Genet 2004;74:1303-1308. 3. Riley LG, Cooper S, Hickey P, Rudinger-Thirion J, McKenzie M, Compton A, Lim SC, Thorburn D, Ryan MT, Giege R, Bahlo M, Christodoulou J. Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia-MLASA syndrome. Am J Hum Genet 2010;87:52-59. 4. Burrage LC, Tang S, Wang J, Donti TR, Walkiewicz M, Luchak JM, Chen LC, Schmitt ES, Niu Z, Erana R, Hunter JV, Graham BH, Wong LJ, Scaglia F. Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 gene. Mol Genet Metab 2014;113:207-212. 5. Zeharia A, Fischel-Ghodsian N, Casas K, Bykhovskaya Y, Tamari H, Lev D, Mimouni M, Lerman-Sagie T. Mitochondrial myopathy, sideroblastic anemia, and lactic acidosis: an autosomal recessive syndrome in Persian Jews caused by a mutation in the PUS1 gene. J Child Neurol 2005;20:449-452. 6. Fernandez-Vizarra E, Berardinelli A, Valente L, Tiranti V, Zeviani M. Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA). J Med Genet 2007;44:173-180. 7. Metodiev MD, Assouline Z, Landrieu P, Chretien D, Bader-Meunier B, Guitton C, Munnich A, Rötig A. Unusual clinical expression and long survival of a pseudouridylate synthase (PUS1) mutation into adulthood. Eur J Hum Genet 2015;23:880-882. 8. Cao M, Donà M, Valentino ML, Semplicini C, Maresca A, Cassina M, Torraco A, Galletta E, Manfioli V, Sorarù G, Carelli V, Stramare R, Bertini E, Carozzo R, Salviati L, Pegoraro E. Clinical and molecular study in a long-surviving patient with MLASA syndrome due to novel PUS1 mutations. Neurogenetics 2016;17:65-70. 9. Viscomi C, Bottani E, Zeviani M. Emerging concepts in the therapy of mitochondrial disease. Biochim Biophys Acta 2015;1847:544-557

Address for Correspondence/Yazışma Adresi: Çiğdem Seher KASAPKARA, M.D., Gazi University Faculty of Medicine, Division of Metabolism Diseases, Ankara, Turkey E-mail : cskasapkara@gmail.com ORCID-ID: orcid.org/0000-0002-3569-276X

Received/Geliş tarihi: June 12, 2017 Accepted/Kabul tarihi: August 22, 2017 DOI: 10.4274/tjh.2017.0231

377


LETTERS TO THE EDITOR

Turk J Hematol 2017;34:356-381

Frequency and Risk Factors for Secondary Malignancies in Patients with Mycosis Fungoides Mikozis Fungoidesli Hastalarda Sekonder Malignite Sıklığı ve Risk Faktörleri Fatma Pelin Cengiz, Nazan Emiroğlu, Nahide Onsun Bezmialem Vakıf University Faculty of Medicine, Department of Dermatovenereology, İstanbul, Turkey

To the Editor, Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL), has an incidence of 6.4 per million people [1]. Patients with CTCL have an increased risk of the development of secondary malignancies, particularly lymphomas [2,3]. We conducted a 20-year population-based cohort study to assess the risk factors of secondary cancers in MF patients from our center. From 1998 to 2015, a total of 143 cases of CTCL were documented in our database. In this same time period, 13 cases (9.1%) of secondary malignancy excluding non-melanoma skin cancer were diagnosed at least 3 months following the

diagnosis of CTCL (Table 1). MF patients were grouped by their tumor stage from I to IV. Statistical analysis was performed with SPSS 15. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Risk factors significantly associated with secondary cancers in univariate analyses were entered into a multivariate logistic regression model. Significance was set at p<0.05. The vast majority of patients had early-stage disease: 64 (45.35%) stage IA, 30 (20.97%) stage IB, 24 (16.78%) stage IIA, 13 (9.09%) stage IIB, 4 (2.79%) stage IIIA, 3 (2.09%) stage IIIB, 4 (2.79%) stage IVA, and 1 (1.43%) stage IVB.

Table 1. Clinical features of mycosis fungoides patients with secondary malignancies.

Sex

Age at diagnosis of MF (years)

Age at diagnosis of malignancy (years)

Stage of MF

Type of malignancy

Presence of lymphomatoid papulosis

Systemic treatment for MF

Patient 1

Male

8

15

IVA

Non-Hodgkin

No

Interferon, acitretin

Patient 2

Male

63

65

IB

Adult T-cell leukemia

No

Interferon, acitretin

Patient 3

Male

62

63

IIA

Lung cancer

No

None

Patient 4

Male

59

59

IIIB

Nasopharynx

Yes

Acitretin

Patient 5

Male

69

68

IIA

Lung cancer + adult T-cell leukemia

Yes

None

Patient 6

Female

56

59

IB

Renal cell carcinoma

Yes

None

Patient 7

Male

60

60

IB

Lung cancer

No

None

Patient 8

Female

18

30

IB

Hodgkin

Yes

Interferon, acitretin

Patient 9

Male

46

50

IIB

Bladder cancer

No

Acitretin

Patient 10

Female

48

51

IIB

Superficial spreading malignant melanoma

No

None

Patient 11

Female

22

23

IIA

Hodgkin

No

None

Patient 12

Female

36

36

IB

Hodgkin

Yes

None

Patient 13

Female

33

35

IIA

Non-Hodgkin lymphoma

No

Interferon, UVA1

MF: Mycosis fungoides.

378


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LETTERS TO THE EDITOR

Stage IV disease, the presence of lymphomatoid papulosis, and duration of disease (more than 10 years) were shown to be the factors that increased the risk of developing secondary solid tumors (OR: 21.958, 95% CI: 2.039-839.657; OR: 19.926, 95% CI: 2.387-166.362; OR: 0.635, 95% CI: 0.420-0.959, respectively). In the vast majority of the patients, secondary malignancies occurred during the first year of diagnosis of MF (60%).

oncogenes make the host immunosuppressed. We found that older age, stage of MF, and the presence of lymphomatoid papulosis increased the risk of coexistence of two other malignancies besides MF. Therefore, extensive evaluation for secondary malignancies in the adult population would be warranted, particularly if the patient has lymphomatoid papulosis.

Our study supports previous findings about an increased risk of developing a second primary malignancy, especially Hodgkin lymphoma, chronic leukemia, and lung cancer, in patients with MF. In previous epidemiological studies, patients with MF had an elevated risk of secondary neoplasms (mean relative risk: 1.73, range: 1.32-2.4) [2,3]. Some authors have suggested that anti-lymphoma drugs [4] and particularly alkylating agents may lead to leukemia [5]. MF and hematological malignancies may have the same genetic origin, carcinogens, or viruses that affect lymphocyte precursors, and additionally the production of cytokines by the first neoplasm may induce the development of the secondary neoplasm [5]. It was shown that MF is a T helper cell 2 (Th2) mediated disease and is associated with human leukocyte antigen 2 alleles. The antigens causing inappropriate antigens presenting to T lymphocytes are still unknown. Viruses (Epstein-Barr virus, herpes simplex virus), deficiency of vitamin D, and medications are possible causative agents. In addition to these factors, increased levels of transforming growth factor-β, interleukin-10, and Th2 cytokines and the activation of STAT-3

Keywords: T-cell neoplasms, Non-Hodgkin Oncogenes, T-cell mediated immunity

lymphoma,

Anahtar Sözcükler: T hücreli neoplazmlar, Hodgkin dışı lenfoma, Onkogenler, T hücre aracılı immünite

References 1. Bradford PT, Devesa SS, Anderson WF, Toro JR. Cutaneous lymphoma incidence patterns in the United States: a population-based study of 3884 cases. Blood 2009;113:5064-5073. 2. Vakeva L, Pukkala E, Ranki A. Increased risk of secondary cancers in patients with primary cutaneous T cell lymphoma. J Invest Dermatol 2000;115:6265. 3. Scarisbrick JJ, Child FJ, Evans AV, Fraser-Andrews EA, Spittle M, RussellJones R. Secondary malignant neoplasms in 71 patients with Sezary syndrome. Arch Dermatol 1999;135:1381-1385. 4. Au WY, Ma SK, Chung LP, Chim CS, Kwong YL. Two cases of therapy-related acute promyelocytic leukemia (t-APL) after mantle cell lymphoma and gestational trophoblastic disease. Ann Hematol 2002;81:659-661. 5. Green MH, Young RC, Merrill JM, De Vita VT. Evidence of a treatment dose response in acute nonlymphocytic leukemias which occur after therapy of non-Hodgkin’s lymphoma. Cancer Res 1983;43;1891-1898.

Address for Correspondence/Yazışma Adresi: Fatma Pelin CENGİZ, M.D., Bezmialem Vakıf University Faculty of Medicine, Department of Dermatovenereology, İstanbul, Turkey Phone : +90 506 701 54 06 E-mail : fpelinozgen@hotmail.com ORCID-ID: orcid.org/0000-0003-0669-6232

Received/Geliş tarihi: June 12, 2017 Accepted/Kabul tarihi: August 22, 2017 DOI: 10.4274/tjh.2017.0234

379


LETTERS TO THE EDITOR

Turk J Hematol 2017;34:356-381

Leishmaniasis: Bone Marrow Aspirate Smear and Rapid Antibody Test Layşmanyazis: Kemik İliği Aspirasyon Yayması ve Hızlı Antikor Testi Beuy Joob1, Viroj Wiwanitkit2 Sanitation 1 Medical Academic Center, Bangkok, Thailand DY Patil University Faculty of Medicine, Pune, India

1 2

To the Editor, The publication by Dorji et al. [1], “Microscopic Image of Leishman-Donovan Bodies in Bone Marrow Aspirate Smear of Patient Suffering from Unexplained Intermittent Low-Grade Fever and Cough”, is very interesting. Indeed, the clinical presentation of leishmaniasis is usually nonspecific and it is hard to discriminate it from other tropical infections [2]. However, the common presentation that might be useful for the inclusion of leishmaniasis in differential diagnosis is splenomegaly [2]. In the present report, an important observation is a negative antibody test for Leishmania pathogens. The gold standard for the diagnosis of leishmaniasis is usually the examination of the blood or marrow and identification of the parasite. However, it is considered harmful. A new rapid test might be a solution and a less invasive technique. Nevertheless, the problem of the diagnostic properties of the available rapid test is frequently reported [3]. There are many possible explanations for false negative results. One important explanation is the prozone phenomenon [4] due to heavy infection. In the present case, there might be parasites and excessive antigens that could induce false negative results due to the immunological testing and the dilution of the blood sample before the test could be a simple method to reduce the problem of the prozone effect. Focusing on the examination of bone marrow a spirates, a similar problem with diagnostic false negatives can be expected [5]. At present, the test with the best diagnostic properties is the polymerase chain reaction-based test [5].

Keywords: Leishmaniasis, Bone marrow, Aspirate, Smear, Antibody Anahtar Sözcükler: Layşmanyazis, Kemik iliği, Aspirasyon, Yayma, Antikor Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Dorji K, Tobgay T, Jamtsho R, Samal PD, Rai P. Microscopic image of Leishman-Donovan bodies in bone marrow aspirate smear of patient suffering from unexplained intermittent low-grade fever and cough. Turk J Hematol 2017;34:266-267. 2. Wiwanitkit V. Bone marrow leishmaniasis: a review of situation in Thailand. Asian Pac J Trop Med 2011;4:757-759. 3. Khan MG, Alam MS, Bhuiyan AT, Jamil MA, Saha B, Islam M, Haque R, Hossain M, Jamil KM. Short communication: evaluation of a new rapid diagnostic test for quality assurance by kala azar elimination programme in Bangladesh. J Parasitol Res 2011;2011:862475. 4. Itoh Y, Yamaguchi T. Factors that affect analytical results in an enzyme immunoassay. Nihon Rinsho 1995;53:2143-2148. 5. Piarroux R, Gambarelli F, Dumon H, Fontes M, Dunan S, Mary C, Toga B, Quilici M. Comparison of PCR with direct examination of bone marrow aspiration, myeloculture, and serology for diagnosis of visceral leishmaniasis in immunocompromised patients. J Clin Microbiol 1994;32:746-749.

Address for Correspondence/Yazışma Adresi: Beuy JOOB, M.D., Sanitation 1 Medical Academic Center, Bangkok, Thailand E-mail : beuyjoob@hotmail.com ORCID-ID: orcid.org/0000-0002-5281-0369

Received/Geliş tarihi: August 04, 2017 Accepted/Kabul tarihi: August 22, 2017 DOI: 10.4274/tjh.2017.0291

380


Turk J Hematol 2017;34:356-381

LETTERS TO THE EDITOR

Receiver Operating Characteristic Curve Analysis May be Helpful to Study the Prognostic Value of miR-155 in B-Cell Non-Hodgkin Lymphoma İşlem Karakteristik Eğrisi Analizi B-Hücre Non-Hodgkin Lenfomada miR-155’in Prognostik Değerini Çalışmada Yardımcı Olabilir Long Su The First Hospital of Jilin University, Department of Hematology, Changchun, China

To the Editor, Aberrant expression of microRNA (miR-155) has been reported previously in several hematological malignancies [1,2,3,4]. Recently, Bedewy et al. [5] published their excellent findings in this journal. They reported that miR-155 expression was significantly upregulated in patients with B-cell non-Hodgkin lymphoma (NHL) compared with normal controls. In patients with B-cell NHL, a high level of miR-155 expression was associated with the presence of B symptoms, involvement of extranodal sites, and high Eastern Cooperative Oncology Group score. In patients with diffuse large B-cell lymphoma (DLBCL), high miR-155 levels were related to non-germinal B-cell-like type and higher International Prognostic Index scores. High miR-155 expression was also associated with inferior event-free survival. Accordingly, the authors concluded that miRNA-155 might be a potential biomarker of prognosis and monitoring in B-cell NHL, and especially that of the DLBCL type [5]. Receiver operating characteristic (ROC) curve analysis is a graphical plot that illustrates the diagnostic ability of a binary classifier system as its discrimination threshold is varied. The best cutoff value can be calculated by ROC analysis for continuous variables to predict dichotomous variables with the best sensitivity and specificity. In Bedewy et al.’s [5] study, the patients were divided into high-expression and low-expression groups based on the median level of miR-155 relative expression units. I wonder if the median value of miR-155 expression level in this study was the cutoff value with the best sensitivity and specificity between patients and normal controls by ROC analysis. Similarly, in patients with B-cell NHL, whether there is another cutoff value that is associated with clinical stage,

treatment response, long-term outcomes, and so on remains to be determined. Keywords: Non-Hodgkin lymphoma, miR-155, Prognostic value Anahtar Sözcükler: Non-Hodgkin lenfoma, miR-155, Prognostik değer Conflict of Interest: The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

References 1. Jones K, Nourse JP, Keane C, Bhatnagar A, Gandhi MK. Plasma microRNA are disease response biomarkers in classical Hodgkin lymphoma. Clin Cancer Res 2014;20:253-264. 2. Wallace JA, Kagele DA, Eiring AM, Kim CN, Hu R, Runtsch MC, Alexander M, Huffaker TB, Lee SH, Patel AB, Mosbruger TL, Voth WP, Rao DS, Miles RR, Round JL, Deininger MW, O’Connell RM. miR-155 promotes FLT3-ITDinduced myeloproliferative disease through inhibition of the interferon response. Blood 2017;129:3074-3086. 3. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J Jr, Lu L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage JO, Warnke R, Levy R, Wilson W, Grever MR, Byrd JC, Botstein D, Brown PO, Staudt LM. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503-511. 4. Marcucci G, Maharry KS, Metzeler KH, Volinia S, Wu YZ, Mrózek K, Nicolet D, Kohlschmidt J, Whitman SP, Mendler JH, Schwind S, Becker H, Eisfeld AK, Carroll AJ, Powell BL, Kolitz JE, Garzon R, Caligiuri MA, Stone RM, Bloomfield CD. Clinical role of microRNAs in cytogenetically normal acute myeloid leukemia: miR-155 upregulation independently identifies high-risk patients. J Clin Oncol 2013;31:2086-2093. 5. Bedewy AML, Elmaghraby SM, Shehata AA, Kandil NS. Prognostic value of miRNA-155 expression in B-cell non-Hodgkin lymphoma. Turk J Hematol 2017;34:207-212.

Address for Correspondence/Yazışma Adresi: Long SU, M.D., The First Hospital of Jilin University, Department of Hematology, Changchun, China Phone : +86 0431 887 821 57 E-mail : sulongjdyy@163.com ORCID-ID: orcid.org/0000-0002-5360-468X

Received/Geliş tarihi: August 09, 2017 Accepted/Kabul tarihi: August 22, 2017 DOI: 10.4274/tjh.2017.0297

381


AUTHOR INDEX 2017

34th Volume Index / 34. Cilt Dizini AUTHOR INDEX - YAZAR DİZİNİ 2017

A. Emre Eşkazan................................. 233, 291 Abdulkadir Ünsal......................................... 116 Abdurrahman Kara........................................ 40 Abraham Pouliakis......................................... 52 Ageliki Tirogala............................................... 52 Ahmad Antar................................................358 Ahmed A. Shehata....................................... 207 Ahmed M. L. Bedewy.................................. 207 Ahmet Emin Kürekçi..................................... 64 Ahmet Koluman............................................. 93 Ahmet Muzaffer Demir............................... 89 Ahmet Pekoğlu............................................... 64 Ahmet Uygun................................................359 Ahu Senem Demiröz......................................81 Aida Berenjian.............................................. 118 Aidin Meshksar.............................................250 Ajlan Tükün................................................... 151 Aleksandar Jankovic...................................268 Alessandro Allegra.......................................184 Alexandra Tsoka............................................. 52 Ali Eser............................................................ 118 Ali Fettah....................................... 40, 183, 372 Ali Şahin Küçükaslan..................................174 Ali Ünal............................................................. 27 Alişan Yıldıran...............................................345 Ana Bulatovic...............................................268 Ana Catarina Oliveira.................................372 Ana K. Nuñez-Cortes..................................239 Anastasia Livada............................................ 52 Andaç Salman............................................... 118 Andreia Lopes...............................................372 Andrés A. Leon-Peña..................................239 Anna J. Kopinska..........................................186 Anna Koclega................................................186 Anurag Gupta............................................... 370 Argyrios Tsantes............................................. 52 Arie C. Van Der Spek...................................124 Arzu Akyay..................................................... 114 Arzu Ekici.......................................................264 Ashutosh Kumar..........................................369 Aslı Akın Belli...................................... 122, 192 Aslı Korur....................................196, 280, 324 Aspasia Argyrou............................................. 52 Asude Kara........................................... 122, 192 Aşkın Şen........................................................ 114 Ayfer Gedük..................................................190 Ayla Eker Sarıboyacı................................... 213 Aylin Okçu Heper.........................................194 Aynur Küçükçongar Yavaş........................264 Aysel Pekel....................................................... 64 Ayşe Salihoğlu.................................... 233, 291 Ayşegül Üner................................................... 34 Aytekin Ünlü................................................... 64 Azhar Sattar..................................................278 Bahattin Tunç........................................40, 183 Baohua Huang..............................................126 Baoshan Liu...................................................200

Barış Malbora................................................179 Behnaz Valibeigi..........................................300 Bengi Öztürk................................................... 16 Bengü Nisa Akay..........................................194 Berna Atabay................................................179 Betül Orhaner...............................................264 Betül Tavil........................................................ 40 Beuy Joob..............................................101, 380 Bin Wang........................................................200 Buket Kosova................................................174 Burcu Belen...................................................179 Burcu Yazıcıoğlu..........................................143 Burçin Tezcanlı Kaymaz.............................174 Can Boğa........................... 196, 270, 280, 324 Carl Hsieh.......................................................328 Caterina Musolino.......................................184 Celalettin Üstün............................................. 16 Celeste Bento................................................372 Cengiz Bayram................................................ 40 Cengiz Beyan................................................359 Cengiz Ceylan...............................................197 Cengizhan Açıkel........................................... 64 Chen Fangping................................................10 Cheng-Wei Chou.........................................159 Chieh-lin Jerry Teng...................................159 Chingiz Asadov.............................................258 Chunhua Song..............................................366 Çağatay Taşkapan........................................ 114 Çağdaş Aktan................................................174 Çağla Sarıtürk...............................................324 Çetin Demir..................................................... 34 Çiğdem Gereklioğlu.................196, 280, 324 Çiğdem Seher Kasapkara...........................376 Demet Aydın.................................................167 Demet Çekdemir............................................ 27 Deniz Kızmazoğlu.......................................... 46 Deniz Yılmaz Karapınar..............................254 Deniz Yücelten.............................................. 118 Devrim Dündar.............................................340 Dildar Bahar Genç....................................... 109 Dilek Gürlek Gökçebay...............................183 Dimitra Agoritsa............................................. 52 Dimitrios Maltezos........................................ 52 Ebru Yılmaz Keskin......................................372 Eda Ataseven................................................... 46 Eda Güngörürler.......................................... 213 Ekaterini Kontopanou.................................. 52 Ekaterini Manaka........................................... 52 Eldar Abdulalimov.......................................258 Eleonora Lamantea.....................................376 Elias Kyriakou.................................................. 52 Elias Markopoulos......................................... 52 Elif Azarsız.....................................................254 Elif Gülsüm Ümit........................................... 89 Elisavet Grouzi................................................ 52 Elise Schapkaitz.............................................. 59 Emel Özyürek................................................345

Emin Kaya........................................................ 27 Emin Sami Arısoy.........................................340 Emine Begüm Gençer................................. 314 Emine Dağıstan............................................120 Emine Zengin................................................340 Emmanouil Lysikatos.................................... 52 Emre Ali Acar................................................ 181 Erdal Karaöz.................................................. 213 Erdal Kurtoğlu.................................... 122, 192 Erden Atilla........................................1, 16, 244 Erkan Maytalman........................................280 Esra T. Demirsoy...........................................190 Fadıl Vardar...................................................254 Fahri Şahin..............................................27, 174 Fatih Demircioğlu........................................120 Fatih Ezgü......................................................376 Fatih Kandemir.............................................280 Fatma Demir Yenigürbüz............................ 46 Fatma Demirel................................................ 40 Fatma Dilek Kaymakçı................................ 118 Fehmi Hindilerden......................................... 72 Ferda Perçin...................................................143 Ferit Avcu......................................................... 64 Fevzi Fırat Yalnız..........................................233 Feyza Bayrakdar Çağlayan........................ 116 Figen Kuloğlu.................................................. 89 Filiz Vural.......................................................... 27 Funda Ceran..................................................226 Funda Şimşek................................................167 Füsun Özdemirkıran...................................... 27 George Karam...............................................358 Gökhan Güngör............................................356 Gökhan Özgür...............................................359 Gönül Oğur....................................................345 Grzegorz Helbig...........................................186 Guifang Ouyang...........................................362 Guillermo J. Ruiz-Argüelles......................239 Guillermo J. Ruiz-Delgado........................239 Gunay Aliyeva...............................................258 Gül Nihal Özdemir......................................... 99 Gülay Erman.................................................. 213 Güldane Seval...............................................137 Gülsüm Çağlıyan............................................ 27 Gülsüm Özet..................................................226 Gülşen Bolat.................................................... 16 Günhan Gürman............................................ 16 Güray Saydam.......................................27, 174 Gürcan Dikme................................................. 99 Gürsel Güneş................................................. 103 Habip Gedik...................................................167 Hakan Erdoğan.............................................264 Hakan Göker................................................. 103 Hakan Hanımoğlu........................................ 113 Hakan Özdoğu................. 196, 270, 280, 324 Haldun Öniz...................................................179 Hale Ören......................................................... 46 Halil Gürhan Karabulut.............................. 151


AUTHOR INDEX 2017

Haluk Demiroğlu.......................................... 103 Hamdi Akan..................................................... 16 Hande Kızılocak.............................................. 99 Hasan Özdil.................................................... 107 Hatice Ilgın Ruhi.......................................... 151 Hatice Şanlı...................................................194 Hayri Özsan...................................................282 Hayriye Mine Miskioğlu............................. 181 He Qun...............................................................10 Hilal Aydın.....................................................120 Hong Qian......................................................334 Hsin-Chen Lin...............................................159 Hu Sun............................................................200 Hüseyin Önay................................................120 Hüseyin Tokgöz............................................360 Ioannis Tsolakis............................................... 52 Işık Atagündüz.....................................118, 352 İbrahim C. Haznedaroğlu............27, 93, 103 İbrahim Eker.................................................... 64 İbrahim Kulaç.................................................. 34 İdil Yenicesu..................................................143 Ilana Levy.......................................................289 İlknur Kozanoğlu......................196, 280, 324 İpek Yönal Hindilerden................................. 72 İsmail Yaşar Avcı............................................ 64 İsmet Aydoğdu............................................. 181 Jan E. Dyr........................................................111 Jaroslav Cermak............................................111 Jasmita Dass..................................................188 Jelena Tosic Dragovic.................................268 Jia Meng.........................................................334 Jiajia Jin..........................................................334 Jian Tong........................................................334 Jianning Zhao...............................................334 Jie Zhang........................................................200 Jing Huang........................................................10 Juan Carlos Olivares-Gazca......................239 Jung-Kuang Yu.............................................374 Junqing Xu.....................................................126 Kanay Yararbaş............................................. 105 Katarina Markovic.......................................268 Kazuo Wakayama........................................354 Klara Dalva..................................................... 314 Klara Pecankova............................................111 Konstantinos Malekas.................................. 52 Konstantinos Stamoulis............................... 52 Kuang-Hsi Chang........................................159 Kuenzang Dorji.............................................266 Lale Olcay....................................................... 151 Leontini Fountoulaki-Paparisos................ 52 Levent Karapınar..........................................179 Leyan Wang......................................................10 Leyla Cinel...................................................... 118 Leyla Tümer...................................................376 Liming Chen..................................................126 Long Su........................................................... 381 Lu Chen..............................................................10 Madhu Mati Goel........................................369 Mahmut Yeral.................. 196, 270, 280, 324 Mani Ramzi....................................................300 Manu Goyal................................................... 370 Maria Baka....................................................... 52 Maria Gavalaki............................................... 52 Maria Mouratidou......................................... 52

Mariana A. Mendez Huerta......................239 Marianna Politou........................................... 52 Marije M. Van Santen.................................124 Mario Soliman..............................................366 Massimo Zeviani..........................................376 Maurice Kfoury............................................358 Mehdi Khansalar..........................................300 Mehmet Can Uğur.......................................197 Mehmet Gündüz............................................ 16 Mehmet Halil Çeliksoy...............................345 Mehmet Özen................................................. 16 Mehmet Yılmaz.............................................. 27 Mehran Karimi..............................................250 Melda Cömert........................................27, 174 Melek Erdem................................................... 46 Meliha Nalçacı.......................................72, 131 Melikşah Uzakgider.....................................179 Meltem Aylı...................................................137 Meral Beksaç..........................................16, 314 Meral Türker..................................................179 Meriban Karadoğan....................................340 Meriç Kaymak Cihan................................... 151 Mesude Falay................................................226 Michael Halefom Mezgebe......................... 59 Milan Pantelic...............................................268 Mili Jain..........................................................369 Mine Hekimgil..............................................122 Mohammad Mostafa Safarpour.............250 Mónica Leon-Gonzalez..............................239 Muhammad Ikram Ullah...........................278 Muhit Özcan................................................... 16 Muhlis Cem Ar.................................... 233, 291 Murat Akyıldız..............................................356 Murat Büyükdoğan.....................................360 Murat Dayangaç..........................................356 Murat Elli.......................................................345 Murat Sarı...................................................... 131 Murat Seval...................................................137 Murat Sünbül................................................ 107 Murat Yıldırım..............................................359 Musa Barış Aykan........................................359 Mustafa Erkoçoğlu......................................120 Mustafa Nuri Yenerel................................... 72 Mutlu Arat..............................................16, 350 Mürüvvet Seda Aydın................................. 103 Naciye Demirel.............................................167 Nada Dimkovic.............................................268 Nadia Zanetti................................................376 Nadim El-Majzoub......................................358 Nafiye Urgancı.............................................. 109 Namık Özbek.................................................183 Nannan Li.......................................................126 Narges Rezaei...............................................300 Nargess Arandi.............................................300 Nazan Dalgıç................................................. 109 Nazan Sarper................................................340 Nazif Zeybek................................................... 64 Nejat Akar...............................................93, 105 Neşe Yaralı..............................................40, 183 Neval Özkaya...................................................81 Nevin Kılıç......................................................264 Nihal Karadaş Özdemir..............................254 Nihan Akkoç..................................................233 Niki Vgontza.................................................... 52

Nilgün Sayınalp............................................ 103 Ning Xu...........................................................362 Nirong Bao.....................................................334 Nishant Verma..............................................369 Nitin Sood......................................................272 Noha S. Kandil.............................................. 207 Nur Efe İris........................................... 167, 350 Nur Soyer................................................27, 174 Nuray Başsüllü.................................................81 Nurhilal Büyükkurt.....................................324 Nuri Barış Hasbal......................................... 116 Nüket Yürür Kutlay..................................... 151 Nükhet Tüzüner..................................... 81, 291 Oktay Bilgir...................................................... 27 Orhan Gürsel................................................... 64 Osman İlhami Özcebe................................. 103 Osman İlhan............................................. 16, 27 Osman Kara...................................................352 Osman Yokuş.................................................167 Önder Arslan..........................................16, 244 Önder Şirikçi..................................................352 Özcan Çeneli.................................................364 Özge Özer Kaya............................................197 Özlem Çakıcı..................................................179 Özlem Özdemir.............................................264 Özlen Bektaş..................................................364 Paraskevi Papadopoulou.............................. 52 Pavel Majek....................................................111 Pelin Aytan.......................................... 196, 280 Pervin Topçuoğlu........................ 16, 194, 244 Petar Djuric....................................................268 Pınar Çetinalp Demircan........................... 213 Pınar Genç.....................................................264 Pınar Yurdakul.............................................. 314 Pratap Rai......................................................266 Puja Devi Samal...........................................266 Qi Han.............................................................366 Recep Sancak................................................345 Reyhan Diz-Küçükkaya............. 72, 350, 356 Rıza Aytaç Çetinkaya.................................... 64 Ritesh Sachdev.............................................272 Rixin Jamtsho...............................................266 Roger K. Schindhelm..................................124 Rongdi Yan....................................................200 Rosalba Morreale.........................................184 Ruchika K Goel.............................................272 Sabina Langer...............................................188 Sabina Russo.................................................184 Salih Aksu...................................................... 103 Sanjay Mishra...............................................369 Saqib Mahmood...........................................278 Sebahattin Yılmaz......................................... 64 Seçil Saral.......................................................194 Seda Yılmaz...................................................364 Selami Koçak Toprak.............................. 1, 244 Selim Öncel....................................................340 Selim Sayın....................................................359 Selin Berk....................................................... 291 Selma Sarı...................................................... 131 Sema Aylan Gelen.......................................340 Sema Vural..................................................... 109 Semra Şen......................................................254 Sena Sert........................................................ 107 Serena Valsami............................................... 52


AUTHOR INDEX 2017

Serkan Güvenç.............................................360 Serkan İşgören..............................................190 Sezaneh Haghpanah......................... 250, 300 Sezen Güntekin Ergün...............................143 Sezgin Etgül.................................................. 103 Shabbir Hussain...........................................278 Shahida Mohsin...........................................278 Shalini Goel...................................................272 Shao-Min Han..............................................159 Shereen M. Elmaghraby............................. 207 Shinya Kimura..............................................354 Shyam Aggarwal..........................................188 Sıdıka Fındık..................................................364 Simten Dağdaş.............................................226 Sinan Demircioğlu.......................................364 Sinan Özkavukçu.........................................137 Sinem Civriz Bozdağ...................................244 Slavisa Sekulic..............................................268 Slawomira Kyrcz Krzemien.......................186 Smeeta Gajendra............................... 100, 272 Soner Solmaz................................................324 Soner Yılmaz................................................... 64 Songül Tepebaşı...........................................324 Sora Yasri........................................................ 102 Sousanna Pefani............................................ 52 Sreeja Thandilath Thekkelakayil.............. 370 Stavros Nousis................................................ 52 Stavroula Tsitlakidou.................................... 52 Stephan Borte...............................................345 Suar Çakı Kılıç...............................................340 Suchi Mittal...................................................188 Surmaya Gafarova.......................................258 Süheyl Asma...............................196, 280, 324 Süheyla U. Bozkurt............................ 190, 352 Sülen Sarıoğlu..............................................282 Süreyya Bozkurt........................................... 103 Şadiye Mehtat Ünlü....................................282 Şebnem Yılmaz Bengoa............................... 46 Şencan Acar..................................................356 Şeniz Öngören.................................... 233, 291

Şöhret Aydemir............................................254 Şule Toprak....................................................372 Şükrü Kasap...................................................122 Şükrü Nail Güner.........................................345 Tahira Mammadova....................................258 Tamar Tadmor...............................................289 Taner Baştürk.....................................1, 16, 116 Taner Yıldırmak............................................167 Tao Yuan.........................................................334 Tashi Tobgay..................................................266 Tayfur Toptaş.................................................352 Teoman Soysal.................................... 233, 291 Tezer Kutluk..................................................... 34 Tiraje Celkan.................................................... 99 Tuğçe Kurtaraner......................................... 109 Tuğrul Elverdi................................................ 291 Tuncay Aslan................................................. 103 Tuncay Sahutoğlu........................................ 116 Tunç Fışgın.....................................................345 Tülin Ergun.................................................... 118 Tülin Fıratlı Tuğlular....................................352 Türkiz Gürsel..................................................143 Tze-Kiong Er..................................................374 Ufuk Demirci................................................. 181 Uğur Demirsoy..............................................340 Uğur Muşabak................................................ 64 Ülker Koçak...................................................143 Ümit Yavuz Malkan..................................... 103 Ümran Çalışkan............................................360 Ünsal Özgen.................................................. 114 Vanessa Innao...............................................184 Vani Rajashekharaiah.................................328 Vassiliki Pournara........................................... 52 Vassiliki Rekari................................................ 52 Veysel Sabri Hançer....................................360 Vildan Çulha..................................................183 Viroj Wiwanitkit........................ 101, 102, 380 Volkan Karakuş................................... 122, 192 Wen-Li Hwang..............................................159 Wen-shuang Sun.........................................334

Wen-xiang Chen..........................................334 Xiao-Ping Huang.........................................362 Xiaoqian Liu...................................................126 Xiaoxia Chu...................................................126 Xilian Zhou....................................................366 Xing Cui..........................................................200 Xu Yajing...........................................................10 Yahveth Cantero-Fortiz.............................239 Yahya Büyükaşık...................................34, 103 Yaman Tokat..................................................356 Yan Gu.............................................................366 Yanyan Luan..................................................126 Yasemin Ardıçoğlu....................................... 105 Yasushi Kubota.............................................354 Yaşar Bekir Kutbay......................................197 Yegana Guliyeva..........................................258 Yelda Dere............................................ 122, 192 Yener Koç....................................................... 116 Yıldız Aydın.......................................... 233, 291 Yi-Hong Liu...................................................374 Yinghui Liu.....................................................126 Yuanfeng Zhang...........................................126 Yuka Hirakawa..............................................354 Yüksel Pabuşçu............................................. 181 Zafer Başlar..........................................113, 291 Zafer Gülbaş..................................................190 Zahide Eriş.....................................................350 Zehra Işık Haşıloğlu....................................... 99 Zerrin Ertaş...................................................... 64 Zeynep Kayra Tanrıverdi............................ 114 Zhao Xielan.......................................................10 Zheng Ge........................................................366 Zhi-Yuan Shi.................................................159 Zhuan-Bo Luo...............................................362 Zoran Milojevic............................................268 Zühre Kaya.....................................................143 Zümrüt Şahbudak Bal................................254


SUBJECT INDEX 2017

34th Volume Index / 34. Cilt Dizini SUBJECT INDEX - KONU DİZİNİ 2017

Acute Leukemia Relapsed/refractory leukemia / Nüks/refrakter lösemi, 46 FLAG regimen / FLAG tedavisi, 46 Chemotherapy / Kemoterapi, 46 Childhood / Çocukluk çağı, 46 Hypogammaglobulinemia / Hipogamaglobulinemi, 89 Leukemia / Lösemi, 89, 109, 126, 186, 190 Lymphoma / Lenfoma, 89 Myeloma / Miyelom, 89, 181, 182 Vancomycin-resistant Enterococcus / Vankomisin dirençli entekok, 89 Wernicke’s encephalopathy / Wernicke ensefalopatisi, 99 Thiamine deficiency / Tiamin eksikliği, 99 Pediatric leukemia / Pediatrik lösemi, 99, 340 Trisomy 6 / Trizomi 6, 103 Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 340, 300, 307 Breast cancer / Meme kanseri, 103 Granulocytic sarcoma / Granülositik sarkom, 122 Acute monoblastic leukemia / Akut monoblastik lösemi, 122 CD34 / CD34, 122 Myeloperoxidase / Miyeloperoksidaz, 122 Monosomal karyotype / Monozomal karyotip, 126 Prognosis / Prognoz, 126, 143, 207 Methotrexate / Metotreksat, 143 Toxicity / Toksisite, 143 Folate-related gene polymorphisms / Folat-ilişkili gen polimorfizmleri, 143 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 40, 151, 366 Glucocorticoid receptor gene / Glukokortikoid reseptör geni, 151 BclI and N363S polymorphisms / BclI ve N363S polimorfizmler, 151 TET2 / TET2, 174 ASXL1 / ASXL1, 174 IDH1 / IDH1, 174 IDH2 / IDH2, 174 Single nucleotide polymorphisms / Tek nükleoitid polimorfizm, 174 Ph-negative myeloproliferative neoplasms / Ph-negatif Miyeloproliferatif neoplaziler, 174 Granulocytic sarcoma / Granülositik sarkom, 190 Lymphadenopathy / Lenfadenopati, 190 Azacitidine / Azasitidin, 192 Leukemia cutis / Lösemi kutis, 192 Febrile neutropenia / Netropenik Ateş, 109, 167, 254, 340 Infection / Enfeksiyon, 340 Acute myelogenous leukemia / Akut myeloid lösemi, 350 Bullous pyoderma gangrenosum / Büllöz piyoderma gangrenosum, 350 Pathergy / Paterji, 350 EZH2 / EZH2, 366 Adult / Erişkin, 366 T-cell / T-hücre, 366 Gene mutation / Gen mutasyonu, 300 FLT3 / FLT3, 300 NPM1 / NPM1, 300

Autoantibody / Otoantikor, 307 Cancer / Kanser, 307 Carbonic anhydrase / Karbonik anhidraz, 307

Anemia Schistocyte / Şistosit, 59 Thrombotic microangiopathy / Trombotik mikroanjiopati, 59 Microscopy / Mikroskop, 59 ADVIA 2120 / ADVIA 2120, 59 Standardization / Standardizasyon, 59 Refractory anemia with ring sideroblasts / Halka sideroblastlı refrakter anemi, 100 RARS with thrombocytosis / Trombositoz ile birlikte RARS, 100 Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis / Halka sideroblast ve trombositoz ile birlikte miyelodisplastik sendrom/miyeloproliferatif neoplaz, 100 Abnormal hemoglobins / Anormal hemoglobinler, 114 Hb Kansas / Hb Kansas 114 Children / Çocuk, 120 Griscelli syndrome / Griscelli sendromu, 120 Status epilepticus / Status epileptikus, 120 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiyositoz, 120 Adult / Erişkin, 196, 366 Sickle cell anemia / Orak hücreli anemi, 196, 358 Genetic counseling / Genetik danışmanlık, 196 Transplantation / Transplantasyon, 102, 196, 345 Blood components / Kan içeriği, 244 Blood processing / Kan ürünlerini işleme, 244 Donors / Verici, 244 Transfusion strategy / Transfüzyon stratejisi, 244 Thalassemia / Talasemi, 258 Sickle/β-thalassemia / Orak/β talasemi, 258 Codon / Kodon, 258 Genotype / Genotip, 258 Phenotype / Fenotip, 258 Osteomyelitis / Osteomiyelit, 358 Salmonella typhi / Salmonella typhi, 358 Myopathy / Miyopati, 376 Lactic acidosis / Laktik asidoz, 376 Sideroblastic anemia / Sideroblastik anemi, 376 Hidden blood loss / Gizli kan kaybı, 334 Antioxidants / Antioksidanlar, 334 Proanthocyanidin / Proantosiyanidin, 334 Hydrogen water / Hidrojenli su, 334

Bleeding Disorders Hemophilia A / Hemofili A, 113 Intradiploic hematoma / İntradiploik hematom, 113 Coagulopathy / Koagülopati, 113 Intraosseous / İntraosseoz, 113 Children / Çocuk, 120 Griscelli syndrome / Griscelli sendromu, 120


SUBJECT INDEX 2017

Status epilepticus / Status epileptikus, 120 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiyositoz, 120 Afibrinogenemia / Afibrinojenemi, 183 Bone cysts / Kemik kistleri, 183 Child / Çocuk, 183 Rare / Nadir, 183 Acquired hemophilia / Edinsel hemofili, 184 Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Factor VIII / Faktör VIII, 184 Autoantibody / Otoantikor, 184 Coagulation disorder / Koagülasyon bozukluğu, 184 Congenital / Konjenital, 250 Factor V deficiency / Faktör V eksikliği, 250 Phenotype / Fenotip, 250 Rare bleeding disorders / Nadir kanama bozuklukları, 250 Psychogenic purpura / Psikojenik purpura, 274 Gardner-Diamond syndrome / Gardner-Diamond sendromu, 274 Antidepressant / Antidepresan, 274 Adolescent / Adölesan, 274 Hemophilia A / Hemofili A, 278 FVIII gene / FVIII geni, 278 Intron 1 / İntron 1, 278 Polymerase chain reaction / Polimeraz zincirleme reaksiyonu, 278 Pakistan / Pakistan, 278 Dysfibrinogenemia / Disfibrinojenemi, 356 Liver transplantation / Karaciğer nakli, 356 Subdural hematoma / Subdural hematom, 356

Chronic Leukemia Imatinib / İmatinib, 10 Sokal score / Sokal skoru, 10 Hasford score / Hasford skoru, 10 European Treatment Outcome Study score / Avrupa Tedavi İzlem Çalışması skoru, 10 Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197, 362 Allogeneic transplantation / Allojenik transplantasyon, 16 Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 16 Hematologic remission / Hematolojik remisyon, 16 TP53 / TP53, 34 Immunohistochemistry / İmmünohistokimya, 34 Chronic lymphocytic lymphoma / Kronik lenfositik lenfoma, 34 Proliferation centers / Proliferasyon merkezleri, 34 Nilotinib / Nilotinib, 137, 197, 362 Fertility / Doğurganlık, 137 Mouse / Fare, 137 Gonads / Gonad, 137 Richter’s syndrome / Richter sendromu, 188 Flow cytometry / Akım sitometri, 188 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188 CD5-positive / CD5- pozitif, 188 Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188 Cytogenetic / Sitogenetik, 197 Molecular hematology / Moleküler hematoloji, 197 Life-quality / Yaşam kalitesi, 197 Dasatinib / Dasatinib, 197 Hairy cell leukemia / Saçlı hücreli lösemi, 270, 289, 291 Splenectomy / Splenektomi, 270, 291 Sweet’s syndrome / Sweet’s sendromu, 270

Systemic mastocytosis / Sistemik mastositoz, 276 Systemic mastocytosis with an associated hematological neoplasm / Hematolojik malignite ile seyreden sistemik mastositozis, 276 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 276 KIT D816V / KIT D816V, 276 Purine Analogs / Pürin Analogları, 289 Cladribine / Kladribin, 289, 291 Pentostatin /Pentostatin, 289 Imatinib mesylate / İmatinib mesilat, 362 Interstitial pneumonitis / İnterstisyel pnömonitis, 362 Tuberculosis / Tüberküloz, 362 Interferon / İnterferon, 291

Coagulation Ankaferd Blood Stopper / Ankaferd Blood Stopper, 93 Shiga-toxigenic Escherichia coli / Shigatoksijenik Escherichia coli, 93 Salmonella / Salmonella, 93 Campylobacter / Campylobacter, 93 Listeria monocytogenes / Listeria monocytogenes, 93 Monitoring / İzlem, 101 International normalized ratio / Uluslararası normalleştirilmiş oran, 101 Hemostasis / Hemostaz, 101 Acute myocardial infarction / Akut miyokard infarktüsü, 107 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 107 Eltrombopag / Eltrombopag, 107 Hemophilia A / Hemofili A, 113 Intradiploic hematoma / İntradiploik hematom, 113 Coagulopathy / Koagülopati, 113 Intraosseous / İntraosseoz, 113 Afibrinogenemia / Afibrinojenemi, 183 Bone cysts / Kemik kistleri, 183 Child / Çocuk, 183 Rare / Nadir, 183 Acquired hemophilia / Edinsel hemofili, 184 Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Factor VIII / Faktör VIII, 184 Autoantibody / Otoantikor, 184 Coagulation disorder / Koagülasyon bozukluğu, 184 Sticky platelet syndrome / Yapışkan trombosit sendromu, 239 Thrombophilia / Trombofili, 239 Miscarriages / Düşükler, 239 Congenital / Konjenital, 250 Factor V deficiency / Faktör V eksikliği, 250 Phenotype / Fenotip, 250 Rare bleeding disorders / Nadir kanama bozuklukları, 250 Hemophilia A / Hemofili A, 278 FVIII gene / FVIII geni, 278 Intron 1 / İntron 1, 278 Polymerase chain reaction / Polimeraz zincirleme reaksiyonu, 278 Pakistan / Pakistan, 278 Dysfibrinogenemia / Disfibrinojenemi, 356 Liver transplantation / Karaciğer nakli, 356 Subdural hematoma / Subdural hematom, 356

Hematological Malignancies Imatinib / İmatinib, 10 Sokal score / Sokal skoru, 10 Hasford score / Hasford skoru, 10 European Treatment Outcome Study score / Avrupa Tedavi


SUBJECT INDEX 2017

İzlem Çalışması skoru, 10 Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197, 362 Allogeneic transplantation / Allojenik transplantasyon, 16 Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 16 Hematologic remission / Hematolojik remisyon, 16 Chronic myeloproliferative neoplasms / Kronik miyeloproliferatif neoplaziler, 27 Treatment / Tedavi, 27 Survival / Sağkalım, 27 JAK2 mutation / JAK2 mutasyonu, 27 Relapsed/refractory leukemia / Nüks/refrakter lösemi, 46 FLAG regimen / FLAG tedavisi, 46 Chemotherapy / Kemoterapi, 46 Childhood / Çocukluk çağı, 46 Hypogammaglobulinemia / Hipogamaglobulinemi, 89 Leukemia / Lösemi, 89, 109, 126, 186, 190 Lymphoma / Lenfoma, 89 Myeloma / Miyelom, 89, 181, 182, 352 Vancomycin-resistant Enterococcus / Vankomisin dirençli entekok, 89 Trisomy 6 / Trizomi 6, 103 Breast cancer / Meme kanseri, 103 Children / Çocuk, 120 Griscelli syndrome / Griscelli sendromu, 120 Status epilepticus / Status epileptikus, 120 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiyositoz, 120 Granulocytic sarcoma / Granülositik sarkom, 122 Acute monoblastic leukemia / Akut monoblastik lösemi, 122 CD34 / CD34, 122 Myeloperoxidase / Miyeloperoksidaz, 122 Monosomal karyotype / Monozomal karyotip, 126 Prognosis / Prognoz, 126, 143, 207 Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Prognostic factors / Prognostik faktörler, 131 Serum immunoglobulins / Serum immunoglobulin, 131 Nephelometric measurement / Nefelometrik ölçüm, 131 M-protein / M-protein, 131 Nilotinib / Nilotinib, 137, 197, 362 Fertility / Doğurganlık, 137 Mouse / Fare, 137 Gonads / Gonad, 137 Methotrexate / Metotreksat, 143 Toxicity / Toksisite, 143 Folate-related gene polymorphisms / Folat-ilişkili gen polimorfizmleri, 143 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 40, 151, 366 Glucocorticoid receptor gene / Glukokortikoid reseptör geni, 151 BclI and N363S polymorphisms / BclI ve N363S polimorfizmler, 151 TET2 / TET2, 174 ASXL1 / ASXL1, 174 IDH1 / IDH1, 174 IDH2 / IDH2, 174 Single nucleotide polymorphisms / Tek nükleoitid polimorfizm, 174 Ph-negative myeloproliferative neoplasms / Ph-negatif Miyeloproliferatif neoplaziler, 174 Myeloma / Miyelom, 89, 181, 182 Infection / Enfeksiyon, 182, 340 Atlas / Atlas, 182 Fracture / Kırık, 182

Acquired hemophilia / Edinsel hemofili, 184 Factor VIII / Faktör VIII, 184 Autoantibody / Otoantikor, 184, 307 Coagulation disorder / Koagülasyon bozukluğu, 184 Bing-Neel syndrome / Bing-Neel sendromu, 186 Waldenström’s macroglobulinemia / Waldenström makroglobulinemisi, 186 Central nervous system / Merkezi sinir sistemi, 186 MYD88 L265P mutation / MYD88 L265P mutasyonu, 186 Cerebrospinal fluid / Beyin-omurilik sıvısı, 186 Leukemia / Lösemi, 89, 109, 126, 186, 190 Richter’s syndrome / Richter sendromu, 188 Flow cytometry / Akım sitometri, 188 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188 CD5-positive / CD5- pozitif, 188 Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188 Granulocytic sarcoma / Granülositik sarkom, 190 Lymphadenopathy / Lenfadenopati, 190 Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 340, 300, 307 Azacitidine / Azasitidin, 192 Leukemia cutis / Lösemi kutis, 192 Mycosis fungoides / Mikozis fungoides, 194 Ichthyosiform / İktiyotik, 194 Sezary syndrome / Sezary sendromu, 194 Anaplastic / Anaplastik, 194 CD3/TCR / CD3/TCR, 194 Cytogenetic / Sitogenetik, 197 Molecular hematology / Moleküler hematoloji, 197 Life-quality / Yaşam kalitesi, 197 Dasatinib / Dasatinib, 197 Myelodysplastic syndromes / Miyelodisplastik sendromlar, 200 Telomere-binding proteins / Telomer-bağlayıcı proteinler, 200 Reverse transcription-polymerase chain reaction / Ters transkripsiyon-polimeraz zincir reaksiyonu, 200 International Prognostic Scoring System / Uluslararası Prognoz Skorlama Sistemi, 200 World Health Organization Prognostic Scoring System / Dünya Sağlık Örgütü Prognoz Skorlama Sistemi, 200 MicroRNA-155 / MikroRNA-155, 207 Non-Hodgkin lymphoma / Hodgkin dışı lenfoma, 207, 378, 381 CD56 / CD56, 226 CD117 / CD117, 226 Flow cytometry / Akım sitometri, 226 Multiple myeloma / Multipl miyeloma, 131, 184, 226, 233, 282 Relapse refractory / Relaps refrakter, 233 Bendamustine / Bendamustin, 233 Distal arteriovenous fistula / Distal arteriyovenöz fistül, 268 Multislice computer tomography/ Çok kesitli bilgisayarlı tomografi, 268 Hemodialysis / Hemodiyaliz, 268 Hairy cell leukemia / Saçlı hücreli lösemi, 270, 289, 291 Splenectomy / Splenektomi, 270, 291 Sweet’s syndrome / Sweet’s sendromu, 270 Hodgkin’s lymphoma / Hodgkin lenfoma, 272 Metastatic adenocarcinoma / Metastatik adenokarsinom, 272 Reed-Sternberg cells / Reed-Sternberg hücreleri, 272 PET/CT / PET/BT, 272 Systemic mastocytosis / Sistemik mastositoz, 276 Systemic mastocytosis with an associated hematological neoplasm / Hematolojik malignite ile seyreden sistemik mastositozis, 276


SUBJECT INDEX 2017

Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 276 KIT D816V / KIT D816V, 276 Pediatric leukemia / Çocukluk çağı lösemileri, 340 Febrile neutropenia / Nötropenik Ateş, 109, 167, 254, 340 Purine analogs / Pürin analogları, 289 Cladribine / Kladribin, 289, 291 Pentostatin / Pentostatin, 289 Acute myelogenous leukemia / Akut myeloid lösemi, 350 Bullous pyoderma gangrenosum / Büllöz piyoderma gangrenosum, 350 Pathergy / Paterji, 350 Plasmacytoma / Plazmasitom, 352 Intracranial / İntrakraniyal, 352 Essential thrombocythemia / Esansiyel trombositemi, 360 Primary myelofibrosis / Primer miyelofibroz, 124, 360 Calreticulin / Kalretikulin, 360 Imatinib mesylate / İmatinib mesilat, 362 Interstitial pneumonitis / İnterstisyel pnömonitis, 362 Tuberculosis / Tüberküloz, 362 Follicular lymphoma / Foliküler lenfoma, 364 Extranodal / Ekstranodal, 364 Prostatic involvement / Prostat Tutulumu, 364 EZH2 / EZH2, 366 Adult / Erişkin, 366 T-cell / T-hücre, 366 Abnormal hemoglobins / Anormal hemoglobinler, 372 Hemoglobin San Diego / Hemoglobin San Diego, 372 Hemoglobin variant / Hemoglobin varyantı, 372 T-cell neoplasms / T hücreli neoplazmlar, 378 Oncogenes / Onkogenler, 378 T-cell mediated immunity / T hücre aracılı immünite, 378 miR-155 / miR-155, 381 Prognostic value / Prognostik değer, 381 Interferon / İnterferon, 291 Gene mutation / Gen mutasyonu, 300 FLT3 / FLT3, 300 NPM1 / NPM1, 300 Cancer / Kanser, 307 Carbonic anhydrase / Karbonik anhidraz, 307 Monoclonal gammopathy of renal significance / Renal öneme sahip monoklonal gammopati, 282 Plasma cell disorders / Plazma hücre hastalıkları, 282 Renal involvement / Böbrek tutulumu, 282 Kidney / Böbrek, 282 Cast nephropathy / Kast nefropati, 282

Immunohematology Leukemia / Lösemi, 89, 109, 126, 186, 190 Febrile neutropenia / Febril nötropeni, 109, 167, 254, 340 Candida / Candida, 109 Immune response inflammatory syndrome / İmmün yanıt enflamatuvar sendromu, 109 Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Prognostic factors / Prognostik faktörler, 131 Serum immunoglobulins / Serum immunoglobulin, 131 Nephelometric measurement / Nefelometrik ölçüm, 131 M-protein / M-protein, 131 Neutrophil / Nötrofil, 167 CD64 / CD64, 167 Bacteremia / Bakteriyemi, 167, 254

Diagnostic parameter / Diyagnostik parametre, 167 Acute compartment syndrome / Akut kompartman sendromu, 179 Inflammation / Enflamasyon, 179 Immune deficiency / İmmün yetmezlik, 179 Richter’s syndrome / Richter sendromu, 188 Flow cytometry / Akım sitometri, 188 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188 CD5-positive / CD5-pozitif, 188 Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188 Immunoregulatory effect / Bağışıklık baskılayıcı etki, 213 Co-culture / Ko-kültür, 213 Mesenchymal stem cells / Mezenkimal kök hücre, 213 T cells / T hücre, 213 CD56 / CD56, 226 CD117 / CD117, 226 Flow cytometry / Akım sitometri, 226 IL-6 / IL-6, 254 IL-8 / IL-8, 254 IL-10 / IL-10, 254 Hematopoitic stem cell / Hematopoetik kök hücre, 345 Transplantation /Transplantasyon, 102, 196, 345 Children / Çocuk, 345 Immunodeficiency / İmmün yetmezlik, 345 Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 340, 300, 307 Autoantibody / Otoantikor, 307 Cancer / Kanser, 307 Carbonic anhydrase / Karbonik anhidraz, 307 Cord blood / Göbek kordon kanı, 314 Aldehyde dehydrogenase / Aldehit dehidrogenaz, 314 Colony-forming unit-granulocyte/macrophage / Koloni oluşturan birim granülositer/makrofaj, 314 L-Carnitine / L-Karnitin, 328 Plasma / Plazma, 328 Antioxidant enzymes / Antioksidan enzimler, 328 Lipid peroxidation / Lipid peroksidasyonu, 328 Protein oxidation / Protein oksidasyonu, 328 Hidden blood loss / Gizli kan kaybı, 334 Antioxidants / Antioksidanlar, 334 Proanthocyanidin / Proantosiyanidin, 334 Hydrogen water / Hidrojenli su, 334

Iron Disorder Iron overload / Demir yüklenmesi, 1 Hematopoietic stem cell transplantation / Hematopoietik kök hücre nakli, 1 Ferritin / Ferritin, 1 Iron chelation / Demir şelasyonu, 1 Iron / Demir, 102 Overload / Aşırı yüklenme, 102 Hematopoietic stem cell / Hematopoietik kök hücre, 102 Transplantation / Transplantasyon, 102, 196, 345

Infection Disorders Hypogammaglobulinemia / Hipogamaglobulinemi, 89 Leukemia / Lösemi, 89, 109, 126, 186, 190 Lymphoma / Lenfoma, 89 Myeloma / Miyelom, 89, 181, 182, 352 Vancomycin-resistant Enterococcus / Vankomisin dirençli entekok, 89 Ankaferd Blood Stopper / Ankaferd Blood Stopper, 93 Shiga-toxigenic Escherichia coli / Shigatoksijenik Escherichia coli, 93


SUBJECT INDEX 2017

Salmonella / Salmonella, 93 Campylobacter / Campylobacter, 93 Listeria monocytogenes / Listeria monocytogenes, 93 Febrile neutropenia / Febril nötropeni, 109, 167, 254, 340 Candida / Candida, 109 Immune response inflammatory syndrome / İmmün yanıt enflamatuvar sendromu, 109 Allogeneic hematopoietic stem cell transplantation / Allojeneik hematopoietik kök hücre transplantasyonu, 159 Cytomegalovirus / Sitomegalovirüs, 159 Graft-versus-host disease / Graft versus host hastalığı, 159 Taiwan / Tayvan, 159 Neutrophil / Nötrofil, 167 CD64 / CD64, 167 Bacteremia / Bakteriyemi, 167, 254 Diagnostic parameter / Diyagnostik parametre, 167 Acute compartment syndrome / Akut kompartman sendromu, 179 Inflammation / Enflamasyon, 179 Immune deficiency / İmmün yetmezlik, 179 IL-6 / IL-6, 254 IL-8 / IL-8, 254 IL-10 / IL-10, 254 Leishman-Donovan body / Leishman-Donovan cismi, 266 Bone marrow aspirate smear / Kemik iliği aspirasyonu yayması, 266 Microscopic image / Mikroskobik görüntü, 266 Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 300, 307, 340 Pediatric leukemia / Çocuk çağı lösemileri, 340 Infection / Enfeksiyon, 340 Leukoagglutination / Lökoaglütinasyon, 354 Cold agglutinin / Soğuk aglütinin, 354 Mycoplasma pneumoniae / Mycoplasma pneumoniae, 354 Eosinophilia / Eozinofili, 354 Pseudoleukopenia / Psödolökopeni, 354 Sickle cell anemia / Orak hücreli anemi, 196, 358 Osteomyelitis / Osteomiyelit, 358 Salmonella typhi / Salmonella typhi, 358 Cholangitis / Kolanjit, 359 Döhle bodies / Döhle cisimciği, 359 May-Hegglin anomaly / May-Hegglin anomalisi, 359 Imatinib mesylate / İmatinib mesilat, 362 Interstitial pneumonitis / İnterstisyel pnömonitis, 362 Chronic myeloid leukemia / Kronik miyeloid lösemi, 362 Nilotinib / Nilotinib, 137, 197, 362 Tuberculosis / Tüberküloz, 362 Leishmaniasis / Layşmanyazis, 380 Bone marrow / Kemik iliği, 380 Aspirate / Aspirasyon, 380 Smear / Yayma, 380 Antibody / Antikor, 380

Lymphoma Diagnosis of lymphoma / Lenfoma tanısı, 81 World Health Organization lymphoma classification / Dünya Sağlık Örgütü lenfoma sınıflaması, 81 Discrepancies in diagnosis / Tanı tutarsızlıkları, 81 Hematopathology / Hematopatoloji, 81 Bing-Neel syndrome / Bing-Neel sendromu, 186 Waldenström’s macroglobulinemia / Waldenström makroglobulinemisi, 186

Central nervous system / Merkezi sinir sistemi, 186 MYD88 L265P mutation / MYD88 L265P mutasyonu, 186 Cerebrospinal fluid / Beyin-omurilik sıvısı, 186 Leukemia / Lösemi, 89, 109, 126, 186, 190 Richter’s syndrome / Richter sendromu, 188 Flow cytometry / Akım sitometri, 188 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188 CD5-positive / CD5- pozitif, 188 Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188 Mycosis fungoides / Mikozis fungoides, 194 Ichthyosiform / İktiyotik, 194 Sezary syndrome / Sezary sendromu, 194 Anaplastic / Anaplastik, 194 CD3/TCR / CD3/TCR, 194 MicroRNA-155 / MikroRNA-155, 207 Non-Hodgkin lymphoma / Hodgkin dışı lenfoma, 207, 378, 381 Prognosis / Prognoz, 126, 143, 207 Hodgkin’s lymphoma / Hodgkin lenfoma, 272 Metastatic adenocarcinoma / Metastatik adenokarsinom, 272 Reed-Sternberg cells / Reed-Sternberg hücreleri, 272 PET/CT / PET/BT, 272 Follicular lymphoma / Foliküler lenfoma, 364 Extranodal / Ekstranodal, 364 Prostatic involvement / Prostat Tutulumu, 364 T-cell neoplasms / T hücreli neoplazmlar, 378 Oncogenes / Onkogenler, 378 T-cell mediated immunity / T hücre aracılı immünite, 378 miR-155 / miR-155, 381 Prognostic value / Prognostik değer, 381

Molecular Hematology Trisomy 6 / Trizomi 6, 103 Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 340, 300, 307 Breast cancer / Meme kanseri, 103 Thalassemia / Talasemi, 105 Variant / Varyant, 105 Genetic counseling / Genetik danışmanlık, 105 Prenatal diagnosis / Prenatal tanı, 105 Beta globin gene / Beta globin geni, 105 Turkish / Türk, 105 Monosomal karyotype / Monozomal karyotip, 126 Prognosis / Prognoz, 126, 143, 207 Leukemia / Lösemi, 89, 109, 126, 186, 190 Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197 Nilotinib / Nilotinib, 137, 197, 362 Fertility / Doğurganlık, 137 Mouse / Fare, 137 Gonads / Gonad, 137 Methotrexate / Metotreksat, 143 Toxicity / Toksisite, 143 Folate-related gene polymorphisms / Folat-ilişkili gen polimorfizmleri, 143 Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 40, 151, 366 Glucocorticoid receptor gene / Glukokortikoid reseptör geni, 151 BclI and N363S polymorphisms / BclI ve N363S polimorfizmleri, 151 TET2 / TET2, 174 ASXL1 / ASXL1, 174 IDH1 / IDH1, 174 IDH2 / IDH2, 174


SUBJECT INDEX 2017

Single nucleotide polymorphisms / Tek nükleoitid polimorfizm, 174 Ph-negative myeloproliferative neoplasms / Ph-negatif Miyeloproliferatif neoplaziler, 174 Bing-Neel syndrome / Bing-Neel sendromu, 186 Waldenström’s macroglobulinemia / Waldenström makroglobulinemisi, 186 Central nervous system / Merkezi sinir sistemi, 186 MYD88 L265P mutation / MYD88 L265P mutasyonu, 186 Cerebrospinal fluid / Beyin-omurilik sıvısı, 186 Leukemia / Lösemi, 89, 109, 126, 186, 190 Adult / Erişkin, 196, 366 Sickle cell anemia / Orak hücreli anemi, 196, 358 Genetic counseling / Genetik danışmanlık, 196 Transplantation / Transplantasyon, 102, 196 Myelodysplastic syndromes / Miyelodisplastik sendromlar, 200 Telomere-binding proteins / Telomer-bağlayıcı proteinler, 200 Reverse transcription-polymerase chain reaction / Ters transkripsiyon-polimeraz zincir reaksiyonu, 200 International Prognostic Scoring System / Uluslararası Prognoz Skorlama Sistemi, 200 World Health Organization Prognostic Scoring System / Dünya Sağlık Örgütü Prognoz Skorlama Sistemi, 200 MicroRNA-155 / MikroRNA-155, 207 Non-Hodgkin lymphoma / Hodgkin dışı lenfoma, 207, 378, 381 Immunoregulatory effect / Bağışıklık baskılayıcı etki, 213 Co-culture / Ko-kültür, 213 Mesenchymal stem cells / Mezenkimal kök hücre, 213 T cells / T hücre, 213 Thalassemia / Talasemi, 258 Sickle/β-thalassemia / Orak/β talasemi, 258 Codon / Kodon, 258 Genotype / Genotip, 258 Phenotype / Fenotip, 258, 374 Wolman disease / Wolman hastalığı, 264 Hemophagocytic lymphohistiocytosis / Hemafagositik lenfohistiyositozis, 264 Hemophagocytosis / Hemofagositoz, 264 Hemophilia A / Hemofili A, 278 FVIII gene / FVIII geni, 278 Intron 1 / İntron 1, 278 Polymerase chain reaction / Polimeraz zincirleme reaksiyonu, 278 Pakistan / Pakistan, 278 Essential thrombocythemia / Esansiyel trombositemi, 360 Primary myelofibrosis / Primer miyelofibroz, 124, 360 Calreticulin / Kalretikulin, 360 EZH2 / EZH2, 366 T-cell / T-hücre, 366 Abnormal hemoglobins / Anormal hemoglobinler, 372 Hemoglobin San Diego / Hemoglobin San Diego, 372 Hemoglobin variant / Hemoglobin varyantı, 372 Para-Bombay / Para-Bombay, 374 FUT1 gene / FUT1 geni, 374 Blood transfusion / Kan transfüzyonu, 374 Myopathy / Miyopati, 376 Lactic acidosis / Laktik asidoz, 376 Sideroblastic anemia / Sideroblastik anemi, 376 miR-155 / miR-155, 381 Prognostic value / Prognostik değer, 381 Gene mutation / Gen mutasyonu, 300

FLT3 / FLT3, 300 NPM1 / NPM1, 300

Multiple Myeloma Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Prognostic factors / Prognostik faktörler, 131 Serum immunoglobulins / Serum immünglobulin, 131 Nephelometric measurement / Nefelometrik ölçüm, 131 M-protein / M-protein, 131 Myeloma / Miyelom, 89, 181, 182, 352 Infection / Enfeksiyon, 181 Atlas / Atlas, 181 Fracture / Kırık, 181 Acquired hemophilia / Edinsel hemofili, 184 Factor VIII / Faktör VIII, 184 Autoantibody / Otoantikor, 184 Coagulation disorder / Koagülasyon bozukluğu, 184 CD56 / CD56, 226 CD117 / CD117, 226 Flow cytometry / Akım sitometri, 226 Relapse refractory / Relaps refrakter, 233 Bendamustine / Bendamustin, 233 Distal arteriovenous fistula / Distal arteriyovenöz fistül, 268 Multislice computer tomography / Çok kesitli bilgisayarlı tomografi, 268 Hemodialysis / Hemodiyaliz, 268 Plasmacytoma / Plazmasitom, 352 Intracranial / İntrakraniyal, 352 Monoclonal gammopathy of renal significance / Renal öneme sahip monoklonal gammopati, 282 Plasma cell disorders / Plazma hücre hastalıkları, 282 Renal involvement / Böbrek tutulumu, 282 Kidney / Böbrek, 282 Cast nephropathy / Kast nefropati, 282

Myelodysplastic Syndromes Refractory anemia with ring sideroblasts / Halka sideroblastlı refrakter anemi, 100 RARS with thrombocytosis / Trombositoz ile birlikte RARS, 100 Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis / Halka sideroblast ve trombositoz ile birlikte miyelodisplastik sendrom/miyeloproliferatif neoplazi, 100 Myelodysplastic syndromes / Miyelodisplastik sendromlar, 200 Telomere-binding proteins / Telomer-bağlayıcı proteinler, 200 Reverse transcription-polymerase chain reaction / Ters transkripsiyon-polimeraz zincir reaksiyonu, 200 International Prognostic Scoring System / Uluslararası Prognoz Skorlama Sistemi, 200 World Health Organization Prognostic Scoring System / Dünya Sağlık Örgütü Prognoz Skorlama Sistemi, 200

Myeloproliferative Disorders Chronic myeloproliferative neoplasms / Kronik miyeloproliferatif neoplaziler, 27 Treatment / Tedavi, 27 Survival / Sağkalım, 27 JAK2 mutation / JAK2 mutasyonu, 27 Primary myelofibrosis / Primer miyelofibroz, 124, 360 Internuclear bridging / Nükleuslar arası köprüleşme, 124 Erythrocytes / Eritrositler, 124


SUBJECT INDEX 2017

TET2 / TET2, 174 ASXL1 / ASXL1, 174 IDH1 / IDH1, 174 IDH2 / IDH2, 174 Single nucleotide polymorphisms / Tek nükleoitid polimorfizm, 174 Ph-negative myeloproliferative neoplasms / Ph-negatif Miyeloproliferatif neoplaziler, 174 Essential thrombocythemia / Esansiyel trombositemi, 360 Calreticulin / Kalretikulin, 360

Stem Cell Transplantation Iron overload / Demir yüklenmesi, 1 Hematopoietic stem cell transplantation / Hematopoietik kök hücre nakli, 1 Ferritin / Ferritin, 1 Iron chelation / Demir şelasyonu, 1 Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197 Allogeneic transplantation / Allojenik transplantasyon, 16 Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 16 Hematologic remission / Hematolojik remisyon, 16 Iron / Demir, 102 Overload / Aşırı yüklenme, 102 Hematopoietic stem cell / Hematopoietik kök hücre, 102, 345, 321 Transplantation / Transplantasyon, 102, 196, 345 Allogeneic hematopoietic stem cell transplantation / Allojeneik hematopoietik kök hücre transplantasyonu, 159 Cytomegalovirus / Sitomegalovirüs, 159 Graft-versus-host disease / Graft versus host hastalığı, 159 Taiwan / Tayvan, 159 Bing-Neel syndrome / Bing-Neel sendromu, 186 Waldenström’s macroglobulinemia / Waldenström makroglobulinemisi, 186 Central nervous system / Merkezi sinir sistemi, 186 MYD88 L265P mutation / MYD88 L265P mutasyonu, 186 Cerebrospinal fluid / Beyin-omurilik sıvısı, 186 Leukemia / Lösemi, 89, 109, 126, 186, 190 Immunoregulatory effect / Bağışıklık baskılayıcı etki, 213 Co-culture / Ko-kültür, 213 Mesenchymal stem cells / Mezenkimal kök hücre, 213 T cells / T hücre, 213 Mycosis fungoides / Mikozis fungoides, 194 Ichthyosiform / İktiyotik, 194 Sezary syndrome / Sezary sendromu, 194 Anaplastic / Anaplastik, 194 CD3/TCR / CD3/TCR, 194 Adult / Erişkin, 196 Sickle cell anemia / Orak hücreli anemi, 196, 358 Genetic counseling / Genetik danışmanlık, 196 Hematopoietic stem and progenitor cells / Hematopoietik stem ve progenitor hücreler, 280 Stem cell mobilization / Stem hücre mobilizasyonu, 280 Apheresis / Aferez, 280 ISBT 128 / ISBT 128, 280 JACIE / JACIE, 280 Labeling / Etiketleme, 280 Traceability / Takip edilebilirlik, 280 Children / Çocuk, 345 Immunodeficiency / İmmün yetmezlik, 345 Cord blood / Göbek kordon kanı, 314

Aldehyde dehydrogenase / Aldehit dehidrogenaz, 314 Colony-forming unit-granulocyte/macrophage / Koloni oluşturan birim granülositer/makrofaj, 314 Donor / Donör, 321 Informed consent / Bilgilendirilmiş onam, 321 Audiovisual method / Audovizuel yöntem, 321 Bone marrow transplantation / Kemik iliği nakli, 321 L-Carnitine / L-Karnitin, 328 Plasma / Plazma, 328 Antioxidant enzymes / Antioksidan enzimler, 328 Lipid peroxidation / Lipid peroksidasyonu, 328 Protein oxidation / Protein oksidasyonu, 328

Thalassemia Thalassemia / Talasemi, 105 Variant / Varyant, 105 Genetic counseling / Genetik danışmanlık, 105 Prenatal diagnosis / Prenatal tanı, 105 Beta globin gene / Beta globin geni, 105 Turkish / Türk, 105

Thrombosis Monitoring / İzlem, 101 International normalized ratio / Uluslararası normalleştirilmiş oran, 101 Hemostasis / Hemostaz, 101 Acute myocardial infarction / Akut miyokard infarktüsü, 107 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 107 Eltrombopag / Eltrombopag, 107 Vasculitis / Vaskülit, 116 Anticoagulants / Antikoagülan, 116 Rivaroxaban / Rivaroksaban, 116 Sticky platelet syndrome / Yapışkan trombosit sendromu, 239 Thrombophilia / Trombofili, 239 Miscarriages / Düşükler, 239

Thrombocytopenia Platelet / Trombosit, 64 Cryopreservation / Kriyoprezervasyon, 64 Microparticle generation / Mikropartikül, 64 Hemostatic activity / Hemostatik aktivite, 64 Immune thrombocytopenia / İmmün trombositopeni, 72 Rituximab / Rituksimab, 72 Early response / Erken yanıt, 72 Late response / Geç yanıt, 72 Sustained response / Sürekli yanıt, 72 Acute myocardial infarction / Akut miyokard infarktüsü, 107 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 107 Eltrombopag / Eltrombopag, 107 Children / Çocuk, 120 Griscelli syndrome / Griscelli sendromu, 120 Status epilepticus / Status epileptikus, 120 Hemophagocytic lymphohistiocytosis / Hemofagositik lenfohistiyositoz, 120 Psychogenic purpura / Psikojenik purpura, 274 Gardner-Diamond syndrome / Gardner-Diamond sendromu, 274 Antidepressant / Antidepresan, 274 Adolescent / Adölesan, 274 Thrombocytopenia / Trombositopeni, 370


SUBJECT INDEX 2017

Megakaryocytic emperipolesis / Megakaryositik emperipolez, 370 GATA1 / GATA1, 370

Other

Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 40, 151, 366 Endocrine / Endokrin, 40 Late effects / Geç yan etkiler, 40 Children / Çocuk, 40 Ankaferd Blood Stopper / Ankaferd Blood Stopper, 93 Shiga-toxigenic Escherichia coli / Shigatoksijenik Escherichia coli, 93 Salmonella / Salmonella, 93 Campylobacter / Campylobacter, 93 Listeria monocytogenes / Listeria monocytogenes, 93 Wernicke’s encephalopathy / Wernicke ensefalopatisi, 99 Thiamine deficiency / Tiamin eksikliği, 99 Pediatric leukemia / Pediatrik lösemi, 99 Hemophilia A / Hemofili A, 113 Intradiploic hematoma / İntradiploik hematom, 113 Coagulopathy / Koagülopati, 113 Intraosseous / İntraosseoz, 113 Bullous / Büllöz, 118 Figurate erythema / Figüre eritem, 118 Myelodysplastic syndrome / Miyelodisplastik sendrom, 118 Sweet’s syndrome / Sweet sendromu, 118 Glucocorticoid receptor gene / Glukokortikoid reseptör geni, 151 BclI and N363S polymorphisms / BclI ve N363S polimorfizmleri, 151 Acute compartment syndrome / Akut kompartman sendromu, 179 Inflammation / Enflamasyon, 179 Immune deficiency / İmmün yetmezlik, 179 Myeloma / Miyelom, 89, 181, 182, 352 Infection / Enfeksiyon, 181 Atlas / Atlas, 181 Fracture / Kırık, 181 Afibrinogenemia / Afibrinojenemi, 183 Bone cysts / Kemik kistleri, 183 Child / Çocuk, 183 Rare / Nadir, 183 Cytogenetic / Sitogenetik, 197 Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197 Molecular hematology / Moleküler hematoloji, 197 Life-quality / Yaşam kalitesi, 197 Dasatinib / Dasatinib, 197 Nilotinib / Nilotinib, 137, 197, 362 Immunoregulatory effect / Bağışıklık baskılayıcı etki, 213 Co-culture / Ko-kültür, 213 Mesenchymal stem cells / Mezenkimal kök hücre, 213 T cells / T hücre, 213 Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282 Relapse refractory / Relaps refrakter, 233 Bendamustine / Bendamustin, 233 Sticky platelet syndrome / Yapışkan trombosit sendromu, 239 Thrombophilia / Trombofili, 239 Miscarriages / Düşükler, 239 Wolman disease / Wolman hastalığı, 264 Hemophagocytic lymphohistiocytosis / Hemafagositik lenfohistiyositozis, 264 Hemophagocytosis / Hemofagositoz, 264 Hairy cell leukemia / Saçlı hücreli lösemi, 270, 289, 291 Splenectomy / Splenektomi, 270, 291 Sweet’s syndrome / Sweet’s sendromu, 270

Psychogenic purpura / Psikojenik purpura, 274 Gardner-Diamond syndrome / Gardner-Diamond sendromu, 274 Antidepressant / Antidepresan, 274 Adolescent / Adölesan, 274 Hematopoietic stem and progenitor cells / Hematopoietik stem ve progenitor hücreler, 280 Stem cell mobilization / Stem hücre mobilizasyonu, 280 Apheresis / Aferez, 280 ISBT 128 / ISBT 128, 280 JACIE / JACIE, 280 Labeling / Etiketleme, 280 Traceability / Takip edilebilirlik, 280 Leukoagglutination / Lökoaglütinasyon, 354 Cold agglutinin / Soğuk aglütinin, 354 Mycoplasma pneumoniae / Mycoplasma pneumoniae, 354 Eosinophilia / Eozinofili, 354 Pseudoleukopenia / Psödolökopeni, 354 Dysfibrinogenemia / Disfibrinojenemi, 356 Liver transplantation / Karaciğer nakli, 356 Subdural hematoma / Subdural hematom, 356 Neuroblastoma / Nöroblastoma, 369 Circulating tumor cells / Dolaşan tümör hücreleri, 369 Metastasis / Metastaz, 369 Myopathy / Miyopati, 376 Lactic acidosis / Laktik asidoz, 376 Sideroblastic anemia / Sideroblastik anemi, 376 T-cell neoplasms / T hücreli neoplazmlar, 378 Non-Hodgkin lymphoma / Hodgkin dışı lenfoma, 207, 378, 381 Oncogenes / Onkogenler, 378 T-cell mediated immunity / T hücre aracılı immünite, 378 Hematopoietic stem cell / Hematopoietik kök hücre, 321 Donor / Donör, 321 Informed consent / Bilgilendirilmiş onam, 321 Audiovisual method / Audovizuel yöntem, 321 Bone marrow transplantation / Kemik iliği nakli, 321 Hidden blood loss / Gizli kan kaybı, 334 Antioxidants / Antioksidanlar, 334 Proanthocyanidin / Proantosiyanidin, 334 Hydrogen water / Hidrojenli su, 334 Monoclonal gammopathy of renal significance / Renal öneme sahip monoklonal gammopati, 282 Plasma cell disorders / Plazma hücre hastalıkları, 282 Renal involvement / Böbrek tutulumu, 282 Kidney / Böbrek, 282 Cast nephropathy / Kast nefropati, 282

Pathology TP53 / TP53, 34 Immunohistochemistry / İmmünohistokimya, 34 Chronic lymphocytic lymphoma / Kronik lenfosittik lenfoma, 34 Proliferation centers / Proliferasyon merkezleri, 34 Schistocyte / Şistosit, thrombotic microangiopathy / trombotik mikroanjiopati, 59 Microscopy / Mikroskop, 59 ADVIA 2120 / ADVIA 2120, 59 Standardization / Standardizasyon, 59 Diagnosis of lymphoma / Lenfoma tanısı, 81 World Health Organization lymphoma classification / Dünya Sağlık Örgütü lenfoma sınıflaması, 81


SUBJECT INDEX 2017

Discrepancies in diagnosis / Tanı tutarsızlıkları, 81 Hematopathology / Hematopatoloji, 81 Refractory anemia with ring sideroblasts / Halka sideroblastlı refrakter anemi, 100 RARS with thrombocytosis / Trombositoz ile birlikte RARS, 100 Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis / Halka sideroblast ve trombositoz ile birlikte miyelodisplastik sendrom/ miyeloproliferatif neoplazi, 100 Vasculitis / Vaskülit, 116 Anticoagulants / Antikoagülan, 116 Rivaroxaban / Rivaroksaban, 116 Bullous / Büllöz, 118 Figurate erythema / Figüre eritem, 118 Myelodysplastic syndrome / Miyelodisplastik sendrom, 118 Sweet’s syndrome / Sweet sendromu, 118 Granulocytic sarcoma / Granülositik sarkom, 122 Acute monoblastic leukemia / Akut monoblastik lösemi, 122 CD34 / CD34, 122 Myeloperoxidase / Miyeloperoksidaz, 122 Primary myelofibrosis / Primer miyelofibroz, 124, 360 Internuclear bridging / Nükleuslar arası köprüleşme, 124 Erythrocytes / Eritrositler, 124 Richter’s syndrome / Richter sendromu, 188 Flow cytometry / Akım sitometri, 188 Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188 CD5-positive / CD5- pozitif, 188 Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188 Granulocytic sarcoma / Granülositik sarkom, 190 Leukemia / Lösemi, 89, 109, 126, 186, 190 Lymphadenopathy / Lenfadenopati, 190 Leishman-Donovan body / Leishman-Donovan cismi, 266 Bone marrow aspirate smear / Kemik iliği aspirasyonu yayması, 266 Microscopic image / Mikroskobik görüntü, 266 Distal arteriovenous fistula / Distal arteriyovenöz fistül, 268 Multislice computer tomography / Çok kesitli bilgisayarlı tomografi, 268 Hemodialysis / Hemodiyaliz, 268 Hairy cell leukemia / Saçlı hücreli lösemi, 270, 289, 291 Splenectomy / Splenektomi, 270, 291 Sweet’s syndrome / Sweet’s sendromu, 270 Hodgkin’s lymphoma / Hodgkin lenfoma, 272 Metastatic adenocarcinoma / Metastatik adenokarsinom, 272 Reed-Sternberg cells / Reed-Sternberg hücreleri, 272 PET/CT / PET/BT, 272 Acute myelogenous leukemia / Akut miyeloid lösemi, 350 Bullous pyoderma gangrenosum / Büllöz piyoderma gangrenosum, 350

Pathergy / Paterji, 350 Cholangitis / Kolanjit, 359 Döhle bodies / Döhle cisimciği, 359 May-Hegglin anomaly / May-Hegglin anomalisi, 359 Follicular lymphoma / Foliküler lenfoma, 364 Extranodal / Ekstranodal, 364 Prostatic involvement / Prostat Tutulumu, 364 Neuroblastoma / Nöroblastoma, 369 Circulating tumor cells / Dolaşan tümör hücreleri, 369 Metastasis / Metastaz, 369 Thrombocytopenia / Trombositopeni, 370 Megakaryocytic emperipolesis / Megakaryositik emperipolez, 370 GATA1 / GATA1, 370 Leishmaniasis / Layşmanyazisis, 380 Bone marrow / Kemik iliği, 380 Aspirate / Aspirasyon, 380 Smear / Yayma, 380 Antibody / Antikor, 380

Autoimmune Disorders Immune thrombocytopenia / İmmün trombositopeni, 72 Rituximab / Rituksimab, 72 Early response / Erken yanıt, 72 Late response / Geç yanıt, 72 Sustained response / Sürekli yanıt, 72 Acute myocardial infarction / Akut miyokard infarktüsü, 107 Immune thrombocytopenic purpura / İmmün trombositopenik purpura, 107 Eltrombopag / Eltrombopag, 107

Transfusion Red blood cell / Kırmızı kan hücresi, 52 Transfusion practice / Transfüzyon uygulamaları, 52 Blood storage age / Kan depolama yaşı, 52 Platelet / Trombosit, 64 Cryopreservation / Kriyoprezervasyon, 64 Microparticle generation / Mikropartikül, 64 Hemostatic activity / Hemostatik aktivite, 64 Blood components / Kan içeriği, 244 Blood processing / Kan ürünlerini işleme, 244 Donors / Verici, 244 Transfusion strategy / Transfüzyon Stratejisi, 244 Para-Bombay / Para-Bombay, 374 Phenotype / Fenotip, 374 FUT1 gene / FUT1 geni, 374 Blood transfusion / Kan transfüzyonu, 374

Tjh 2017 4  
Tjh 2017 4  
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