Clinical Laboratory Hematology, 2E by Shirlyn B. McKenzie Solution Manuals

Type:

Solution Manual

Resource:

Clinical Laboratory Hematology

Edition:

2nd Edition

Author(s):

Shirlyn B. McKenzie J. Lynne Williams

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Instructor’s Resource Manual Linda F. Comeaux, BS, CLS(NCA) Dean, Health, Sciences, and Engineering Arapahoe Community College

to Accompany

Clinical Laboratory Hematology Second Edition

Shirlyn B. McKenzie, PhD, CLS(NCA), MT(ASCP)SH Department of Clinical Laboratory Sciences University of Texas Health Science Center at San Antonio

J. Lynne Williams, PhD, CLS(NCA), MT(ASCP) Medical Laboratory Sciences Program Oakland University

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CONTENTS

Introduction 1 2 3 4 5 6 7 8

Introduction to Hematology The Hematopoietic System The Anemias Nonmalignant Disorders of Leukocytes Neoplastic Hematologic Disorders Body Fluids Hemostasis Hematology/Hemostasis Procedures

1 20 33 49 82 97 117 129 147

Appendix

169

Laboratory Worksheet—Differential Worksheet

170

Laboratory Worksheet—Blood Smear Evaluation

171

Answers to Practice Questions

172

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Introduction

This Instructor’s Resource Manual is a supplement to the Clinical Laboratory Hematology textbook and is part of the complete teaching and learning package compiled by its authors. Like the textbook, the Instructor’s Resource Manual is created to support instruction and facilitate learning for both CLT/MLT and CLS/MT students.

The Clinical Laboratory Hematology textbook and Instructor’s Resource Manual have been thoughtfully prepared to assist the educator in promoting “learner-centered” activities. An ancient Chinese proverb states: “I listen, I forget Á I see, I remember Á I do, I understand.”

The Clinical Laboratory Hematology textbook has a unique blend of features to enhance student comprehension and learning. Likewise, the Instructor’s Resource Manual provides guidance and information to assist the educator and encourage the students’ active participation in the learning process.

ORGANIZATION OF THE INSTRUCTOR’S RESOURCE MANUAL This Instructor’s Resource Manual includes active learning and retention tips, teaching strategies, a sample syllabus, transition grids, suggested student activities, additional practice questions and case studies, word searches, and crossword puzzles. It is organized as noted below by the same content sections as found in the Clinical Laboratory Hematology textbook.

Section 1

Introduction to Hematology

Chapter 1 Chapter 2 Chapter 3

Introduction to Hematology Cellular Homeostasis Hematopoiesis

Section 2

The Hematopoietic System

Chapter 4 Chapter 5

Structure and Function of Hematopoietic Organs The Erythrocyte

Copyright © 2010 by Pearson Education, Inc.

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Chapter 6 Chapter 7

Hemoglobin The Leukocyte

Section 3

The Anemias

Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18

Introduction to Anemia Anemias of Disordered Iron Metabolism and Heme Synthesis Hemoglobinopathies: Qualitative Defects Thalassemia Megaloblastic and Nonmegaloblastic Macrocytic Anemias Hypoproliferative Anemias Introduction to Hemolytic Anemia Hemolytic Anemia: Membrane Defects Hemolytic Anemia: Enzyme Deficiencies Hemolytic Anemia: Immune Anemias Hemolytic Anemia: Nonimmune Defects

Section 4

Nonmalignant Disorders of Leukocytes

Chapter 19 Chapter 20

Nonmalignant Granulocyte and Monocyte Disorders Nonmalignant Lymphocyte Disorders

Section 5

Neoplastic Hematologic Disorders

Chapter 21 Chapter 22 Chapter 23 Chapter 24 Chapter 25 Chapter 26 Chapter 27

Introduction to Hematopoietic Neoplasms Myeloproliferative Disorders Myelodysplastic Syndromes Acute Myeloid Leukemias Acute Lymphoblastic Leukemias Lymphoid Malignancies: Chronic Lymphoid Leukemias, Lymphomas, and Plasma Cell Neoplasms Hematopoietic Stem Cell Transplantation

Section 6

Body Fluids

Chapter 28

Morphologic Analysis of Body Fluids in the Hematology Laboratory

Section 7

Hemostasis

Chapter 29 Chapter 30 Chapter 31 Chapter 32 Chapter 33

Primary Hemostasis Secondary Hemostasis and Fibrinolysis Disorders of Primary Hemostasis Disorders of Secondary Hemostasis Thrombophilia

Section 8

Hematology Procedures

Chapter 34 Chapter 35 Chapter 36 Chapter 37 Chapter 38 Chapter 39 Chapter 40 Chapter 41

Hematology Procedures Bone Marrow Examination Automation in Hematology and Hemostasis Flow Cytometry Chromosome Analysis of Hematopoietic and Lymphoid Disorders Molecular Analysis of Hematologic Diseases Laboratory Testing in Coagulation Quality Assessment in the Hematology Laboratory

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UNIQUE FEATURES Each of the eight sections in the Instructor’s Resource Manual has a number of unique features, which are described below for both Level I and Level II learners.

• Transition Grid The Transition Grid provides the educator with an easy cross-reference between Clinical Laboratory Hematology and other hematology textbooks.

• Objectives

All chapter objectives, known as checklist items, are listed.

• Active Learning A variety of “learner-centered” activities are presented. Detailed information concerning implementation of these activities can be found in the Teaching Tips section of the Instructor’s Guide.

• Laboratory Activities Most sections include suggested activities to perform in the student laboratory. A sample differential and blood smear evaluation worksheet is provided in the Instructor’s Guide appendix.

• Practice Questions Additional practice questions and one case study for each level (I and II) are included per section. Three questions from each Taxonomy (recall, application, and analysis) of cognitive learning are integrated throughout the questions. Answers to the practice questions may be found in the Instructor’s Guide appendix.

• Word Search and Crossword Puzzles Copy-ready word search and crossword puzzles are provided at the end of each section. All sections have both a Level I and Level II word search and crossword puzzle with a correlating answer sheet.

SUPPLEMENTAL WEB SITE An innovative supplement to the Clinical Laboratory Hematology textbook is the Companion Website found at www.pearsonhighered.com/mckenzie. Educators are encouraged to guide their learners to this additional teaching and assessment resource. This website is dynamic and will be periodically updated.

Educators and learners are provided with additional multiple choice questions at both the Level I and Level II abilities. To challenge students, there are problem-solving/critical-thinking questions that can be attempted by students as guided by their instructor. For additional support and information, students can visit the following websites.

Other suggested sites to visit include: http://www.beckman.com/lars/personnel/webinars.asp http://www.lymphoma.org.uk http://dpalm.med.uth.tmc.edu/faculty/bios/nguyen/Webcoag/Webcoag.htm http://www.whonamedit.com/ http://www.bloodline.net www.clot-ed.com www.phd.msu.edu/Education/InteractiveHemostasisAnimations.aspx http://ashimagebank.hematologylibrary.org/ Active Learning sites to visit: http://honolulu.hawaii.edu/intranet/committees/FacDevCom/guidebk/teachtip/active.htm http://trc.ucdavis.edu/TRC/ta/tatips/activelearning.pdf http://www.acu.edu/cte/activelearning/whyuseal2.htm ..

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INSTRUCTOR’S RESOURCE DVD-ROM The Instructor’s Resource DVD-ROM (0-13-513737-3) contains many resources in an electronic format.

It includes the complete test bank that allows instructors to design customized quizzes and exams. The TestGen wizard guides you through the steps to create a simple test with drag-and-drop or point-andclick transfer. You can select test questions either manually or randomly and use online spell-checking and other tools to quickly polish your test content and presentation. The question formats include multiple choice, fill-in-the-blank, true/false, and essay. You can save your test in a variety of formats both locally and on a network, print up to 25 variations of a single test, and publish your tests in an online course. For more information, please visit www.pearsonhighered.com/testgen.

It also includes a PowerPoint Lecture package that contains key discussion points, along with figures and tables for each chapter. This feature provides dynamic, fully designed, integrated lectures that are ready to use and allows instructors to customize the materials to meet their specific course needs.

An Image Library of images and tables in the book is also available to you. We feel this offering will help you to be more successful in the classroom as these images can be projected on-screen during a class lecture or inserted into handouts or quizzes. You have permission from Pearson to copy and paste these images into your PowerPoint lectures, printed documents, and web site, as long as you have adopted and are using Clinical Laboratory Hematology, 2nd edition, as your course textbook.

A Bonus Image Library (an instructor’s dream) also is available on the DVD-ROM. This library includes high resolution color microscopic pictures of normal and abnormal hematopoietic cells. It is organized by chapter and each slide is indexed to help you locate a particular slide quickly. The slides can be used in digital presentations in the classroom or in on-line courses if you have adopted the book as your course textbook.

An electronic version of this Instructor’s Resource Manual in PDF and Word formats can be accessed on the Instructor’s Resource DVD-ROM.

TEACHING TIPS This Teaching Tips section provides brief information on writing objectives, test item writing, explanations of the suggested activities, and a sample syllabus. This material is intended to assist the clinical laboratory educator at all levels of instruction.

Writing Objectives Objectives are specific performance or behavioral statements describing what the “student will be able to do” after completing a particular instructional activity. Learners will be guided and directed by the objectives written by the instructor. These instructional objectives should be measurable and consistent with the instruments designed to assess student learning, that is, exams, laboratory practicals, and so on.

Instructional or learning objectives are generally classified into three different domains: cognitive, affective, and psychomotor.

• Cognitive domain involves mental operations from recall (lowest level) to evaluation (highest level).

• Affective domain involves the attitudes, values, or feelings from acquisition (lower level) to action and internalization (higher level).

• Psychomotor domain ranges from simple manipulation to the highest level of creative performance.

Effective learning objectives should be written within each domain to address and fulfill the five areas of developmental needs. Generally, the intellectual needs are within the cognitive domain, the 4

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physical developmental needs are found within the psychomotor domain, and the psychological, social, and moral/ethical needs are within the affective domain. This particular Teaching Tip section focuses on the cognitive domain.

Intellectual or cognitive domain objectives are composed according to the complexity of the skills.

Benjamin Bloom and associates have created the most widely used and accepted taxonomy (classification) of cognitive objectives, known as “Bloom’s Taxonomy.” Bloom’s Taxonomy includes the following six categories.

1. Knowledge (recalling or recognizing information) 2. Comprehension (understanding the meaning of information) 3. Application

(applying or using the information)

4. Analysis

(comprehending information into its component parts)

5. Synthesis

(generating new ideas by putting the components together)

6. Evaluation

(judging the worth of an idea, opinion, or information)

This Taxonomy can be further condensed into three categories: • Taxonomy I

Knowledge

• Taxonomy II

Application

• Taxonomy III

Evaluation

Taxonomy I is the simple recall of information that has been presented. Taxonomy II is the interpretation of facts and an application of the information that is given. Taxonomy III involves problem solving at the highest level of learning. This Instructor’s Guide provides examples of the Taxonomy I, II, and III cognitive process in the practice question sections.

Finally, when writing learning objectives the educator should strive to compose a statement with verbs that are measurable or observable. Action verbs such as explain, discuss, or formulate give overt instructions to the learner. Instructors should avoid verbs that are covert or not obviously measurable such as understand, realize, learn, know, believe, indicate, comprehend, or appreciate. Direct, measurable objectives may include some of the following action verbs.

Taxonomy I choose, complete, classify, define, describe, explain, express, identify, recall, recognize, state, or summarize Taxonomy II apply, analyze, appraise, calculate, compute, demonstrate, develop, diagram, differentiate, distinguish, establish, interpret, perform, provide, relate, solve, submit, or utilize Taxonomy III assess, compile, compare and contrast, create, design, evaluate, formulate, forecast, generate, justify, predict, propose, recommend, resolve, revise, synthesize, validate Note: Action verbs may cross over categories depending on the content covered and the question being asked.

Reference: Kellough, R., and Kellough, N. A Guide to Methods and Resources, Planning for Competence. Upper Saddle River, New Jersey: Merrill/Prentice Hall, 1999.

Test Item Writing It is not always difficult to write test questions, however, it is sometimes challenging to compose assessment items that are meaningful and effective. Here are a few hints on how to successfully assess your student’s learning while creating exam questions. ..

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Multiple choice questions, a type of “selected response test item,” are some of the more common items used in written exams. These items are composed of the item stem (the question or statement) and possible options to complete or answer the question. Options or choices that are incorrect are called distracters.

Overall, test items should: • Test one, relevant concept.

• Correlate with the learning objectives and Taxonomy level. The action verbs listed above can also be used in test item writing. The appropriate action verb will determine the Taxonomy or cognitive level required to answer each question.

• Be well written in terms of vocabulary, sentence structure, spelling, and grammar.

• Avoid unimportant information that will make the question too long or difficult to comprehend.

Item stems should: • Be written clearly and concisely so the students know what is being asked—avoid being “tricky.”

• Use different phrasing than what is in the textbook.

• Not educate or teach concepts within the context of the stem.

• Not include unessential information as an introduction.

• Not use vague or absolute terms, that is, generally, never, or always.

Options/distracters should: • Be consistent with the content of the stem but should not repeat the wording or phrasing.

• Avoid the use of “all of the above”—recognizing one wrong option will eliminate this choice.

• Include four to five responses (four options seems preferable).

• Be listed in alphabetical or chronological order.

• Have a consistent length—test-wise students can identify longer options as generally correct.

• Be plausible.

• Be created in a manner that represents errors in thinking that students might commonly make.

Active Learning and Suggested Activities Teaching and learning experts acknowledge that an environment in which students play a role and take responsibility for their learning and the teacher performs as facilitator rather than lecturer is far more conducive to learning retention. The “Learning Pyramid” states a learner will retain only 10% of information read and only 20% seen in audiovisual activities. Furthermore, demonstrations provide only 30% retention, discussions 50%, hands-on practice retains 75%, and teaching others allows for 90% retention of information.1 Every good instructor strives to teach in a way that will optimize student learning. This section provides tools to assist the instructor in creating “learner-centered” activities to enhance student learning. Some educators believe that performing such activities will sacrifice too much time or content.

Although it is true a guided activity may take more time, it is also true that the student will develop more critical-thinking skills and comprehend the information much more efficiently.

1

National Training Laboratories, Bethel, Maine, Average Learning Retention Rates http://www.tcde.tehama.k12 .ca.us/pyramid.pdf.

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Although the Clinical Laboratory Science profession is composed of many introverted individuals, it is essential to communicate openly and clearly. Many of the suggested activities in this Guide provide opportunities for the learner to experience and practice conveying a subject or message.

Each section of the Instructor’s Resource Manual presents a list of suggested learner-centered activities to enhance learning of the content in that section. The following information explains how to create and execute each exercise.

1. Clear the Mud At the end of any class period, the instructor will pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. The students should place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor can then have each student present their explanation during the next class period to the whole class.

2. Create/Construct The “Create” sections are self-explanatory. These exercises are provided to assess the learners “synthesis” cognitive skills. For instance, the instructor may ask the students to create a case study for a disease condition that includes laboratory results and patient clinical presentation.

3. Diagram The “Diagram” sections are also self-explanatory. These exercises assess the students’ ability to “apply” and “analyze” at the Taxonomy II level. Drawing or diagramming a hemoglobin molecule or the coagulation cascade can be an effective learning tool.

4. Extra, Extra, Read All About It This activity will encourage the students’ creativity while assessing their comprehension, knowledge, and writing ability. It is simply a fun and different way to have the learner relate their understanding of the material. For example, section II of this Guide suggests the following exercise: Have the students write a newspaper or magazine article about their interview with the well-known celebrity group, “Erythropoietin.” Include in this article where “our stars” are from, what influences them, what affect they have on their environment, and describe “their” most important contribution.

5. Group Discussion Whether teaching in the classroom or on-line, it is invaluable to engage the learner in an active discussion. Set the ground rules for discussion prior to revealing the topic. Use such rules as: • Only professionally delivered comments may be made.

• There will be mutual respect for everyone’s opinion—everyone’s opinion is valued.

• No name calling or attacking comments may be made. In electronic discussions no inflammatory messages may be posted.

• Everyone must participate—no person may dominate the discussion.

• Establish a time limit for each comment and for the overall discussion.

Instructors: Allow students to facilitate the discussion. At the end of the discussion provide a summary of the comments offered. It is invaluable for the learners to know that their opinions have been heard and are valid.

For example, at the end of a discussion on “human cloning” the instructor would summarize and say, “I have appreciated everyone’s view on this topic. I heard that some say it would be unacceptable to clone a human for any reason while others thought cloning for medical or research purposes could be considered,” and so on.

6. Hematology Bowl This activity is also similar to the classic television game show, Jeopardy.

The Instructor • Divides the class into three to four small groups (four to five students per group). One student from each group is to be the contestant. ..

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• Creates six to eight categories, that is, RBC, Leukemia, Coagulation, and so on.

• Constructs five to seven “answers” per category. ° Each answer should be assigned a point value.

• Generally, the point value and degree of difficulty increase for each answer in the category.

Hematology Bowl Rules • A selected student will choose the first category.

• The instructor reads the answer.

• The contestants “buzz in” or simply raise their hand if they think they know the correct answer. The first contestant to do this will have the option to respond and may consult with the other members of their group.

• Contestants must formulate their response in the form of a question.

• If the contestant provides a correct answer, their group is given the designated point value of the answer, and they get to select the next category. ° If the contestant does NOT provide a correct answer, the remaining contestants may “buzz in” and attempt a response.

Sample Answer • (Under the category “RBC”—response point value equals 100.) Answer: “Stimulates the production of red blood cells.” Contestant’s response: “What is erythropoietin?”

7. Match-Up Game This learning activity is identical to the Concentration card game (also known as Memory Match) where a full deck of cards are shuffled and laid face down on a flat surface.

The first player turns over two random cards attempting to make a match. If the match is successful, the player removes the two cards, places them to their side, and takes another turn. If the match is unsuccessful, the player returns the cards to the facedown position, and the next player takes their turn.

The laboratory Match-Up Game is similar in process; however, the game can be altered by using clinical laboratory terms and definitions, disease names and conditions/characteristics, or test parameters and laboratory results, and so on.

The instructor can create the Match-Up Game (usually a minimum of 10–15 matches is ideal) and have the students “play” it. To further enhance learning, a small group of 2–3 learners can “create” the cards themselves.

• Divide the class into small groups and assign a particular topic to each group. The students should use blank/identical index cards and should refrain from making any distinguishable markings on the backside of the cards. After the groups of learners “create” the game, they can exchange cards with another group of learners and “play” the game.

8. Mapping Mapping exercises are effective in identifying the student’s ability to assemble and organize various pieces of information. The students are instructed to: • Take out a clean piece of paper.

• Start at the center of the paper and write down the topic to map.

• Draw lines extending from the central topic. At the end of each short line, write another word that relates to the central topic. From this extension draw another line and write a word or short phrase that relates to this extended topic and so on and so on.

Here’s a short (incomplete) example of a map on the subject of “leukemia.”

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FAB Classification CLL

Seen in children ALL

Chronic leukemias

Leukemia

Proliferation of lymphoid cells

Acute leukemias

may see Auer rods AML CML Greater than 30% blast seen

M1-without maturation

FAB Classification

M2-with maturation

Each open line represents an area where additional information can be attached. First, have the students create a map without the assistance of textbooks or notes. This will assess their current level of comprehension. Instructors may or may not choose to then allow the students to complete the map using the text.

9. Mystery Box The Mystery Box activity allows the students to assemble or arrange separate “pieces of information” into one.

• Divide the class into small groups of two to four students.

• A decorated or cardboard box may be used.

• For each box place all of the necessary information on separate pieces of paper.

• Have the students empty the box and combine, arrange, or assemble the information according to the activity instructions given in each section.

For example: Section I of the Instructor’s Guide has the following Mystery Box activity.

Create a Mystery Box with the different components and reference ranges. Be sure to include at least: • Leukocytes • Erythrocytes • Thrombocytes • Plasma • Whole blood • 4.0 – 11.0 * 109/L • 4.0 – 5.5 * 1012/L • 150 – 450 * 109/L • 55% of blood volume

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• 5–6 liters in adults • And so on The instructor places each of the above components or reference ranges on a separate piece of paper and places all pieces in the Mystery Box. The students will empty the box and match the component with its appropriate reference range.

Additional Notes 1. The instructor should decide if textbooks or other reference materials may be used during this exercise.

2. The student may be directed to read a section of the textbook and, without additional instruction, perform the Mystery Box activity.

3. A time limit for completing this activity may be implemented.

4. Students may perform these exercises individually or in teams.

5. As an alternate activity, the students may be asked to create their own Mystery Box, then give it to another student or group to solve.

10. One Minute Paragraph The one minute paragraph activity is used to assess students’ understanding of a particular topic. It may be used several times during the course. Students are requested to write all that they know about a particular subject for exactly one minute without any assistance from textbooks or notes. Instructors should read the students’ work, evaluate their level of comprehension on the topic, and provide feedback.

The one minute paragraph may be performed at any time during the class. Composing the one minute paragraph at the end of a class period, however, gives the instructor guidance on how well the students have grasped the information presented that day. The instructor may or may not choose to give advanced notice of this activity.

11. Mnemonic Device Learners need some process or technique to assist their information retention and memory. Mnemonic devices are one example of such a technique. An example of a mnemonic device is Princess Brown’s Pet Ox Ran Everywhere. In this case the first letter of each word in the phrase represents the first letter of each cell in the erythrocyte maturation series, starting with the most immature, Pronormoblast, Basophilic normoblast, and so on.

Instructors should allow the students to create their own mnemonic device. Instructors may choose to select a particular phrase that the whole class will utilize.

12. Pyramid Game This exercise is similar to the game show, $25,000 Pyramid, and promotes a fun/enjoyable environment for learning.

Activity Preparation • A pyramid of boxes or index cards are created (any number of cards may be used depending on how long the activity will proceed).

• The index cards are placed face down in a pyramid shape.

• On the back side of the card is a category.

• The cards are turned over, starting with the lower left side of the pyramid and proceeding in the following manner.

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Pyramid Game Rules • One student provides the clues (Student A). The other student provides the answers (Student B).

• Student A turns over the first card and looks at the category. Student A then begins to list words or characteristics included in the category without using any of the words listed on the card.

• Student B tries to name the category as soon as it is recognized.

• If Student B is successful, Student A moves to the next card. Student A has the option to pass on a category but must eventually return to it in order to complete the pyramid.

• A specific time limit should be set for each pyramid, that is, one to two minutes.

• The instructor and fellow classmates should act as time keepers and monitor for giving appropriate clues.

• Each team of students (Students A and B = a team) can record their time, and the team completing the pyramid in the shortest amount of time would be considered the winner.

Sample category and clues Category:

6 5

4

Neutrophil maturation

2

3

Clues: Myeloblast. . .promyelocyte. . .myelocyte. . .metamyelocyte. . .band. . .seg (The word “neutrophil” cannot be used as a clue.)

13. Short Story This activity is similar to the “Extra, Extra, Read All About It” exercise. Creating a “Short Story” will encourage the students’ creativity while learning the subject content. For instance, Section I of the Instructor’s Guide invites the learner to create a story entitled “The Life and Development of a Cell.” This exercise asks the learner to personify a cell and discuss its journey of development.

Instructors may choose to give this exercise as an in-class activity, as a group assignment, or even as a take-home exercise.

14. Small Group Teaching Often learners cannot appreciate the amount of time and work needed to prepare a lecture. This activity gives the student an opportunity to experience the depth and attention to detail needed in order to produce an effective presentation.

Each student group should be given this assignment with adequate time to research and prepare. The instructor may act as facilitator for this exercise and should provide direction on what topics need to be included. The instructor may also give direction or hints on how to address an audience.

The instructor may consider planning which lectures throughout the course will be taught by a small group of learners. Each student should have the opportunity to participate in this activity.

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15. Think-Pair-Share Think-Pair-Share is an in-class type of activity. A subject or topic is introduced, and the students are asked to discuss their understanding of the subject with one other student.

Selected pairs of students may be asked to share their discussion and information. The instructor should designate a particular amount of time for this activity, that is, one to five minutes.

This activity may also be used as an Ice Breaker exercise on the first day of a new class.

Students are asked to pair with another student. Students share with each other a small amount of information about themselves. After a designated amount of time, each student shares with the class information they learned about their partner. Generally, it appears to be easier to talk about someone else instead of yourself, especially on the first day of class. This activity is a gentle and effective way of getting to know the students in class.

16. Thumbs Up/Thumbs Down To get immediate feedback on any topic during the class period the instructor can quickly ask for a “Thumbs Up or Thumbs Down” so students can indicate if they comprehend the information presented. As the instructor presents topics that are potentially difficult he or she can stop momentarily and ask students to show either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

17. Tic-Tac-Toe This activity is constructed just like the television game show. Arrange your students in the following manner.

• Two contestants—one is “X”; one is “O.” • Seat nine students in chairs to form a square—three rows of three chairs each.

• Each student should have two fairly large cards—one marked with an “X” and the other marked with an “O.” • The instructor or a designated student is the “host.”

Proceed with the following activities: • The instructor creates a series of questions prior to the activity.

• The contestant (usually “X” goes first) is asked to pick a particular student in the square.

• The host reads the first question to the contestant and the selected student in the square. For example, “Auer rods are found in what type of leukemia?”

• The student in the square provides an answer. The contestant decides to either agree or disagree with this answer.

• If the contestant makes a correct decision, then their symbol (X or O) is placed in the square.

If the contestant makes an incorrect decision, then the opponent’s symbol is placed in the square (the student in that square should now hold up the appropriate symbol).

• Play continues in the same manner for the other contestant.

• The object of the game is to create a tic-tac-toe. The first contestant to do this wins the round. **Rotate students as contestants and square participants.

18. Web Search In addition to the distinctive Web site feature of the Clinical Hematology Textbook, instructors may encourage students to conduct their own Web search on a selected topic. The activity of researching and discovering information is an invaluable learning tool. To make the students accountable, the instructor may also request a short report on their findings.

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SAMPLE SYLLABUS Each course should include a syllabus. This document acts as a stated agreement between the instructor and learner and provides valuable information to guide and direct the student to success.

Basic information in a course syllabus includes: 1. Course title, number, and credits 2. Instructor contact information 3. Course description 4. Prerequisite requirements 5. Textbook or materials required 6. Course or program policies and procedure 7. Grading scales and policies 8. Course objectives Each component of the course syllabus should be thoughtfully and accurately written. Course objectives, in particular, are vital to guide the students’ learning. These objectives should be written to address the affective, psychomotor, and cognitive domains. Suitable action verbs are included in each objective to address the appropriate skill or cognitive level (see the “Writing Objectives” section of this Teaching Tips document).

Note: To assist instructors in the development of course objectives, Level I and Level II Objectives found in each chapter of Clinical Laboratory Hematology may be utilized.

Sample CLT 131—Introduction to Hematology Hi!—Welcome to the 2-credit on-line hybrid course of an “Introduction to Hematology” (the study of blood).

A calendar with assignments, tests, and due dates is listed under the “Assignments” link. Course materials are found under “Course Documents,” and a student manual that describes how to use the on-line features is found under the “Tools” button.

This CLT 131 syllabus includes the following information: • Course description • Participation • Corequisite • Laboratory makeup policy • Course technology requirement • Due dates • Textbooks/required materials • Academic integrity policy • Instructor contact information • Withdrawal policy ..

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• Learner expectations • Disability services • Classroom environment • Grading • Attendance • Course objectives Course Description CLT 131—Introduction to Hematology is designed for anyone interested in the laboratory sciences. It is especially offered for learners interested in the Clinical Assistant Certificate and/or the Clinical Laboratory Technology degree.

This course will cover the topics of: 1. Laboratory safety 2. OSHA (Occupational Safety and Health Administration) standards 3. An introduction to phlebotomy (blood drawing) 4. Basic hematology test theory and procedures such as: • Hemoglobin and hematocrit determinations • White blood cell (WBC) and red blood cell (RBC) structure and function • Manual hematocrit test procedures • Peripheral slide preparation Corequisite There are no “required” prerequisites; however, completion of or current enrollment in Biology 201 and/or Biology 202 (Anatomy and Physiology) is recommended. Contact the CLT Program Director for more information about general education and CLT courses needed to complete the AAS degree.

Course Technology Requirements A basic working knowledge of the following will be necessary to succeed in this on-line course.

• Completing Internet browsing • Operating a word processor program (i.e., Microsoft Word) • Sending and receiving e-mail with attachments • Participating in chat room discussions • Accessing, reading, and posting to bulletin boards Textbooks/Required Materials 1. **Textbook: Clinical Laboratory Hematology, 2/e, Shirlyn B. McKenzie and J. Lynne Williams, Pearson publisher, copyright 2010, ISBN 0-13-513732-2 2. **CLT 131 Laboratory Manual **All of the above books are required and can be purchased from the college bookstore.

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3. Lab coat (for student laboratory sessions) 4. Black sharpie marker 5. Computer with Internet access and an Internet browser that is 4.0 or higher. You may also access the college’s open computer lab at no additional cost.

Contact Information Instructor: Name ###-###-#### (phone) ###-###-#### (fax) e-mail address College, hospital, or University Address City, State zip Technical Support: Name ###-###-#### (phone) ###-###-#### (fax) e-mail address Learner Expectations To be successful in this on-line hybrid course, you will need to set aside at least six to eight hours per week in order to complete reading assignments, course documents, answer unit questions, participate in on-line activities and discussions, and take quizzes. You are strongly encouraged to designate a specific block of time each week to work on these materials. Although your activities and participation are asynchronous (not at the same time), it is essential that you stay current with the pace of the course offerings. When attending scheduled laboratory sessions, you are expected to have reviewed and completed all required assignments. Doing this will promote a successful learning experience.

Additionally, all learners are expected to adhere to the following terms throughout this course.

Please send the instructor an e-mail at “e-mail address” by “month, date, year” with your acceptance of these terms of agreement.

When e-mailing, please always put CLT 131 in the subject line in order to identify your message as part of the Introduction to Hematology course. The instructor will respond to e-mails and phone messages within 24 hours (excluding weekends and holidays).

Terms of Agreement Classroom Environment It is crucial that the on-line classroom be an atmosphere that is stimulating and safe for learning. Each learner is encouraged and welcomed to offer their thoughts and opinions. In the on-line environment, each learner should expect trust and mutual respect that celebrates diversity of thought, culture, and intellect. The instructor and classmates will work constructively while maintaining a professional demeanor and consideration for everyone.

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Attendance Students are appreciated for the time and effort they give to participating in the on-line activities and attending all on campus laboratory class sessions. Five points/hour for each laboratory session will be offered to students that are present. Attendance points will be used in the final grade calculations.

Please be courteous and notify the CLT Program Director and/or Instructor if your attendance is prohibited by an emergency. (CLT office ###-###-####) Participation Interaction and participation in on-line and laboratory sections is essential for a successful learning experience. A portion of your evaluation and grade is based upon your participation and thoughtful contributions to class discussions and activities. Ten points will be given for each discussion activity based upon the depth and quality of your response posted by the assigned due date. Likewise, 5–20 points/unit will be offered for successful completion of unit questions by the assigned due date.

Laboratory Makeup Policy On campus, laboratory classes will NOT be repeated. Open laboratory times will be available during the semester for student practice. Missed laboratory practical exams cannot be made up.

Due Dates Access the course “Assignments” navigational button for a listing of course assignments and a calendar of due dates. There are established deadlines for assignments, group work, unit questions, and tests. The instructor will be flexible with the due dates for unit questions and assignments if notified in advance. However, out of respect for each participant, extensions cannot be provided for group projects, discussion activities, or quizzes. It is truly in your best interest to stay current with the materials as they are presented. If you are having difficulties, please contact the instructor as soon as possible, and arrangement for tutorial sessions can be made.

Academic Integrity You demonstrate academic integrity when you submit your own work or properly acknowledge the work of others. Violating this principle constitutes academic dishonesty and may result in disciplinary action. The College’s Student Handbook determines academic misconduct and dishonesty as: “Cheating or knowingly assisting another student in committing an act of cheating or other forms of academic dishonesty. Cheating includes but is not limited to: 1) use of any unauthorized assistance in taking quizzes, tests, or examinations; 2) dependence upon the aid of sources authorized by the instructor in writing papers, preparing reports, solving problems, or carrying out other assignments; or acquisition, without permission, of tests or other academic material belonging to a member of the college faculty or staff.”

Plagiarism or knowingly assisting another student in committing an act of plagiarism.

Plagiarism includes, but is not limited to the use Á of the published or unpublished work of another person without full and clear acknowledgment. It also includes submitting examinations, themes, reports, drawings, laboratory notes, undocumented quotations, or other material as one’s own work when such work has been prepared by another person or copied from another person. Unauthorized possession of examinations, answer keys, reserve library materials, computer software, computer codes, or laboratory materials is unacceptable behavior.

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Please refer to the College Student Handbook for more information on Code of Conduct and Students Rights and Responsibilities. (The CLT Program Guide and College Student Handbook will be mailed to you the first week of class.)

Withdrawal Policy Students are responsible for meeting the deadline dates and for initiating a withdrawal either through the college Web site or calling the Office of Admissions and Records (###-###-####). The deadline for receiving a refund is before the first 15 percent of the course is completed. After the first 15 percent of the class and before 80 percent of the class is complete, students may still withdraw. However, no refund will be granted.

• Last day to drop this course and receive a refund: month date, year • Last day to withdraw from this course and receive a “W” with NO refund: month date, year Disability Services Support services are available to students with disabilities who have a documentable need for accommodation in accordance with the Americans with Disabilities Act and the Rehabilitation Act of 1973, Section 504. To access services at the College, contact The Center for Academic Resources in Room #### or call ###-###-####/tty.

Grading Required Projects and/or Papers 2–3 On-line Journal Entries 2 Discussion Activities 1–2 Group Projects/Cooperative Learning Exercises Submission of Laboratory Manual Unit questions each week Number of Examinations LECTURE: 3–4 ON-LINE EXAMS (On-line): 1 FINAL WRITTEN EXAM LABORATORY: 2–3 PRACTICAL EXAMS (On Campus): LABORATORY COMPETENCIES 1 Final Practical Exam Scale 93%– 100% = A 85%– 92% = B 75%– 84% = C 65%– 74% = D 6 65% = F

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Grade Determinations Lecture exams = 20% Class participation/Assignments = 15% Lecture final exam = 15% Laboratory practicals = 15% Competencies = 20% Lab final practical = 15% STUDENTS MUST RECEIVE AT LEAST 75 PERCENT IN BOTH THE LECTURE AND LABORATORY SECTIONS IN ORDER TO SUCCESSFULLY COMPLETE THIS COURSE. Objectives Each individual unit of instruction in the Laboratory Manual has specific objectives to guide the learner.

The following are overall course objectives for the CLT 131—Introduction to Hematology course.

After successful completion of this course the learner should be able to On-line Sections 1. Discuss OSHA safety regulations for blood-borne pathogens.

2. Discuss fire, chemical, electrical, and radiation safety, as well as laboratory safety rules.

3. Evaluate Medical Ethics scenarios.

4. Describe the microscope parts and functions.

5. Explain the proper venipuncture technique and describe all appropriate phlebotomy equipment.

6. Calculate basic metric system problems.

7. Compare and contrast the morphology and function of whole blood cellular elements.

8. Describe all parameters of the complete blood count (CBC).

9. Explain hemoglobin and hematocrit test procedures and results.

10. Classify the criteria for making a quality peripheral blood smear.

On Campus Laboratory Sections 1. Demonstrate compliance with OSHA safety regulations for blood-borne pathogens.

2. Properly utilize fire, chemical, electrical, radiation, and laboratory safety guidelines.

3. Demonstrate proper use of personal protective equipment.

4. Demonstrate acquired responsibility by: a. Arriving on time to lab and setting up equipment or supplies as listed. b. Being ready to listen attentively to the instructor’s directions. c. Cleaning and turning off equipment as instructed. d. Disinfecting bench tops before and after the lab period. e. Returning and storing all equipment (i.e., microscopes), supplies, and reagents to specified area in a correct and orderly fashion.

5. Practice proper venipuncture and finger stick procedures and evaluate fellow classmates’ technique.

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6. Determine suitability of hematology specimens and dispose of them in the appropriate biohazard containers.

7. Correctly identify and date each specimen and reagent to minimize the chance of misunderstanding.

8. Properly store, clean, and operate the laboratory microscopes.

9. Determine hematocrit values on whole blood samples.

10. Prepare acceptable peripheral blood smears and view for slide and staining quality.

11. Apply and adhere to quality control standards for all student laboratory testing.

12. Recognize technical testing errors for all laboratory tests and suggest possible reasons for and solutions to errors.

Laboratory Competencies Complete and document 100 percent of the following tasks with a minimum competency of 75 percent.

1. Complete all laboratory assignments.

2. Prepare 10 acceptable peripheral blood smears.

3. Perform and read five manual hematocrits.

4. Obtain three successful venipunctures.

5. Obtain two successful finger sticks.

References Kellough, R., and N. Kellough. A Guide to Methods and Resources, Planning for Competence. Upper Saddle River, New Jersey: Merrill Prentice Hall, 1999.

National Credentialing Agency (NCA) Test Item Writing Review.

Test Item Writing at http://www.jmu.edu/assessment/itemformats.htm.

Frary, R. “More Multiple-Choice Item Writing Do’s and Don’ts.” ERIC Digest at http://www.ed.gov, 1995.

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1 Introduction to Hematology

A.

TRANSITION GRID

Information concerning Chapter 1, “Introduction; ” Chapter 2, “Cellular Homeostasis;” and Chapter 3, “Hematopoiesis” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 1

Chapter 2

Chapter 3

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 1 n/a n/a n/a

Chapter 2 n/a Chapter 6 Chapter 3

Chapter 2 Chapter 1 Chapter 7 Chapter 4

B.

OBJECTIVES

Chapter 1 – Introduction Levels I and II 1. Compare the reference intervals for hemoglobin, hematocrit, erythrocytes, and leukocytes in infants, children, and adults.

2. Identify the function of erythrocytes, leukocytes, and platelets.

3. Describe the composition of blood.

4. Explain the causes of change in the steady state of blood components.

5. Describe clinical pathway, critical pathway, reflex testing, and disease management and identify the laboratory’s role in developing these models.

6. Compare capitated payment, prospective payment, and fee-for-service and describe the impact of capitation on the laboratory.

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Chapter 2 – Cellular Homeostasis Level I 1. Describe the location, morphology, and function of subcellular organelles of a cell.

2. Describe the lipid asymmetry found in the plasma membrane of most hematopoietic cells.

3. Differentiate the parts of the mammalian cell cycle.

4. Define R (restriction point) and its role in cell cycle regulation.

5. Define apoptosis and explain its role in normal human physiology.

6. Classify and give examples of the major categories of initiators and inhibitors of apoptosis.

7. List the major events regulated by apoptosis in hematopoiesis.

Level II 1. Explain the significance of SNPs, introns, exons, UTRs, post-translational protein modifications.

2. List the components and explain the function of the Ubiquitin-Proteosome system.

3. Define cyclins and Cdks and their role in cell cycle regulation; describe the associated Cdk partners and function of cyclins D, E, A, and B. 4. Define CAK (Cdk-activating kinase) and the two major classes of CKIs (cyclin-dependent kinase inhibitors) and describe their function.

5. Compare the function of cell-cycle checkpoints in cell-cycle regulation.

6. Describe/illustrate the roles of p53 and pRb in cell-cycle regulation.

7. Propose how abnormalities of cell-cycle regulatory mechanisms can lead to malignancy.

8. Define caspases and explain their role in apoptosis.

9. Differentiate the extrinsic and intrinsic pathways of cellular apoptosis.

10. Define and contrast the roles of proapoptotic and antiapoptotic members of the Bcl-2 family of proteins.

11. Describe apoptotic regulatory mechanisms.

12. Give examples of diseases associated with increased apoptosis and inhibited (decreased) apoptosis.

13. Define, and give examples of, epigenetics, oncogenes and tumor suppressor genes and their roles in cell biology.

14. Differentiate using morphologic observations, the processes of necrotic cell death and apoptotic cell death.

Chapter 3 – Hematopoiesis Level I 1. Describe the basic concepts of cell differentiation and maturation.

2. Compare and contrast the categories of hematopoietic precursor cells: hematopoietic stem cells, hematopoietic progenitor cells, and maturing cells, including proliferation and differentiation potential, morphology, and population size.

3. Describe the hierarchy of hematopoietic precursor cells and the relationships of the various blood cell lineages to each other (including the concept of colony-forming units/CFUs).

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4. Discuss the general characteristics of growth factors and identify the major examples of early acting (multilineage), later acting (lineage restricted), and indirect acting growth factors.

5. Differentiate between paracrine, autocrine, and juxtacrine regulation.

6. List examples of negative regulators of hematopoiesis.

7. Define hematopoietic microenvironment.

Level II 1. Identify the phenotypic characteristics differentiating the hematopoietic stem cells and progenitor cells.

2. Identify the key cytokines required for lineage-specific regulation.

3. Describe the structure and role of growth factor receptors.

4. Summarize the concept of signal transduction pathways.

5. Discuss the roles of transcription factors in the regulation of hematopoiesis and differentiation.

6. Outline current clinical uses of cytokines.

7. Describe the cellular and extracellular components of the hematopoietic microenvironment.

8. Discuss the proposed mechanisms used to regulate hematopoietic stem/progenitor cell proliferation/differentiation.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leaves, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Diagrams a. Using the information provided in Chapter 2, have the learners create their own explicit diagram of the: • Cell structure • Stages of the cell cycle • **Hematopoietic precursor cell model 3. Group Discussion a. Create a group discussion on the topic of the Medical Ethics concerning stem cell transplants.

4. Mystery Box a. Create a Mystery Box with the different blood components and corresponding reference ranges. Be sure to include at least • Leukocytes • Erythrocytes

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• Thrombocytes • Plasma • Whole blood • 4.5 - 11.0 * 109/L • 4.5 - 5.5 * 1012/L • 150 - 450 * 109/L • 55 percent of blood volume • 5 – 6 liters in adults • And so on 5. One-Minute Paragraph Have learners submit a one minute paragraph on the: a. Composition of blood b. **Abnormal tissue homeostasis and cancer 6. Short Story Have the learners, individually or as a group, write: a. The story of “The Life and Development of a Cell” b. A “Murder Mystery” of a Cell 7. Think-Pair-Share a. **Discuss the value and need for “clinical” and “critical” pathways. b. **Discuss Necrosis versus Apoptosis.

8. Thumbs Up/Thumbs Down Remember to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

D.

LABORATORY ACTIVITIES

1. **Perform WBC and RBC counts on each learner in the class, and create a “class reference range” for WBC and RBC. You might want to have students read the section “Reference Interval Determination” in Chapter 41 if you include this activity.

E.

PRACTICE QUESTIONS

1. List the components of whole blood and state the reference range for each. [Taxonomy 1] 2. **Explain why “reflex testing protocols” are designed. [Taxonomy 1].

3. A patient experiencing a viral infection is likely to demonstrate an increase in which of the following? [Taxonomy 1] a. Erythrocytes b. Hematocrit c. Leukocytes d. Thrombocytes 4. Patient Aaron had an RBC count of 4.0 * 1012/L. Explain why a hospital in Alabama might consider this value “normal,” whereas a facility in Utah may recognize Aaron’s RBC as “below” normal. [Taxonomy 2] 5. **In the text Clinical Laboratory Hematology, differentiate “precursor cells” from “maturing cells.”

[Taxonomy 2]

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6. Analyze each set of results and answer the following questions. [Taxonomy 2] A. WBC = 18.6 * 109/L RBC = 3.50 * 1012/L Hgb = 10.2 g/dL Hct = 31.1% Plt = 202 * 109/L

B. WBC = 0.6 * 109/L RBC = 2.12 * 1012/L Hgb = 7.5 g/dL Hct = 24.3% Plt = 89 * 109/L

C. WBC = 6.5 * 109/L RBC = 4.79 * 1012/L Hgb = 16.4 g/dL Hct = 49.6% Plt = 543 * 109/L

Which set of results demonstrates a a. Leukocytosis b. Erythrocytopenia c. Thrombocytopenia d. Critically decreased H & H e. Leukopenia f. Thrombocytosis 7. **Compare and contrast a “fee-for-service” reimbursement system to a “capitated payment” plan.

[Taxonomy 3] 8. Compare and contrast the processes of cell “self-renewal” versus “cell differentiation.” [Taxonomy 3] 9. Evaluate the following patient results and conclude which body function (mechanism) will be adversely affected. Explain why. [Taxonomy 3] WBC = 0.8 * 109/L RBC = 2.5 * 1012/L Hgb = 8.2 g/dL Hct = 25.1% Plt = 222 * 109/L 10. A 26-year-old male, Type I diabetes mellitus patient is seen in the emergency department. The patient’s blood chemistry results confirm the need for renal dialysis. Explain the etiology for this patient’s hematology results. [Taxonomy 3] WBC = 11.8 * 109/L RBC = 3.2 * 1012/L Hgb = 9.5 g/dL Hct = 27.6% Plt = 321 * 109/L

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Introduction to Hematology—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

V N O I T A R E F I L

H L Y H R P R Z L O Q

E P Y R E V H L N C T

H N J N H D T R F S C T D T S S S Z R E Y L T E E M Y G Y D E N N H C N C Y M E I K O B K M C G K P B L N L E O O G M A Q B N C T S O S W I X T Y L R T P Q R D L T H E M A T O L O T X P L Z S X Q L

O R P R L

K D Q N S

K G L N I

Y Z G G F E R M S E

T L U K I

V V C C O

R T H N P

D N R A O

N E M M T X E S S N M I U M B S E M R O L N A T C M N P U M E O N H W P G Y T A C W M M

I X Y V W F L P S M R W Y V O M A T I N D L B U M I N C T A M E H M W

• cell division—repeated reproduction • another term for hematopoietic growth factor • study of formed cellular blood elements • process of forming a blood clot • outer boundary of a cell • the earliest morphologically recognizable cell of each blood cell lineage • the process of blood cell formation • how a blood cell passes through vessel walls • part of cell that contains the genetic material • genes that induce tumor formation • main constituent of plasma • programmed cell death • platelet • lightly stained portions of chromatin • undifferentiated, pleuripotential hematopoietic cell (2 words)

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Introduction to Hematology—Level I V N O I T A R E F I L

H L Y H R P R Z L O Q

E P Y R E V H L N C T

H N J N H D T R F S C T D T S S S Z R E Y L T E E M Y G Y D E N N H C N C Y M E I K O B K M C G K P B L N L E O O G M A Q B N C T S O S W I X T Y L R T P Q R D L T H E M A T O L O T X P L Z S X Q L

N E M M T X E S S N M I U M B S E M R O L N A T C M N P U M E O N H W P G Y T A C W M M

O K K Y Z T V R D I X Y V W F L R D G G G L V T N P S M R W Y V P Q L F E U C H R O M A T I N D R N N R M K C N A L B U M I N C L S I S E I O P O T A M E H M W • cell division—repeated reproduction (proliferation) • another term for hematopoietic growth factor (cytokines) • study of formed cellular blood elements (hematology) • process of forming a blood clot (hemostasis) • outer boundary of a cell (membrane) • the earliest morphologically recognizable cell of each blood cell lineage (blast) • the process of blood cell formation (hematopoiesis) • how a blood cell passes through vessel walls (diapedese) • part of cell that contains the genetic material (nucleus) • genes that induce tumor formation (oncogenes) • main constituent of plasma (albumin) • programmed cell death (apoptosis) • platelet (thrombocyte) • lightly stained portions of chromatin (euchromatin) • undifferentiated, pleuripotential hematopoietic cell (2 words) (stem cell)

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Introduction to Hematology—Level I 1

2 3 4

5

6

7

8 9 10 11

12

13 14

15

ACROSS

DOWN

1 Growth and development of lymphoid cells 4 Stage during which microtubular spindles align during mitosis 7 Cells that defend against foreign antigens 8 Where the metabolic activities of the cell takes place 12 Alternate copies (alleles) of a gene 13 Type of cell that differentiates into mature, functional cells 15 Cell murder

2 Outside of cell 3 Study of the entire genome of an organism 5 Arrest of bleeding 6 Study of the formed cellular blood elements 9 Vital protein inside erythrocytes 10 Organelles that assemble amino acids into protein 11 Group of proteins responsible for apoptotic cell death 14 Complete blood count (abrv)

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Introduction to Hematology—Level I L Y M P H O P O I E S I S X G M E T A P H A S E H H R N L E U K O C Y T E S A O M M C M O A E I S C Y T O P L A S M C H T O L S E A R L U M S I O L C O I B G A A G P O L Y M O R P H I S M S L S P O P R O G E N I T O R A B C M S I B E E S N E C R O S I S

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Introduction to Hematology—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

L X Z B K S N E F Q S C B L Z E W F T F N G B W K I C B L R K L B V V O X R M H T P R M I Y M C L I C T C T Q

R L N X E C T N O Y G

F C K N

F T S N D P I T A T S O F H H T N L T Y

Y A L X

N B L N

N C M F

K A H X

D L N S D I P I H Q T R T E F N S L N N E F V R C E M S E R N G G P A R N O W A B H E C P S W B P N W E W L O O T S W T P H R I K B T C T P A P O E P O Y M K E D J T F M

L N T B

I G A F T A D Q K N N

L C T N T I R C O T A

J T K L

M E H J

• stage in mitosis where cells condense their chromosomes • tumor suppressor gene • payment plan whereby the provider is reimbursed a fixed amount for a service • growth factor that promotes proliferation and differentiation of progenitor cells (abrv) • process for generating diverse cell populations • programmed cell death • cell membranes are composed of proteins and ______ • packed red cell volume • testing done based on results of screening tests • regulatory subunit of Cdks • proteins that direct apoptosis • hematopoietic growth factors • hematopoietic growth factor for erythroid cells (abrv) • precursor of all hematopoietic cells (abrv)

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Introduction to Hematology—Level II L X Z B K S N E F Q S C B L Z E W F T F N G B W K I C B L R K L B V V O X R M H T P R M I Y M C L I C T C T Q

R L N X E C T N O Y G

D L N S D I P I H Q T R T E F N S L N N E F V R C E M S E R N G G P A R N O W A B H E C P S W B P N W E W L O O T S W T P H R I K B T C T P A P O E P O Y

I G A F T A D Q K N N

L C T N T I R C O T A

F Y N N K F T S N D M K L J M C A B C A P I T A T E D N T E K L L M H S O F H H J T T K H N X N F X T N L T Y F M B L J • stage in mitosis where cells condense their chromosomes (prophase) • tumor suppressor gene (antioncogene) • payment plan whereby the provider is reimbursed a fixed amount for a service (capitated) • growth factor that promotes proliferation and differentiation of progenitor cells (abrv) (scf) • process for generating diverse cell populations (differentiation) • programmed cell death (apoptosis) • cell membranes are composed of proteins and ______ (lipids) • packed red cell volume (hematocrit) • testing done based on results of screening tests (reflex) • regulatory subunit of Cdks (cyclin) • proteins that direct apoptosis (caspases) • hematopoietic growth factors (cytokines) • hematopoietic growth factor for erythroid cells (abrv) (epo) • precursor of all hematopoietic cells (abrv) (hsc)

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Introduction to Hematology—Level II 1

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4 Type of cells that differentiate into mature, functional cells 5 Multiprogenitor cell capable of differentiating into leukocytes, erythrocytes, and megakaryocytes (2 abrv) 6 Gene that can transform cells into malignant cells 8 Physical manifestation of an individual’s genotype 12 Describes a cell that has exited the cell cycle—nonproliferative state 13 Specialized type of paracrine signaling— cytokine is not secreted by the cell that produced it 14 Thrombocytes

1 Lineage specific marker for erythrocytes 2 Cells of the bone marrow that include adipocytes, endothelial cells, and fibroblasts 3 Process of producing granulocytes 4 Describes characteristic in which growth factors act on more than one cell type 7 Platelets are derived from _________ 9 “On top of genetics” 10 Fat cell 11 Small polypeptide that tags molecules for destruction

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Introduction to Hematology—Level II G S G L P R O G E N I T O R R Y L R A C F U G E M M O N C O G E N E O I M M U P H E N O T Y P E A L H P T G L O O I R A A P R G O K U D O I E P A B I I N N I R I P E A E C T Q O S T O U C I Q U I E S C E N C E I Y S C Y T T S J U X T A C R I N E E N P L A T E L E T S

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2 The Hematopoietic System

A.

TRANSITION GRID

Information concerning Chapter 4, “Structure and Function of Hematopoietic Organs”; Chapter 5, “The Erythrocyte”; Chapter 6, “Hemoglobin”; and Chapter 7, “The Leukocyte” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 4

Chapter 5

Chapter 6

Chapter 7

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 3 Chapter 1 Chapter 7 Chapter 4

Chapter 4 Chapter 3 Chapters 8, 9 Chapter 5

Chapter 5 Chapter 3 Chapter 10 Chapter 5

Chapter 6 Chapter 15 Chapter 12 Chapters 14, 16

B.

OBJECTIVES

Chapter 4 – Structure and Function of Hematopoietic Organs Level I 1. Identify the sites of hematopoiesis during embryonic and fetal development as well as in childhood and adulthood.

2. Identify organ/tissue sites in which each hematopoietic cell type differentiates.

3. Explain the difference between primary and secondary lymphoid tissue.

4. Describe the function of bone marrow, spleen, lymph nodes, and thymus.

Level II 1. Associate physical findings (hypersplenism, lymphadenopathy) with the presence of hematologic disease.

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2. Describe the pathophysiologic changes that lead to bone marrow hyperplasia or extramedullary hematopoiesis.

3. Identify sites of extramedullary hematopoiesis.

4. Describe the structure of bone marrow, spleen, lymph nodes, and thymus.

Chapter 5 – The Erythrocyte Level I 1. List and describe the stages of erythrocyte maturation in the marrow from youngest to most mature cells.

2. Explain the maturation process of reticulocytes and the cellular changes that take place.

3. Identify the normal range for reticulocytes.

4. Explain the function of erythropoietin; include the origin of production, bone marrow effects, and normal values.

5. Describe the function of the erythrocyte membrane.

6. Name the energy substrate of the erythrocyte.

7. Diagram the mechanism of extravascular erythrocyte destruction and hemoglobin catabolism.

8. Diagram the mechanism of intravascular erythrocyte destruction and hemoglobin catabolism.

9. State the average dimensions and life span of the normal erythrocyte.

10. Describe the function of 2,3-BPG and its relationship to the erythrocyte.

Level II 1. Summarize the mechanisms involved in the regulation of erythrocyte production.

2. Describe the structure of the erythrocyte membrane, including general dimensions and features; assess the function of the major membrane components.

3. Explain the mechanisms used by the erythrocyte to regulate permeability to cations, anions, glucose, and water.

4. Compare and contrast three pathways of erythrocyte metabolism and identify key intermediates as well as the relationship of each to erythrocyte survival and longevity.

5. Generalize the metabolic and catabolic changes within the erythrocyte that occur with time that “label” the erythrocyte for removal by the spleen.

6. Compare and contrast erythrocyte extravascular destruction with intravascular destruction and identify which process is dominant given laboratory results.

7. Predict the effects of increased and decreased erythropoietin levels in the blood.

Chapter 6 – Hemoglobin Level I 1. Diagram the quaternary structure of a molecule of hemoglobin identifying the heme ring, globin chains, and iron.

2. Assemble fetal and adult hemoglobin molecules with appropriate globin chains.

3. Explain how pH, temperature, 2,3-BPG, and PO2 affect the oxygen dissociation curve (ODC).

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4. List the types of hemoglobin normally found in adults and newborns and give their approximate concentration.

5. Summarize hemoglobin’s function in gaseous transport.

6. Define normal hemoglobin values.

7. Explain how the fine balance of hemoglobin concentration is maintained.

8. Compare HbA with HbA1c and explain what an increased concentration of HbA1c means.

Level II 1. Construct a diagram to show the synthesis of a hemoglobin molecule.

2. Describe the ontogeny of hemoglobin types; contrast differences in oxygen affinity of HbF and HbA, and relate them to the structure of the molecule.

3. Explain the molecular control of heme synthesis.

4. Given information on pH, 2,3-BPG, temperature, and HbF concentration, interpret the ODC and translate it into the physiologic effect on oxygen delivery.

5. Contrast the structures and functions of relaxed and tense hemoglobin and propose how these structures affect gaseous transport.

6. Describe how abnormal hemoglobins are acquired, and select a method by which they can be detected in the laboratory.

7. Assess the oxygen affinity of abnormal acquired hemoglobins and reason as to how this affects oxygen transport.

8. Compare and contrast the exchange of O2, CO2, H+ and Cl- at the level of capillaries and the lungs.

Chapter 7 – The Leukocyte Level I 1. Identify terms associated with increases and decreases in leukocytes.

2. Differentiate morphologically the leukocyte precursors found in the proliferative compartment of the bone marrow.

3. Compare and contrast the development including distinguishing maturation and cell features of the granulocytic, monocytic-macrophage, and lymphocytic cell lineages.

4. Compare and contrast the morphologic and other distinguishing cell features of each of the leukocytes found in the peripheral blood.

5. Compare and contrast the function of each of the leukocytes found in the peripheral blood.

6. Summarize the process of neutrophil migration and phagocytosis.

7. List the adult reference ranges for the leukocytes found in the peripheral blood.

8. Calculate absolute cell counts from data provided.

9. Differentiate absolute values and relative values of cell count data.

10. List causes/conditions that increase or decrease absolute numbers of individual leukocytes found in the peripheral blood.

11. Compare and contrast pediatric and newborn reference ranges and adult reference ranges.

12. Explain immunoglobulin diversity.

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Level II 1. Summarize the kinetics of the granulocytic, monocytic-macrophage, and lymphocytic cell lineages.

2. Describe the processes that permit neutrophils to leave the peripheral blood circulation and move to a site of infection and propose how defects in these processes affect the body’s defense mechanism.

3. Compare and contrast the immunologic features and functions of each of the leukocytes found in the peripheral blood.

4. Summarize lymphocyte membrane characteristics and molecular changes used to differentiate subtypes.

5. Differentiate between polyclonal and monoclonal antibodies and discuss each type in relationship to a patient’s clinical condition.

6. Compare and discriminate morphological features of Russell bodies, Mott cells, and flame cells.

7. Design systems to evaluate laboratory data of leukocytes for error detection.

8. Formulate pathways to correlate laboratory data of leukocytes with clinical knowledge of the patient.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Diagram a. The mechanism of extravascular hemoglobin catabolism b. The mechanism of intravascular hemoglobin catabolism c. The quaternary structure of a molecule of hemoglobin d. **Graphically design all erythroid precursors.

3. Extra, Extra, Read All About It a. Have each learner write a newspaper or magazine article about the celebrity group, “Erythropoietin,” they interviewed. Include in this article where “our stars” are from, what influences them, what affect they have on their environment, and describe “their” most important contribution. b. **You are a traveller through the bone marrow. Describe what you see when you come upon the Erythroblastic Island. Tell the story of the erythroblast inhabitant. c. **Have the student ceate a travel log for a neutrophil’s experience after it leaves the bone marrow. Instruct the learner to include the adventures during adherence, migration, and phagocytosis.

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4. Mapping Create a Map for: a. The development of hematopoiesis from fetus to adult b. **The chemical synthesis of heme and globin chains c. **Lymphocytes, including function, development, and markers d. **Metabolic pathways in the erythrocyte 5. Match-Up Game Create a Concentration Game with: a. The names of different types of hemoglobin and their corresponding globin chain pairs. May also include in this activity the definitions and names of hemoglobin when combined with different substances (e.g., oxyhemoglobin, sulfhemoglobin, etc.). b. Divide the learners into small groups. Have each group “create” their own concentration game for the following. When completed have each group play the other groups’ game.

• The list of leukocytes and their reference ranges • The list of leukocytes and their morphology • The list of leukocytes and conditions in which each would be increased or decreased • **This same exercise can be adjusted to a Level II activity if cells such as promonocytes, prolymphocytes, immunoblasts, and so on are included. ** • The following cells and their functions • Megakaryocytes • Mast cells • Osteoblasts • Osteoclasts • Macrophages • Reticular cells • Adipocytes 6. Mystery Box a. Provide information on hematopoiesis • Time of gestation (e.g., third month of fetal life) • Type of cell produced (e.g., primitive erythroblast, T cell, etc.)

• Organ or location (e.g., yolk sac, thymus, etc.)

• Have the learners pull each piece of paper and match the time of gestation, type of cell produced, and organ to solve the hematopoiesis mystery. b. Provide pieces of paper that have a basic disease condition listed on them, that is, bacterial infection, flu, and so on.

• Have the learners select a disease condition and create correlating CBC and manual differential results. c. Provide the total WBC counts and differential percents for the normal leukocytes.

• Have the learners calculate the absolute values for each set of results provided.

• Also have learners evaluate the absolute values using the appropriate medical terms, that is, lymphocytosis, neutropenia, and so on.

• An alternate Mystery Box is to provide the medical terms, that is, agranulocytosis and basophilia, and ask the learners to create corresponding differential and CBC results. ** d. On separate pieces of paper write the: • Size and N:C ratio • Cytoplasmic characteristics • Nuclear characteristics Have students combine the appropriate characteristics and identify the correct name of the erythroid precursor. To extend this exercise, the instructor may consider asking students to draw, or graphically design, each erythroid precursor and then match each with the appropriate characteristic.

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7. One-Minute Paragraph a. **Without prior learning or lecture, have the learners read the appropriate sections and ask them to write a one-minute paragraph on the following topics.

• Discuss the negative consequences of hyperplasia.

• Explain why blood cells must have the ability to “deform.”

• Explain the reason for a hypoxic, acidic, and hypoglycemic environment in the spleen. b. Describe the function of bone marrow, spleen, thymus and lymph nodes. Give one minute for each site. c. After the appropriate lecture, give the learners one minute to explain the structure of the hemoglobin molecule. d. ** Explain the Bohr effect. e. ** Describe the relationship between lymphocytes and macrophages in the immune system.

8. Mnemonic Device Have each learner develop and share a mnemonic device to learn the: a. Erythrocyte maturation stages, e.g., Princess Brown’s Pet Ox Ran Everywhere b. Neutrophilic maturation stages 9. Small Group Teaching Assign a small group of learners the following topics. They should be instructed to present and teach these concepts to the rest of the class. a. Erythrocyte destruction (extravascular and intravascular) b. Oxygen dissociation curve and how pH, temperature, 2,3-BPG, and PO2 affect the curve c. **The role of the iron responsive element-binding protein (IRE_BP) d. **Differentiate the marginating and circulating pools of neutrophils. Explain how the two pools freely exchange neutrophils.

10. Think-Pair-Share a. Distinguish between primary and secondary lymphoid tissues. b. Describe the morphology that differentiates a band from a segmented neutrophil. c. **In detail, explain the hemoglobin-2,3 BPG interaction. d. **Discuss the relationship between neutrophils and coagulation. e. **Differentiate between polyclonal and monoclonal antibodies and describe clinical conditions where each is found.

11. Thumbs Up/Thumbs Down Remember to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

12. Web Search a. Have each learner conduct a Web search on the following topics and write two to three paragraphs on the information they collected.

• Glycolytic pathway (Embden-Meyerhoff pathway) • Hexose monophosphate shunt • Methemoglobin reductase pathway • Rapoport-Leubering shunt b. Have each learner find and share a Web site that provides leukocyte morphology.

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LABORATORY ACTIVITIES

1. Discuss specimen requirements, preparation, and handling for WBC, RBC, and Hgb. 2. Discuss pre-analytical, analytical, and post-analytical causes for unexpected results.

3. Perform manual cell counts for • WBC • RBC 4. Perform automated procedures on learner/patient or quality control samples to evaluate: • WBC • RBC • Hgb 5. Discuss interfering substances for WBC, RBC, and Hgb analysis.

6. **Morphologically identify RBC stages of maturation using prepared peripheral smears, Kodachrome, or CD-ROM programs.

7. Morphologically identify normal WBC using prepared peripheral smears, Kodachrome, or CDROM programs.

E.

PRACTICE QUESTIONS

1. The role of the macrophage in the erythroblastic island is to [Taxonomy 1] a. Insulate the maturing erythrocytes b. Protect the erythroblast from other invading cells c. Provide nutrients to the erythroblast d. Regulate erythropoiesis by secreting various cytokines 2. **What is the rate limiting enzyme in heme synthesis? [Taxonomy 1] a. ALAS b. Ferrochelatase c. Glucose-6-phosphatase d. Peptidase 3. **Describe how oxygen diffuses from the blood into the tissues. [Taxonomy 1] 4. **List two sites of extramedullary hematopoiesis and describe the structure of each. [Taxonomy 1] 5. Distinguish between the “hematopoietic compartment” and the “vascular compartment.” [Taxonomy 2] 6. Tommy moved from a location at sea level where his red count = 4.90 * 1012/L to a mountain home at 10,000 feet. Select the red blood cell count that might reflect his living in the mountains for 12 months. [Taxonomy 2] a. 3.70 * 1012/L b. 4.80 * 1012/L c. 5.70 * 1012/L d. 6.90 * 1012/L

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7. **Explain the “high mortality” of neutrophils during a sustained inflammatory response. [Taxonomy 2] 8. **Compare and contrast the content and structure of the red and white pulp in the spleen. [Taxonomy 3] 9. **Estimate the change in the factors that affect oxygen affinity of an individual at rest and one that has completed 20 minutes of strenuous exercise. [Taxonomy 3] 10. Compare and contrast the nucleus and cytoplasmic morphology in mature and immature neutrophils. [Taxonomy 3] 11. A 15-year-old female patient was seen at a city medical clinic. For the past three days, she has complained of an “upset stomach” and excessive vomiting. Her guardian gave her a drink of sodium bicarbonate and water each day to calm her stomach. Laboratory results revealed a condition of metabolic alkalosis. How would this patient’s acid-base condition affect her oxygen affinity and the oxygen dissociation curve? [Taxonomy 3]

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The Hematopoietic System—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

B Z T X H Y H J H Y J B P F L E D N R N K S W F E C R G G L M R C E N D N R N T Y Z E L R D L D T L N P X R

N W D T Y N H B Q T W T S J J D P E R P L A S I A T K L T N T E V S Y L P L C F Z L I K S T B O T I L K E Y C M K H Q M A J T X T S P I B U K T P D Q J L X P Y X O Q D H W K O X C K E B R R H C T T M W P N O V K X L O R L E V Y T N H I O F P T Y N E R D M M C G Q S P N R R E O J T M D O X O R O Z M B A T R N L S H G G T G E C C D M R M U L I O B J L G A P X L E K O D E E H A K X O Z H H M D M B R C K Z N K V Q B M P D U Q L L T R W I P R V W I C Y L W A P S R N N B D P X L N D L T S Z A J E T Y C O N O M C A N T G M K M M T K B D L E B T M E T A M Y E L O C Y T E K Y Y

Q Y D P C V N

I

B O L G O M E H T E M

• cell process in which cell engulfs foreign substances • cell that is increased in parasitic infections • excessive proliferation of normal cells within an organ • hemoglobin without oxygen • decrease in leukocytes • earliest morphologically recognizable erythrocyte precursor • largest mature cell in peripheral blood • neutrophil with >5 lobes • type of hematopoiesis outside the bone marrow • most numerous WBC in adult peripheral blood • cells involved in the formation of calcified bone • precursor granulocytic cell with kidney bean shaped nucleus • tissue basophil (2 words) • organ that produces EPO • Hb with Fe+++

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The Hematopoietic System—Level I B Z T X H Y H J H Y J B P F L E D N R N K S W F E C R G G L M R C E N D N R N T Y Z

N W D T Y N H B Q T W T S J J D P E R P L A S I A T K L T N T E V S Y L P L C F Z L I K S T B O T I L K E Y C M K H Q M A J T X T S P I B U K T P D Q J L X P Y X O Q D H W K O X C K E B R R H C T T M W P N O V K X L O R L E V Y T N H I O F P T Y N E R D M M C G Q S P N R R E O J T M D O X O R O Z M B A T R N L S H G G T G E C C D M R M U L I O B J L G A P X L E K O D E E H A K X O Z H H M D M B R C K Z N K V Q B M P D U Q L L T R W I P R V W I

E D T P Q

C D A M P

L L L X Y

R D N R D

Y L N E C

L W A T S Z T G M T A M V N I

P A K Y B

S R N J E T M M T E L O O L G

N B D Y C O K B D C Y T O M E

P N L E H

• cell process in which cell engulfs foreign substances (phagocytosis) • cell that is increased in parasitic infections (eosinophil) • excessive proliferation of normal cells within an organ (hyperplasia) • hemoglobin without oxygen (deoxyhemoglobin) • decrease in leukocytes (leucopenia) • earliest morphologically recognizable erythrocyte precursor (pronormoblast) • largest mature cell in peripheral blood (monocyte) • neutrophil with >5 lobes (hypersegmented) • type of hematopoiesis outside the bone marrow (extramedullary) • most numerous WBC in adult peripheral blood (neutrophil) • cells involved in the formation of calcified bone (osteoblasts) • precursor granulocytic cell with kidney bean shaped nucleus (metamyelocyte) • tissue basophil (2 words) (mast cell) • organ that produces EPO (kidney) • Hb with Fe+++ (methemoglobin)

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The Hematopoietic System—Level I 1

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3 Increase in neutrophils 5 Iron-protein complex visualized with Prussianblue stain 7 Unique molecule produced by B lymphocytes and plasma cells 9 Reddish-purple primary granules 11 Inadequate oxygen at the tissue level 12 Plasma glycoprotein that transports free hemoglobin 13 Type of lymphocyte with nucleus eccentrically placed (2 words) 14 Type of hemoglobin with two alpha and two beta globin chains is the major Hb of _______ 15 Non-nucleated RBC with residual RNA

1 Mature granulocyte with presence of large basophilic granules 2 Type of immunity produced by antibodies by B lymphocytes 4 Earliest morphologically recognizable granulocyte precursor 6 Sulfur atom combined with hemoglobin 8 Type of Hb with glucose irreversibly attached 10 During fetal development, major site of T cell production

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The Hematopoietic System—Level I B H A N E U T R O P H I L I A S M O O M P H E M O S I D E R I N Y H U A E I M M U N O G L O B U L I N G L L F L O A Z U R O P H I L I C Y B E C L T M H Y P O X I A H A P T O G L O B I N S S Y G Y T M P L A S M A C E L L U O A D U L T S B T R E T I C U L O C Y T E N D

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The Hematopoietic System—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

E P F F O H R E Y E M N E D B M E T O K M M Q G R S P E C T R I N H Y L D G V X Z N M T J G F R F A T C Y C U L L I N G K N R S N G M S O C Z C G L N R L Z Z I T O J S A L H K M N R G X K A S Y C B I Q L E R N Y X V E F H E T Y D S T H C Y O C V J T C R D C T E O X R G O M M T M K C C E Y O Z T F K P H E O A Q C G R P P S T Y N Q X N B T R T Y Z T A D I X C H R D R H H S P O N W I W S B O L Z R P F Y D O Z P P D N G X K A Z B B O N K R T R H P J W K U M Q N W R H N K M T L I Z R K E W N C X D Z R P W T G R L F C L G Q M C N W Y M G P C T L I H W N I T E I O P O R H T Y R D C Z T F I H S E D I R O L H C B

P S I S O T Y C O L U N A R G A E Q

• first granulocytic precursor with presence of azurophilic granules • last normoblast stage capable of mitosis • increase in leukocytes • filtering and destruction of damaged red cells by the spleen • granulocyte with a horseshoe shaped nucleus • cell process in which cell engulfs foreign substances • pathway for metabolism of glucose in RBC (2 words) • hormone that stimulates production of RBC • type of hemoglobin that constitutes <2% of total Hb in adults • cell involved in resorption and remodeling of calcified bone • Cl—diffuses into the cell from the plasma (2 words) • passage of blood cells through the unruptured capillary wall • predominant skeletal protein • totality of all stages of erythrocytes • absence of granulocytes

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The Hematopoietic System—Level II E P F F O H R E Y E M N T O K M M Q G R S P E C Y L D G V X Z N M T J G C Y C U L L I N G K N R O C Z C G L N R L Z Z I L H K M N R G X K A S Y E R N Y X V E F H E T Y Y O C V J T C R D C T E M M T M K C C E Y O Z T O A Q C G R P P S T Y N R T Y Z T A D I X C H R P O N W I W S B O L Z R Z P P D N G X K A Z B B R H P J W K U M Q N W R L I Z R K E W N C R L F C L G Q M C L I H W N I T E I D C Z T F I H S E

E T F S T C D O F Q D P O H

D R R N O B S X K X R F N N

B M E I N H F A T G M S J S A I Q L T H C R G O P H E N B T H H S Y D O K R T K M T

X D Z R P W T N W Y M G P C O P O R H T Y D I R O L H C

• first granulocytic precursor with presence of azurophilic granules (promyelocyte) • last normoblast stage capable of mitosis (polychromatophilic) • increase in leukocytes (leukocytosis) • filtering and destruction of damaged red cells by the spleen (culling) • granulocyte with a horseshoe shaped nucleus (band) • cell process in which cell engulfs foreign substances (phagocytosis) • pathway for metabolism of glucose in RBC (2 words) (Embden Meyerhoff) • hormone that stimulates production of RBC (erythropoietin) • type of hemoglobin that constitutes <2% of total Hb in adults (fetal) • cell involved in resorption and remodeling of calcified bone (osteoclast) • Cl—diffuses into the cell from the plasma (2 words) (chloride shift) • passage of blood cells through the unruptured capillary wall (diapedesis) • predominant skeletal protein (spectrin) • totality of all stages of erythrocytes (erythron) • absence of granulocytes (agranulocytosis)

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G T R B

P S I S O T Y C O L U N A R G A E Q

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The Hematopoietic System—Level II 1 2

3

4

5

6

7

8

9

10

11

12

13

ACROSS

DOWN

2 Abnormal RBC shape due to an alteration in membrane lipids 4 Neutrophil migration guided by chemoattractant gradients 6 Syndrome; lack of thymus development in the fetus 7 Form by which most CO2 is transported by the blood 8 Inactive X chromosome appears as an appendage of the neutrophil nucleus (2 words) 11 Neutrophilic precursor; kidney-bean shaped nucleus 12 Projection of calcified bone extending from cortical bone into the marrow space 13 Normoblast with abundant gray-blue cytoplasm and low N:C ratio

1 Microenvironment that supports hematopoietic cell proliferation in the bone marrow 3 Characteristics of most growth factors in which they act on more than one cell type 4 Result of excess deoxygenated hemoglobin in the blood 5 Type of cell in which the cytoplasm is replaced with a single fat vacuole 8 The effects of pH on hemoglobin-oxygen affinity (2 words) 9 Plasma cell filled with globules containing immunoglobulins 10 First enzyme in the heme synthetic pathway (abrv)

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The Hematopoietic System—Level II S A C A N T H O C Y T E P R L C H E M O T A X I S D I G E O R G E Y M D I A A I B I C A R B O N I C N P T O O B A R R B O D Y S C O O I M Y A H P S O T L R I T M E T A M Y E L O C Y T E T S S F C F E T R A B E C U L A L C P O L Y C H R O M A T O P H I L I C

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3 The Anemias

A.

TRANSITION GRID

Information concerning Chapter 8, “Introduction to Anemia;” Chapter 9, “Anemias of Disordered Iron Metabolism and Heme Synthesis;” Chapter 10, “Hemoglobinopathies: Qualitative Defects;” Chapter 11, “Thalassemia;” Chapter 12, “Megaloblastic and Nonmegaloblastic Macrocytic Anemias;” Chapter 13, “Hypoproliferative Anemias;” Chapter 14, “Introduction to Hemolytic Anemia;” Chapter 15, “Hemolytic Anemia: Membrane Defects;” Chapter 16, “Hemolytic Anemia: Enzyme Deficiencies;” Chapter 17, “Hemolytic Anemia: Immune Anemias;” and Chapter 18, “Hemolytic Anemia: Nonimmune Defects” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 8

Chapter 9

Chapter 10

Chapter 11

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 10 Chapters 4,5 Chapter 18 Chapters 6,7

Chapter 11 Chapter 6 Chapter 11 Chapter 10

Chapter 12 Chapter 11 Chapter 26 Chapter 13

Chapter 13 Chapter 12 Chapter 27 Chapter 13

Clinical Laboratory Hematology

Chapter 12

Chapter 13

Chapter 14

Chapter 15

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 14 Chapter 7 Chapter 20 Chapter 11

Chapter 15 Chapter 8 Chapter 21 Chapter 9

Chapter 16 Chapter 9 Chapter 24 Chapter 12

Chapter 17 Chapter 9 Chapter 23 Chapter 12

Clinical Laboratory Hematology

Chapter 16

Chapter 17

Chapter 18

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 18 Chapter 10 Chapter 23 Chapter 12

Chapter 19 Chapter 13 Chapter 25 Chapter 12

Chapter 20 Chapter 13 Chapter 24 Chapter 12

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OBJECTIVES

Chapter 8 – Introduction to Anemia Level I 1. Describe and identify specific anisocytes and poikilocytes.

2. Classify and describe erythrocytes based on erythrocyte indices.

3. Calculate absolute reticulocyte count, corrected reticulocyte count and reticulocyte production index from reticulocyte results, hematocrit, and RBC count.

4. Describe and identify erythrocyte inclusions including staining characteristics.

5. Describe and recognize abnormal variation in erythrocyte distribution on stained smears.

6. Identify laboratory tests to evaluate erythrocyte destruction.

7. Given CBC and RPI results, categorize an anemia according to morphologic classification.

8. Correlate polychromatophilia on a blood smear with other laboratory results of erythrocyte production and destruction.

Level II 1. Relate adaptations to anemia with patient symptoms.

2. Correlate patient history and clinical symptoms with laboratory results in anemia.

3. Interpret RDW results.

4. Assess bone marrow response to anemia given CBC and reticulocyte results.

5. Correlate poikilocytes with the mechanism of formation and assess their clinical significance.

6. Correlate CBC results with findings on a blood smear and troubleshoot discrepancies.

7. Determine the clinical significance of erythrocyte inclusions and select methods to differentiate the inclusions.

8. Evaluate the distribution of erythrocytes on stained smears and determine its clinical significance.

9. Assess and interpret bone marrow findings in the presence of anemia.

10. Compare the sensitivity and specificity of tests used to screen and confirm a differential diagnosis of anemia.

11. Compare the morphologic and functional classification of anemia.

12. Given laboratory results, classify an anemia in terms of morphology and pathophysiologic mechanism (function).

13. Choose appropriate follow-up tests to determine anemia classification and evaluate the results.

Chapter 9 – Anemias of Disordered Iron Metabolism and Heme Synthesis Level I 1. Define sideropenic and sideroachrestic as they relate to anemias.

2. Diagram the transport of iron from ingestion to incorporation into heme.

3. Define the following terms and explain their role in iron metabolism: transferrin, hemosiderin, ferritin, TIBC. 4. Describe physiologic factors that affect the amount of iron needed by the body.

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5. Compare and contrast the typical blood features and iron studies associated with iron deficiency anemia (IDA), anemia of chronic disease (ACD), lead poisoning, and sideroblastic anemia.

6. Explain the etiology and pathophysiology of iron deficiency anemia, anemia of chronic disease, and sideroblastic anemia.

7. Define hemosiderosis.

8. Calculate transferrin saturation and UIBC. Level II 1. List the three stages of iron deficiency and define characteristic RBC morphology of each stage.

2. Compare and contrast iron stores, hemoglobin, serum iron, TIBC, saturation, serum ferritin, and RBC morphology in the three stages of iron deficiency.

3. Describe the function of the proteins involved in iron metabolism including hepcidin, HFE, transferrin receptor, hemojuvelin, divalent metal transporter 1, duodenal cytochrome–B reductase, hephaestin, and ferroportin.

4. Explain the molecular control of total body iron and cellular iron.

5. Describe how genetic defects in the iron metabolism proteins can affect the body’s iron homeostasis.

6. Contrast the basic defects in iron deficiency anemia, sideroblastic anemia, and anemia of chronic disease, and describe how these defects affect hemoglobin synthesis.

7. Recognize the clinical features associated with iron deficiency.

8. Correlate the following laboratory features with iron deficiency anemia and sideroblastic anemia: erythrocyte morphology and protoporphyrin studies, iron studies, and bone marrow.

9. Select laboratory tests and discuss test results that help differentiate iron deficiency anemia, anemia of chronic disease, and sideroblastic anemia.

10. Summarize the results of bone marrow analysis in sideroblastic anemia and anemia of chronic disease, and contrast them with those found in iron deficiency anemia.

11. Outline the classification of sideroblastic anemias and describe the differentiating feature of the hereditary type.

12. Describe the relationship of the anemias associated with alcoholism and malignant disease to sideroblastic anemia.

13. Describe the role of molecular diagnostics in hereditary sideroblastic anemia.

14. Explain the significance of finding microcytic anemia in the presence of lead poisoning, and suggest reflex testing that would help define an accurate diagnosis.

15. Explain how lead poisoning and alcohol affect erythropoiesis and their relationship to sideroblastic anemia, and recognize the abnormal peripheral blood and clinical features that can be associated with these disorders.

16. Discuss the treatment for iron deficiency, sideroblastic anemia, and anemia of chronic disease and expected laboratory findings associated with successful therapy.

17. Differentiate primary (hereditary) and secondary hemochromatosis and summarize typical results of iron studies in this disease.

18. Describe the genetic abnormality associated with hereditary hemochromatosis and identify the screening and diagnostic tests for this disease.

19. Describe the basic defect in porphyria and its effect on the blood. ..

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20. Develop a reflex-testing pathway for an effective and cost-efficient diagnosis when microcytic and/or hypochromic cells are present.

21. Evaluate laboratory test results and use them to identify the etiology and pathophysiology of the anemias that have a defective heme synthesis component.

Chapter 10 – Hemoglobinopathies: Qualitative Defects Level I 1. Define hemoglobinopathy.

2. Explain the basis of defects resulting in the production of abnormal hemoglobins.

3. Explain the basis of the hemoglobin electrophoresis method in identifying abnormal hemoglobins.

4. Describe the epidemiology of sickle cell anemia (SCA) and other hemoglobinopathies.

5. Identify the globin chain defects causing SCA and hemoglobin C disease.

6. Associate laboratory analyses with their use in detecting and identifying hemoglobinopathies.

7. Recognize and identify abnormal laboratory test results, including peripheral blood findings and screening and confirmatory tests, typically associated with homozygous and heterozygous conditions involving HbS, HbC, HbD, HbE, and compound heterozygous conditions involving hemoglobin S and other abnormal hemoglobins.

8. List major clinical findings typically associated with the hemoglobinopathies listed in Objective 7.

Level II 1. Compare the synthesis and concentration of abnormal hemoglobins in homozygous and heterozygous conditions.

2. Compare the prevalence of hemoglobins S, C, D, and E. 3. Compare and contrast the pathophysiology of hemoglobin variants in terms of altered solubility, function, and stability.

4. Analyze the structure of the hemoglobin molecule in sickle cell anemia (SCA) and relate it to the pathophysiology of the disease.

5. Contrast clinical findings in persons who are homozygous and heterozygous for hemoglobins S, C, D, and E and in those who have compound heterozygotes for these abnormal hemoglobins.

6. Identify and explain current therapies for SCA. 7. Evaluate and interpret mobility patterns obtained on cellulose acetate and citrate agar gel hemoglobin electrophoresis when structurally abnormal hemoglobins are present.

8. Select, evaluate, and interpret tests used in detecting and identifying abnormal hemoglobins.

9. Design a laboratory testing algorithm for optimizing tests used in detecting and identifying abnormal hemoglobins.

10. Evaluate laboratory test results and medical history of a clinical case for a patient with a hemoglobinopathy and suggest a possible diagnosis.

11. Explain the physiologic abnormality resulting in unstable hemoglobins, methemoglobinemia, and hemoglobin variants with increased or decreased oxygen affinity.

12. Interpret laboratory findings associated with the disorders in Objective 11.

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Chapter 11 – Thalassemia Level I 1. Define thalassemia.

2. Differentiate thalassemias from hemoglobinopathies based on definition and basic pathophysiology.

3. Describe the typical peripheral blood morphology associated with thalassemia.

4. Compare and contrast the etiology of a- and b-thalassemia.

5. For each of the four genotypes of a-thalassemia describe the: a. Number of affected alleles b. Individuals affected c. Basic pathophysiology d. Symptoms e. Laboratory results including blood cell morphology and hemoglobin electrophoresis 6. For each of the six genotypes of b-thalassemia describe the: a. Individuals affected b. Basic pathophysiology c. Symptoms d. Laboratory results including blood cell morphology and hemoglobin electrophoresis Level II 1. List and describe five genetic defects found in thalassemias.

2. Compare and contrast a- and b-thalassemia 3. Correlate the outcomes in hemoglobin synthesis resulting from the five genetic defects in thalassemia.

4. For all four genotypes of a-thalassemia: a. Correlate all three nomenclature systems: genotype, genotype description, and phenotype. b. Explain advanced pathophysiology. c. Describe treatment and prognosis.

5. For all four phenotypes of b-thalassemia: a. List expected genotypes. b. Explain advanced pathophysiology. c. Describe treatment and prognosis.

6. Correlate clinical severities of both a- and b-thalassemia with their respective genotypes.

7. Compare and contrast other thalassemia and thalassemia-like conditions to include: a. db-thalassemia b. gdb-thalassemia c. Hemoglobin Constant Spring d. Hereditary persistence of fetal hemoglobin (HPFH) e. Hemoglobin Lepore f. Thalassemia/hemoglobinopathy combination disorders 8. Differentiate iron deficiency anemia and HPFH from thalassemia based on results of laboratory tests and clinical findings.

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Chapter 12 – Megaloblastic and Nonmegaloblastic Macrocytic Anemias Level I 1. Explain the cause and process of megaloblastic maturation in the bone marrow.

2. Describe the body’s requirements for vitamin B12 and folate and their physiologic role.

3. List the laboratory tests used to confirm a diagnosis of vitamin B12 deficiency and give expected results.

4. List the laboratory tests used to confirm a diagnosis of folic acid deficiency and give expected results.

5. Recognize the six most common disorders that result in a macrocytic anemia.

6. Name four causes of a vitamin B12 deficiency and give two distinguishing clinical or laboratory characteristics of each.

7. Describe the etiology and pathophysiology of pernicious anemia, clinical symptoms, and clinical subtypes.

8. Name three causes of a folate deficiency and give two distinguishing clinical or laboratory characteristics of each.

9. Differentiate the pathophysiology and peripheral blood findings of nonmegaloblastic anemia from those of megaloblastic anemias.

10. Summarize the common or typical blood picture seen with a folate or vitamin B12 deficiency.

Level II 1. Summarize the process of vitamin B12 and folic acid metabolism and explain how a deficiency can result in megaloblastosis.

2. Compare macrocytosis associated with a normoblastic marrow and macrocytosis associated with a megaloblastic marrow on the basis of physiological defect or cause, and differentiate based on the laboratory blood picture.

3. Summarize the mechanism of maturation defects that lead to megaloblastosis and recognize the morphologic blood cell abnormalities.

4. Compare and contrast the various clinical forms and causes of a vitamin B12 and folate deficiency on the basis of clinical symptoms and laboratory results.

5. Categorize the causes and clinical variations of pernicious anemia.

6. Compare and contrast the various clinical forms and causes of a folic acid deficiency.

7. Demonstrate how a folate or vitamin B12 deficiency results in megaloblastic maturation.

8. Choose and briefly explain four laboratory tests used to identify the cause of a macrocytic anemia; give the expected results of these four tests in a patient with an autoantibody directed against intrinsic factor.

9. Assess Schilling’s test results and provide a differential diagnosis.

10. Compare and contrast the causes of macrocytosis with a normoblastic marrow.

11. Construct an algorithm of laboratory testing to distinguish between a megaloblastic anemia and a macrocytic, normoblastic anemia.

12. Evaluate a case study from a patient with anemia. Determine the most probable diagnosis from the medical history and laboratory results.

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Chapter 13 – Hypoproliferative Anemias Level I 1. Define hypoproliferative anemia.

2. Cite the diagnostic criteria for aplastic anemia.

3. Describe the epidemiology and etiology of aplastic anemia.

4. Explain the pathophysiology of aplastic anemia.

5. Explain the difference between acquired and constitutional aplastic anemia.

6. List the major clinical and laboratory characteristics of aplastic anemia.

7. Identify environmental factors associated with the development of aplastic anemia.

8. Describe the etiology, bone marrow, and peripheral blood in pure red cell aplasia.

9. Identify peripheral blood findings associated with the following: aplastic anemia, pure red cell aplasia, and anemia due to renal disease.

Level II 1. List and explain possible causes of aplastic anemia.

2. Discuss prognosis in aplastic anemia.

3. Compare methods of treatment and management of patients with aplastic anemia.

4. Contrast aplastic anemia with other causes of pancytopenia on the basis of clinical findings and peripheral blood and bone marrow findings.

5. Compare and contrast pure red cell aplasia with aplastic anemia and other causes of erythroid hypoproliferation.

6. Compare and contrast major characteristics of Diamond-Blackfan syndrome and transient erythroblastopenia of childhood.

7. Explain the pathophysiology of anemia due to renal disease.

8. Evaluate laboratory test results and medical history of a clinical case for a patient with hypoproliferative anemia and suggest a possible diagnosis.

Chapter 14 – Introduction to Hemolytic Anemia Level I 1. List the laboratory tests that can be used to assess a hemolytic anemia and give expected results.

2. Define hemolysis and reconcile a normal hemoglobin in compensated hemolytic disease.

3. Assess laboratory results in intravascular and extravascular hemolysis.

4. Summarize the clinical findings associated with a hemolytic anemia.

5. Explain the difference between intrinsic and extrinsic erythrocyte defects and give an example of each.

Level II 1. Evaluate clinical findings of hemolytic anemia and differentiate those associated with acute and chronic disease.

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2. Compare and contrast the processes of intravascular and extravascular hemolysis and explain how laboratory results can be used to differentiate.

3. Interpret laboratory and clinical findings in hemolytic anemia and determine the type of hematologic defect present.

4. Recommend follow-up tests that could be necessary for a diagnosis of hemolytic disease.

Chapter 15 – Hemolytic Anemia: Membrane Defects Level I 1. List the hereditary membrane disorders involved with erythrocyte skeletal protein abnormalities.

2. List the hereditary erythrocyte membrane disorders involved with abnormal membrane permeability.

3. Describe the pathophysiology and recognize laboratory features associated with hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis.

4. Discuss the principle of the osmotic fragility test and interpret the results.

5. Describe the etiology, pathophysiology, and laboratory features of paroxysmal nocturnal hemoglobinuria (PNH).

6. Explain the principle of the sugar water (sucrose hemolysis) test and interpret its results.

Level II 1. Differentiate the protein defects associated with each hereditary membrane defect discussed in the chapter.

2. Create a flow chart using laboratory tests to differentiate hereditary spherocytosis, elliptocytosis, pyropoikilocytosis, stomatocytosis syndromes, and paroxysmal nocturnal hemoglobinuria.

3. Explain the role of decay-accelerating factor (DAF) and membrane inhibitor of reactive lysis (MIRL) in PNH. 4. Explain the results of the sucrose hemolysis test and immunophenotyping to determine a diagnosis of paroxysmal nocturnal hemoglobinuria (PNH).

5. Evaluate a clinical case study and determine the type of membrane disorder present by correlating clinical history and laboratory features.

Chapter 16 – Hemolytic Anemia: Enzyme Deficiencies Level I 1. Identify the two main pathways by which erythrocytes catabolize glucose.

2. Explain the role of erythrocyte enzymes in maintaining the cell’s integrity, and describe how deficiencies in these enzymes lead to anemia.

3. Identify the most common erythrocyte enzyme deficiency.

4. Describe the inheritance pattern for glucose-6-phosphate dehydrogenase (G6PD).

5. Explain how the diagnosis of G6PD deficiency is made.

6. List the tests used to detect G6PD deficiency and describe their principles.

7. Recognize the erythrocyte morphology in a Romanowsky-stained blood smear associated with G6PD deficiency.

8. Identify common compounds that induce anemia in G6PD deficiency.

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Level II 1. Recommend appropriate laboratory testing and interpret results for suspected G6PD deficiency following a hemolytic episode.

2. Explain the function of glutathione in maintaining cellular integrity.

3. Associate the mechanisms of hemolysis with defects in the glycolytic and hexosemonophosphate shunt pathways.

4. Correlate clinical and laboratory findings with the common G6PD isoenzyme variants.

5. Diagram the reaction catalyzed by pyruvate kinase, and explain how a defect of this enzyme can cause hemolysis.

6. Recognize erythrocyte morphology associated with pyruvate kinase deficiency.

7. Review and interpret laboratory findings in a case study presentation of G6PD deficiency.

Chapter 17 – Hemolytic Anemia: Immune Anemias Level I 1. List the antibody systems or specificities usually involved in: a. cold agglutinin syndrome b. paroxysmal cold hemoglobinuria (PCH) c. warm autoimmune hemolytic anemias (WAIHA) 2. Describe the DAT, its purpose and identify the typical DAT profile (polyspecific AHG, anti-IgG, anti-C3) found in patients with cold agglutinin syndrome and warm autoimmune hemolytic anemia.

3. Describe and recognize the characteristic hematologic findings associated with the following conditions: a. cold agglutinin syndrome b. warm autoimmune hemolytic anemia (WAIHA) 4. Describe and recognize the typical hematologic laboratory findings in ABO and Rh hemolytic disease of the fetus and newborn (HDFN).

5. Contrast alloimmune and autoimmune hemolytic anemias including stimulus for antibody production, site of hemolysis, type of antibody involved, and direct antiglobulin test (DAT) and indirect antiglobulin test (IAT) reactions.

6. List the most common antibody specificities involved in immediate and delayed hemolytic transfusion reactions.

Level II 1. Compare the pathophysiology of extravascular and intravascular hemolysis in immune hemolytic anemia including site of destruction, immunoglobulin class of antibody, and underlying mechanism.

2. Given a set of laboratory data, determine the underlying mechanism of hemolysis and suggest confirmatory tests.

3. Contrast the different mechanisms of drug-induced immune hemolytic anemia and identify the drugs commonly involved.

4. Describe the mechanism of hemolysis, select the confirmatory tests for paroxysmal cold hemoglobinuria (PCH), and evaluate the results.

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6. Interpret the results of laboratory tests for HDFN and determine whether evidence of hemolysis is present.

7. Compare and contrast the pathophysiology and clinical findings of an immediate transfusion reaction with those of a delayed reaction.

8. List the causes of the secondary types of cold agglutinin syndrome and warm autoimmune hemolytic anemia and identify key laboratory results linking the cause and the autoimmune condition.

9. Compare the typical laboratory findings in immediate versus delayed transfusion reactions.

Chapter 18 – Hemolytic Anemia: Nonimmune Defects Level I 1. Define microangiopathic hemolytic anemia (MAHA); list several associated disorders and the age group most commonly affected.

2. Describe the general morphology and hematologic values associated with MAHA and criteria that distinguish disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS).

3. Recognize the characteristic erythrocyte morphology of MAHA on a stained blood film.

4. Identify several organisms that can cause erythrocyte hemolysis.

Level II 1. Summarize the general pathophysiology for MAHA. 2. Compare and contrast the clinical findings, underlying cause, treatment, and characteristic findings for erythrocytes, platelet count, and coagulation tests for each of the following types of MAHA: a. Hemolytic uremic syndrome (HUS) b. Thrombotic thrombocytopenic purpura (TTP) c. Disseminated intravascular coagulation (DIC) 3. Define exercise-induced hemoglobinuria.

4. Given a set of data and clinical history, determine whether MAHA is a probable diagnosis, or the possible etiology, and propose follow-up tests that should be performed.

5. Compare the cause of hemolysis by the following infectious agents: a. Plasmodium parasites (malaria) b. Babesia parasites c. Bartonella bacteria d. Clostridium bacteria

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation 58

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to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Create/Construct a. Have the students create a complete case study to reflect an aplastic anemia that includes clinical and laboratory characteristics. b. Have the students create a complete case study to reflect a hemolytic anemia that includes clinical and laboratory characteristics. c. **Have the students create a high cognitive level (Taxonomy 2 or 3) test question concerning skeletal protein abnormalities. d. **Have each student create a complete case study (pathophysiology, clinical findings and laboratory findings, therapy) for one of the hemolytic anemias caused by membrane defects.

After each case is created, have students exchange their cases with other students in the class who then have to solve the new case study by the next class period.

3. Diagram a. The transport of iron from ingestion to incorporation into heme b. Draw images that represent the process of extravascular hemolysis including the breakdown of hemoglobin.

4. Extra, Extra, Read All About It a. Write a newspaper article describing the life of the Macrocytosis sisters, Megaloblastic (known as “Meg”) and Nonmegaloblastic (known as “Nonmeg”). Include the ways they are different and alike.

5. Group Discussion Create a group discussion regarding the: a. Appearance of the peripheral smear for all of the different types of anemias b. Explanation of nuclear-cytoplasmic asynchrony c. Causes for Vitamin B12 deficiency and folic acid deficiency d. **Appearance of the bone marrow for all of the different types of anemias e. **Pathophysiology and characteristics of hemoglobin variants such as HbM, Lepore, and unstable hemoglobins such as Hb Koln and Hb Hammersmith. f. **The differentiation between immune and nonimmune hemolytic anemias g. Intravascular versus extravascular cell destruction • The instructor may also consider giving a case study for students to evaluate and analyze prior to this discussion.

6. Hematology Bowl The instructor should create the following jeopardy categories: a. Red cell morphology b. Red cell inclusions c. Macrocytic anemias d. Microcytic/hypochromic anemias e. Heme synthesis disorders f. Thalassemias g. Hemoglobinopathies h. Laboratory features of anemias i. Megaloblastic and nonmegaloblastic anemias j. Hypoproliferative anemias 7. Mapping Create a map for: a. Variations in red blood cell shape b. RBC inclusions c. Iron deficiency d. Hemoglobinopathies ..

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e. Anemias characterized by intravascular hemolysis f. Hemolytic anemias • Or have the students map each of the categories of hemolytic anemias • Membrane defects • Enzyme deficiencies • Immune anemias • Nonimmune defects 8. Match-Up Game Create a concentration game for the: a. Type of cell shape variation with a description of its appearance, e.g. cigar-shaped RBC = elliptocyte b. Type of anemia with the appearance of the red cells on the peripheral smear, e.g. iron deficiency = microcytic/hypochromic c. Types of hemolytic anemias and their corresponding causes and/or characteristics, for example, include • Hereditary spherocytosis • Hereditary elliptocytosis • Hereditary pyropoikilocytosis • Hereditary stomatocytosis • Hereditary xerocytosis • Abetalipoproteinemia • G6PD deficiency • Pyruvate kinase deficiency • Warm autoimmune hemolytic anemia • Cold autoimmune hemolytic anemia • Alloimmune hemolytic anemia • Microangiopathic hemolytic anemia 9. Mystery Box a. On separate pieces of paper write the • Name of variant types of hemoglobin, e.g. HbS • Peripheral blood results, e.g. normocytic, normochromic, reticulocytosis, etc.

• Electrophoresis, e.g. Homozygous = HbS, F, A2 • Genetic mutation, e.g. B6 (A3) Glu : Val Have the students combine all of the appropriate components together, then create a case study using the above information and adding medical background for an associated disease condition. b. Create a mystery box for each of the four genotypes of alpha thalassemia. On separate pieces of paper give the following specific information and have the students compile the information then identify the alpha thalassemia.

• Number of affected alleles • Individuals affected • Basic pathophysiology • Symptoms • Laboratory results, including blood cell morphology and hemoglobin electrophoresis • **treatment and diagnosis Note: an alternate exercise is to perform the same mystery box for beta thalassemia. c. Create a mystery box with each of the following components listed on separate pieces of paper. Have a small group of students combine the appropriate components then identify the disease state.

• Type of hemolytic anemia • Laboratory findings 60

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• Clinical findings • Source of defect • **Therapy (optional) 10. One-Minute Paragraph Write a one-minute paragraph on the: a. General characteristics of each type of anemia discussed • this could be done at the conclusion of each lecture b. Three stages of iron deficiency and characteristic RBC morphology c. Differences between major and minor thalassemias d. How the Schilling test is used to distinguish Vitamin B12 deficiency due to malabsorption, dietary deficiency, or absence of intrinsic factor e. Each type of hemolytic anemia • Hereditary spherocytosis • Hereditary elliptocytosis • Myeloperoxidase deficiency • Autoimmune hemolytic anemia • Hemolytic disease of the newborn • Infectious agents • Burns • And so on f. **The different laboratory tests • Fluorescent spot test • Dye reduction test • Ascobate cyanide test g. Mechanisms of hemolysis h. **Causes for positive DAT in normal individuals i. **Underlying disorders most frequently associated with secondary AIHA 11. Mnemonic Have each learner develop and share a mnemonic device to learn the: a. Types of red cell inclusions (e.g., Papa Made Charlie Hand Josie Several Hangers for pappenheimer, malaria, cabot ring, howell-jolly, stippling, heinz) b. Variations in red cell shape c. Types of hemoglobin variants d. Different types of anemia found in each chapter e. Other thalassemias (besides a and b ) and thalassemia-like conditions • db-thalassemia • gdb-thalassemia • Hemoglobin Constant Spring • Hereditary persistence of hemoglobin • Hemoglobin Lepore f. Types of organisms that can cause red blood cells to lyse (e.g., Mama Caused Baby’s Blues: Malaria, Clostridium, Babesia, and Bartonella) g. **Types of Microangiopathic hemolytic anemia h. Nonimmune causes of red cell destruction 12. Short Story Have the learners prepare a short story on: a. How red cells become sickle shaped in a sickle cell anemia patient b. Red cell intravascular and extravascular destruction c. **Red cell destruction using one of the MAHA causes 13. Small Group Teaching Assign a small group of learners the following topics. They should be instructed to present and teach these concepts to the rest of the class. a. **Reticulocyte count, methods for counting reticulocytes and quantitation of reticulocytes ..

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b. Compare and contrast the etiology of alpha and beta thalassemia c. G6PD Deficiency d. **Differentiate Cold Agglutinin Disease from Benign Cold Agglutinins e. Hemolytic uremic syndrome 14. Think-Pair-Share a. The appearance of the peripheral smear for various types of anemias (i.e., B12/folate, iron deficiency, and beta thalassemia) b. **The appearance of the bone marrow for various types of anemias c. ** Laboratory findings for the following as it relates to hemoglobinopathies • Bone Marrow • Hemoglobin Electrophoresis • Solubility Test • Sickling Test d. **Discuss the various types of microangiopathic hemolytic anemias. e. The cause of the anemia • **Discuss the causes of membrane defects, that is, differentiate inherited (HS) and acquired (PNH).

• **Compare the prenatal and postnatal pathophysiology of hemolytic disease of the newborn. f. Reticulocytosis as it relates to hemolytic anemias g. Explain how elliptocytes are formed. h. **Explain the differential diagnosis of hereditary spherocytosis and the presence of spherocytes from warm AIHA. 15. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

16. Tic-Tac-Toe a. A game of tic-tac-toe can be created for anemias in general or on specific chapter content such as: • physiologic factors that affect the amount of iron needed by the body • pathophysiology of iron deficiency, anemia of chronic disease, and sideroblastic anemia • blood features and iron studies associated with iron deficiency anemia, anemia of chronic disease, lead poisoning and sideroblastic anemia • effects of disease on tissue iron • **bone marrow analysis in iron deficiency anemia contrasted with sideroblastic anemia and anemia of chronic disease • **functions of the proteins involved in iron metabolism • **classification and characteristics of the porphyrias b. A tic-tac-toe game can be created using the characteristics of hemoglobinopathies and thalassemias.

D.

LABORATORY ACTIVITIES

Perform the following suggested laboratory activities: 1. Manual hemoglobin, hematocrit, and red cell counts • These values can be compared with the values obtained with a hematology analyzer.

2. RBC indices calculations using the results found in #1.

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3. Manual reticulocyte count.

• Calculate the corrected reticulocyte and reticulocyte production index.

4. Manual differentials on selected anemias.

• Including thalassemia, folate or B12 deficiency, aplastic anemia, and iron deficiency anemia • Differential results should check within the acceptable range of results that were obtained by the instructor or clinical facility.

• **A comparison between different types of anemias should also be done. The comparisons should include beta-thalassemia and iron deficiency anemia.

5. Scattergrams evaluation from the hematology instrument 6. **A hypoplastic bone marrow evaluation 7. **Myeloid:erythroid ratio calculation 8. **Hemoglobin electrophoresis using samples of sickle cell anemia and beta-thalassemia • If samples are not available, the student should evaluate the gel patterns or the densitometer patterns obtained from prepared samples.

9. Manual differentials on patients with aplastic anemia, iron deficiency anemia, beta-thalassemia, lead poisoning, B12/folate deficiency, and blood loss • **Student should make a tentative diagnosis and determine what additional testing should be performed to confirm the diagnosis. This may be done using the textbook and other reference books as a guide.

10. Differential on a patient with increased nucleated red cells. Correct WBC if appropriate.

11. **Special stains for iron and Heinz bodies could be performed as time permits.

12. Sickle cell screening test 13. Differential on a patient with hereditary spherocytosis and a microangiopathic hemolytic anemia 14. **Special stains that include the fluorescent spot test for G6PD and a test for pyruvate kinase deficiency 15. ** Ham test for PNH 16. **Donath-Landsteiner test for PCH 17. **Osmotic fragility test for hereditary spherocytosis

E.

PRACTICE QUESTIONS

1. List 3 conditions that could cause a disproportionately low (relative increase in plasma volume) hematocrit. [Taxonomy 1] 2. The type of cell development in folic acid deficiency is _____. [Taxonomy 1] 3. Match the following red cell inclusions with their composition: [Taxonomy 1] Red Cell Inclusion a. Heinz body b. Howell-Jolly body c. Pappenheimer body d. Spindle fiber

Composition 1. DNA 2. Hemoglobin 3. Iron

4. True or False: A Hb value of 11.8 g/dL and Hct of 35% is considered anemic for a 7-year-old male?

[Taxonomy 2] ..

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5. The following was observed on a peripheral smear: red cells microcytic, hypochromic. There were no other abnormalities observed. This would be consistent with: [Taxonomy 2] a. Thalassemia b. Folate deficiency c. Aplastic anemia d. Iron deficiency anemia 6. **Discuss the appearance of the bone marrow in aplastic anemia and B12 deficiency. Correlate the appearance with the appearance of the peripheral smear. [Taxonomy 2] 7. The two hemoglobin variants in an alkaline electrophoretic procedure that have the same positive charge are: [Taxonomy 2] a. HbS and HbC b. HbC and HbE c. HbS and HbE d. HbD and HbC 8. A 56-year-old female patient has the following iron studies results. [Taxonomy 2] Hemoglobin = 10.0 g/dL MCV = 85 fL Serum Iron = 40 ug/dL TIBC = 261 ug/dL Ferritin = 220 ug/L % Saturation = 15% Transferrin = 5.70 mg/L a. Calculate the UIBC b. These results are generally consistent with what type of condition?

9. **Compare and contrast the laboratory findings for beta-thalassemia major and hemoglobin Lepore.

[Taxonomy 3] 10. A 3-year-old boy of Greek decent was admitted with a hemoglobin level of 8 gm/dL. The appearance of the red cells on the smear were microcytic and hypochromic. What additional laboratory tests would you expect to be abnormal? What would you expect to find? Explain. [Taxonomy 3] 11. **Compare and contrast the bone marrow and peripheral blood appearance of aplastic anemia and pure red cell aplasia. [Taxonomy 3] 12. The defect that is found in hereditary spherocytosis is _____. [Taxonomy 1] 13. Match the following type of hemolytic anemia with the cause. Answers may be used more than once [Taxonomy 1] Hemolytic Anemia a. Hereditary Elliptocytosis b. HDN c. Cold Hemagglutinin Disease d. Myeloperoxidase Deficiency

Cause 1. Enzyme defect 2. Membrane defect 3. Immune destruction 4. Nonimmune destruction 5. None of the above causes

14. The test performed at delivery that would detect if a baby had HDN would be the _______.

[Taxonomy 1] 15. Discuss the importance of the membrane structure and enzymes in the inherited hemolytic anemias. [Taxonomy 2] 16. Discuss the coagulation test abnormalities found in DIC. [Taxonomy 2] 17. Identify and explain the mechanisms that could cause a drug to destroy a red cell. [Taxonomy 2]

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18. **A 5-year-old girl was admitted to the hospital with a fever and back pain. Upon examination of the peripheral smear, burr cells, nucleated red cells, and schistocytes were observed. Which of the following tests is most likely normal? [Taxonomy 3] a. Haptoglobin b. Creatinine c. Urine hemoglobin d. Platelet count 19. **Compare and contrast HUS and PNH. [Taxonomy 3] 20. **Compare and contrast autoimmune hemolytic anemia and hemolytic transfusion reactions [Taxonomy 3] Case Study #1 A 55-year-old Caucasian male, LT had been well up until four months ago. He started to feel tired and had no energy. LT went to his physician where a complete blood count was done. The following results were obtained: Hb 10 grams/dL, Hematocrit 31%, red count 4.2 * 1012/L, MCV was 73.8fL. The white cell count and platelet count were normal. A manual differential was requested. The red cells were slightly microcytic and hypochromic. Some of the red cells had coarse basophilic stippling. Answer the following questions regarding this case study: 21. What is basophilic stippling? [Taxonomy 1] 22. In what types of anemia may basophilic stippling be found? [Taxonomy 1] 23. What is the most probable cause of the anemia? [Taxonomy 2] 24. What tests should be ordered to confirm the suspected diagnosis of this patient? [Taxonomy 2] 25. What would the results be for the tests identified in the previous question? [Taxonomy 1] Case Study #2 MD, a 65-year-old female, went to her physician because she was tired. Upon doing a CBC, it was noted that her hemoglobin was 8.8 g/dL and her hematocrit was 29%. The red cells had a normal appearance. Her physician ordered the following tests: iron, TIBC, ferritin, B12, and folate. All these tests were within normal limits.

26. List two additional possibilities for a cause of her anemia. [Taxonomy 2] 27. For each additional cause, list one test that should be used to determine the type of anemia present. [Taxonomy 1]

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The Anemias—Level I (Nonhemolytic Anemias) Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

C M S I L O H O C L A X A B L S H I B D N T V K C K U R I E W I J V M T X W P Y V A M D N T P C M S N O N H L N E P P Z E Y R K Z R I W R H B L V B T K P C N L X R M S H H U M R M N J O O L E B L A Y O O C M K L C F T N R C V L L L R T W O N N N H Y A V E J W A K W L Y G P I R T C P G L B N B N C R Z C T Y N M N E H L B F O N M Q T I O B H B A R T T J M C L G J R T A L L V P D H R K M Y X N R V R P K I W C Z M A K R C Z E K T C C I N K Y J X I V L T F M S W B J W C F I W L T A P L A S T I C X N X A C O W P D P H A G O C Y T O S I S M P K • another name for Vitamin B12 • RBC alignment resembling a stack of coins • cell process in which cell engulfs foreign substances • an unusual craving for ingesting unnatural items as food • disease that has both HbA and HbS (3 words) • Hb with 4 gamma chains • common cause of nonanemic macrocytosis • RBC with larger than normal central pallor • another name for a sickle cell • storage form of iron • type of anemia characterized by pancytopenia • condition of increased iron stores • ratio of hemoglobin mass to volume • variation in RBC shape • decrease in all blood cells

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The Anemias—Level I (Nonhemolytic Anemias) C M S I L O H O C L A X A H I B D N T V K C K U R I J V M T X W P Y V A M D N M S N O N H L N E P P Z E Z R I W R H B L V B T K P X R M S H H U M R M N J O B L A Y O O C M K L C F T V L L L R T W O N N N H Y J W A K W L Y G P I R T C B N B N C R Z C T Y N M N B F O N M Q T I O B H B A J M C L G J R T A L L V P K M Y X N R V R P K I W C K R C Z E K T C C I N K Y

B L S E W I T P C Y R K C N L O L E N R C A V E P G L E H L R T T D H R Z M A J X I

H E M O C H R O M A T O S I

V L T F M S W B J W C F I W L T S A P L A S T I C X N X A C O W P X D P H A G O C Y T O S I S M P K P • another name for Vitamin B12 (cobalamin) • RBC alignment resembling a stack of coins (rouleaux) • cell process in which cell engulfs foreign substances (phagocytosis) • an unusual craving for ingesting unnatural items as food (pica) • disease that has both HbA and HbS (3 words) (sickle cell trait) • Hb with 4 gamma chains (barts) • common cause of nonanemic macrocytosis (alcoholism) • RBC with larger than normal central pallor (hypochromic) • another name for a sickle cell (drepanocyte) • storage form of iron (ferritin) • type of anemia characterized by pancytopenia (aplastic) • condition of increased iron stores (hemochromatosis) • ratio of hemoglobin mass to volume (mchc) • variation in RBC shape (poikilocytosis) • decrease in all blood cells (pancytopenia)

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The Anemias—Level I (Nonhemolytic Anemias) 1

2

3

4

6

5

7 8 9 10

11 12

13

14

15

ACROSS

DOWN

3 Type of destruction when RBCs are destroyed in the spleen 4 Lead poisoning may demonstrate this RBC inclusion (2 words) 7 Disease characterized by quantitative defect in hemoglobin production 10 Megaloblastic anemia resulting from a lack of intrinsic factor (2 words) 14 Description of RBC where MCV = 94 fL 15 Macrocytic anemias are generally classified as _____

1 Iron deposits in erythrocytes (2 words) 2 First nonnucleated stage of RBC development in the bone marrow 5 Major long-term storage form of iron 6 Disease that results from an inherited abnormality of the structure or synthesis of the globin portion of the Hb molecule 8 Protein that binds and transports iron in the blood 9 Type of anemia caused by Vit B12 deficiency 11 Teardrop cells are also known as _____ 12 Description of RBC size in Iron Deficiency 13 Group of inherited disorders characterized by a block in porphyrin synthesis

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The Anemias—Level I (Nonhemolytic Anemias) P R E X T R A V A S C U L A R E P T B A S O P H I L I C S T I P P L I N G E E C H T H A L A S S E M I A N U E O T H L M S R M E O O P E R N I C I O U S A N E M I A C G D N G M Y L D E S A E T O M A R P F L R E B I C I O E O B I C R N R R B O N O R M O C Y T I C P R L D O O O H I A I P C C Y N S E A Y T R T S T T Y I I H I M E G A L O B L A S T I C Y C S S

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The Anemias—Level II (Nonhemolytic Anemias) Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

X L C F J M W D R K T L H I M A C R O C Y T I N T R I N S I C F A V T S J Z X P F M P T A R E K D S Q O W K P C M R P I N E K R J F H Z C T O C L T R P Y L W A Z L X V R Y C H O V O P P R Q K O C L R N R L I M J L K I O H B E H D P E M N M A

Z L Q M X I C G M L C T O R J L M F Q D Q E W E H T C M L N X Y M I H E G M W F Y A W C Q L R H M C Z T I T R

Y D R I A

H Q M Q C

K B C Q P

D I S R T

R N R M O N Q O N J

Y K I R R

K T V X T

A K C R B

T B O I C P M R T M

T L F X K

I A L A S

• number of chromosomes in somatic cells that is 2n • granulocytic precursor with first presence of azurophilic granules • needed for the absorption of Vitamin B12 (2 words) • group of inherited disorders characterized by a block in porphyrin synthesis • sex-linked SA is due to an abnormality in this enzyme (abrv) • neurological disease in vitamin B12 deficiency is due to this • calculated indices of anisocytosis (abrv) • cells with more than two concavities • test to determine effective red cell production (abrv) • the absence of hydrochloric acid in stomach gastric secretions • a defect in iron utilization • another name for Vit B12 • type of anemia found with abnormal liver function

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T N H V H

A X K K N

R S Y K K

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The Anemias—Level II (Nonhemolytic Anemias) X L C F J M W D R K T L H I M A C R O C Y T I N T R I N S I C F A V T S J Z X P F M P T A R E K D S Q O W K P C M R P I N E K R J F H Z C T O C L T R P Y L W A Z L X V R Y C H O V O P P R Q K O C L R N R L I M J L K I O H B E H D P E M N M A

Z L Q M X I C G M L C T O R J L M F Q D Q E W E H T C M L N X Y M I H E G M W F Y A W C Q L R H M C Z T I T R

Y D R I A

H Q M Q C

K B C Q P

D I S R T

R N R M O N Q O N J

Y K I R R

K T V X T

A K C R B

T B O I C P M R T M

T L F X K

I A L A S

T N H V H

A X K K N

R S Y K K

• number of chromosomes in somatic cells that is 2n (diploid) • granulocytic precursor with first presence of azurophilic granules (promyelocyte) • needed for the absorption of Vitamin B12 (2 words) (intrinsic factor) • group of inherited disorders characterized by a block in porphyrin synthesis (porphyrias) • sex-linked SA is due to an abnormality in this enzyme (abrv) (alas) • neurological disease in vitamin B12 deficiency is due to this (demyelination) • calculated indices of anisocytosis (abrv) (rdw) • cells with more than two concavities (knizocytes) • test to determine effective red cell production (abrv) (rpi) • the absence of hydrochloric acid in stomach gastric secretions (achlorhydria) • a defect in iron utilization (sideroacrestic) • another name for Vit B12 (cobalamin) • type of anemia found with abnormal liver function (macrocytic)

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The Anemias—Level II (Nonhemolytic Anemias) 1

2

3

4

5 6

7

8

9

10

11

12

13

14

15

ACROSS

DOWN

1 Type of test to definitively distinguish vitamin B12 deficiency due to malabsorption, dietary deficiency, or absence of IF. 5 Nuclear characteristic of neutrophils found in patients with pernicious anemia 6 A calculated index that can be used as an early indicator of response to iron therapy in IDA (abrv) 8 The failure of hematopoietic cells to generate and develop in the bone marrow 10 Anemia characterized by the triad of macrocytosis, Howell-Jolly bodies, and hypersegmented PMNs 11 Decreased production of any cell type 14 Type of anemia associated with diffuse cancer of the prostate, breast, and stomach 15 Rapid test for HbS in the heterozygous or homozygous state

2 Disorders that have an absence of α and β chain synthesis that is compensated by increased γ chain production into adult life 3 Master iron regulating hormone; acts by inhibiting activity of genes involved in intestinal iron transport 4 Extensive splenic damage secondary to infarction 7 Autosomal recessive disorder characterized by abnormal chromosomal fragility (2 words) 9 Cellular protein that combines with iron to form ferritin 12 Acquired stem cell disease in which a blood cell membrane abnormality makes the cell susceptible to in vivo complement hemolysis (abrv) 13 One or two or more genes that correspond to the same trait and occupy the same position on paired chromosomes

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The Anemias—Level II (Nonhemolytic Anemias) S C H I L L I N G P F H A H Y P E R S E G M E N T E D U I R F P T A C O N A P L A S I A I S C P D P O M E G A L O B L A S T I C L N F N E I E N A H Y P O P R O L I F E R A T I V E N N R L C M Y E L O P H T H I S I C L T M T E O I S O L U B I L I T Y L M A N E Y

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The Anemias—Level I (Hemolytic Anemias) Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

C H A I R U N I R E D I I M W M H K L H K T G R T V C N M E W E I N D I Y T K M G Q M M D E K N C T H R O M B O C Y T O O Z Y J L N B G L J M X M G H X L Q T L M Y S L R G F K W D N O N C S M O R C R R F D B H L Z I N B D Y B M G I X W F P L K Q X T H S N G G K L D C G T N T G U J N L L M G H K O N J R V B V E I T M C R Z T I J G J H G Q Y H M V T A M L G N K T K B T C J J Y R N I E J Q J K Q B T W N D Q

S N R I P H F R L S D L Z T C K Z

O M E H K P L N E C T H L X E S E N I A N N A N K I L W E B L Q R O O C N L I Q I G M B C O M F T T U Y E P N F R A E Y U H N T S T V K

• type of syndrome found with obstetric complication (abrv) • type of MAHA with decreased fibrinogen level • platelet finding in TTP • destruction of erythrocytes; release of hemoglobin • presence of hemoglobin in the urine • type of red cell found in MAHA • inheritance pattern for G6PD deficiency (2 words) • RBC index elevated in hereditary spherocytosis (abrv) • protein that is decreased in intravascular hemolysis • most warm AIHA are associated with this type of antibody (abrv) • type of bilirubin increased in hemolytic anemias • presence of iron in the urine • HDFN is classified as a _____ disease • excess unconjugated bilirubin may lead to _____ • type of anemia in HUS (RBC size)

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The Anemias—Level I (Hemolytic Anemias) C H A I R U N I R E D I I M W M H K L H K T G R T V C N M E W E I N D I Y T K M G Q M M D E K N C T H R O M B O C Y T O O Z Y J L N B G L J M X M G H X L Q T L M Y S L R G F K W D N O N C S M O R C R R F D B H L Z I N B D Y B M G I X W F P L K Q X T H S N G G K L

S N R I P H F R L S D

O M E H K P L N E C T H L X E S E N I A N N A N K I L W E B L Q R O O C N L I Q I G M B

D C G M G H I T M G Q Y K T K E J Q

L Z T C K Z

C T E R U S

T K C H B J

N T O N R Z M V T C K Q

G J T T J B

U J N R V B I J G A M L J Y R T W N

L V J G N D

L E H N I Q

O M T U P N A E H N T V

F Y F Y T K

• type of syndrome found with obstetric complication (abrv) (hellp) • type of MAHA with decreased fibrinogen level (dic) • platelet finding in TTP (thrombocytopenia) • destruction of erythrocytes; release of hemoglobin (hemolysis) • presence of hemoglobin in the urine (hemoglobinuria) • type of red cell found in MAHA (schistocyte) • inheritance pattern for G6PD deficiency (2 words) (sex linked) • RBC index elevated in hereditary spherocytosis (abrv) (mchc) • protein that is decreased in intravascular hemolysis (haptoglobin) • most warm AIHA are associated with this type of antibody (abrv) (igg) • type of bilirubin increased in hemolytic anemias (indirect) • presence of iron in the urine (hemosiderinuria) • HDFN is classified as a _____ disease (alloimmune) • excess unconjugated bilirubin may lead to _____ (kernicterus) • type of anemia in HUS (RBC size) (normocytic)

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The Anemias—Level I (Hemolytic Anemias) 1

2

3 4 5

6 7

8

9

10

11

12

13

14

15

ACROSS

DOWN

1 This type of hemolysis is associated with spherocytes 3 Protozoan disease that causes hemolysis after an individual is bitten by a tick 6 These intracellular bodies are found in G6PD deficiency 8 Hereditary _____ is a type of hemolytic anemia characterized by a deficiency of spectrin 9 Syndrome characterized by the presence of clumps of erythrocytes on the blood smear (2 words) 10 Test which will detect antibodies of the IgG class and/or complement (2 words) 12 A cold reacting IgG class antibody is associated with this hemolytic anemia (abrv) 13 The sucrose hemolysis test is a screening test for this disease (abrv) 14 A hemolytic anemia caused by an immune reaction against self-antigens 15 This type of hemolysis occurs within the blood vessels

2 This therapy may result in a delayed or immediate immune reaction 4 Presence of excessive hemoglobin in the plasma 5 The most common erythrocyte enzyme deficiency is ____ (abrv) 7 This clinical condition is associated with an increase in heme catabolism 11 Deficiency of oxygen to the cells

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The Anemias—Level I (Hemolytic Anemias) E X T R A V A S C U L A R R B A B E S I O S I S N H S G H E I N Z F 6 M J U S P H E R O C Y T O S I S A S D G U I C O L D A G G L U T I N I N O O D A N T I H U M A N G L O B U L I N I Y I C P C H P N H E O E X A U T O I M M U N E I I A I N T R A V A S C U L A R

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The Anemias—Level II (Hemolytic Anemias) Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

C N N R A L U C S E N K F X G Z Z E N W N N L D R F I O M K W N C R K D I H A P T O G L O H Y T D D R R Y B T Q F A T M R W I A H T A Y K M J T T D T R V U V D N U N F J I I W P A L R C B Z R S M O G N C Q Q A O M T P M F M H T L G U R R Z A S M W G A L M M E T Y C O T L T R I A N G U L

A V A R K N K G P H Y M Y W Z X B I N R F C M Y T B C T N O R B T Z B K F C N C V Z R I C I R K K C G T G C T M S I H C O C Y T

T D F T T F J F X V N E R N S E

• antibodies against their own erythrocytes • type of erythrocyte found in MAHA • RBC with slit-like area of pallor • serum glycoprotein that transports free plasma hemoglobin to the liver • fragmented RBC that are triangle shaped • sensitivity to Vicia faba beans • alloimmune disease caused by feto-maternal blood group incompatibility (abrv) • test used to help diagnose warm AIHA (abrv) • protects the RBC from oxidant damage • these bodies are pitted from the erythrocyte to form bite cells • a type of hemolytic anemia caused by microcirculatory lesions (abrv) • antibody class associated with cold agglutinins (abrv) • a type of RBC found in immune hemolytic anemias • type of hemolysis associated with march hemoglobinuria • the reserve iron binding capacity of transferring (abrv)

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The Anemias—Level II (Hemolytic Anemias) C N N R A E N K F X N W N N L O M K W N I H A P T H Y T D D T Q F A T A H T A Y T D T R V U N F J I L R C B Z

L U C S G Z Z E D R F I C R K D O G L O R R Y B M R W I K M J T U V D N I W P A R S M O

G N C Q Q A O M P M F M H T L G R R Z A S M W G L M M E T Y C O L T R I A N G U

T U A T L

A V A R K N K G P H Y M Y W Z X B I N R F C M Y T B C T N O R B T Z B K F C N C V Z R I

T D F T T F J F X V N

N I H C R S E N T S C P K H M E M R T O X C

C K G S O

E R N S E

V H N Q S

I C C I C

R K G T T M H C Y T

Y T E P T

• antibodies against their own erythrocytes (autoantibodies) • type of erythrocyte found in MAHA (schistocyte) • RBC with slit-like area of pallor (stomatocytes) • serum glycoprotein that transports free plasma hemoglobin to the liver (haptoglobin) • fragmented RBC that are triangle shaped (triangulocytes) • sensitivity to Vicia faba beans (favism) • alloimmune disease caused by feto-maternal blood group incompatibility (abrv) (hdn) • test used to help diagnose warm AIHA (abrv) (dat) • protects the RBC from oxidant damage (glutathione) • these bodies are pitted from the erythrocyte to form bite cells (heinz) • a type of hemolytic anemia caused by microcirculatory lesions (abrv) (maha) • antibody class associated with cold agglutinins (abrv) (igm) • a type of RBC found in immune hemolytic anemias (spherocyte) • type of hemolysis associated with march hemoglobinuria (intravascular) • the reserve iron binding capacity of transferring (abrv) (uibc)

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The Anemias—Level II (Hemolytic Anemias) 1

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6 7

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1 Hemolytic anemia associated with an episode of gastroenteritis (3 words) 3 Hereditary spherocytosis may be confirmed by the ___ test (2 words) 5 ____ fever is found in patients who are infected with P. falciparum which is complicated by the presence of massive acute intravascular hemolysis 7 This test helps differentiate CHD from PCH (2 words) 9 The injection of this substance into Rh-negative mothers prevents isoimmunization (abrv) 12 Type of hemolytic anemia which occurs as a result of antibody development to an RBC antigen that the individual lacks 14 Transient hemolytic anemia after strenuous exercise involving contact with a hard surface (2 words) 15 Disorders that affect the composition of the membrane lipid bilayer lead to the formation of

2 Formation of bilestones in the gallbladder or the bile duct 4 Nonimmune infectious agent that causes a direct parasitization of erythrocytes (species) 6 In infections with Streptococcus pneumonia the bacterial enzyme responsible for the capillary damage 8 Antihypertensive drug which can cause a positive DAT 10 Immune mediated process when RBC are destroyed prematurely (abrv) 11 This clinical condition is associated with an increase in heme catabolism 13 Test used to detect antibodies in the serum of an individual (abrv)

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The Anemias—Level II (Hemolytic Anemias) H E M O L Y T I C U R E M I C S Y N D R O M E H O S M O T I C F R A G I L I T Y P L B L A C K W A T E R A L S I N M D O N A T H L A N D S T E I N E R O H L U D I D R H I G I J A L L O I M M U N E A H U A S M M A M U I E I N S T N D I I I D M A R C H H E M O G L O B I N U R I A T E S A C A N T H O C Y T E S

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4 Nonmalignant Disorders of Leukocytes

A.

TRANSITION GRID

Information concerning Chapter 19, “Nonmalignant Granulocyte and Monocyte Disorders” and Chapter 20, “Nonmalignant Lymphocyte Disorders” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 19

Chapter 20

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 21 Chapter 15 Chapter 28 Chapter 15

Chapter 22 Chapter 15 Chapter 28 Chapter 17

B.

OBJECTIVES

Chapter 19 – Nonmalignant Granulocyte and Monocyte Disorders Level I 1. Recognize neutrophilia from hematologic data and name the common disorders associated with neutrophilia.

2. Explain the quantitative and qualitative neutrophil response to acute bacterial infections.

3. Identify immature granulocytes and morphologic changes (toxic granulation, Döhle bodies, intracellular organisms, and vacuoles) often seen in reactive neutrophilia.

4. Define and recognize leukemoid reaction, leukoerythroblastosis, and pyknotic nuclei.

5. Distinguish leukemoid reaction from chronic myelogenous leukemia based on laboratory data including the leukocyte alkaline phosphatase stain.

6. Identify neutropenia from hematologic data and list the common disorders associated with neutropenia.

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7. Recognize the conditions associated with spurious, or false, neutropenia.

8. Identify neutrophil nuclear alterations including Pelger-Huët, hypersegmentation, and pyknotic forms.

9. Recognize as abnormal and seek assistance for identification of rare or unusual cytoplasmic abnormalities such as morulae, Alder-Reilly granules, or Chédiak-Higashi inclusions.

10. State the common conditions associated with abnormal eosinophil, basophil, and monocyte counts.

11. Define Gaucher and Niemann-Pick diseases.

Level II 1. Assess the etiology, associated conditions, and peripheral blood findings for immediate, acute, chronic, and reactive neutrophilia.

2. Contrast the hematologic and clinical features for leukemoid reaction and chronic myelogenous leukemia (CML).

3. Organize neutropenia to include etiology and associated conditions as well as blood and bone marrow findings.

4. Recognize, evaluate, and select appropriate corrective action for spurious or false neutropenia.

5. Appraise the nuclear abnormalities of neutrophils including Pelger-Huët, pseudo-Pelger-Huët, hypersegmentation, and pyknotic nuclei, and reconcile them with the appropriate clinical conditions of the patient.

6. Appraise the cytoplasmic abnormalities of neutrophils including toxic granulation, Döhle bodies, vacuoles, intracellular organisms, and morulae and reconcile them with the appropriate clinical conditions of the patient.

7. Recognize and summarize the clinical features of the inherited granulocyte functional disorders (Chédiak-Higashi, Alder-Reilly, May-Hegglin, and chronic granulomatous diseases) and differentiate their cellular abnormalities.

8. Evaluate alterations in the relative and/or absolute numbers of eosinophils, basophils, and monocytes and associate them with the clinical condition of the patient.

9. Evaluate the etiology, laboratory findings, and clinical features of the lysosomal storage disorders.

10. Identify and differentiate the abnormal macrophages seen in Gaucher disease, Niemann-Pick disease, and sea-blue histiocytosis.

11. Construct an efficient and cost-effective reflex testing pathway for follow-up neutrophilia, neutropenia, and qualitative granulocyte abnormalities.

12. Evaluate a case study from a patient with a nonmalignant granulocyte disorder.

Chapter 20 – Nonmalignant Lymphocyte Disorders Level I 1. Identify the infectious agent and describe the clinical symptoms and corresponding leukocyte differential in infectious mononucleosis.

2. Describe and recognize the reactive morphology of lymphocytes found in infectious mononucleosis.

3. Relate the heterophile antibody test to infectious mononucleosis.

4. Given a differential and leukocyte count, calculate an absolute lymphocyte count and differentiate it from a relative lymphocyte count. ..

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5. Identify reactive cell morphology associated with viral infections and compare it with normal lymphocyte morphology.

6. Describe clinical symptoms of disorders in which a leukocytosis is caused by lymphocytosis.

7. State the complications associated with CMV infections.

8. Identify absolute and relative lymphocytopenia and lymphocytosis, and list conditions associated with these abnormal counts.

9. Explain the pathophysiology of HIV infections and describe how it affects lymphocytes.

10. Describe the abnormal hematological findings associated with AIDS. Level II 1. Assess and resolve/explain conflicting results between peripheral blood morphology and serologic tests in suspected Epstein-Barr virus (EBV) infection.

2. Describe the pathophysiology of infectious mononucleosis.

3. Assess and correlate antibody titers found in infectious mononucleosis with respect to the various EBV viral antigens.

4. State the pathophysiology of toxoplasmosis infections, and explain the resulting lymphocytosis.

5. Differentiate benign lymphocytic leukemoid reactions from neoplastic lymphoproliferative disorders by laboratory results and characteristics of cell morphology.

6. Define the pathophysiology of CMV infections, and give clinical findings associated with it.

7. In Bordetella pertussis infections, propose the cause of lymphocytosis and recognize laboratory features associated with it.

8. Assess a patient case using the CDC AIDS case surveillance criteria and recommend appropriate laboratory testing.

9. Explain the cytopenia, identify the defect, and recognize the laboratory features in congenital qualitative disorders of lymphocytes.

10. Evaluate a case study from a patient with a lymphoproliferative disorder and conclude from the medical history and laboratory results the most likely diagnosis for the disorder.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Create Have each learner create: a. A set of CBC and differential results that demonstrate a “shift-to-the-left.”

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b. **A case study demonstrating a condition of “alloimmune neonatal neutropenia.” c. **Have each learner create a set of (interdisciplinary) patient laboratory results for the following conditions: • Wiskott-Aldrich Syndrome • DiGeorge Syndrome • Agammaglobulinemia • Hereditary Ataxia-Telangiectasia 3. Group Discussion Create a group discussion: a. Distinguishing leukemoid reaction from chronic myelogenous leukemia b. Describing Wiskott-Aldrich Syndrome c. Explaining DiGeorge Syndrome d. A family has a child with an inherited lipid storage disorder. Identify advantages/disadvantages of performing genetic screening on all members of the child’s immediate family.

4. Mapping Have each learner create a “map” of: a. Leukemoid reaction b. Causes for a “true neutropenia” c. **Lipid storage diseases d. Disorders associated with a reactive lymphocytosis e. Quantitative leukocyte disorders f. Qualitative leukocyte disorders 5. Match Up Create a match up game or have the learners create the game using the following: a. Inherited qualitative neutrophil abnormalities b. Qualitative or morphologic neutrophil abnormalities c. Eosinophil disorders d. Basophil disorders e. Monocyte/macrophage disorders f. Infectious lymphocytosis 6. Mystery Box a. In this Mystery Box, write on individual pieces of paper the appropriate terms and specific laboratory results that correlate with these terms. Have the learners match the pieces and explain why they chose to place them together.

For example: • Leukocytosis matches with the result WBC = 12.3 * 109/L • Absolute neutrophilia matches with the result 8.4 * 109/L (abs) • Lymphocytosis matches with the result 62% • And so on b. With this Mystery Box, list the definitions and individual characteristics of the following conditions on separate pieces of paper. Have the learners match the definition and all of the characteristics appropriately with each condition.

• Leukemoid reaction • Chronic myelocytic leukemia • Leukoerythroblastic reaction • Pelger-Huët • Hypersegmentation • Infectious mononucleosis • Toxoplasmosis • Cytomegalovirus • Bordetella pertussis

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c. Have a small group of learners create a Mystery Box for their classmates which includes each white blood cell and an associated disease condition, for example, monocytosis— inflammatory conditions.

7. Short Story a. Have the learners prepare a short story about the cause for development of “Neutrophilia” and her cousin “Neutropenia.” b. Create the story of the “Neutrophilic Abnormality” family. List all family members such as Pelger-Huet, Döhle Body, Alder-Reilly, May-Hegglin, etc. Describe their basic anomaly, how they are related to each other, and how they are different.

8. Small Group Teaching Have a small group of learners research and teach the following: a. **Compare and contrast the causes for malignant and benign quantitative abnormalities of neutrophils. b. **Congenital and familial neutropenia c. **Immune neutropenia d. The three recognized stages of a patient infected with HIV • This exercise could also incorporate a “Web Search.”

9. Think-Pair-Share a. List and discuss the factors affecting neutrophil concentration in peripheral blood. b. Explain the affect of active exercise on the peripheral neutrophil count. c. Discuss why “40 percent of new leukemia cases present with a low rather than high total white count.” d. Explain why “morphologically heterogeneous populations” of lymphocytes may be seen on a stained peripheral smear. e. **Distinguish the characteristics of hypereosinophilic syndrome and eosinophilic leukemia. f. List factors affecting neutrophil concentration in peripheral blood.

10. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

11. Tic-Tac-Toe Use the following examples of sample questions in a complete Hollywood Squares game.

• Explain why LAP scores are elevated in reactive chronic neutrophilia.

• Explain how a “neutropenia” may result from a severe infection.

• What affect can exercise, emotional stimuli, and exposure to extreme temperatures have on the circulating neutrophils?

• Describe how a neutropenia develops due to decreased bone marrow production.

• List the nuclear morphologic abnormalities of neutrophils.

• List the cytoplasmic morphologic abnormalities of neutrophils seen with bacterial infections.

• Distinguish between Pelger-Huët and psuedo-Pelger-Huët.

• List the inherited functional abnormalities of neutrophils.

• **Describe the abnormality in: ° Alder-Reilly anomaly ° Chédiak-Higashi syndrome ° May-Hegglin anomaly ° Chronic granulomatous disease

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• List three causes of an eosinophilia.

• Name the lipid storage diseases as they relate to abnormal macrophages.

• **Describe the abnormality in: ° Gaucher disease ° Niemann-Pick disease ° Sea-Blue histiocytosis

D.

LABORATORY ACTIVITIES

Perform the following suggested laboratory activities: 1. View prepared slides, kodachrome slides, or Bonus Image Library on DVD that accompanies this text for images of cells found in: • Neutrophilic cytoplasmic abnormalities • Pelger-Huët • CML • Leukemoid reactions • Infectious mononucleosis • **Inherited functional abnormalities • **Qualitative lipid storage disorders 2. Set up a practical examination (microscope stations) with slides from a selection of the above conditions.

E.

PRACTICE QUESTIONS

1. What morphologic abnormality may be seen as an artifact in blood smears made from blood collected and stored in EDTA anticoagulant? [Taxonomy 1] a. hypersegmentation b. pyknotic nucleus c. toxic granulation d. vacuoles 2. Damaged tissue releases _____. [Taxonomy 1] a. bacteria b. cytokines c. granules d. neutrophils 3. The “CDC recommends initiation of antiretroviral therapy within _____ of a needle stick injury from a potential HIV source.” [Taxonomy 1] a. 30 minutes b. 2 hours c. 2 days d. 1 week 4. Use the following set of results to calculate the absolute values and use the appropriate terminology to interpret each, that is, neutropenia, lymphocytosis, and so on. [Taxonomy 2] WBC ⴝ 9.8 : 109/L

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Manual Differential Segs ⴝ 78% Lymphs ⴝ 12% Monos ⴝ 3% Eos ⴝ 7% Baso ⴝ 0%

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Absolute Value Interpretation

5. **Explain the affect of corticosteroid therapy on the peripheral neutrophil count and the migration of neutrophils to the tissues. [Taxonomy 2] 6. **Differentiate a lymphocytic leukemoid reaction from chronic lymphocytic leukemia. [Taxonomy 2] 7. **Compare and contrast pseudoneutropenia and pseudoneutorphilia. [Taxonomy 3] 8. **Compare and contrast the characteristics of an immunoblast and a lymphoblast. [Taxonomy 3] 9. Recommend legislation (a bill) to eradicate the threat of HIV infections. [Taxonomy 3] Note: This may require additional research.

10. A 2-year-old female patient is in her physician’s office with superficial cuts on her arms and legs that have not healed within two weeks. Ever since she was three months old, she has been treated for a recurring ear infection. The current physical exam revealed an enlarged liver and spleen.

The hematology laboratory results indicated a decreased neutrophil and platelet count. It was also noted that a few neutrophils with large gray granules in the cytoplasm were seen. What abnormality might be suspected? [Taxonomy 3]

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Nonmalignant Disorders of Leukocytes – Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

L Y M O N O Y M Q D B D M N K X Q Z P V D N P G H K M R M T O A L P K X C C I B C T Y D R L I Q T N V C I O O L J A S H S G B I C O I W S S R R C C S T O V E L R V H N Y C N B O R

C Y T O D C R T R N H Y K L K Q J N E O O V C U N Y N L T D I N N H W Q P B Q F P M T R

G N U W O I V K O Y V T T Y L Y W J N K M R X K B N L M M K E I C I F D A T E L

X R Q E G C E L

L X T L S I D A

P P C N P I K R G J N

E N D M O G J L A N X T H V O M R C E C H D

I L L Y H F L T Y Y F

A R M S K M V I M B R S E Y Y O N R M T L L D Y J N B C M N T O V N T G M M Y A T Q N H

F C K P X O X L U W G X W S M A X P C D M U G R T V Q D X E K Y Q W Z P N N A M E I O N U M M I G P E R T U S S

P C I L F N H I

H R A G R A N U L O C Y T O S I S D S

• lipid storage disease; deficiency of glucocerebrosidase • causes infectious mononucleosis (abrv) • decreased number of monocytes • increased WBC • increased number of lymphocytes • absolute monocyte count > 0.8 x 109 / L • lipid storage disease–deficiency of sphingomyelinase (2 words) • increased number of neutrophils • impaired ability to mount an immune response • causative agent for whooping cough (2 words) • process in which cells ingest solid substances • absolute neutrophil count below 0.5 x 109 / L • increase in eosinophils • appear as clear, unstained areas in the cytoplasm • degenerated nuclei

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Nonmalignant Disorders of Leukocytes – Level I L Y M O N O C Y T O P E N I A R M S Y M Q D B D D C R T P D M L K M V I M N K X Q Z R N H Y C O G L M B R S P V D N P G K L K Q N J L Y E Y Y O H K M R M T J N E O P A N H N R M T O A L P K X O V C U I X T F L L D Y C C I B C T N Y N L K H V L J N B C Y D R L I Q T D I N R O M T M N T O T N V C I O N H W Q G R C Y V N T G O L J A S H P B Q F J E C Y M M Y A S G B I C O P M T R N H D F T Q N H I W S G N U W O X L F C K P X O X P S R R I V K O Y R X L U W G X W S C C C S V T T Y L Q T M A X P C D M I T O V Y W J N K E L U G R T V Q D L E L R M R X K B G S X E K Y Q W Z F V H N N L M M K C I P N N A M E I N Y C N E I C I F E D O N U M M I G H B O R D A T E L L A P E R T U S S I • lipid storage disease; deficiency of glucocerebrosidase (Gaucher) • causes infectious mononucleosis (abrv) (ebv) • decreased number of monocytes (monocytopenia) • increased WBC (leukocytosis) • increased number of lymphocytes (lymphocytosis) • absolute monocyte count > 0.8 x 109 / L (monocytosis) • lipid storage disease–deficiency of sphingomyelinase (2 words) (niemann pick) • increased number of neutrophils (neutrophilia) • impaired ability to mount an immune response (immunodeficiency) • causative agent for whooping cough (2 words) (bordatella pertussis) • process in which cells ingest solid substances (phagocytosis) • absolute neutrophil count below 0.5 x 109 / L (agranulocytosis) • increase in eosinophils (eosinophilia) • appear as clear, unstained areas in the cytoplasm (vacuoles) • degenerated nuclei (pyknotic)

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Nonmalignant Disorders of Leukocytes—Level I 1 2 3

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2 Anomaly with giant gray-green peroxidasepositive bodies found in cytoplasm of leukocytes (2 words) 7 Decreased number of neutrophils 8 Herpes virus that replicates only in human cells 10 Neutrophil with 6 or more segments 11 Disease caused by infection with HIV-1 (abrv) 12 Large blue-black granules in neutrophil cytoplasm associated with infection (2 words) 13 Self-limiting lymphoproliferative disease; caused by infection with EBV: infectious ____ 14 An increase in the concentration of immature forms of leukocytes in circulation (4 words)

1 Anomaly in which neutrophil nucleus does not segment beyond 2 lobes (2 words) 3 Marked decrease of all blood cells in the peripheral blood 4 Passage of WBC through blood vessel walls into extravascular tissue 5 Benign leukocyte proliferation-total WBC > 25 x 109/L-circulating immature leukocyte precursors (2 words) 6 Decreased number of lymphocytes 9 Light grayish-blue cytoplasmic inclusions (2 words)

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Nonmalignant Disorders of Leukocytes—Level I P C H E D I A K H I G A S H I L P D G L A I L E N E U T R O P E N I A Y R U C P C M V H K Y E D P U E T D O H Y P E R S E G M E N T E D O E H O T O P S L P A I D S E I E E D N S B N R I T O X I C G R A N U L E S A D A A I M O N O N U C L E O S I S E T S I S H I F T T O T H E L E F T N

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Nonmalignant Disorders of Leukocytes—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

D D E S S E R P P J O D N N L L W J D B H S I S I H T B I Z L R M D L Q A M G R E N A N I S M R W D B G S Z O E Z V Y N O E T P D L T K W G D Y H I K J G R M C Y I A J F O Z O Y U L T H E K H L C C S E G B P W H I K M I L M L E D T R D Z Y K R S N E U J L W F R P D L G B D T L X D F R G L E U K E M O I D

U S O N U M M I T H Y Y G Y H T H P O L E Y M R N T K V B L N A P M M R T R K L H E K V R T N U T P M P F G M L O G O R X T Y L A C T R G P V E S E Y Z E N M C J A W T V T R B P L N J O K E T K U K J T S C H T R O P E N I A F O F M R R C S K M L D M R R M R E A C T I O N

• type of immunity needed to limit infection by EBV • type of cell infected by EBV (2 words) • decreased numbers of neutrophils • increased number of mast cells • increase in these cells is found in immediate hypersensitivity reactions • congenital disorders resulting in severe qualitative immune defects (abrv) • replacement of normal hematopoietic tissue in bone marrow by fibrosis • type of antibodies found in acute EBV infection • leukocyte inclusions associated with infections of Ehrlichia sp • immune deficiency syndrome characterized by the absence of a thymus • condition of increased leukocytes with a shift to the left (2 words) • serologic results may not be positive in individuals who are ____ • leukocyte inclusion associated with bacterial infection (2 words) • type of neutrophilia caused by a transient change in number of leukocytes circulating • inherited disease characterized by a deficiency of glucocerebrosidase

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Nonmalignant Disorders of Leukocytes—Level II D D E S S E R P P J O D N N L L W J D B H S I S I H T B I Z L R M D L Q A M G R E N A N I S M R W D B G S Z O E Z V Y N O E T P D L T K W G D Y H I K J G R M C Y I A J F O Z O Y U L T H E K H L C C

U S O N T H Y Y H P O L N T K V P M M R H E K V T P M P O G O R A C T R S E Y Z J A W T

S M D J B L

P K T F K R

E G B I L M Z Y K L W F D T L E U K

P W H L E D R S N R P D X D F E M O

I T E L R I

K R U G G D

U M M G Y H E Y M B L N T R K R T N F G M X T Y G P V E N M V T R

L N J O U K J T R O P E O F M R M L D M E A C T

K S N R R I

I T R A L U L L E C B

E T C H I A C S R M O N

• type of immunity needed to limit infection by EBV (cellular) • type of cell infected by EBV (2 words) (b lymphocyte) • decreased numbers of neutrophils (neutropenia) • increased number of mast cells (mastocytosis) • increase in these cells is found in immediate hypersensitivity reactions (basophils) • congenital disorders resulting in severe qualitative immune defects (abrv) (scids) • replacement of normal hematopoietic tissue in bone marrow by fibrosis (myelophthisis) • type of antibodies found in acute EBV infection (heterophil) • leukocyte inclusions associated with infections of Ehrlichia sp (morulae) • immune deficiency syndrome characterized by the absence of a thymus (digeorge) • condition of increased leukocytes with a shift to the left (2 words) (leukemoid reaction) • serologic results may not be positive in individuals who are ____ (immunosuppressed) • leukocyte inclusion associated with bacterial infection (2 words) (dohle body) • type of neutrophilia caused by a transient change in number of leukocytes circulating (immediate) • inherited disease characterized by a deficiency of glucocerebrosidase (gaucher)

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Nonmalignant Disorders of Leukocytes—Level II 1 2 3 5

4

6 7

8

9

10

11

12

13

14

15

ACROSS

DOWN

4 Chronic condition where neutrophils engulf but do not kill microorganisms 7 Syndrome characterized by persistent blood eosinophilia with no apparent cause 8 Majority of reactive lymphocytes in EBV infection are of this type (2 words) 11 Inherited condition associated with Dohle-like cytoplasmic inclusions in all granulocytes (2 words) 12 Persistent blood eosinophilia with tissue infiltration and no apparent cause (abrv) 13 A transformed, fully differentiated B lymphocyte normally found in the bone marrow (2 words) 14 These cells in patients with viral infection must be distinguished from malignant lymphoblasts 15 Contagious condition of young children marked by leukocytosis and lymphocytosis (2 words)

1 Reaction demonstrating the presence of NRBC and a neutrophilic shift to the left in the peripheral blood 2 Immediate but transient neutrophilia as a result of cells from the marginating pool entering the circulating pool 3 Anomaly with blue Döhle-like cytoplasmic inclusions in all granulocytes 5 Autosomal recessive disease characterized by a deficiency of sphingomyelinase and the presence of foamy macrophages in bone marrow 6 Membrane bound sac in the cytoplasm that contain various hydrolytic enzymes 9 Cytoplasmic enzyme increased in leukemoid reactions and decreased in CML (abrv) 10 Disease caused by infection with the intracellular protozoan Toxoplasma gondii and associated with eosinophilia.

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Nonmalignant Disorders of Leukocytes—Level II L E P M G R A N U L O M A T O U S N L A K E I Y H Y P E R E O S I N O P H I L I C U E S H E D T L Y M P H O C Y T E T A L D E R R E I L L Y O A A S G O Y N P N O G X T E N M L O H E S U P E I P R T I N L O R C A B O K S L P M P L A S M A C E L L H O S I I M M U N O B L A S T S T L I I I I N F E C T I O U S L Y M P H O C Y T O S I S A

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A.

TRANSITION GRID

Information concerning Chapter 21, “Introduction to Hematopoietic Neoplasms”; Chapter 22, “Myeloproliferative Disorders”; Chapter 23, “Myelodysplastic Syndromes”; Chapter 24, “Acute Myeloid Leukemias”; Chapter 25, “Acute Lymphoblastic Leukemias”; Chapter 26, “Lymphoid Malignancies”; and Chapter 27, “Hematopoietic Stem Cell Transplantation” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 21

Chapter 23

Chapter 24

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 26 Chapter 27 Chapter 28 Chapter 16 Chapters 17, 18 Chapter 19 Chapters 29, 30 Chapter 34 Chapter 35 Chapters 18–20 Chapter 21 Chapter 22

Chapter 29 Chapter 16 Chapter 36 Chapter 19

Clinical Laboratory Hematology

Chapter 25

Chapter 26

Chapter 27

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 30 Chapter 16 Chapter 36 Chapter 19

Chapter 31 Chapter 22 Chapter 37 Chapter 20

Chapter 32 Chapters 16, 19 Chapters 29, 34 Chapters 9, 20, 21

B.

Chapter 22

OBJECTIVES

Chapter 21 – Introduction to Hematopoietic Neoplasms Level I 1. Define and differentiate the terms neoplasm and malignant and identify hematopoietic disorders that can be included in each category.

2. Compare and contrast the general characteristics of the myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute and chronic leukemias. ..

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3. Name and describe the classification systems used for MDS, MPD, and the leukemias.

4. List the various methods used to categorize the leukemias.

5. Compare and contrast the laboratory findings of the acute and chronic leukemias and myeloid and lymphoid leukemias.

6. Differentiate proto-oncogenes and oncogenes and summarize their relationship to neoplastic processes.

7. Correlate patient age to the overall incidence of the hematopoietic neoplasms.

8. Explain the usefulness of immunological techniques, chromosome analysis, molecular analysis, and cytochemistry in the diagnosis and prognosis of hematopoietic neoplasms.

9. Predict the prognosis and survival rates of the hematopoietic neoplasms.

Level II 1. Explain how proto-oncogenes are activated and the role that oncogenes and tumor suppressor genes and their protein products play in the etiology of hematopoietic neoplasms.

2. Describe the effects of radiation on the incidence of leukemia.

3. Differentiate between the acute and chronic leukemias based on their clinical and hematologic findings.

4. Reconcile the use of chemotherapy for treatment of leukemia.

5. Compare and contrast treatment options for the hematopoietic neoplasms including possible complications.

6. Name the leukemogenic factors of leukemia and propose how each contributes to the development of leukemia.

7. Compare and contrast laboratory features of MDS, MPD, and acute leukemia (AL), and justify a patient diagnosis based on these features.

8. Define the principles of, explain the applications of, and select appropriate cytochemical stains for bone marrow evaluation.

9. Select laboratory procedures appropriate for confirming cell lineage and diagnosis in hematopoietic neoplasms.

10. Explain the role of epigenetics in cancer.

11. Define cancer stem cell and explain its similarities to the hematopoietic stem cell (HSC).

Chapter 22 – Myeloproliferative Disorders Level I 1. Interpret the leukocyte alkaline phosphatase (LAP) activity found in patients with chronic myelogenous leukemia (CML).

2. Identify the major lineages involved with the various myeloproliferative disorders (MPD): chronic myelogenous leukemia, CML; polycythemia vera, PV; essential thrombocythemia, ET; and chronic idiopathic myelofibrosis, CIMF. 3. Recognize abnormal complete blood count (CBC) results that suggest an MPD. 4. Explain the diagnostic chromosome abnormality associated with CML and its significance in acute lymphoblastic leukemia (ALL).

5. Identify the peak incidence of CML according to age and sex distribution.

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6. Describe and recognize the peripheral blood findings in CML patients.

7. List and recognize laboratory findings typically associated with CIMF. 8. Define criteria that indicate a transformation of an MPD into a blast crisis.

9. Compare the lab findings in primary PV to secondary polycythemia.

10. Describe and recognize the characteristic peripheral blood picture found in essential thrombocythemia (ET).

Level II 1. Differentiate the subgroups of myeloproliferative disorders (MPD) from other reactive and neoplastic diseases based on laboratory findings in the peripheral blood, bone marrow, and other diagnostic laboratory tests.

2. Contrast laboratory findings in MPD and myelodysplastic syndromes (MDS) as well as acute leukemia (AL).

3. Describe molecular gene mutations in MPD and hypothesize how these changes in the pluripotential stem cell lead to MPD and blast crisis (Ph-chromosome; BCR/ABL1 rearrangement; p53, p16 mutations, FIP1L1/PDGFRA, and JAK2 (V617F) mutations and expression of growth factor receptors).

4. Differentiate chronic myelogenous leukemia (CML) from a benign leukemoid reaction using laboratory tests.

5. Assess the role of platelet-derived growth factor (PDGF) in the fibrosis found with CIMF. 6. Assess laboratory results including the evaluation of peripheral blood and bone marrow smears using the diagnostic criteria associated with CIMF. 7. Compare clinical and laboratory findings, and interpret laboratory findings in relative polycythemia and absolute polycythemia.

8. Infer the significance of finding one form of the glucose-6-phosphate dehydrogenase (G6PD) isomer in myeloproliferative disorders.

9. Use laboratory results and clinical findings to differentiate essential thrombocythemia from nonmalignant conditions that result in thrombocytosis.

10. Use the criteria suggested for diagnosing variants of CML, chronic eosinophilic leukemia, CEL; chronic basophilic leukemia, CBL; and chronic neutrophilic leukemia, CNL to evaluate laboratory results of patients.

11. Differentiate unclassifiable myeloproliferative disease from other subgroups of MPD based on laboratory features.

12. Describe systemic mastocytosis by giving the criteria for diagnosis.

13. Using peripheral blood and bone marrow findings and patient medical history, determine the classification of MPD. Chapter 23 – Myelodysplastic Syndromes Level I 1. Define myelodysplastic syndromes (MDS) and list general characteristics of these diseases.

2. List the six subgroups of MDS recognized by the World Health Organization (WHO) Classification System and identify key morphological and clinical criteria that distinguish each group.

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3. Describe laboratory findings and recognize changes in morphology that are characteristic of this group of disorders.

4. Define the WHO category of myelodysplastic/myeloproliferative diseases, and list its general/ characteristics.

5. List the four subgroups of myelodysplastic syndromes/myeloproliferative diseases (MDS/MPD), and identify key morphological and clinical criteria as well as laboratory findings that distinguish each group.

Level II 1. Describe the pathophysiology of MDS. 2. Distinguish among Type I, Type II, and Type III blasts and promyelocytes.

3. Summarize the treatment and prognosis of MDS. 4. Explain the relationship between MDS and acute leukemia.

5. Assess the results of cytogenetic and molecular tests and correlate them with a diagnosis of MDS. 6. List and briefly describe the MDS variants not listed in the six WHO subgroups.

7. Differentiate MDS from myeloproliferative disorders, acute leukemia, and other hematologic abnormalities using peripheral blood, bone marrow, and cytogenetic characteristics.

8. Differentiate MDS/MPD from MDS, MPD, acute leukemia, and other hematologic abnormalities using peripheral blood, bone marrow, and cytogenetic characteristics.

9. Select laboratory tests that are helpful in diagnosing and differentiating MDS and MDS/MPD. 10. Evaluate a patient’s laboratory and clinical findings, and propose a diagnostic MDS or MDS/MPD subgroup.

Chapter 24 – Acute Myeloid Leukemias Level I 1. Define acute leukemia, and explain the difference between acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS).

2. List and define the common variants of AML as defined by the WHO classification.

3. Describe and recognize the typical peripheral blood picture (erythrocytes, leukocytes, and thrombocytes) seen in AML. 4. Describe the M:E ratio in bone marrow in acute leukemia (AL).

5. Give the typical results of cytochemical stains in AML. 6. Identify Auer rods and describe their significance.

Level II 1. Compare and contrast the various presentations of AML. 2. Predict the most likely leukemia type based on patient history, physical assessment, and laboratory findings.

3. Correlate cellular presentation of AML with prognosis and common complications.

4. Correlate Wright-stain morphology of the AML subgroups with cytochemical stains, flow cytometry, and genetic testing.

5. Evaluate peripheral blood results in relation to oncological therapy (i.e., complete or partial remission, relapse, engraftment).

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6. Evaluate patient data from the medical history and laboratory results to determine whether a disorder can be classifed, and, if not, specify the additional testing to be performed.

7. Compare the WHO and FAB classifications of AML. Chapter 25 – Acute Lymphoblastic Leukemias Level I 1. Define acute lymphoblastic leukemia (ALL) and differentiate it from acute myeloid leukemia (AML).

2. List and define the common variants seen in ALL as defined by the FAB and WHO classifications.

3. Describe and recognize the typical peripheral blood picture (erythrocytes, leukocytes, blasts and thrombocytes) seen in ALL. 4. Give the typical results of cytochemical stains in ALL. 5. Summarize the clinical signs and symptoms and the most frequent age groups associated with ALL. 6. Define the rare acute leukemias that are not included in AML and ALL groups.

Level II 1. Compare and contrast the various presentations of ALL. 2. Predict the most likely WHO or immunophenotype subgroup based on patient history, physical assessment, and laboratory findings.

3. Correlate cellular presentation with prognosis and common complications in ALL. 4. Correlate Wright stain blast morphology in the ALL subgroups with cytochemical stains, flow cytometry, and genetic testing results.

5. Evaluate peripheral blood results in relation to oncological therapy (i.e., complete or partial remission, relapse).

6. Identify AL from a peripheral blood smear and recommend laboratory tests that may be useful in differentiation of AML and ALL and in classification of subtypes.

Chapter 26 – Lymphoid Malignancies: Chronic Lymphoid Leukemias, Lymphomas, and Plasma Cell Neoplasms Level I 1. Describe the clinical presentation of patients with lymphoid malignancies.

2. Describe how the diagnosis of lymphoid malignancy is made.

3. Differentiate among chronic lymphocytic leukemia (CLL), lymphoma, and multiple myeloma based on peripheral blood findings and ancillary studies.

4. Describe the histology of a normal lymph node.

5. Summarize the causes of lymphadenopathy.

6. Describe the morphologic differences between Hodgkin and non-Hodgkin lymphoma.

7. List and describe the chronic leukemic lymphoproliferative disorders.

8. Recognize and differentiate abnormal and normal lymphocytes on a stained peripheral blood smear and associate their presence with a clinical diagnosis.

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10. Compare the laboratory and clinical findings of multiple myeloma and Waldenström macroglobulinemia.

11. Define monoclonal gammopathy.

Level II 1. Describe the use of immunophenotyping and genotyping in detecting clonality and list the results characteristic of the more common lymphoid malignancies.

2. Contrast the features of low-grade and high-grade lymphoma.

3. Compare and contrast the laboratory features characteristic of the following non-Hodgkin lymphomas: a. Small lymphocytic lymphoma b. Follicular lymphoma c. Mantle cell lymphoma d. MALT lymphoma e. Waldenström macroglobulinemia f. Diffuse large B cell lymphoma g. Burkitt lymphoma h. Lymphoblastic lymphoma i. Peripheral T cell lymphoma j. Anaplastic large cell lymphoma 4. Compare and contrast the laboratory features characteristic of Hodgkin lymphoma (HL) subtypes.

5. Compare and contrast the clinical and laboratory features characteristic of the following chronic leukemic lymphoproliferative disorders: a. Chronic lymphocytic leukemia (CLL) b. Prolymphocytic leukemia (PLL) c. Hairy cell leukemia (HCL) d. Circulating lymphoma e. Large granular lymphocyte leukemia (LGLL) f. Sézary syndrome 6. Compare and contrast the laboratory features characteristic of the following plasma cell disorders: a. Plasmacytoma b. Multiple myeloma c. Monoclonal gammopathy of undetermined significance 7. Recognize and identify the peripheral blood abnormalities associated with CLL, PLL, HCL, LGLL, Sézary syndrome, multiple myeloma, and non-Hodgkin lymphoma.

8. Describe the etiology and pathogenesis of lymphoid neoplasms.

9. Differentiate reactive from malignant proliferations of lymphoid cells using clinical and laboratory data.

Chapter 27 – Hematopoietic Stem Cell Transplantation Level I 1. Describe the sources of hematopoietic stem cells and characteristics of each source.

2. Identify the diseases that can be treated with different sources of hematopoietic stem cells.

3. List characteristics of stem cells.

4. Describe the significance of ABO and HLA antigens for stem cell transplant.

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5. Identify the infections that are serious complications during the peritransplant period.

6. Describe the significance and effects of graft-versus-host disease (GVHD) and compare them to those of the graft-versus-leukemia (GVL) process.

Level II 1. Summarize the collection and processing of hematopoietic stem cells and assess the success of these procedures.

2. Explain the role of the clinical laboratory professional in stem cell transplantation.

3. Select and outline methods to enumerate hematopoietic stem cells.

4. Explain the complications of stem cell transplant, and assess the patient’s risk of developing them.

5. Formulate the sequence of events for a patient who will receive a stem cell transplant.

6. Select laboratory tests used to determine engraftment and assess engraftment given results of these tests.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Construct/Create a. **Have each team construct a justification/argument for why each of the different synonyms should be used to describe chronic idiopathic myelofibrosis (CIMF) • myelofibrosis with myeloid metaplasia • Agnogenic myeloid metaplasia • Idiopathic myelofibrosis • Primary myelofibrosis • Aleukemic myelosis • Myelosclerosis • Splenomegalic myelophthisis • Leukoerythroblastic anemia b. **Create a comprehensive case study with laboratory results for a patient diagnosed with MDS, AML, ALL, CML. c. **Create a marketing campaign to either encourage or discourage umbilical cord stem cell donations. d. **Develop a comprehensive case study including cytogenetic studies for precursor T-Cell Leukemia. e. **Create a job description for the role of a clinical laboratory professional in stem cell transplantation. ..

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3. Group Discussion Use the following topics to create active discussion: a. Compare and contrast the general characteristics of myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), and acute and chronic leukemias. b. Compare and contrast myeloid and lymphoid leukemias. c. Discuss the laboratory procedures for diagnosing and classifying neoplasms. d. Compare the characteristics of ALL and ANLL. e. **Discuss the genetic analysis used to identify characteristics of the acute leukemias and some MPD and MDS. f. Compare and contrast the peripheral blood features of CML and leukemoid reactions. g. Compare and contrast the FAB and WHO classification systems for acute leukemias and/ or MDS. h. **Discuss the characteristics of Waldenström macroglobulinemia. i. **Discuss the characteristics of both Hodgkin and Non-Hodgkin lymphoma. j. **Discuss the advantages and disadvantages of using cord blood stem cells versus marrow stem cell donors.

4. Mapping Have each learner create a “map” of the: a. Tumor development b. Survival rates for patients with leukemia c. Neoplastic disorders of hematopoietic cells d. Types of leukemia e. Laboratory findings in AML and ALL f. Ann Arbor staging system for malignant lymphoma g. **Types of stem cell transplants h. **Juvenile forms of CML (JCML) i. **Myelodysplastic syndrome classifications j. **Acute myeloid leukemias including morphologic, immunophenotypic, and cytochemical results k. **WHO classification of lymphoid neoplasms 5. Match-Up Game Create a concentration game using: a. The terms and their definitions • Dyspoiesis • Clonality • Anaplastic • Ataxia telangiectasia • Neoplasm • Prolymphocytes • Smudge cell • TRAP • Allogeneic • Autologous • Apheresis • Purging • Cryopreserved • Chimerism • **Leukemic hiatus • **Hybridomas b. The subgroups and variants of myelodysplastic syndromes and their basic descriptions. c. The FAB and/or WHO classification of acute myelocytic and lymphocytic leukemias. d. **Common genetic abnormalities in hematopoietic neoplasms.

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e. Cytochemical stains used to differentiate acute myeloid leukemia from acute lymphoblastic leukemia. f. **Key features of subgroups of chronic leukemic lymphoid malignancies or key features of malignant lymphomas.

6. Mystery Box a. Create a Mystery Box filled with laboratory results (may also include scatterplots and histograms) for CML, MMM, PV, and ET. Have students choose a set of results and identify the disease state. Then also have the students create the “case study” or clinical condition for a particular patient with this disease. b. Create a Mystery Box filled with the different types of leukemia, their clinical characteristics, laboratory results, and disease prognosis. c. Create a Mystery Box filled with the classification, peripheral blood, bone marrow, genetics, and immunophenotype characteristics of myelodysplastic and myeloproliferative diseases. d. Place in the Mystery Box the different cytochemical stains, immunophenotyping, and cytogenetic results, and their associated disease conditions of the acute myelocytic leukemias and/or the acute lymphocytic leukemias. e. Place in the Mystery Box the following chronic leukemic lymphoid malignancies, malignant lymphomas, and plasma cell neoplasms. Have each small group of students pick one of the malignancies and create the CBC, differential, other laboratory results (i.e., immunophenotyping), and patient clinical condition typical for the disease.

• B-cell lymphocytic leukemia • Prolymphocytic leukemia • Hairy cell leukemia • Large granular lymphocyte leukemia • Sézary’s syndrome • Hodgkin’s lymphoma • Non-Hodgkin’s lymphoma • Multiple myeloma • Waldenström’s macroglobulinemia 7. One-Minute Paragraph Write a one-minute paragraph. a. Explain the association of renal failure with chemotherapy treatment. b. Describe the Philadelphia chromosome. c. Explain how polycythemia vera can result in a microcytic/hypochromic anemia. d. Describe the therapy or treatment for each of the major disease conditions covered in this section (CML, MMM, PV, ET, MDS, AML, ALL, etc.). e. Discuss non-Hodgkin’s lymphoma. f. Discuss the value/benefit of hematopoietic stem cell transplantation (HSCT). g. **List and describe the types of stem cell transplants. h. **Compare and contrast “low-grade” and “high-grade” types of lymphoma. i. **Explain the role of the tumor suppressor gene (TSG) in tumor growth. j. **List and describe the various ways to differentiate leukemic blasts. k. Compare and contrast the characteristics of acute and chronic leukemia. l. Discuss the age groups typically found in acute and chronic leukemia.

8. Mnemonic Device a. Create a mnemonic device for the five subgroups of myelodysplastic syndromes and the ** WHO classifications of lymphoid neoplasms. b. Create a mnemonic device (perhaps a song) for the WHO classification for acute leukemias. c. **Create a mnemonic device for the different malignant lymphomas. ..

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9. Pyramid Game Create the Pyramid Game using the following as possible categories: a. Myeloproliferative disorders b. Myelodysplastic syndromes c. WHO classification of acute nonlymphocytic leukemias d. **Hematologic abnormalities in MDS e. Classifications of acute myeloid leukemias f. Types of lymphoid malignancies g. Acute lymphoblastic leukemias 10. Small Group Teaching Have a small group of learners research and teach the following: a. The differentiation of myeloid and lymphoid leukemias b. **Compare and contrast cytochemical, immunologic, and cytogenetic analysis of myeloid and lymphoid leukemias. c. Compare and contrast Hodgkin’s and non-Hodgkin’s lymphoma. d. **Compare and contrast the effects of antibiotic, antimetabolic, and alkylating agent therapies. e. **Explain “Ras proteins.” f. **The four classifications of secondary polycythemia vera g. **Describe the genetic abnormalities found in MDS. h. **The role of the clinical laboratory professional in stem cell transplantation i. **Complications associated with stem cell transplantation j. **Discuss the acute leukemias of ambiguous lineage 11. Think-Pair-Share a. Compare and contrast malignant neoplasms and benign neoplasms. b. Explain the reason for an increased serum uric acid test result in leukemia patients. c. Discuss the limitations of molecular genetic analysis. d. Discuss the biologic properties of hematopoietic stem cells that make them beneficial for transplantation. e. Explain the difference between an absolute and relative erythrocytosis. f. Describe the bone marrow assessment found in AML. g. Explain why the peripheral leukocyte count can be increased, decreased, or normal in patients with ALL. h. **Compare and contrast T-cell and B-cell acute lymphoblastic leukemia. i. **Define myelophthisic anemia. j. **List and describe the plasma cell neoplasms. k. **List the normal functions of proto-oncogenes and what activates them.

12. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

13. Tic-Tac-Toe Create a Tic-Tac-Toe exercise using the: a. Cytochemical stains, their descriptions, reactions, and the diseases they differentiate. b. Myeloproliferative disorders, their characteristics and clinical findings.

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LABORATORY ACTIVITIES Perform the following suggested laboratory activities:

1. View prepared slides, kodachrome slides, or images or Bonus Image Library textbook that accompanies this text for: • Chronic myelocytic leukemia • Myelofibrosis with myeloid metaplasia • Polycythemia vera • Essential thrombocythemia • Acute myelocytic leukemias • Acute lymphocytic leukemia 2. Set up a practical examination (microscope stations) with slides from a selection of the above conditions.

3. **Perform any of the procedures for cytochemical stains. See Chapter 34 for discussion of these stains.

• Myeloperoxidase • Sudan Black B • Chloroacetate esterase • Alpha-naphthyl esterase • Periodic acid-Schiff • Leukocyte alkaline phosphatase • Tartrate resistant acid phosphatase 4. **Analyze and evaluate prepared cytochemical stains. (See Chapter 34) 5. **Perform immunologic analysis using a flow cytometer (if available). (See Chapter 37)

E.

PRACTICE QUESTIONS

1. Patients diagnosed with myelodysplastic syndrome have a predisposition for leukemia.

[Taxonomy 1] a. True b. False 2. Hematopoietic stem cells are not routinely taken from cadavers. Explain why. [Taxonomy 1] 3. Which of the following characteristics can be used to distinguish a leukemoid reaction from chronic myelocytic leukemia? [Taxonomy 1] a. Hypercellular bone marrow b. Increased number of bands on a peripheral smear c. Presence of cytoplasmic vacuoles/Döhle bodies d. Presence of myelocytes and metamyelocytes on the blood smear 4. Differentiate oncogenes and proto-oncogenes. [Taxonomy 2] 5. A 57-year-old male patient has been diagnosed with AML. Explain to this patient the criteria for a state of “remission” and what his chances are of reaching it. [Taxonomy 2] 6. Distinguish between myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS).

[Taxonomy 2] 7. **Recommend future uses of hematopoietic stem cell transplants. [Taxonomy 3] 8. **Formulate a hypothesis for the reason radiation can induce leukemia. [Taxonomy 3]

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9. **“Exactly how viruses induce leukemia is unclear, but it is suspected that the incorporation of the viral genome into host DNA may lead to activation of proto-oncogenes.” Generate another hypothesis of how viruses induce leukemia. [Taxonomy 3] 10. A 53-year-old male patient is seen by his primary care physician. He complains of excessive tiredness and a lack of stamina in activities he typically performs, that is, work, gardening, and sports.

His physician orders a CBC with differential. [Taxonomy 3] WBC = 25.0 * 109/L RBC = 3.11 * 1012/L Hgb = 8.6 g/dL Hct = 26.3% MCV = 84.6 fL MCH = 27.6 pg MCHC = 32.7 g/dL RDW = 16.2% Plt = 89 * 109/L Neutrophils = 18% Bands = 2% Lymphocytes = 28% Monocytes = 13% Eosinophils = 6% Basophils = 4% Blasts = 19% NRBC = 4 Few large platelets seen One day later the physician orders the following cytochemical stains and receives these results.

Myeloperoxidase = positive Sudan Black B = positive Chloroacetate esterase = positive Nonspecific esterase = negative List the possible disease conditions for this patient considering these laboratory results. Suggest further testing and give the expected results for the diseases you listed.

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Neoplastic Hematologic Disorders—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

P S U D A N B L A C K B C Y Z N R N H W Y M Q A J K M N M G F M W B G H L X R K B T K R D M K I Q T V K F N N W G L M R H R R L L N A N Y X Y M X N L B M L T N K R X M E R L K H L T E B C M E N A X U Y H N N J P T N D B R X L L L B G P N H L N G R T B Y Z Q J D L W Z N U P O N R X C J T Z G R K L T D K J Y Y K R L D R G D L N J X M Y Y I G I V S R B N G W N Y E G E E A Q J H T B C Z L G T J A T B Y C W M M J N G I T M K N K T O R A L T K L B T N Y P A D O P H K Y S G F F Z B L M Y L Q B T T G H R D U G P D W W K D C K D M Q X L R L O L L O T I S L L J F N P M J E X M O G P T M N R X M X K Q M E Z H T K T M B P E B R R R N G X B A E T F M D M R T R R K Q E C L R H T N Q K H I D D R X A O P U T T P F V N F D B T R L Q N G F V W N L F L N C N K K V F I F M J L K T K P J D J M I M V L X O M O N L R G Q G J A B Q M D K L M H K H C H N K B R L H J N T J Y I Q G B P P B P R P N N J D C P L L O F L F T F T D Y C R L E U K E M I A B L V G A H G D C B K N Y P A R E H T O M E H C M Z T R L S L I T J J E D L T Q P P Q X L P Q D Y G J F N Z T J C Q R S D M K Y Q Z W P Q N M N G B X L L J W I K R B S L W V K W W K M X V P X J W W V M M M X C K M E • cell in the bone marrow that produces reticular fibers • hematopoiesis occurring outside the bone marrow • cytochemical stain that stains phospholipids (3 words) • FAB classification M5 is an acute ____ leukemia • nonmalignant • solid tumor found in lymphatic tissue • treatment of choice for leukemia • characteristic cell found in Hodgkin’s disease (2 words) • opposite of benign • chromosome-reciprocal translocation between chromosome 9 and 22 • malignant bone marrow neoplasm • MPD subgroup-overproduction of platelets (2 words) • type of analysis based on identifying specific membrane antigens • type of ALL in which the malignant cell frequently has prominent vacuolation • type of leukemia derived from the CFU-GEMM stem cell ..

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Neoplastic Hematologic Disorders—Level I P S U D A N J K M N M G Q T V K F N X N L B M L N A X U Y H L N G R T B Z G R K L T Y I G I V S Z L G T J A R A L T K L L M Y L Q B Q X L R L O G P T M N R R R N G X B

B L A C K B C Y F M W B G H L X N W G L M R H R T N K R X M E R N N J P T N D B Y Z Q J D L W Z D K J Y Y K R L R B N G W N Y E T B Y C W M M J B T N Y P A D O T T G H R D U G L L O T I S L L X M X K Q M E Z A E T F M D M R

Z R R L R N D G N P P J H T

N R N H W Y M Q A K B T K R D M K I L L N A N Y X Y M K H L T E B C M E X L L L B G P N H U P O N R X C J T R G D L N J X M Y E E A Q J H T B C G I T M K N K T O H K Y S G F F Z B D W W K D C K D M F N P M J E X M O T K T M B P E B R R R K Q E C L R H

T N Q K H I D D R X A O P U T T P F V N R L Q N G F V W N L F L N C N K K V F I K T K P J D J M I M V L X O M O N L R G B Q M D K L M H K H C H N K B R L H J N Q G B P P B P R P N N J D C P L L O F L D Y C R L E U K E M I A B L V G A H G D Y P A R E H T O M E H C M Z T R L S L I D L T Q P P Q X L P Q D Y G J F N Z T J D M K Y Q Z W P Q N M N G B X L L J W I L W V K W W K M X V P X J W W V M M M X • cell in the bone marrow that produces reticular fibers (fibroblast) • hematopoiesis occurring outside the bone marrow (extramedullary) • cytochemical stain that stains phospholipids (3 words) (sudan black b) • FAB classification M5 is an acute ____ leukemia (monoblastic) • nonmalignant (benign) • solid tumor found in lymphatic tissue (lymphoma) • treatment of choice for leukemia (chemotherapy) • characteristic cell found in Hodgkin’s disease (2 words) (reed sternberg) • opposite of benign (malignant) • chromosome-reciprocal translocation between chromosome 9 and 22 (Philadelphia) • malignant bone marrow neoplasm (leukemia) • MPD subgroup-overproduction of platelets (2 words) (essential thrombocythemia) • type of analysis based on identifying specific membrane antigens (immunologic) • type of ALL in which the malignant cell frequently has prominent vacuolation (burkitt) • type of leukemia derived from the CFU-GEMM stem cell (myelogenous)

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Neoplastic Hematologic Disorders—Level I 1

2

3 4

5

6 7 8

9

10

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14 15

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4 Abnormal condition in which there is an increase in serum immunoglobulins 6 MPD subgroup-overproduction of erythrocytes (2 words) 8 Disorder characterized by panhypercellularity, erythrocytosis, granulocytosis, or thrombocytosis in the peripheral blood 9 MDS subgroup in which anemia is the primary clinical finding 13 General type of leukemia; accumulation of immature and nonfunctional hematopoietic cells in the bone marrow 15 Type of transplant where patient’s own marrow is removed, treated, and given back to the patient

1 General type of leukemia when lymphoid cells predominate 2 Syndrome characterized by dysplasia of cells in a hypercellular bone marrow 3 Abnormal enlargement of lymph nodes 5 Enzyme found in primary granules of neutrophils, eosinophils, and monocytes (also used as a cytochemical stain) 7 Arises as a consequence of unregulated proliferation of a single transformed cell 10 MPD subgroup-demonstrating progressive bone marrow fibrosis (abrv) 11 Pink-staining granular inclusions found in blasts and promyelocytes of some AML (2 words) 12 Unsuccessful bone marrow aspiration is described as a ___ ___ (2 words) 14 Enzyme found in secondary granules of maturing granulocytes (abrv)

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Neoplastic Hematologic Disorders—Level I L M L Y Y G A M M O P A T H Y M E Y M P O L Y C Y T H E M I A V E R A P H O L N H O D M Y E L O P R O L I F E R A T I V E C Y P O D Y S E P E T P R L N I L R E F R A C T O R Y A O A C A I X S P U S D M I M A C U T E T R F D T R I L Y A H R C A U T O L O G O U S Y O P A E D P S

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Neoplastic Hematologic Disorders—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

D Y C P I O T L S Y A C L Y P T A H N E A M V I Q A J A H M N C Q Q X P

S G R A N U L O P O B F L L W M J R L T R K Z X H R T M N D Y X E J E C T B Z S R J L R Y N R C A C E R R Y Y T D N F E H W C N B T I O G R C Z L J Q G H O M T T J V K A L L R O I U K U R G E Y C O N D K A V Y N Y P E P T P N M E T R G T J S V W T O N O K W L D L Q S D C I G Q H P M I V N T D L G P K S T O H K F D E T N R E E D S T E R N K K C I E N E G N Y

I E S I S Z X R S V E G U X F N O P J N N N M F Z L A M K X S G K Q L T N C Z D E Q T F R T W W T N Y O K F V O G S B G N I Y I B Y Q E Y S X B T T K T F R W I B E R G R S W Q W Q

K N K R N N O N S P E C I F I C N N

• intranuclear membrane bound inclusion bodies found in plasma cells • an altered gene that contributes to the development of cancer • abnormal granulocyte maturation • nuclear DNA synthesis without cytoplasmic division • general term to describe erythrocytosis • increase of mast cells • type of bone marrow or PBSC transplantation from an identical twin • a DNA polymerase found in cell nuclei of thymus lymphocytes (abrv) • dedifferentiated or loss of differentiation • type of leukemia derived from the CFU-GEMM stem cell • characteristic cell found in Hodgkin’s disease (2 words) • type of CML found in 10–14 year old patients • monoblasts are positive for ____ esterase • E. coli enzyme class of chemotherapeutic agent • hormone; regulates the production of erythrocytes

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Neoplastic Hematologic Disorders—Level II D C I T S A L P A N A

Y P O L Y C Y T H E M

V I Q A J A H M N C Q Q X P

S G R B F L R K Z Y X E R J L E R R H W C C Z L T J V U K U D K A T S D P K N K

A N U L W M X H R J E C R Y N Y Y T N B T J Q G K A L R G E V Y N

P N M E V W T O L Q S D M I V N S T O H R E E D K C I E

T N C T K S N

L J T T R D I H L Y Y

O P O R L T M N D B Z S C A C N F E O G R O M T R O I C O N P E P

I E S I Z X R S E G U X N O P J N N M F L A M K S G K Q T N C Z E Q T F T W W T Y O K F

S V F N Z X L D R N V

K N K R N N O N S P E

R O I D F T E

G T J K W L G Q H L G P D E T E R N G N Y

O G S B G N I Y I B Y Q E Y S X B T T K T F R W I B E R G R S W Q W Q

C I F I C N N

• intranuclear membrane bound inclusion bodies found in plasma cells (ditcher) • an altered gene that contributes to the development of cancer (oncogene) • abnormal granulocyte maturation (dysgranulopoiesis) • nuclear DNA synthesis without cytoplasmic division (endomitosis) • general term to describe erythrocytosis (polycythemia) • increase of mast cells (mastocytosis) • type of bone marrow or PBSC transplantation from an identical twin (syngeneic) • a DNA polymerase found in cell nuclei of thymus lymphocytes (abrv) (tdt) • dedifferentiated or loss of differentiation (anaplastic) • type of leukemia derived from the CFU-GEMM stem cell (myelogenous) • characteristic cell found in Hodgkin’s disease (2 words) (reed sternberg) • type of CML found in 10–14 year old patients (juvenile) • monoblasts are positive for ____ esterase (nonspecific) • E. coli enzyme class of chemotherapeutic agent (asparaginase) • hormone; regulates the production of erythrocytes (erythropoietin)

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Neoplastic Hematologic Disorders—Level II 1 3

2

4

5 6

7

8 9

10 11 12 13

14

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DOWN

1 Localized, tumorous collection of monoclonal plasma cells 6 Sign of myeloid dysplasia-hyposegmentation of PMN nucleus (3 words) 9 Type of immunity needed to limit infection by EBV 11 Fusion gene created in CML from the translocation between chromosomes 9 and 22 (abrv) 12 Hairy cells that are not inhibited by tartrate are known as ____ (abrv) 13 Abnormal development of blood cells; characterized by asynchrony in nuclear to cytoplasmic maturation 14 Myeloproliferative disorders are generally characterized by _____ 15 Small, abnormal megakaryocyte sometimes found in the peripheral blood of MDS

2 Stain that is positive for basophils and mast cells; useful in the diagnosis of mast cell disease (2 words) 3 Term to describe a blast population with both myeloid and lymphoid markers on the same cell 4 ____ leukemia describes when abnormal cells are found only in the bone marrow 5 Type of analysis using DNA technology to identify genetic defects 7 Immature lymphoid cell with abundant pale staining cytoplasm and a large central prominent nucleolus 8 Cytochemical stain helpful in diagnosising ALL and acute erythroid and megakaryoblastic leukemias (abrv) 10 Stain generally used to differentiate monoblasts and myeloblasts

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Neoplastic Hematologic Disorders—Level II P L A S M A C Y T O M A B A O M I L L O P S E U D O P E L G E R H U E T L H U R I E E K O P D C N E C E L L U L A R I U O M Y S N L T I M E E A Y C P B C R A B L S T R A P H L T I D Y S P O I E S I S U E C C E R P A N M Y E L O S I S A T S M I C R O M E G A K A R Y O C Y T E

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6 Body Fluids

A.

TRANSITION GRID

Information concerning Chapter 28, “Morphologic Analysis of Body Fluids in the Hematology Laboratory” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 28

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 33 Chapter 30 Chapter 17 Chapter 25

B.

OBJECTIVES

Chapter 28 – Morphologic Analysis of Body Fluids in the Hematology Laboratory Level I 1. List the types of body fluids studied in the clinical laboratory, and describe the body cavities in which they are found.

2. List and describe the normal tissue cells seen in each body fluid type.

3. Describe various artifact types that can be seen in body fluid preparations including those in cytocentrifuged specimens.

4. Define transudate, exudate, and chylous fluid, and differentiate among these fluid types given laboratory data.

5. Describe the appearance of bacterial and fungal organisms in Wright-stained preparations, and suggest methods to confirm them.

6. List the morphologic features of malignant cells on Wright-stained preparations.

7. List the types of crystals that can be found in joint fluids, and associate them with pathologic conditions. ..

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Level II 1. Identify the procedure used to obtain each type of body fluid.

2. Describe the production of cerebrospinal fluid (CSF).

3. Compare and contrast how each of the following fluids is formed: transudate, exudate, and chylous fluid.

4. Evaluate the significance of microorganisms present in Wright-stained body fluid preparations.

5. Compare and contrast the morphologic distinction between benign tissue cells and malignant cells in cytocentrifuged, Wright-stained preparations of body fluids, and identify these cells when seen.

6. Recognize erythrophagocytosis, and explain its significance in CSF. 7. Define birefringence, and explain its use in examining body fluids.

8. Identify and differentiate the various types of crystals that can be found in joint fluids, and associate them with particular disorders.

9. Use peripheral blood and CSF cell counts to differentiate true hemorrhage from a traumatic tap.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Hematology Bowl Create a Hematology Bowl using the following categories: • Cerebrospinal fluid • Serous fluid • Synovial fluid • Artifacts seen in fluids • Cells in fluids • Crystals seen in fluids • Microorganisms seen in fluids 3. Mapping a. Have the learners create a map of “body fluids in the hematology laboratory.”

4. Match-Up Game Create a concentration game using: a. The types of body fluids and the area in the body they are found b. Terms and their definitions/characteristics • CSF white count reference ranges • Total synovial fluid WBC and percent neutrophil with the general diagnosis • Crystal identification with its appearance

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c. Fluid artifacts seen with Wright-stained cytocentrifuge prepared slides and potential mistaken interpretation.

5. Mystery Box a. **Create mystery box entries, including the types of benign mesothelial cells and malignant cells and several of their general morphologic findings (see Table 28-6).

6. One-Minute Paragraph Have the learners prepare a one minute paragraph on the: a. Formation of each type of body fluid 7. Think-Pair-Share a. Discuss the types of cells found in all types of fluids (both normal and abnormal) and the significance of each. b. **Discuss the artifacts found in the various types of fluids. c. **Discuss the differences between transudates and exudates. d. Discuss the morphologic features of reactive mesothelial cells versus malignant cells.

8. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

D.

LABORATORY ACTIVITIES

1. Examine prepared cytospin smears of all types of fluids. Alternatively, show pictures of body fluid cells from Bonus Image Library on DVD that accompanies this text. Divide students into teams and have teams identify those cells you have designated as “unknowns”.

2. Perform a differential on a CSF slide.

• If differentials are not performed, have the student write a paragraph explaining the types of cells that are observed.

E.

PRACTICE QUESTIONS

1. List the reference ranges of the red and white cells normally found in CSF. [Taxonomy 1] 2. _____ crystals would be found in the _____ fluid in a patient with gout. [Taxonomy 1] 3. Match the following crystals with the appearance. [Taxonomy 1] Crystal a. Monosodium Urate b. Calcium pyrophosphate c. Cholesterol d. Talc

Appearance 1. short, rectangular 2. Maltese cross shaped 3. long, thin, needle-like 4. amorphous 5. large, platelike, notched

4. Discuss the differences found in CSF samples for a traumatic tap and cerebral hemorrhage.

[Taxonomy 2] 5. In the examination of a spinal fluid, it appeared that yeasts were present, including some budding yeast. In doing the tests for confirmation of yeast, they were all negative. What might this yeastlike substance be? What test would confirm this? [Taxonomy 2]

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6. **In a pleural fluid, there was an increased number of granulocytes. These granulocytes contained toxic granulation and cytoplasmic vacuoles. What type of organism might be causing this reactive change? [Taxonomy 2] a. viral b. fungal c. bacterial d. mycobacterial e. all of these could cause these changes 7. **Compare and contrast transudates and exudates. [Taxonomy 3] 8. **Compare and contrast the cells seen in a patient with viral or fungal infection versus lymphoma in an effusion. [Taxonomy 3] 9. A pleural fluid that had a very milky appearance was received by the laboratory. What tests would be used to confirm the cause of the appearance? [Taxonomy 3] CASE STUDY DJ., a 6-year-old girl, is admitted to the hospital with a fever and a stiff neck. A spinal fluid was obtained with the following results: CSF Cell Counts WBC 130/uL RBC 5,000/uL

Peripheral Cell Counts 3.6 * 109/L 3.9 * 1012/L

Because both the red cell count and white cell count were increased, the physician wanted to determine if these results were due to a traumatic tap or if meningitis was present. From the above, answer the following questions.

10. What would be the corrected white cell count on this patient? [Taxonomy 2] 11. Is this WBC count indicative of a pathologic state? [Taxonomy 2] 12. If this is a case of meningitis, what would help differentiate a bacterial meningitis from a viral meningitis? [Taxonomy 2] 13. What is the normal ratio of red cells to white cells in the peripheral blood? [Taxonomy 2]

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Body Fluids—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

G T K L X C D A C R P I G R T D V Z M X N L C J H X M G L A

E T A D U S C H Y L O U V G Q G F R G H W O Q F N D Z U P T J T D T I J T K H C L J N A S O T L G A M R C G L C W U I H P K L L A O N W A L U R K M M S W K T P E R I C I V O N Y S

N S F E K B F N B R R T T A R

A R T C H F S C T Q W R K R T Q N Q A H J K S Z E X R F H K G H Q O T F J M R R D I G Q X

V G T Q N V R Q T N B L Z L I H C H C G P R L O A Y R T L T S P U P R E N T N A L K A M K

• common artifact found in body fluids • in disease states, the cell layers will thicken and fluid may accumulate • sac that encloses the heart • ____ indicates a yellowing color to the CSF • disease that is crystal induced • fluid from around the heart or lungs • an ultrafiltrate of plasma • fluid found in a joint • a ___ tap may elevate the CSF protein • WBC found in bacterial infections • benign cells must be differentiated from ___ cells in any body fluid • a milky or lipemic fluid

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Body Fluids—Level I G K X D C P G T V M N

T L C A R I R D Z X L

C J H X M G L A

E C V G N J T N G L P

T A D H Y L G Q G H W O D Z U T D T K H C A S O A M R C W U K L L

N W A K M M T P E I V O

U O F Q P I L T C I A

S U R F T J J L G H O

N S F E K B F N B R R

A R T C H F S C T Q W R K R T Q N Q A H J K S Z E X R F H K G H Q

V Q R N Z H C R A T S

G N Q B L C G L Y L P

T V T L I H P O R T U

L U R S W K R I C N Y S

T T A R

O T J M R D G Q

P N A A

R T L M

E N K K

F R I X

• common artifact found in body fluids (starch) • in disease states, the cell layers will thicken and fluid may accumulate (ascites) • sac that encloses the heart (pericardial) • ____ indicates a yellowing color to the CSF (xanthochromia) • disease that is crystal induced (gout) • fluid from around the heart or lungs (serous) • an ultrafiltrate of plasma (transudate) • fluid found in a joint (synovial) • a ___ tap may elevate the CSF protein (traumatic) • WBC found in bacterial infections (neutrophil) • benign cells must be differentiated from ___ cells in any body fluid (malignant) • a milky or lipemic fluid (chylous)

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Body Fluids—Level I 1

2

3 4

5

6

7

8

9 10 11

12

ACROSS

DOWN

1 Cavity that consists of the space between the inside of the abdominal wall and the outside of the stomach 6 Concentration of CSF glucose represents what percent of blood glucose (2 words) 8 A demyelinating disorder (2 words) 9 Fluid name; saline is inserted into a cavity and then removed for examination 10 The fluid where most pathological yeast are found (abrv) 11 Cavity between the lung and the inside portion of the chest wall 12 Crystals should be examined using ____ light

2 The type of fluid that has a specific gravity of > 1.015 3 Location of the cerebral spinal fluid (2 words) 4 Joint fluid contains ___ ___, which makes it different from other fluids (2 words) 5 Reactive cells in a pleural fluid that may have multinuclei and may be found in clusters 7 Type of syphilis that invades the brain

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Body Fluids—Level I P E R I T O N E A L S X U H U B Y D M A A A E R L T S A U F O R T Y F I V E O C R E T H O R H N N M U L T I P L E S C L E R O S I S I L I C L A V A G E I D A R A S C S F Y P L E U R A L P I A D C P O L A R I Z E D

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Body Fluids—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

W T M K S Q E S X K Q U T T Y M B F A V M N P I D L F N O L E X T M T V S L K Y M O N T T K T G A R X S Z H R P I D Y V T H W L K E Z Y O V C C K U M I R M S L R W G J Q E N T Z K P K V S I S G G C R R P

R Q F Z N A H N V M X V M J P O E W Q S Y S G I X L A Q E P D S R L C U P S K U P E D U I F M F I O F S N J C F C U E R M D M E V T M Y I V N I N V Y G X N D U L Q V T C R G T F N M W A L X E F K M C M K N K Y M I F L N Q H K R X D N H T G E T N E C O R H R Q M M A S C I

R D M T G E K L S K T O N B T H K H L M E U B L L M I V D A O X L F R B N C K L C C H K E T R A T E S

• abnormal fluid collection • thin membrane that provides a covering of the pericardial cavity • in disease states, the cell layers will thicken and fluid may accumulate • special membrane that lines some bony joints of the body • reactive cells in a pleural fluid that may have multinuclei and may be found in clusters • a milky fluid that results from long standing tuberculosis • procedure used to obtain fluid from the joint space • one way to differentiate between mast cells and basophils in fluids (2 words) • ability of a particular material to reflect light • fluid that results from an increased capillary hydrostatic pressure • procedure to obtain fluid from the peritoneal cavity • a type of yeast found in a fluid that is intracellular with a non-staining cell wall

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Body Fluids—Level II W T M K S Q E S X K Q U T T Y M B F A V M N P I D L F N O L E X T M T V S L K Y M O N T T K T G A R X S Z H R P I D Y V T H W L K E

R Q F Z N V M X V M E W Q S Y X L A Q E R L C U P P E D U I I O F S N C U E R M V T M Y I N V Y G X L Q V T C

Z K S Q P G

F F Y K E R

Y O V C C U M I R M L R W G J E N T Z K K V S I S G C R R P

A J S P S F J D V N R

N M W A L K M C M K M I F L N R X D N H T N E C O Q M M A S

H N P O G I D S K U M F C F M E N I D U G T

R D M T G E K L S K T O N B T H K H L M E U B L L M I V D A O X L

X N Q T R C

F N L H T T

E K H G H I

R C C K R E

B K C E A S

• abnormal fluid collection (effusion) • thin membrane that provides a covering of the pericardial cavity (pericardium) • in disease states, the cell layers will thicken and fluid may accumulate (ascites) • special membrane that lines some bony joints of the body (synovium) • reactive cells in a pleural fluid that may have multinuclei and may be found in clusters (mesothelia) • a milky fluid that results from long standing tuberculosis (pseudochylous) • procedure used to obtain fluid from the joint space (arthrocentesis) • one way to differentiate between mast cells and basophils in fluids (2 words) (round nucleus) • ability of a particular material to reflect light (birefringence) • fluid that results from an increased capillary hydrostatic pressure (transudate) • procedure to obtain fluid from the peritoneal cavity (pericentesis) • a type of yeast found in a fluid that is intracellular with a non-staining cell wall (histoplasma)

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Body Fluids—Level II 1

3

2

4

5 6

7 8 9

10

11

ACROSS

DOWN

5 The process in which a phagocytic cell ingests red cells found in autoimmune diseases 7 One of the most common nonhematopoietic malignancy found in body fluids 9 Artifactual change seen in body fluid neutrophils 10 Portion of the brain containing CSF (2 words) 11 Cells that produce CSF (2 words)

1 Type of immunity needed to limit infection by EBV 2 The type of fluid formed from a leakage of lymphatic vessels 3 Artifactual change seen in lymphocytes (2 words) 4 The process of the nucleus on one cell conforming to the shape of an adjacent cell (2 words) 6 Procedure used to obtain fluid from the pleural cavity 8 Common contaminate in all types of fluid

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Body Fluids—Level II C C E H L Y L L H U N O Y L U U E R Y T H R O P H A G O C Y T O S I S T E R L H R E O S A R A D E N O C A R C I N O M A O S G M C Y T O P L A S M I C P R O J E C T I O N S E A E L N R N D T C T I E H S U B A R A C H N O I D S P A C E S T G I I S C H O R O I D P L E X U S N

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7 Hemostasis

A.

TRANSITION GRID

Information concerning Chapter 29, “Primary Hemostasis”; Chapter 30, “Secondary Hemostasis and Fibrinolysis”; Chapter 31, ”Disorders of Primary Hemostasis”; Chapter 32, ”Disorders of Secondary Hemostasis”; and Chapter 33, ”Thrombophilia” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 29

Chapter 30

Chapter 31

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 34 Chapter 24 Chapter 40 Chapter 23

Chapter 35 Chapter 24 Chapter 40 Chapter 23

Chapter 38 Chapter 28 Chapter 42 Chapter 24

Clinical Laboratory Hematology

Chapter 32

Chapter 33

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 36 Chapter 25 Chapters 41, 43 Chapter 24

Chapter 37 Chapters 26, 27 Chapters 42 Chapter 24

B.

OBJECTIVES

Chapter 29 – Primary Hemostasis Level I 1. Define hemostasis, blood coagulation, and thrombosis.

2. Explain the general interaction of the systems involved in maintaining hemostasis.

3. Distinguish the events that occur in primary hemostasis from those that occur in secondary hemostasis. ..

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4. Differentiate the primary hemostatic plug from the secondary hemostatic plug.

5. Name the three types of blood vessels and explain the general roles of the vasculature and normal endothelial cells in aiding and preventing activation of the hemostatic system.

6. Describe the normal morphology and number of platelets on a peripheral blood smear and state the normal concentration in the blood.

7. Name and describe the cell that is the precursor of platelets in the bone marrow.

8. Identify and define the steps in the normal sequence of events of platelet activation following injury to the endothelium.

9. Describe the role of the primary hemostatic plug in the cessation of bleeding.

Level II 1. Identify key histologic features of each type of blood vessel and explain the metabolic functions of endothelial cells in hemostasis.

2. Describe the development of megakaryocytes and platelets in the bone marrow to include the action of humoral factors, the stem cell and progenitor cell compartment, the recognizable features of the morphologic stages, and the mechanism of release from the marrow to peripheral blood.

3. Define endomitosis and polyploidy.

4. Identify three key substances that are stored in the platelet dense bodies and five key substances stored in the platelet alpha granules and explain the role of each in hemostasis.

5. Outline steps the platelets undergo in forming the primary hemostatic plug, including the biochemical mediators necessary for platelet adhesion, platelet aggregation, and platelet secretion.

6. Correlate various platelet ultrastructural features with their functions.

7. Identify platelet agonists and predict their effect on platelet function.

8. Describe the biochemical roles of the secreted contents of the platelet granules in hemostasis.

9. Correlate activation with changes in the platelet ultrastructure and biochemistry.

10. Describe the roles of the platelet in secondary hemostasis.

11. Identify the life span of platelets in the peripheral blood.

Chapter 30 – Secondary Hemostasis and Fibrinolysis Level I 1. Define hemostasis. Identify the three physiologic compartments involved in the hemostatic mechanism.

2. Differentiate primary and secondary hemostasis.

3. List the coagulation factors using Roman numerals and determine how each is evaluated in lab testing.

4. Classify the coagulation factors into groups and discuss their characteristics.

5. Evaluate the importance of vitamin K in hemostasis.

6. Describe the mechanism of action of the coagulation proteins.

7. Diagram the sequence of reactions in the coagulation cascade according to the historic concepts of intrinsic, extrinsic, and common pathways.

8. Identify the factors involved in contact activation.

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9. Diagram the physiologic pathway of blood coagulation.

10. Define fibrinolysis, identify the major components of the fibrinolytic system, and explain why fibrinolysis is a necessary component of hemostasis.

11. List the fragments resulting from fibrinolytic degradation; compare and contrast the fragments resulting from the degradation of fibrinogen and fibrin.

12. List the major biochemical inhibitors that regulate secondary hemostasis.

Level II 1. Explain the interactions of the three physiologic compartments involved in hemostasis.

2. Describe the domain structure of the coagulation factors, determine how this structure affects the action of the serine proteases, and explain the significance of the noncatalytic regions.

3. Summarize the formation of complexes on a phospholipid surface and explain the significance of these complexes to hemostasis.

4. Integrate the role of contact factors with other systems (complement activation, fibrinolysis, inflammation).

5. Describe the multiple roles of thrombin in hemostasis.

6. Explain the physiologic functions of ADAMTS-13, LRP, EPCR, UPAR. 7. Compare and contrast physiologic and systemic fibrinolysis.

8. Describe the physiologic controls of hemostasis including blood flow, feedback inhibition, liver clearance, and inhibitors (antithrombin, tissue factor pathway inhibitor, protein C, protein S, and TAFI).

9. Evaluate a case study from a patient with a defect in hemostasis and, using the medical history and laboratory results, determine the diagnosis.

Chapter 31 – Disorders of Primary Hemostasis Level I 1. Define and differentiate among thrombocytopenia, thrombocytosis, and thrombocythemia and state an expected range of platelet count in each.

2. Define and differentiate among petechiae, purpura, ecchymosis, hematoma, and easy bruisability.

3. Identify laboratory tests that can be ordered to screen for abnormalities of the hemostatic system.

4. Explain the expected clinical consequences when a patient has an abnormality of platelets or blood vessels.

5. Correlate quantitative variations in the platelet count with disease manifestations.

6. Recognize hematologic disorders that are characterized by the presence of thrombocytopenia or thrombocytosis.

7. Describe the etiology, pathophysiology, and laboratory findings of the thrombocytopenias.

8. Differentiate primary (malignant) from secondary (reactive) thrombocytosis.

9. Explain the effect of aspirin and its duration on platelet function.

10. Identify the cause and describe the clinical and laboratory features of hereditary disorders of platelet function.

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Level II 1. Categorize each specific disorder of hemostasis by body system affected (e.g., vasculature, platelets).

2. Predict the type of bleeding symptoms in patients with disorders of primary hemostasis.

3. Describe the expected symptomatology, etiology, pathophysiology, and laboratory test results in patients with disorders of the vasculature.

4. Organize thrombocytopenic and thrombocytosis conditions by etiology and pathophysiology and explain laboratory findings in each condition.

5. Differentiate acute from chronic immune thrombocytopenic purpura by significant clinical and laboratory data.

6. Explain the pathophysiology of thrombocytopenia and thrombocytosis in hematologic disorders.

7. Organize the hereditary and acquired qualitative platelet defects by etiology and pathophysiology, and predict the clinical and laboratory features.

8. Explain the biochemical mechanism of the effect of aspirin on platelet function and recommend a time frame for patients to refrain from taking aspirin and related anti-inflammatory drugs prior to platelet function testing.

9. Summarize the effect of aspirin, alcohol, and antibiotics on platelet function.

Chapter 32 – Disorders of Secondary Hemostasis Level I 1. Define deficiency as it relates to the proteins of secondary hemostasis.

2. Describe the expected results of laboratory screening tests that detect abnormalities of the proteins of secondary hemostasis.

3. Describe the expected laboratory results, pathophysiology, and clinical symptoms in patients with classic (type I) von Willebrand disease.

4. Identify hemostatic proteins that are deficient in hemophilias A and B. 5. Characterize deficiencies of factors VIII and IX by inheritance pattern, clinical symptoms, and laboratory findings.

6. Differentiate classic (type 1) von Willebrand disease and deficiencies of factors VIII and IX based on results of laboratory tests and clinical symptoms.

7. Identify clinical conditions associated with acquired disorders of the hemostatic proteins and describe the expected results in laboratory screening tests for hemostasis.

8. Characterize disseminated intravascular coagulation (DIC) by etiology, pathophysiology, and the results of laboratory testing.

9. Explain physiologic variations in the reference ranges of laboratory hemostatic screening tests in newborns.

10. Describe the expected general clinical consequences in a patient with an abnormality of the proteins of secondary hemostasis.

Level II 1. Explain the clinical symptoms characteristic of deficiencies of the fibrin-forming proteins, and contrast them with the symptoms associated with disorders of primary hemostasis.

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2. Compare and contrast the results of laboratory tests for hemostasis in disorders of primary and secondary hemostasis.

3. Describe genetic mutations and diagram the inheritance pattern that results in deficiencies of the hemostatic proteins.

4. Differentiate von Willebrand disease subtypes, deficiencies of factors VIII and IX, and the inherited deficiencies of the remaining proteins of fibrin formation by inheritance pattern, pathophysiology, clinical symptoms, and laboratory findings.

5. Select and interpret the results of laboratory tests, and identify clinical symptoms that differentiate von Willebrand disease subtypes, deficiencies of factors VIII and IX, Bernard-Soulier disease, and Glanzmann’s thrombasthenia.

6. Describe the pathophysiology of the conditions that result in acquired abnormalities of the hemostatic system, and select confirmatory laboratory procedures.

7. Select and describe the laboratory screening methods for distinguishing between deficiencies and inhibitors of hemostatic proteins.

8. Describe the significance and clinical implications of the development of circulating anticoagulants, and select laboratory procedures that confirm and differentiate between specific and nonspecific factor inhibitors.

9. Choose laboratory methods that differentiate between excessive primary and secondary fibrinolysis, and support your selection.

Chapter 33 – Thrombophilia Level I 1. Define hypercoagulability, thrombophilia, thrombus, and thrombosis.

2. Describe the physiological processes involved in hypercoagulability.

3. Explain how a thrombus becomes a thromboembolus.

4. Contrast a white thrombus and a red thrombus, and list the risk factors predisposing to the formation of each.

5. Define deep vein thrombosis (DVT); explain how it is diagnosed and what clinical conditions can result.

6. Describe the role of heparin in the neutralization of activated coagulation factors by antithrombin.

7. Diagram the relationship of protein C (PC) and protein S (PS) to the coagulation pathway and explain why a deficiency of either might lead to a thrombotic tendency.

8. Explain why both molecular (antigenic) and functional assays should be performed for diagnosing antithrombin, protein C (PC), or protein S (PS) abnormalities in a patient.

9. Describe activated protein C (APC) resistance (APCR), and explain its contribution to thrombophilia.

10. List two side effects of heparin therapy and what hematology procedures should be monitored to prevent or limit these complications.

11. Contrast unfractionated heparin (UFH) and low molecular weight heparin (LMWH) including anticoagulant mechanisms and monitoring.

12. Discuss how oral anticoagulants such as coumadin decrease a person’s risk for thrombosis, and describe the best way to monitor oral anticoagulation.

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Level II 1. Evaluate the utility of laboratory tests in the differential diagnosis of arterial and venous thrombotic disease.

2. List four clinical manifestations suggestive of an inherited thrombophilia, and explain why many patients are not diagnosed.

3. Explain the differences between type I and type II deficiencies of protein C (PC), protein S (PS), and antithrombin; given values for antithrombin or protein C and S assays, determine the type of deficiency in a patient.

4. List two deficiencies that can cause warfarin- (coumadin-) induced skin necrosis and give potential treatment options.

5. Justify performing both a clotting and molecular assay for diagnosis of activated protein C resistance (APCR).

6. Describe the two most common mutations leading to hyperhomocysteinemia.

7. Explain how increased levels of prothrombin or fibrinogen might predispose to thrombosis.

8. Describe how deficiencies of plasminogen or plasminogen activator or increased levels of plasminogen activator inhibitor could lead to thrombosis.

9. Identify secondary disorders leading to thrombosis.

10. Describe and explain the treatment of a thrombotic episode.

11. Explain how the INR has standardized prothrombin times.

12. Evaluate the use and monitoring of thrombolytic therapy in treatment of thrombosis.

13. Given a patient history and appropriate laboratory results, interpret the data to determine a probable diagnosis.

Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Diagram Have the students diagram the: a. Coagulation cascade b. Major naturally occurring inhibitors in coagulation c. **Fibrinolytic system d. **Positive feedback system in coagulation 3. Extra, Extra, Read All about It Have a small group of learners compose a: a. Newspaper article based on an interview with a Platelet on the “Life of a Platelet Á Its Encounters and Its Activities” 134

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b. Feature story on how a blood clot is formed c. Press release from a press agent for a celebrity that has been identified as “hypercoagulable” 4. Group Discussion Create a group discussion on: a. **Inherited disorders in hemostasis and how each affects the individual b. **Platelet disorders comparing and contrasting the qualitative and quantitative disorders c. **The role of the arachidonic acid pathway d. Laboratory testing in hemostasis e. **Functions of endothelial cells f. Process of fibrin stabilization g. Autoimmune thrombocytopenia 5. Mapping Create a map for: a. **Platelet alpha granule proteins (include function and composition) b. Acquired disorders of the vascular system c. Qualitative platelet disorders 6. Match-Up Game Have the students create a match-up game for: a. Coagulation factors numbers and their common name b. Terms and their definitions • Hemostasis • Thrombosis • Thrombopoietin • Thrombogenic • Glycocalyx • Platelet adhesion • Platelet aggregation • Clot retraction • Prothrombin group • Fibrinogen group • Contact group • Vitamin K dependent factors • Fibrinolysis • Inhibitors • Plasminogen activators • INR • And so on c. Coagulation tests and their descriptions or reference ranges d. **Coagulation factors and their description or function e. **Composition and function of platelet alpha granule proteins f. **Naturally occurring inhibitors and their function g. **Components of the fibrinolytic system and their function (see Table 30-4) 7. Mystery Box a. **On separate pieces of paper (or index card) place the description, T1/2, function, plasma concentration, and chromosome properties (see Table 30-2). Have the learners compile the information and identify which coagulation factors have these properties. b. Have learners create laboratory results for platelet count, PT, APTT, and a template bleeding time for the defects in primary hemostasis. c. **On separate pieces of paper write the results of the different features for Acute ITP and Chronic ITP. Have the learners assemble the results for each condition then explain which feature these results represent and why it is important. ..

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d. **Write the results for the laboratory screening tests for the coagulation factor disorders.

Have the learners identify the factor disorder and create the clinical condition and patient background information that correlates with the screening test results.

8. One-Minute Paragraph Have the students prepare a one minute paragraph on each of the following topics: a. Primary hemostasis b. Secondary hemostasis c. Fibrinolysis d. **Platelet granules e. **Thrombophilia f. Any of the coagulation tests • Bleeding Time • Prothrombin time • Activated partial thromboplastin time • Thrombin time • Mixing studies • **Factor assays • **Reptilase time • **Factor VIII inhibitor assay • And so on g. **Describe the three layers of tissue in the vessel wall. h. **Regulation of platelet production i. Describe the role of thrombin. j. **Explain how the clinical bleeding symptoms differ in patients with coagulation factor deficiencies versus platelet defects.

9. Mnemonic a. Have the students develop a mnemonic for the names of the coagulation factors in order, that is, Frank Provided Tea, Coffee, Pizza, Pretzels, And Pop So Phil’s Hunger Faded (fribrinogen, prothrombin tissue factor, calcium, proaccelerin, proconvertin, antihemophilic factor, plasma thromboplastin component, Stuart-Prower, plasma thromboplastin antecedent, Hageman, fibrin stabilizing factor). b. Other mnemonics might include: • Specific factors found in each of the coagulation groups—contact, fibrinogen, prothrombin, and vitamin K dependent • Portions of the pathway—intrinsic, extrinsic, and common • Naturally occurring inhibitors of coagulation • **Factors leading to thrombophilia 10. Pyramid Game Have two learners create the categories for another pair of learners and work through this activity. Utilize the following topics: • Primary hemostasis • Secondary hemostasis and fibrinolysis • **Disorders of the vascular system • Disorders of primary hemostasis • Disorders of secondary hemostasis • Thrombophilia • Laboratory testing in coagulation 11. Short Story Have the students individually or as a group write a story: a. **Comparing the lives of two or three individuals who have different hemostatic disorders 136

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b. Describing the role of platelets in hemostasis c. About great-uncle “von Willebrand” and the disease that carries his name 12. Small Group Teaching Assign a small group of learners the following topics. They should be instructed to present and teach these concepts to the rest of the class. a. The fibrinolytic system b. Inhibitors of fibrinolysis c. Physiologic control of hemostasis d. **Compare and contrast the laboratory tests results for disorders in primary and secondary hemostasis. e. Normal hemostasis and common bleeding disorders in the newborn f. Thrombus formation 13. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

14. Tic-Tac-Toe Create a Tic-Tac-Toe activity, including the: a. Disorders of Hemostasis • Disorders of primary hemostasis • Disorders of secondary hemostasis • Thrombophilia b. Laboratory testing in coagulation c. **Developmental Stages of Megakaryocytes

D.

LABORATORY ACTIVITIES

These tests are described in Chapter 40. See Chapter 40 Website for laboratory procedures for the PT and APTT tests.

Perform the following suggested laboratory activities: 1. PT and APTT coagulation testing.

• If available, both the fibrometer and an automated instrument should be used.

• Compare the results from each instrument.

• An additional experiment can use samples where the storage conditions were not correct and/or the centrifugation was inadequate.

• Students can use paired samples to determine the effect of abnormal storage or centrifugation.

2. Manual platelet count (This procedure is discussed in Chapter 34) 3. Bleeding times 4. PT and APTT coagulation testing on factor deficient plasma 5. Mixing studies 6. Fibrinogen assay 7. **Specific factor assays 8. **Platelet functional assays (if an aggregometer is available) • Case studies with graphs from the aggregometer may be used if an instrument is not available.

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PRACTICE QUESTIONS

1. List four types of granules found in the platelet. [Taxonomy 1] 2. The cytokine responsible for megakaryocyte maturation is _____. [Taxonomy 1] 3. Match the factor number with the common name: [Taxonomy 1] Factor a. IV b. VIII c. I

Common Name 1. Fibrinogen 2. Calcium 3. Anti-Hemophiliac 4. No common name listed

4. Discuss how protein C and protein S deficiencies contribute to thrombosis. [Taxonomy 2] 5. **Differentiate von Willebrand’s disease and factor VIII deficiency by using laboratory test results.

[Taxonomy 2] 6. **Describe the physiological controls that are found in hemostasis. [Taxonomy 2] 7. **Compare and contrast the laboratory results of DIC and HUS, including both coagulation results and appearance on the peripheral smear. [Taxonomy 3] 8. Compare and contrast protein C and antithrombin III deficiencies. [Taxonomy 3] 9. The following laboratory results were obtained on a patient: PT: 15 seconds, APTT: 60 seconds, D-Dimer: increased with the controls for each test being within acceptable limits. What should the laboratory professional look for on the peripheral blood smear that may help the physician with a diagnosis? [Taxonomy 3] a. Bite cells b. Schistocytes c. Target cells d. Spherocytes CASE STUDY LT, a 26-year-old female, had been hospitalized twice in the past two years with deep vein thrombosis (DVT) in the leg. She had not had any previous problems until she decided to take birth control pills. She remembered that her grandmother and her mother had had episodes of thrombosis which she mentioned to her physician during her second episode of DVT. LT’s physician decided that a work-up for thrombophilia was needed. Discuss the following: 10. Why did LT’s physician decide that a work-up for thrombophilia was needed? [Taxonomy 2] 11. Would this most likely be a primary or secondary disorder? [Taxonomy 1] 12. What tests should be ordered in a thrombophilia work-up? [Taxonomy 1] 13. What tests would you expect to be abnormal? [Taxonomy 2] 14. What are the principles for each of these assays? [Taxonomy 1]

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Hemostasis—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

M F T N H I R D O A M M B U O O C C Y X T L O D S P I K S L

E M I T G N I D J R G D P T M T N R R T D U D L L P P B I I Q I J B T C Q J M Z T Q L Y M Y C E D A K H K N L G C W B D I V T C C M P M Q T G O F N S B R T N D N A G J K T W N L L B N A Q R P T V N C C B K T T M T P Q T T X I V E R F B Z Q

E E L B V R W C T O J L K H N P C S R W C J I O I H P S M L R P Y B L C D L O E Z K O S B R R N I R C A I S A P V R Y N T R B H D G I I B S K Z F R N

• therapeutic anticoagulant • protein that degrades fibrin • the ___ is used to standardize the PT for the reagent and instrument (abrv) • plasmin breaks the fibrin polymer into the ___ fragment • increased number of platelets • the coagulation group that contains factors XII and XI • the hemostatic test that is used to determine platelet function (2 words) • disease in which platelets are destroyed by antibodies (abrv) • dissolution of the clot • coagulation factor IV • disease; patient clots “too much” • site of production of most coagulation factors • disorder in which there is a consumption of coagulation proteins and platelets (abrv) • test used to determine abnormalities in the intrinsic and common pathways (abrv) • ___ is the factor that attaches to coagulation factor VIII (2 words)

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Hemostasis—Level I M F T N H I R D O A M M B U O O C C Y X T L O D S P I K S L

E M I T G N I D J R G D P T M T N R R T D U D L L P P B I I Q I J B T C Q J M Z T Q L Y M Y C E D A K H K N L G C W B D I V T C C M P M Q T G O F N S B R T N D N A G J K T W N L L B N A Q R P T V N C C B K T T M T P Q T T X I V E R F B Z Q

E E L B V R W C T O J L K H N P C S R W C J I O I H P S M L R P Y B L C D L O E Z K O S B R R N I R C A I S A P V R Y N T R B H D G I I B S K Z F R N

• therapeutic anticoagulant (coumadin) • protein that degrades fibrin (plasmin) • the ___ is used to standardize the PT for the reagent and instrument (abrv) (inr) • plasmin breaks the fibrin polymer into the ___ fragment (ddimer) • increased number of platelets (thrombocytosis) • the coagulation group that contains factors XII and XI (contact) • the hemostatic test that is used to determine platelet function (2 words) (bleeding time) • disease in which platelets are destroyed by antibodies (abrv) (itp) • dissolution of the clot (fibrinolysis) • coagulation factor IV (calcium) • disease; patient clots “too much” (thrombosis) • site of production of most coagulation factors (liver) • disorder in which there is a consumption of coagulation proteins and platelets (abrv) (dic) • test used to determine abnormalities in the intrinsic and common pathways (abrv) (apt) • ___ is the factor that attaches to coagulation factor VIII (2 words) (von willebrands)

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Hemostasis—Level I 1

2

3 4

5

6 7 8

9

10

11

12

13 14

15

ACROSS

DOWN

4 Coagulation factor IX 6 Small, purple spots caused by blood escaping from capillaries into intact skin 7 The ___ pathway of coagulation begins with factor X 8 Platelet plug 9 One of the functions of platelets 11 The cell from which platelets are derived 12 Naturally occurring inhibitor of coagulation 13 Part of the coagulation pathway that includes factor VII 14 Platelet function that aspirin inhibits 15 Decreased number of platelets

1 Distinct protein fragments formed from the dissolution of fibrin (abrv) 2 Activated by intrinsic and extrinsic activators to form plasmin 3 After vessel injury, the first step in hemostasis 5 Growth factor responsible for the increase in the number of platelets 10 Factor XII

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Hemostasis—Level I F P V D L C H R I S T M A S T P E T E C H I A E S H S C O M M O N R T H R O M B U S C O I O M A D H E S I O N N B A O S O G M E G A K A R Y O C Y T E S P E E R O M A N T I T H R O M B I N I A C E X T R I N S I C T T A G G R E G A T I O N I O N T H R O M B O C Y T O P E N I A

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Hemostasis—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

L B D Q L V F Q J A F I V S E R V N X T Y Y R K K W B J J T R B Y W V N T T C N G I M P D C U V Z R Q Y A N S Y N I L F S Y L F K W V A D N O I

J K R O R E Z O Z Z I B Y J R R D K N F L K J K C N J M B R I N O G E N E M I A J D O T O N I N K W M L F C I F B C W P T K Q F M L T C M A P M P M C E I X M P A M X C S D R Y I B T H Q C H H L T R E P G R W T U I M E S G O F U C I T W D N C G M I M R R J N O S P O V T A O S V V B O Q T N D N B N G P O K L L B W K I D N M D Y H T W E T P H H C Y A T R W I I B I K H C Q O W C R M L L M X D N A H R S M N P Y N I O G E R T G W A K B P F R A D K N K M R Q V F N Z N T B N P L T A G E R G G A M H N E K M

• after vessel injury, the first step of hemostasis • the pathway inhibited by aspirin (2 words) • no detectable fibrinogen found • stage of hemostasis that generates fibrin • deficiency of factor IX (2 words) • general term used to describe the presence of petechiae and ecchymosis • defect in Glanzmann thrombasthemia • the form of ITP that occurs in young children and is self-limiting • nuclear DNA synthesis without cytoplasmic division • disorder in which there is a consumption of coagulation proteins and platelets (abrv) • substance found in the dense bodies of platelets that promote vasoconstriction • the anemia in which ineffective thrombopoiesis occurs (abrv) • a coagulation variant that results in thrombosis (3 words) • dissolution of the clot • the person who first established the role of platelets in hemostasis

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Hemostasis—Level II L B D Q L V F Q J A F I V S E R V N X T Y Y R K K W B J J T R B Y W V N T T C N G I M P D C U V Z R Q Y A N S Y N I L F S Y L F K W V A D N O I

J K R O R E Z O Z Z I B Y J R R D K N F L K J K C N J M B R I N O G E N E M I A J D O T O N I N K W M L F C I F B C W P T K Q F M L T C M A P M P M C E I X M P A M X C S D R Y I B T H Q C H H L T R E P G R W T U I M E S G O F U C I T W D N C G M I M R R J N O S P O V T A O S V V B O Q T N D N B N G P O K L L B W K I D N M D Y H T W E T P H H C Y A T R W I I B I K H C Q O W C R M L L M X D N A H R S M N P Y N I O G E R T G W A K B P F R A D K N K M R Q V F N Z N T B N P L T A G E R G G A M H N E K M

• after vessel injury, the first step of hemostasis (vasoconstriction) • the pathway inhibited by aspirin (2 words) (arachodonic acid) • no detectable fibrinogen found (afibrinogenemia) • stage of hemostasis that generates fibrin (secondary) • deficiency of factor IX (2 words) (hemophilia b) • general term used to describe the presence of petechiae and ecchymosis (purpura) • defect in Glanzmann thrombasthemia (aggregation) • the form of ITP that occurs in young children and is self-limiting (acute) • nuclear DNA synthesis without cytoplasmic division (endomitosis) • disorder in which there is a consumption of coagulation proteins and platelets (abrv) (dic) • substance found in the dense bodies of platelets that promote vasoconstriction (serotonin) • the anemia in which ineffective thrombopoiesis occurs (abrv) (pnh) • a coagulation variant that results in thrombosis (3 words) (factor v leiden) • dissolution of the clot (fibrinolysis) • the person who first established the role of platelets in hemostasis (Bizzozero)

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Hemostasis—Level II 1

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1 The endothelial surface receptor for thrombin 5 When the balance of hemostasis tips towards clotting 7 Platelet consumptive disorder in which platelet thrombi form due to endothelial cell damage (abrv) 10 The event that is occurring during platelet aggregometry when the curve first moves from the baseline (2 words) 11 Bruise caused by bleeding from arterioles into subcutaneous tissue without disruption of intact skin 12 A circulating substance that is known as the lupus anticoagulant (2 words) 13 Antithrombin inhibits the fibrinolytic system by inhibiting ___ 14 Enzyme; factor XIIIa is the only coagulation protein with this enzymatic activity 15 Serine-protease inhibitor; inhibits target molecules by formation of 1:1 stoichiometric complex

2 Normal levels of fibrinogen but abnormal fibrinogen molecules 3 ___ ___ is the platelet disorder in which there is decreased platelet production and decreased adhesion (2 words) 4 Storage pool disease in which the alpha granules are lacking (3 words) 6 An amorphous coat of glycoproteins and mucopolysaccharides covering the surface of cells 8 In platelet aggregation studies, ___ will give variable results in patients with von Willebrand’s disease 9 Growth factor responsible for the increase in the number of platelets

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Hemostasis—Level II T H R O M B O M O D U L I N B Y G H Y P E R C O A G U L A B L E S R R F A N I Y A G B T T P R L R L R D Y I A I S C N T S O T O O E T U H C G L O L R S H A P E C H A N G E C I O L N T E E C C H Y M O S E S Y E S T R B X M Y I O I N A N T I P H O S P H O L I P I D A N T I B O D Y O R K A L L I K R E I N O E M T R A N S G L U T A M I N A S E E I S E R P I N S

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8 Hematology/Hemostasis Procedures

A.

TRANSITION GRID

Information concerning Chapter 34, “Hematology Procedures”; Chapter 35, “Bone Marrow Examination”; Chapter 36, “Automation in Hematology and Hemostasis”; Chapter 37, “Flow Cytometry”; Chapter 38, “Chromosome Analysis of Hematopoietic and Lymphoid Disorders”; Chapter 39, “Molecular Analysis of Hematologic Diseases”; Chapter 40, “Laboratory Testing in Coagulation”; and Chapter 41, “Qualtiy Assessment in the Hematology Laboratory” may also be referenced in the following hematology textbooks.

Clinical Laboratory Hematology

Chapter 34

Chapter 35

Chapter 36

Chapter 37

McKenzie, 1st edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 7 Chapter 31 Chapters 14, 15 Chapters 26, 27

Chapter 9 Chapter 2 Chapter 16 Chapter 26

Chapter 41 Chapter 32 Chapter 39 Chapter 27

Chapter 9 Chapter 34 Chapter 33 Chapter 27

Clinical Laboratory Hematology

Chapter 38

Chapter 39

Chapter 40

Chapter 41

McKenzie, 2nd edition Harmening, 5th edition Rodak, 3rd edition Turgeon, 4th edition

Chapter 24 Chapter n/a Chapter 31 Chapter n/a

Chapter 25 Chapter 35 Chapter 32 Chapter 3

Chapter 39 Chapter 42 Chapter 33 Chapter 29 Chapters 45, 47 Chapter 5 Chapter 26 Chapter 1

B.

OBJECTIVES

Chapter 34 – Hematology Procedures Level I 1. Identify the three anticoagulants used in the hematology laboratory and give examples of laboratory tests that should be performed on blood anticoagulated with each.

2. Explain the mechanism of preventing coagulation for each anticoagulant. ..

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3. Identify equipment and supplies required for phlebotomy.

4. Describe Occupational Safety and Health Administration (OSHA) standards related to phlebotomy.

5. List factors affecting the collection of a blood sample.

6. Determine sequence of draw of phlebotomy collection tubes and correlate the collection technique of a blood sample with potential problems in sample analysis.

7. Describe proper disposal of contaminated equipment and supplies.

8. Identify the component parts of a microscope and explain their functions.

9. Discuss the microscope’s preventative maintenance procedures.

10. Describe the peripheral blood smear preparation methods.

11. Identify the characteristics of an optimally prepared peripheral blood smear.

12. Describe the Romanowsky-staining technique.

13. Recognize characteristics of a properly stained peripheral blood smear.

14. Discuss potential causes of improperly stained peripheral blood smears.

15. List the components included in a complete peripheral blood smear examination.

16. Correlate erythrocyte indices, leukocyte count, and platelet count with peripheral blood smear observations.

17. Determine the corrected leukocyte count based on the presence of nucleated erythrocytes.

18. State the principle of each test: cell enumeration by hemacytometer, hemoglobin concentration, hematocrit, erythrocyte sedimentation rate, reticulocyte count, solubility test for hemoglobin S, hemoglobin electrophoresis, acid elution for hemoglobin F, osmotic fragility.

19. For each test listed in Objective 18, describe the procedure, identify potential sources of error, determine appropriateness of use including reflex testing, calculate and interpret results, and explain the clinical significance of the test.

20. Calculate the erythrocyte indices.

21. Correlate erythrocyte indices with CBC data and peripheral blood smear examination.

22. Calculate the reticulocyte count.

23. Identify reference ranges for each test in terms of gender and age.

Level II 1. Describe factors that influence brightfield microscopy.

2. Explain the principle of phase contrast microscopy.

3. Choose corrective actions for an improperly prepared or stained peripheral blood smear.

4. Detect abnormalities on peripheral blood smear examination, assess how they can alter cell count results (i.e., presence of nucleated erythrocytes or platelet satellitism), and recommend corrective action to ensure valid results.

5. State the principle of each of the following tests: quantitation of hemoglobin F and hemoglobin A2, heat denaturation test for unstable hemoglobin, Heinz body stain, sugar-water screening test, Donath Landsteiner test for PCH, erythropoietin assay, soluble transferrin receptor, and cytochemical stains (e.g., myeloperoxidase).

6. For each test listed in Objective 5, describe the procedure, identify potential sources of error, determine appropriateness of use including reflex testing, interpret results, and explain the clinical significance of the test.

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7. For each test discussed in this chapter, correlate abnormal values with clinical conditions.

8. Select the cytochemical stain appropriate for confirming cell lineage and diagnosis in hematopoietic neoplasms.

Chapter 35 – Bone Marrow Examination Level I 1. Identify the sites for obtaining bone marrow samples.

2. List indications for the need to perform bone marrow studies.

3. Explain the difference between core biopsy and bone marrow aspirate and the use of each.

4. Describe how to perform a bone marrow differential count.

5. Define the M:E ratio, and explain what can cause an increase or decrease in it.

6. Describe how to estimate bone marrow cellularity and iron stores and interpret results.

7. Explain the role of the hematology laboratory in bone marrow evaluation.

8. List the reasons that special stains are used on bone marrow specimens.

Level II 1. Differentiate benign lymphoid aggregates from malignant lymphoma.

2. Evaluate the bone marrow specimen and select the appropriate specimens for ancillary studies.

3. Perform a bone marrow differential count and calculate and interpret the M:E ratio.

4. Estimate and interpret bone marrow cellularity.

5. Correlate results of marrow iron stores with various diseases.

6. Explain the role of the clinical laboratory professional in the bone marrow procedure.

7. Identify key features of a bone marrow report.

Chapter 36 – Automation in Hematology and Hemostasis Level I 1. Cite the electrical impedance principle of cell counting, and identify the instruments that use this technology.

2. Describe the use of radio frequency in cell counting, and identify the instruments that use this technology.

3. State the principles of light scatter used in cell counting, and identify the instruments that use this technology.

4. List the reported parameters for each blood cell–counting instrument.

5. Categorize cell parameters as directly measured and derived from a histogram, scattergram or cytogram, or calculation.

6. Describe the principle of reticulocyte count enumeration by automated blood cell–counting instruments.

7. State the electromechanical principle of clot detection, and identify instruments that use this technology.

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Level II 1. Compare and contrast the methods of analysis for the described histograms, scatterplots, scattergrams, and cytograms, and interpret the results.

2. Describe and interpret the automated reticulocyte parameters.

3. Describe the application of immunological techniques to coagulation instruments, and identify instruments that use this technology.

Chapter 37 – Flow Cytometry Level I 1. Describe the components of a flow cytometer and the principles of cell analysis.

2. Illustrate by example the clinical applications of flow cytometry.

3. Appraise the use of fluorochrome-labeled antibodies in immunophenotyping by flow cytometry.

4. Give examples of the clinical applications of immunophenotyping by flow cytometry, and interpret single dot plots.

5. Define clonality and identify methods for detecting a monoclonal population of cells by immunophenotyping.

6. List the specimens appropriate for immunophenotyping by flow cytometry.

7. Describe how flow cytometry can be used in cell quantitation.

8. Calculate and interpret the absolute CD4 count.

9. Describe reticulocyte counting by flow cytometry, and define immature reticulocyte fraction (IRF).

10. Explain how flow cytometry can be applied to DNA analysis.

11. List the cells positive for CD34, and explain the purpose of a CD34 count.

Level II 1. Compare and contrast the immunophenotyping results characteristic of chronic lymphocytic leukemia, hairy cell leukemia, and non-Hodgkin lymphoma.

2. Identify the pitfalls that can be encountered in immunophenotyping mature lymphoid malignancies by flow cytometry, and generate potential solutions.

3. Compare and contrast the immunophenotyping results characteristic of acute lymphoblastic leukemia and acute myelogenous leukemia.

4. Identify the pitfalls that can be encountered in immunophenotyping acute leukemia and lymphoma by flow cytometry, and generate potential solutions.

5. Compare and contrast the usefulness of the sucrose hemolysis test, Ham test, and flow cytometry immunophenotyping in diagnosing paroxysmal nocturnal hemoglobinuria.

6. Select and explain the quality measures that must be enforced in quantitative flow cytometry.

7. Compare and contrast the absolute CD4 count and HIV viral load in the surveillance of HIV infection.

8. Calculate and interpret the S phase fraction and the DNA index, and give the principle of DNA analysis by flow cytometry.

9. Assess the findings of flow cytometry cell analysis using two or more dot plots, and select the most likely cell represented.

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10. Evaluate flow cytometry results to identify problems and generate solutions.

11. Compare reticulocyte counting by flow cytometry with manual methods, and assess the advantages of flow cytometry.

12. Interpret the immature reticulocyte fraction (IRF).

13. Summarize the uses of analysis for the CD34 antigen, and choose recommended procedures to analyze this antigen.

Chapter 38 – Chromosome Analysis of Hematopoietic and Lymphoid Disorders Level I 1. Define chromosome and mitosis.

2. List the basic steps of cytogenetic analysis, and select the most appropriate type of specimen for analysis of suspected constitutional and neoplastic (acquired) disorders.

3. Identify two major types of chromosome abnormalities, describe how they occur, and use the appropriate terminology to describe them.

4. List the practical uses of cytogenetics in the diagnosis and prognosis of hematolymphoid disorders.

Level II 1. Describe chromosome morphology and mitosis.

2. Describe each step of the cytogenetic harvest procedure.

3. Define and compare aneuploidy, nondisjunction, and anaphase lag.

4. Define and compare translocation, deletion, inversion, and isochromosome.

5. Identify the general categories of hematopoietic disorders for which cytogenetic analysis is useful for patient care.

6. Correlate diagnostic chromosome aberrations with types of hematolymphoid disorders, and assess their prognostic and therapeutic implications.

7. Assess the prognostic impact of cytogenetic results in acute lymphoblastic leukemia.

8. Explain the clinical utility of cytogenetics in transplantation.

9. Correlate chromosome abnormalities with specific oncogene activation, and assess the role of the oncogene in the neoplasm.

Chapter 39 – Molecular Analysis of Hematologic Diseases Level II 1. Define terms and appropriately use nomenclature associated with molecular pathology.

2. Describe the principles and summarize the procedures for each of the common laboratory tests used in molecular diagnostics.

3. Describe and explain the applications of molecular tests in diagnosing and managing inherited disease, infectious disease, and cancer.

4. Explain the role of genes in acquired versus inherited disorders.

5. Identify common chromosomal translocations and molecular genetic abnormalities associated with chronic myelogenous leukemia, acute leukemia, and lymphoma.

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6. Explain how B and T cell malignancies can be differentiated from other conditions by molecular testing.

7. Select the most appropriate molecular test for a given patient condition or provisional diagnosis.

8. Compare and contrast the various molecular methods with regard to diagnostic applications, tissue requirements, and principles.

9. Compare the advantages and disadvantages of molecular tests with other laboratory tests used in diagnosing and managing hematologic disorders.

Chapter 40 – Laboratory Testing in Coagulation Level I 1. Describe special precautions to take regarding specimen collection and processing for coagulation studies, and determine specimen appropriateness.

2. State the principle of each test: bleeding time (BT), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen assay, factor assays, fibrin degradation products (FDP), D-dimer assay, mixing studies.

3. Describe the procedure for determining the bleeding time (BT), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen assay, fibrin degradation products (FDP) and D-dimer assay.

4. Explain the clinical significance of each test listed in Objective 2.

5. Identify the appropriate laboratory procedure for monitoring heparin therapy and oral anticoagulant therapy.

6. Describe the international normalized ratio (INR).

7. Calculate the INR given patient’s prothrombin time (PT), mean normal PT, and international sensitivity index (ISI).

8. Interpret the results of routine coagulation testing (i.e., prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen assay, thrombin time (TT), fibrinogen degradation products (FDP) and, D-dimer assay).

Level II 1. Correlate collection and processing procedures of the blood specimen with potential problems in coagulation testing.

2. Defend the use of 3.2% sodium citrate as an anticoagulant for coagulation studies.

3. State the principle and determine the appropriate utilization for each of the following tests: platelet aggregation studies, reptilase time, prekallikrein screening test, F-XIII screening test, von Willebrand factor activity assay, von Willebrand factor antigen immunoassay, platelet neutralization procedure (PNP), dilute Russell’s viper venom time, lupus anticoagulants, F-VIII inhibitor assay, euglobulin lysis, antithrombin (AT), protein C (PC), protein S (PS), plasminogen, antiplasmin, activated protein C resistance (APCR), and F-Xa inhibition.

4. Describe the procedure for each test listed in Level II Objective 3.

5. Interpret results and explain the clinical significance of each test listed in Level II Objective 3.

6. Interpret a set of laboratory results, and suggest the appropriate follow-up or reflex test.

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7. Select and defend the most appropriate laboratory tests to monitor anticoagulant therapy, interpret the results, and assess conditions that could affect these tests.

8. Project the potential use of molecular markers in the investigation of a hemostatic problem.

Chapter 41 – Quality Assessment in the Hematology Laboratory Level I 1. Identify the components of a quality assessment program and match sources of error with component.

2. State the importance of a quality assessment program.

3. State the importance of documentation in a quality assessment program.

4. Describe the use of proficiency testing in the clinical laboratory including required frequency.

5. Given data, employ an appropriate method to determine the reference range for an analyte.

6. Define universal precautions and identify their source.

7. Demonstrate knowledge of OSHA standards and their application in the clinical laboratory.

8. Given a material safety data sheet (MSDS), identify critical information.

9. Define accuracy, precision, control material, mean, and standard deviation.

10. Given the appropriate data, calculate the mean and standard deviation and create a quality control chart.

11. Interpret quality control results utilizing established control charts.

12. Given test results, recognize CBC data and/or histogram variations that indicate the presence of white blood cell (WBC), red blood cell (RBC), and platelet abnormalities.

13. Recognize CBC data that indicate the presence of interfering substances such as lipemia, hemolysis, and icteria.

14. Identify coagulation test results that indicate a problem with sample integrity.

Level II 1. Design methods of competency testing.

2. Apply and interpret statistics used in method evaluation.

3. Determine the components and interpret the results of a method evaluation study.

4. Determine and appraise a method’s reportable range.

5. Interpret the Westgard rules and their use in evaluating quality control results.

6. Describe the use of moving averages to monitor red blood cell data.

7. Assess the use of patient samples to monitor daily quality control in hematology.

8. Select the appropriate actions to take when abnormalities are detected in hematology or coagulation results.

9. Recommend procedures to correct for the presence of lipemia, hemolysis, and icteria.

10. Demonstrate the ability to use Delta checks in a quality control program.

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Note: Statements in parts C, D, & E identified with asterisks suggest Level II competencies

C.

ACTIVE LEARNING SUGGESTED ACTIVITIES

{Background information on each suggested activity is provided in the Introduction/Teaching Tips section at the beginning of this Instructor’s Resource Manual.} 1. Clear the Mud At the end of class, pass out index cards to all students and ask each of them to write down any topic, current or past, that is still unclear. Have students place their respective index cards into a container on the instructor’s desk. As students leave, they should randomly pick an index card from the container, research the topic, and prepare an answer or explanation to the “muddy point” written on the card. The instructor may choose to ask a few students to present their explanation during the next class period.

2. Create/Construct Have a small group of learners: a. Using “gum drops” or “dots” candy, construct a model of DNA. b. **Besides hematological disorders, formulate an announcement describing other (currently hypothetical) uses of DNA technology. c. **Design a competency testing program. d. Justify the use of patient samples to monitor daily quality control.

3. Diagram a. Have the learners list and describe the component parts of a flow cytometer. Without using their textbooks or another resource (just their list and descriptions), diagram a flow cytometer. b. Diagram the steps of a complete Quality Assurance program to decrease the number of clotted hematology samples coming from the neonatal intensive care unit.

4. Group Discussion Create a group discussion on the: a. Characteristics of proper peripheral blood smear examination b. **Bone Marrow Procedure c. Corrective actions that should be taken when there are red cell, white cell, platelet, and other (i.e., lipemia, hemolysis, and icterus) abnormalities. d. Various reasons for flags obtained on the automated hematology analyzer report, that is, give different groups the following topics: • Flags on RBC indices • RDW flags • White cell abnormalities • Platelet histogram abnormalities • Bring each group back together and have them present their results.

• Consider having further discussion regarding how each of these “flagged” results might relate to one another. e. Print-out of the automated hematology analyzer results or just the scatterplots • Have each group discuss one or two cases; explain what the expected observations are on the peripheral smear and the possible diagnosis.

• Have each group report to the class. f. Differentiation of coagulation test methodologies, that is, electromechanical, optical density, and clot detection 5. Hematology Bowl Create a Hematology Bowl of the following main topics and categories: a. Special hematology procedures b. Automation in hematology • Impedance instruments • Automated reticulocyte analyzers • Coagulation instruments 154

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c. Quality assurance • Basic components • Proficiency/competency testing • Method evaluation • Safety • Quality control d. Common abnormal hematology results (Table 41-7) and their corrective actions 6. Mapping Have each learner create a “map” of: a. Reticulocyte counting (should include manual and automated methods for counting, description of counting procedures, and a description of reticulocyte parameters such as reticulocyte percent, absolute count, reticulocyte production index, and immature reticulocyte fraction) b. **Current applications of cytogenetics in hematopoietic disorders c. **Laboratory methods in molecular diagnostics d. Laboratory testing used to evaluate primary hemostasis e. Laboratory testing used to evaluate secondary hemostasis f. Laboratory testing used to evaluate the fibrinolytic system g. **Laboratory investigation of hypercoagulable state 7. Match-Up Game Create a concentration game using: a. Abnormality found on peripheral blood smear and the appropriate corrective action b. Thresholds for a three part differential on an impedance counter c. Coulter WBC Histogram Region Flags d. The terms/components related to a flow cytometer and their definitions • Flow chamber • Photomultiplier tube • Side light scatter • Forward light scatter • Compensation • Immunophenotyping e. The definitions and stages of the cell cycle • G1 phase • S phase • G2 phase • Interphase • Prophase • Metaphase • Anaphase • Telophase f. The words and their definitions As an alterative to this exercise, have one group of students create this game and have the other groups work through it.

• Genome • Nucleotides • Transcription • Translation • Gene • DNA probe • Hybridization • Mutation • Genotype ..

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• Clonality • Translocation • MRD—minimal residual disease g. **The neoplastic states and their molecular DNA studies results • CML—BCR gene rearrangement • B-cell neoplasms—IG gene rearrangement • T-cell neoplasms—T-receptor gene rearrangement • Follicular center cell lymphoma—BCL2 gene rearrangement • Acute promyelocytic leukemia—RAR—gene rearrangement h. **Descriptions and characteristics of various test methodologies, e.g., • Hemoglobin electrophoresis • Acid elution for hemoglobin F • Hemoglobin A2 by column chromatography • Heinz body stain • Osmotic fragility test • Fluorescent spot test for G6PD • Sugar-water screening test • Acidified serum test • Donath-Landsteiner test for PCH • And so on i. The principles of each parameter on the automated hematology instrument, including: • WBC count • RBC count • Platelet count • Reticulocyte count • Hemoglobin determination • Hematocrit determination • Automated differential j. **Structural chromosome aberrations with explanations 8. Mystery Box a. **On separate pieces of paper place various combinations of hematology instruments and instruct learners to provide a compare and contrast of the instruments written on the piece of paper they randomly selected. Can also perform the same type of mystery box for the different mechanisms used for coagulation instruments. b. **Place in the Mystery Box the subtypes, characteristics, and descriptions of ways to identify the subtypes of B lineage ALL. For example: on separate pieces of paper list • Precursor B cell ALL • Makes up 80% of childhood ALL • Presence of surface CD19 or cytoplasmic CD22 expression • Etc. • Mature B cell ALL • Associated with a worse prognosis unless treated intensely with combination chemotherapy • Displays Burkitt type ALL morphology • And so on • And so on c. **Place in the Mystery Box, the “chromosome aberrations” found in the different nonHodgkin lymphomas (see Table 38-8), and the known gene loci. Have the students accurately combine the characteristics, and then explain the disease condition.

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d. **Place in the Mystery Box, the components found in Table 39-3 “Chromosomal Abnormalities in Selected Subtypes of Myeloid Leukemia.” On a separate piece of paper, place the type of leukemia, karyotype, genes, and clinical significance. Have a group of students work to match all of the components and solve the translocation mystery. e. Place in the Mystery Box, on separate pieces of paper, the different spurious hematology results and the underlying problem (see Table 41-8). As the students select a piece of paper have them give an explanation for the possible cause of the spurious result.

9. One-Minute Paragraph Have the learner prepare a one minute paragraph on the following topics: a. List the anticoagulants used in the hematology laboratory and give examples of laboratory tests that should be performed on each b. List the equipment and supplies required for phlebotomy and list the appropriate steps for a venipuncture. c. List and describe the component parts of a microscope and explain its function. d. Potential causes of an improperly stained blood smear e. Define the M:E ratio and describe what can cause an increase or decrease in the ratio. f. Explain light scattering in a flow cytometer. g. Histograms, scatterplots, and cytograms h. **Any of the special hematology procedures i. The principles of flow cytometry j. The distinction between polyclonal and monoclonal antibodies k. Explain the steps in mitosis. **Because “cytogenetic analysis must have viable cells,” describe the Harvest Procedure for obtaining mitotically active cells. l. **Specifically explain how cytogenetics can be used to monitor the course of a disease condition. m. **Laboratory methods in molecular diagnostics (one minute for each of the following methods) • Southern blot analysis • Polymerase chain reaction (PCR) • In situ hybridization • Fluorescence in situ hybridization (FISH) • DNA sequencing n. **Methods of detecting the BCR/ABL translocation in CML 10. Mnemonic Device Have each learner develop and share a mnemonic device to learn the: a. Collection tube sequence of draw in phlebotomy 11. Pyramid Game Create a pyramid game with the following categories: a. Reticulocyte count b. Solubility test for hemoglobin S c. **Hemoglobin electrophoresis d. Automated hematology instruments e. Principles of coagulation tests, e.g. prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen assay, D-Dimer, mixing studies 12. Short Story Have the learners create a short story: a. About a vampire named PNH (paroxysmal nocturnal hemoglobinuria). Describe his genetic background and the signs and symptoms of his condition. b. Describing the journey a blood sample takes as it goes through the hematology analyzer c. About how a clot is formed and detected using a fibrometer and other clot detection instruments

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13. Small Group Teaching Have a small group of learners research and teach the following: a. Capillary puncture b. **Laboratory procedures such as: acid elution for hemoglobin F, Heinz body stain, osmotic fragility test, sugar-water screening test c. **Compare and contrast benign lymphoid aggregates versus malignant lymphoma. d. **Describe the components of a flow cytometer and the principles of cell analysis. e. Specimen requirements and preparation for immunophenotyping f. Gating, as it applies to flow cytometry and immunophenotyping g. Explain how to perform a coagulation mixing study. h. **Describe the tests to evaluate circulating inhibitors. i. Define the Westgard Rules and discuss types of error.

14. Think-Pair-Share a. Describe conditions for which a bone marrow evaluation is indicated. b. Discuss the automated reticulocyte count. c. Describe “hydrodynamic focusing.” d. Explain how nucleated red blood cells are detected on a WBC scatterplot. e. Explain how platelet poor plasma is created. f. Specifically, describe how flow cytometry is useful in the monitoring of acquired immunodeficiency disorders. g. Define chimerism. h. **Discuss how flow cytometry immunophenotyping can be used to distinguish a neoplasm from a reactive population of cells. i. **Distinguish between congenital, mosaic, and acquired chromosome abnormalities. j. **Discuss “structural chromosome aberrations” and chromosomal “polymorphic variations.” k. **Discuss the advantages and drawbacks of PCR. l. Discuss the potential sources of error in specimen collection and their effect on test outcomes.

15. Thumbs Up/Thumbs Down Remember: to get immediate feedback on any topic during the class period, call for a quick “Thumbs Up or Thumbs Down.” Ask students to indicate if they comprehend the information presented by showing either a thumbs up, indicating they understand the information, or thumbs down, meaning more explanation or clarification is needed.

16. Tic-Tac-Toe a. **Use the following terms and their definitions in a complete “Hollywood Squares” game (this exercise could also be performed using the Match-Up Game format).

• Diploid • Haploid • Aneuploid • Nondisjunction • Trisomy • Monosomy • Polyploid • Endomitosis • Pseudodiploid

D.

LABORATORY ACTIVITIES

Perform the following suggested laboratory activities (Some procedures are available on the Chapters 34 and 40 Website): 1. Operate an automated hematology analyzer. Run samples, analyze results, and suggest further testing (if necessary).

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2. Perform coagulation testing on a coagulation analyser • Samples that have not been properly prepared could also be used. Discuss abnormal results.

3. **Test for osmotic fragility • Students may work in pairs or larger groups 4. **Hemoglobin electrophoresis.

• Or evaluate the gel and densitometer results on previously screened patients 5. **Do a G-6-PD fluorscent stain.

• Or review previously stained smears 6. **Do a Heinz body stain.

• Or review previously stained smears 7. **Perform a Ham’s sugar-water test.

8. **Perform a bone marrow differential count. Calculate and interpret the M:E ratio.

9. Given CBC and NRBC results, calculate a corrected leukocyte count.

10. Given RBC, Hgb, and HCT results, calculate RBC indices and evaluate.

E.

PRACTICE QUESTIONS

1. What are the three components of a quality assurance program? [Taxonomy 1] 2. Define the term universal precautions. [Taxonomy 1] 3. Two ways that the hematology analyzer results are obtained are _____ and _____. [Taxonomy 1] 4. Discuss the use of electrical impedance and optical light scattering in cell counting procedures.

[Taxonomy 2] 5. **Discuss hemoglobin electrophoresis, including in your answer the principle of the procedure, clinical significance, and sources of error. [Taxonomy 2] 6. The following results were obtained on white counts: 6.4 * 109/L, 9.7 * 109/L, 5.1 * 109/L and 10.2 * 109/L. For these results, calculate the mean. [Taxonomy 2] 7. On review of the Levy-Jennings charts, it was observed that the red cell control had increasing results for the last five days. What should be done regarding this situation? [Taxonomy 3] 8. **Compare and contrast PNH and PCH, including the etiology and the results of appropriate laboratory tests. [Taxonomy 3] 9. Compare and contrast proficiency testing and competency testing. [Taxonomy 3] 10. **List three conditions in which immunophenotyping may “not” be useful in mature lymphoid malignancies. Explain why. [Taxonomy 1] 11. **When the centromere is off-center and the “p” arm is less than the “q” arm, this is defined as: [Taxonomy 1] a. acrocentric b. eccentric c. metacentric d. submetacentric 12. The two strands of DNA can be fully dissociated by which of the following techniques?

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c. placing in a strong acidic solution d. sonicating 13. Differentiate forward and side scatter in flow cytometry methodology. [Taxonomy 2] 14. Can a specimen from a red top tube of blood be used for immunophenotyping tests? Explain why or why not. [Taxonomy 2] 15. **Recommend and justify a molecular diagnostic test procedure to determine the nucleotide sequence of abnormal hemoglobin. [Taxonomy 3] 16. **Compare and contrast the principle and applications for Southern blot analysis and Fluorescence In Situ Hybridization (FISH). [Taxonomy 3] 17. **Predict which hematologic abnormality will be detected by molecular techniques in the future.

Explain the reasons for your prediction. [Taxonomy 3] 18. **Using the following results from a 26-year-old male patient, calculate the “T helper” absolute count. Describe whether these results are normal or abnormal and give possible reasons for this patient’s condition. [Taxonomy 2] WBC = 5.6 * 109/L Automated Differential Segs = 70% Lymph = 19% Mono = 8% Eos = 3% Lymphocyte Subset CD4 = 35% CD8 = 41% CASE STUDY CN, a 48-year-old male, noticed that his urine had a red color when he voided first thing in the morning. During the rest of the day and evening, the urine was normal colored. CN made an appointment with his physician.

19. **From these symptoms, what laboratory test might provide some information for a diagnosis?

[Taxonomy 2] 20. **What would the test results (for the test identified above in question #19) be for the diagnosis for this disease? [Taxonomy 1] 21. ** Explain what is occurring in the test identified in question #19 that causes these results to occur. [Taxonomy 2] 22. **What additional test(s) should be ordered? [Taxonomy 2] 23. **What other disease(s) would have a positive result? [Taxonomy 2]

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Hematology/Hemostasis Procedures—Level I Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

L Y C N E I X K T A P E K I L N N Z K R S N N G L R R Y E O M N W N T V W F E N T R R K M Z G L Z K U D A D N B T C D L Z N A N T B Z R T V B L K H I J F T T W O M N R X H N R M Q

C R F Y K I C O N I R A P E H

I T K T H I S V F R P V N X C

F O R U R E Q R F Y M P U V N R O V C B K I C H N C P L F E N M G L L S H O J Q N I Y R T

P S B T D T T P C N R K D R D

D N L P O W R I L L T H B A H C I F L T D M N H J E Y D R T R E R P F Z L F P S I Z R D W

• anticoagulant found in green top specimen collection tubes • EDTA prevents clotting by chelating which analyte • the Coulter S-Plus has three ___ in the dilution chamber • a CD designation is known as a cluster of ____ • number of chromosomes in somatic cells that is 2n • any change in the nucleotide sequence of DNA • functional segment of DNA • an external evaluation of the lab that is required by CLIA ´88 • reproducibility of a test procedure • a ___ is used to monitor the calibration of an instrument • reference ranges for lab test is plus or minus __ standard deviation • hazardous chemical information can be found on an ___ (abrv)

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Hematology/Hemostasis Procedures—Level I L Y C N E I X K T A P E K I L N N Z K R S N N G L R R Y E O M N W N T V W F E N T R R K M Z G L Z K U D A D N B T C D L Z N A N T B Z R T V B L K H I J F T T W O M N R X H N R M Q

C R F Y K I C O N I R A P E H

I T K T H I S V F R P V N X C

F O R U R E Q R F Y M P U V N R O V C B K I C H N C P L F E N M G L L S H O J Q N I Y R T

P S B T D T T P C N R K D R D

D N L P O W R I L L T H B A H C I F L T D M N H J E Y D R T R E R P F Z L F P S I Z R D W

• anticoagulant found in green top specimen collection tubes (heparin) • EDTA prevents clotting by chelating which analyte (calcium) • the Coulter S-Plus has three ___ in the dilution chamber (apertures) • a CD designation is known as a cluster of ____ (differentiation) • number of chromosomes in somatic cells that is 2n (diploid) • any change in the nucleotide sequence of DNA (mutation) • functional segment of DNA (gene) • an external evaluation of the lab that is required by CLIA ´88 (proficiency) • reproducibility of a test procedure (precision) • a ___ is used to monitor the calibration of an instrument (control) • reference ranges for lab test is plus or minus __ standard deviation (two) • hazardous chemical information can be found on an ___ (abrv) (msds)

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Hematology/Hemostasis Procedures—Level I 1 2 3

4

5

6

7 8 9

10

11

12

ACROSS

DOWN

3 Substance in collection tubes that prevents blood from clotting 5 Comparative analysis of the results of the current lab test from the most recently reported result (2 words) 7 A bone marrow that cannot be aspirated (2 words) 9 In PT test ___ is added to citrated platelet poor plasma and then fibrin formation is recorded 10 Two or more cells passing through an aperture at the same time 11 Identification of antigens using detection antibodies 12 ___ is the increased electrical resistance that occurs when a cell passes through an aperture

1 The guideline that states “all samples should be considered potentially infectious” (2 words) 2 Size distribution curve 4 Human genome is composed of 3 billion pairs of ____ 6 Number of chromosomes in a gamete that is referred to as n 8 Adjustment made to an instrument to correct the results so that they match the standards

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Hematology/Hemostasis Procedures—Level I U N H I A N T I C O A G U L A N T U V S C E D E L T A C H E C K L R O E S G O A H D R Y T A P T L A A C P P T H R O M B O P L A S T I N D R L L E E O I S C O I N C I D E N C E B A D R U A T T I M M U N O P H E N O T Y P I N G O O N I M P E D A N C E

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Hematology/Hemostasis Procedures—Level II Figure out what words the clues represent. Then find the words in the grid. Words can go horizontally, vertically, and diagonally in all eight directions.

L P M W V C G N P R Z Z X K Z D W R P D B M N O I T A L S N A R T N W J H N J N C I N H E M O L Y S A T E N Y F F Z K N R H R T C F X R P Q P X P N N G X C Z P I W L F D J D H T S E C P R R I J Q N S T X P H O T E C R N A B T D V C T G T F N T X N V T D J T R M E L K N R L O O Q O K E H I N Y J H N L X M Y I M G G B L M Y P V R K R C R N T S U S O R T M O P L Q D V N E L I T L I T O L A G S J O G Z L D X L G T R A R D M E M O K I Z D Q R A N I J M L Q E N Y J M Z D N J B N M P M E Y T N O M H O H K W W O R L C H N M M M Z C I Q R X H F L K I L T J R C E K F Y B H H N L C S E M O R H C O R O U L F M C Z K Q R V V N A C R O C E N T R I C C • a bone marrow that cannot be aspirated (2 words) • precursor B lymphocytes present normally in the bone marrow • a ___ is used as the specimen for hemoglobin electrophoresis • size distribution curve • two or more cells passing through an aperture at the same time • ___ tubes are the light detectors in flow cytometers • fluorescent markers • cells from two different zygotes expressed in one individual • nuclear structure seen during mitosis and meiosis • presence of identical cells derived from a single progenitor • chromosome that has the centromere close to the terminal end • one daughter cell with an extra chromosome • cell that has more than 46 chromosomes • the total aggregate of inherited genetic material • synthesis of protein from an RNA template

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Hematology/Hemostasis Procedures—Level II L P M W V C G N P R Z Z X K Z D W R P D B M N O I T A L S N A R T N W J H N J N C I N H E M O L Y S A T E N Y F F Z K N R H R T C F X R P Q P X P N N G X C Z P I W L F D J D H T S E C P R R I J Q N S T X P H O T E C R N A B T D V C T G T F N T X N V T D J T R M E L K N R L O O Q O K E H I N Y J H N L X M Y I M G G B L M Y P V R K R C R N T S U S O R T M O P L Q D V N E L I T L I T O L A G S J O G Z L D X L G T R A R D M E M O K I Z D Q R A N I J M L Q E N Y J M Z D N J B N M P M E Y T N O M H O H K W W O R L C H N M M M Z C I Q R X H F L K I L T J R C E K F Y B H H N L C S E M O R H C O R O U L F M C Z K Q R V V N A C R O C E N T R I C C • a bone marrow that cannot be aspirated (2 words) (dry tap) • precursor B lymphocytes present normally in the bone marrow (hematogones) • a ___ is used as the specimen for hemoglobin electrophoresis (hemolysate) • size distribution curve (histogram) • two or more cells passing through an aperture at the same time (coincidence) • ___ tubes are the light detectors in flow cytometers (photomultiplier) • fluorescent markers (fluorochromes) • cells from two different zygotes expressed in one individual (chimerism) • nuclear structure seen during mitosis and meiosis (chromosome) • presence of identical cells derived from a single progenitor (clonality) • chromosome that has the centromere close to the terminal end (acrocentric) • one daughter cell with an extra chromosome (trisomy) • cell that has more than 46 chromosomes (hyperdiploid) • the total aggregate of inherited genetic material (genome) • synthesis of protein from an RNA template (translation)

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Hematology/Hemostasis Procedures—Level II 1

2

3

4

5

6 7 8

12

9

10

11

13

14

15

ACROSS

DOWN

1 Human genome is composed of 3 billion pairs of ____ 3 This term describes when the centromere is in the center of the chromosome (p=q) 4 Cell that has fewer than 46 chromosomes 6 A statistical analysis that might compare two different analyzers for the same analyte (2 words) 8 One daughter cell with a missing chromosome 13 Procedure that is used to quantitate fetal hemoglobin is based on the principle that fetal hgb is __ __ (2 words) 14 A statistical formula that uses the MCV, MCH, and MCHC to monitor an instrument’s performance (2 words) 15 Extracellular-matrix glycoprotein capable of binding heparin

2 The __ __ test detects biphasic antibody (2 words) 5 Quantitative study of a drug’s disposition in the body over time 7 An error in segregation that occurs in mitosis or meiosis so that sister chromatids do not disjoin 9 Cell has 46 chromosomes but is not normal; has structural or numerical aberrations 10 __ __is the specific stain used to detect Heinz bodies (2 words) 11 The percentage of hgb found on electrophoresis may be read on a ____ 12 Chromosomes with variant morphology that have no clinical significance are known as ___ variants.

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Hematology/Hemostasis Procedures—Level II N U C L E O T I D E S O M E T A C E N T R I C A T H Y P O D I P L O I D H H L I N E A R R E G R E S S I O N N A R O M O N O S O M Y P B D N D A S R E P D S A C I D R E S I S T A N T O I T O U L S L S E K D L I Y J I I O I T M U N N D A O M O V I N G A V E R A G E S I N M R C R T P T E P T I L G T H I C O R E F I B R O N E C T I N S I E R C N D E N

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Appendix

Laboratory Worksheet— Differential Worksheet Laboratory Worksheet— Blood Smear Evaluation Answers to Practice Questions

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Laboratory Worksheet DIFFERENTIAL WORKSHEET

STUDENT NAME:

______________________

DATE:

______________________

SAMPLE ID:

______________________

PLT Estimate: __________ WBC Estimate: __________ DIFFERENTIAL COUNT: Segmented Neutrophils

_____

Bands

_____

Eosinophils

_____

Basophils

_____

Lymphocytes

_____

Monocytes

_____

Immature Cells: _____

_____

_____

_____

_____

_____

RBC MORPHOLOGY:

SMEAR EVALUATION:

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Laboratory Worksheet BLOOD SMEAR EVALUATION STUDENT NAME: ______________________ DATE:

______________________

SAMPLE ID:

______________________

I. RBC Size:

Normocytic ( )

Microcytic ( )

Macrocytic ( )

Color:

Normochromic ( ) Hypochromic ( )

Abnormalities:

Poikilocytosis (quantify) Anisocytosis (quantify) Polychromasia (identify) Nucleated erythrocytes (quantify) Erythrocyte inclusion (identify or describe)

II. WBC Number:

Normal ( )

Neutrophils:

Shift: None ( )

Increased ( )

Decreased ( ) Left ( )

Abnormalities: Lymphocytes: Monocytes: Eosinophils: Basophils: Description of other cells:

III. PLATELETS Number: Normal ( )

Increased ( )

Decreased ( )

Morphology: Interpretation of smear:

Additional testing that should be performed and expected results: .. ..

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Answers to Practice Questions I. INTRODUCTION TO HEMATOLOGY 1. Erythrocytes: 45% of whole blood Leukocytes and platelets: 1% Plasma: 55% 2. Reflex testing helps the physician choose appropriate tests from the expansive list of tests available based on the results of screening tests. This helps contribute to a cost-efficient and effective diagnosis.

3. C. leukocytes 4. Utah is at a higher elevation than Alabama. The partial pressure of oxygen at high altitudes is less than at lower altitudes. As a physiologic response, the body produces more RBCs at higher altitudes to get the available oxygen to the tissues. Thus, you would expect higher reference ranges for erythrocytes and hemoglobin at high altitudes.

5. Precursor cells are inclusive of all cells before the fully mature blood cell. This compartment includes hematopoietic stem cells, progenitor cells, and maturing cells. Maturing cells are those cells that are committed to maturing into a particular cell line and have the characteristics of that cell line that allow them to be identified morphologically. This is the largest group of hematopoietic cells.

6. a. A b. A, B c. B d. B e. B f. C 7. A fee for service is an insurance plan where the patients choose their own doctor and other health care providers and the providers determine the fee. The fee is then reimbursed partially or fully by the insurer. A capitated payment plan is a type of plan where the insurer negotiates with health care providers to provide services for a set fee. The insurer determines who the providers will be based on the provider’s willingness to accept the reimbursements set by the insurer.

8. Self-renewal is the process by which a stem cell can undergo cell division to replace itself. It gives rise to daughter cells identical to itself. These daughter cells have self-renewal ability, as well as the ability to give rise to other more differentiated cells. The pluripotent hematopoietic stem cell (HSC) is a cell that has self-renewal properties. Self-renewal assures that the stem cell population is sustained as long as the individual lives. Cell differentiation, on the other hand, occurs when cells develop different properties that distinguish them from cells that were initially equivalent. This is the process that is responsible for the generation of diverse cell populations necessary for the function and survival of the organism.

9. The WBC count is very low and, thus, compromises the body’s immune defense system. This means the patient will be at risk of infection. The WBC are the cells that fight infection by phagocytosis and production of antibodies and other substances that kill microorganisms. The patient is also anemic as evidenced by the low hemoglobin, hematocrit, and RBC count. This will decrease delivery of oxygen to the tissues. Thus, the patient may have symptoms such as fatigue.

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10. This patient is anemic. Renal disease is a common complication of diabetes. The kidney is the source of erythropoietin, the renal hormone that stimulates production of RBCs in the bone marrow. If this hormone is low, as may happen with kidney disease, the patient may become anemic.

II. THE HEMATOPOIETIC SYSTEM 1. D - regulate erythropoiesis by secreting various cytokines 2. A - ALAS 3. The partial pressure gradient of a gas such as oxygen determines the rate of diffusion across the alveolar-capillary membrane. Gas diffuses from a higher partial pressure gradient to a lower partial pressure gradient. Arterialized blood leaves the lungs with a PO2 of 100 mm Hg and a PCO2 of 40 mm Hg. The PO2 of interstitial fluid is about 40 mm Hg and the PCO2 is about 45 mm Hg. This means that when the blood gets to the tissues, oxygen diffuses out of the blood, and CO2 diffuses into the blood.

4. Thymus: lymphopoietic, bilobular organ demarcated into an outer cortex and central medulla.

Spleen: organ enclosed by a capsule of connective tissue. Has three zones of tissue within the capsule; white pulp, red pulp, and the marginal zone.

5. The hematopoietic compartment is the part of the bone marrow where the hematopoietic cells mature. This compartment also contains the stromal cells that form a support network for the hematopoietic cells and provides a favorable microenvironment for hematopoietic cell proliferation. The vascular compartment is composed of blood vessels that provide a blood supply to the bone marrow, as well as the osseous bone. Mature hematopoietic cells gain access to the blood by crossing the sinus wall in the bone marrow by a receptor-mediated process.

6. C. 5.70 * 1012/L 7. Neutrophils migrate toward the site of inflammation from the blood vessel. This migration is directed by chemotaxins in the tissues. The neutrophil may phagocytose particles, which stimulates degranulation of the cell. During this ingestion, the cells increase consumption of O2 by 2 to 10 fold and generate hydrogen peroxide. Other oxygen-dependent granular proteins are released from the neutrophil. This process has potent microbicidal activity but also is damaging to the cells themselves and surrounding tissue. Thus, as the inflammation continues, more cells are attracted to the site and die as a result of the respiratory burst and degranulation of phagocytes.

8. SPLEEN: The red pulp is composed of sinuses and cords. The sinuses are filled with blood, giving this pulp its red color. The white pulp is the visible grayish white zone that contains lymphocytes, macrophages, and dendritic cells. This is where the immune response is initiated.

9. Due to an increase in the metabolic rate in tissue during exercise there is an increase in CO2, H+, and heat. These factors decrease the affinity of hemoglobin for oxygen, causing an increase in the amount of oxygen released to the tissues.

10. There is a larger nuclear to cytoplasmic ratio in the immature neutrophils. The younger cells, such as the myeloblast and promyelocyte, may have nuclei with nucleoli and young lacey chromatin. As the cell matures, the nucleus indents and segments. The cytoplasm in younger cells is a deeper blue color. Myeloblasts do not have granules. Primary granules begin to appear in the promyelocyte. As the cell matures, secondary granules become abundant, and the cytoplasm takes on a pink-lilac color.

11. In metabolic alkalosis, the oxygen affinity of hemoglobin is increased. This would shift the ODC to the left. ..

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III. THE ANEMIAS 1. Hydremia of pregnancy, overhydration in oliguric renal failure, congestive heart failure, vongestive splenomegaly, chronic diseases, hypoalbuminemia, recumbancy 2. Megaloblastic 3. 1 = B, 2 = A, 3 = C 4. False. The Hb and Hct cutoffs for a 7-year-old male are 11.4 g/dL and 34.5% respectively.

5. D. iron deficiency anemia 6. In aplastic anemia the bone marrow is hypoplastic due to a decrease in all cell lines. Maturation of the cells is normal. In B12 deficiency, the marrow is hyperplastic with megaloblastic maturation. The peripheral blood has a pancytopenia in aplastic anemia due to the hypoproliferation in the bone marrow. The RBCs may be normocytic or macrocytic but are usually normal in shape. In B12 deficiency, many of the abnormal cells in the bone marrow are destroyed before being released into the peripheral blood. This may produce a pancytopenia or a selective decrease in RBCs. The RBCs are usually macrocytic, but contrary to aplastic anemia, the macrocytes often have an oval shape. They may also contain Howell-Jolly bodies. The finding of hypersegmented neutrophils is highly sensitive and specific for megaloblastic anemia. These are not found in the blood in aplastic anemia.

7. B - HbC and HbE 8. UIBC = TIBC - serum iron. 261ug/dL - 40 ug/dL = 221 ug/dL. These results are consistent for a patient with anemia of chronic disease 9. Hematologic findings are similar in homozygous beta-thalassemia and Hb Lepore. The cells are microcytic and hypochromic. Hemoglobin concentration is decreased and there is moderate to marked anisocytosis and poikilocytosis. Hb electrophoresis reveals no HbA in either condition but an increase in HbF. In Hb Lepore there is no HbA2, but Hb Lepore is present. In beta-thalassemia there is some HbA2, but no Hb Lepore.

10. Because the most common cause of hypochromic microcytic anemia is iron deficiency, iron studies should be ordered. If these tests reveal an adequate amount of iron, hemoglobin electrophoresis should be done. Thalassemia is common in individuals of this ethnicity. If the boy had thalassemia, the electrophoresis should show abnormal amounts of HbA, HbA2, and/or HbF and possibly the presence of abnormal hemoglobins like Hb Lepore. The electrophoretic pattern is dependent on the type of thalassemia inherited and whether it is present in the heterozygous or homozygous state. See chapter tables for characteristics of alpha-thalassemia and beta-thalassemia.

11. In aplastic anemia there is a hypoplastic bone marrow that affects precursors of all cell lines— red cells, white cells, and platelets. The peripheral blood reveals a pancytopenia. On the other hand, in pure red cell aplasia there is a selective decrease in erythroid precursors in the bone marrow and in the red cells in the peripheral blood. The WBC and platelets and their precursors in the bone marrow appear normal.

12. Hereditary spherocytosis is due to a defect in the red blood cell membrane. The defect is a primary deficiency of spectrin or a secondary deficiency of spectrin due to defective association with other skeletal proteins.

13. A-2, 4 B-3 C-3 D-1 174

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14. The direct antiglobulin test should be done on the baby and the indirect antiglobulin test on the mother.

15. The membrane is responsible for maintaining the stability and discoid shape of the cell, for preserving cell deformability, and for selective permeability. If the membrane structure is altered, then one or more of these membrane functions may be abnormal, leading to hemolysis of the cell. RBC enzymes are important for protecting the cell against oxidant injury and for providing the cell with energy. Defects in these enzymes can lead to oxidant injury of the cell or impaired ability to maintain normal cation concentrations. This can lead to hemolysis of the cell.

16. DIC is a microangiopathic anemia in which the coagulation process is activated and thrombi form in the microvasculature. This causes consumption of coagulation factors and an increase in fibrinolytic products. Thus, coagulation tests are abnormal, including APTT, PT, TT, FDP, decreased fibrinogen, and a decline in the platelet count.

17. There are four mechanisms suggested that play a role in drug-induced hemolytic anemia. The first is absorption of the drug to the RBC. Antibody is formed against the drug, and the sensitized cell is removed from circulation by the spleen. The second is immune complex formation. In this mechanism, the drug combines with an antibody and this complex adsorbs to the cell membrane.

Complement is activated leading to complement-mediated hemolysis. The third mechanism is autoantibody induction, in which the drug adheres to the RBC membrane, altering the membrane.

This stimulates production of the antibody that attaches to the drug. The sensitized cell is removed from circulation by the spleen. The fourth mechanism is membrane modification. This results in nonspecific binding of normal plasma proteins, including IgG and complement to the membrane. This is a nonimmunologic mechanism and is not associated with hemolysis.

18. B. creatinine 19. In HUS, the RBC is intrinsically normal, and in PNH the cell membrane is abnormal. Both are characterized by hemolytic anemia and are acquired disorders. HUS is primarily found in children, whereas PNH is usually found in adults. HUS is associated with schistocytes but PNH is not. HUS is a form of microangiopathic hemolytic anemia that is characterized by hemolytic anemia with erythrocyte fragmentation, acute renal failure, thrombocytopenia, and variable central nervous system symptoms. It is associated with gastroenteritis, upper respiratory infections, urinary tract infections, or viral disease. The hemolysis is caused by microcirculatory lesions that injure the RBC as it passes through the vessel. PNH is an acquired disorder of the RBC membrane. There is an abnormal clone of differentiated hematopoietic cells that bind abnormally large amounts of complement and that are abnormally sensitive to complement lysis.

The susceptibility to complement is due to the absence of decay accelerating factor (DAF), C8 binding protein (C8bp), and membrane inhibitor of reactive lysis (MIRL) on the cell membrane.

These proteins help cells avoid lysis by complement. The deficiency of these proteins on the cell is due to the absence of a membrane glycolipid that serves as an anchor that attaches the proteins to the cell membrane.

20. AIHA result of autoantibody—directed against self-antigens usually primary (no identifiable cause) may be linked to some diseases (secondary) antibody reacts with cells of almost all persons(directed against high incidence antigen) Copyright © 2010 by Pearson Education, Inc.

HTR result of antibody directed against foreign antigens (alloantibody) due to exposure to foreign antigen on transfused cells antibody reacts only with cells from person possessing the antigen

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antibody screen and panel positive all cells

antibody screen and panel positive only with cells possessing the corresponding antigen specificity

both may cause extra- or intravascular hemolysis depending on the antibody specificity involved both will have positive DAT, but once the transfused cells are destroyed the DAT is negative. In AIHA the autoantibody is continually produced and attaches to RBC as they are produced.

21. Basophilic stippling is found in the RBCs and is composed of RNA and mitochondrial remnants.

22. It can be found in toxic states such as lead poisoning, anemias associated with abnormal hemoglobin synthesis, or thalassemia. It can also be found as a staining artifact (diffuse).

23. Because the RBC count is normal but the hemoglobin and hematocrit, MCV and MCHC, are decreased and the MCV is low, this may be a case of thalassemia minor.

24. To assure a cost-efficient and diagnostically effective laboratory evaluation, iron studies should be done first to rule out iron deficiency. However, the RBC count is rather high for this diagnosis. If iron studies are normal, electrophoresis should be done to search for thalassemia. If the iron studies are abnormal, the physician will probably treat the patient with iron and follow up to see if the hemoglobin rises before ordering an electrophoresis.

25. If this were a thalassemia, the hemoglobin electrophoresis should show abnormalities in the concentrations of HbA, HbA2, HbF, or the presence of abnormal hemoglobins such as HbH or Hb Lepore.

26. Cause

27. Test

Autoimmune Aplastic anemia

DAT Bone marrow (before the bone marrow is done, the patient’s WBC and platelets must be assessed) Bone marrow Assess the WBC and platelets and, if warranted, proceed to bone marrow Patient drug history; DAT Review of blood smear for schistocytes, serum haptoglobin, urine for hemosiderin, platelet count, FDP, D-dimer test Ham test Donath-Landsteiner test

Pure red cell aplasia Leukemia Drug-induced hemolysis Microangiopathic hemolysis

PNH PCH

IV. NONMALIGNANT DISORDERS OF LEUKOCYTES 1. D - vacuoles 2. B - cytokines 3. B - 2 hours 4. Absolute value = WBC * % differential Manual Diff Segs = 78% Lymphs = 12% Monos = 3% Eos = 7% Baso = 0% 176

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Absolute Value 9

7.6 * 10 /L 1.2 * 109/L 0.3 * 109/L 0.7 * 109/L 0.0 * 109/L

Interpretation normal lymphopenia normal normal Normal ..

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5. Corticosteroid therapy increases the bone marrow output and decreases the migration of neutrophils to the tissues, therefore causing a neutrophilia.

6. Lymphocytic leukemoid reactions represent a type of nonmalignant reactive lymphocytosis. This condition is characterized by a relative increased lymphocyte count and the appearance of reactive or immature-appearing lymphocytes.

Chronic lymphocytic leukemia is a type of lymphoid malignancy with an absolute lymphocytosis. The lymphocytes seen on a peripheral smear are small and mature looking. A few prolymphocytes may also be seen.

7. Pseudoneutrophilia: Increased circulating neutrophils that are mature and normal. Increased circulating neutrophils occur without a pathologic stimulus and are probably a simple redistribution of the marginated granulocyte pool to the circulating granulocyte pool.

Pseudoneutropenia: Also produced by an alteration in the circulating and marginated pools of neutrophils. This alteration, however, transfers more neutrophils from the circulating pool to the marginal pool.

8. Immunoblast: The immunoblast is generally larger than the lymphoblast and has a high N:C ratio. A lacy chromatin pattern and nucleoli are visible in the nucleus. The cytoplasm is a deep blue color. These cells proliferate to produce daughter cells.

Lymphoblast: The lymphoblast is 10 – 18 mm in diameter (smaller than an immunoblast) with a high N:C ratio. The nuclear chromatin is fine and lacy with one or two well-defined pale blue nucleoli present. A perinuclear zone may be seen. The scant amount of cytoplasm stains a deep blue.

9. This question is aimed at the students’ knowledge, critical thinking skills, and creativity. There is no one right answer to this question. Students may research the structure of a bill, current legislation, and the federal budget process to complete this question.

10. Chédiak-Higashi anomaly is when a type of inherited functional abnormality is suspected. The gray granules seen in the cytoplasm of the neutrophils are actually the fusion of primary nonspecific and secondary specific neutrophilic granules. This prevents the granules from being delivered into the phagosomes to participate in killing of ingested bacteria.

V. NEOPLASTIC HEMATOLOGIC DISORDERS 1. A - true 2. Usually the donor of hematopoietic stem cells receives cytokines before the marrow is collected to increase the quantity of stem cells in the marrow. The yield of stem cells from cadavers would be low in comparison since they did not receive the cytokines before death.

3. C. presence of cytoplasmic vacuoles/Döhle bodies 4. Proto-oncogenes are genes that are normal and direct normal cellular processes like cellular growth and differentiation but when activated are known as oncogenes. Proto-oncogenes may be activated by gene mutation, rearrangement, or amplification. Oncogenes allow cells to proliferate without the normal controls, resulting in malignancy.

5. Remission means that you have no symptoms or clinical signs of the disease. Blood tests are also normal in remission. The remission rate in AML is about 55–65 percent so chances are good that he will reach remission. Most patients remain in remission for 3 to 10 years.

6. Both are premalignant neoplasms of the bone marrow. Myeloproliferative disorders are characterized by panhypercellularity of the bone marrow and an increase in at least one cell line in the peripheral blood. Myelodysplastic syndromes are usually characterized by a hypercellular bone marrow but the cells are dysplastic. The peripheral blood reveals cytopenia with dysplasia. ..

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7. Hematopoietic stem cell transplants may be used in the future for any hereditary or acquired bone marrow abnormality in which there is an abnormal clone of cells. Also, it could be used if there is permanent damage to hematopoietic cells from drugs, radiation, and so on. However, if the bone marrow stroma is also affected by the damaging agent, it is unlikely that the microenvironment will be conducive to hematopoietic cell regeneration.

8. Radiation causes mutations in a cell’s DNA which may activate a proto-oncogene to an oncogene, which may result in leukemia.

9. Viruses may cause a breakdown in the body’s cell-mediated immunologic self-surveillance system used to destroy abnormal cells. Alternatively, viruses may provide some sort of protection for cells with activated oncogenes.

10. Possible diseases include CML and RAEB2. .

Disease: CML Tests: Bone marrow; cytogenetics for Ph chromosome. The bone marrow would be hypercellular with a predominance of neutrophilic cells and their precursors. The Ph chromosome would be present.

Disease: RAEB 2 Tests: Bone marrow; cytogenetics. The bone marrow is usually hypercellular with 10-19 percent blasts. Cytogenetics is abnormal in up to 50% of cases of MDS.

VI. BODY FLUIDS 1. Normally, no cells are found in the CSF. Even low cell counts in the CSF is an indication of a pathology.

2. Monosodium urate crystals would be found in the synovial (joint) fluid in a patient with gout.

3. A-3 B-1 C-5 D–2 4. Signs of a traumatic tap are decreasing RBC counts after the first tube collected and xanthochromia is usually not present. In cerebral hemorrhage, usually all tubes have the same concentration of RBC and the fluid is xanthochromic. Erythrophagocytosis is a definitive sign of hemorrhage. Hematoidin crystals may be seen in CSFs from older hemorrhages.

5. This might be starch contaminating the fluid. The specimen should be examined with polarized light. If it is starch particles, they will have refractile centers and show birefringence showing up as bright Maltese cross-like figures.

6. E. all of these could cause these changes 7. A transudate is the result of increased capillary hydrostatic pressure or decreased plasma oncotic pressure while an exudate is formed by increased capillary permeability and/or decreased lymphatic resorption. A transudate will have a specific gravity of 61.015 and a total protein of 63 g/dL. The ratio of effusion total protein to serum total protein is less than 0.5. The ratio of effusion lactate dehydrogenase (LD) to serum LD is less than 0.6. The total WBC count is 61,000/mL. In comparison, the exudate has a specific gravity 71.015, total protein 73.0 g/dL, ratio of fluid protein to serum protein 70.5, ratio of fluid LD to serum LD 70.6, and total WBC count 71,000/mL.

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8. The lymphocytes found in a viral or fungal infection are heterogenous with reactive forms present. In lymphoma, the lymphocytes are homogenous. The morphology of the lymphoma cells will depend on the particular type of lymphoma. The nuclear membrane may be irregular and chromatin may be clumped or immature. In cytospin preparations, nucleoli may be seen in either normal lymphocytes or the lymphocytes in lymphoma.

9. Triglycerides should be measured. In a chylous effusion, the triglycerides are elevated (7110 mg/dL) and in a pseudochylous effusion the triglycerides are 650 mg/dL. The cells in the fluid should be examined. In a pseudochylous effusion, there will be a mixed reactive cell population with many inflammatory and necrotic cells, while in a chylous effusion there is a predominance of lymphocytes.

10. 125/mL 11. Yes 12. The type of predominant WBC present gives a clue as to the type of meningitis present. If neutrophils predominate, it is probably bacterial, and if lymphocytes predominate, it is probably viral.

13. There are 1,000 RBCs for every 1–2 WBCs in the peripheral blood.

VII. HEMOSTASIS 1. Dense bodies; alpha granules; lysosomal granules; microperoxisomes.

2. Thrombopoietin 3. A-2; B-3; C-1 4. Protein C is activated to activated protein C (APC) by thrombin. APC with protein S (PS) as a cofactor inactivate factor Va and factor VIIIa. A deficiency in either PC or PS can lead to thrombosis due to a decreased capacity to destroy factors Va and VIIIa. This results in an increase in thrombin generation. Excess thrombin generates fibrin and activates TAFI (antifibrinolytic effect).

5. The bleeding time is increased in VWD but normal in factor VIII deficiency. Platelet aggregation with ristocetin is absent in VWD but normal in factor VIII deficiency. The APTT may be increased in both. The factor VIII assay is decreased in both. The vWf:Ag assay is decreased in VWD but normal in factor VIII deficiency.

6. When a blood vessel is injured, the vessel initially constricts to slow blood flow and enhance clot formation. Return to normal blood flow serves to limit coagulation by continually diluting the concentration of activated factors.

The liver removes activated coagulation factors from the circulation. Positive feedback mechanisms include: thrombin promotes the release of platelet factor Va and the exposure of negatively charged phospholipid surfaces used in the assembly of the clot; thrombin activates factors V and VIII; factor Xa feeds back to activate factor VII; factor Xa can activate factor VIII. There is also negative feedback inhibition of coagulation. Some activated factors have the potential to destroy other factors. Thrombin has the ability to activate factors V and VIII. FDP function as inhibitors of fibrin formation.

Naturally occurring inhibitors prevent the initiation or amplification of the coagulation cascade. These include antithrombin, heparin cofactor II, protein C and protein S, TFPI, a 2-macroglobulin, a 1-antitrypsin, C1-inhibitor, TAFI, and a2-antiplasmin.

7. In both DIC and HUS the platelet count is decreased, although this may only be evident if serial platelet counts are done. In both diseases there are schistocytes on the blood smear. In DIC the

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PT and APTT are usually increased, while in HUS the PT may be normal or slightly prolonged but the APTT is normal. Fibrin split products may be elevated in both.

8. A deficiency in Protein C (PC) and antithrombin (AT) will both predispose an individual to thrombosis due to unregulated coagulation. They are both inhibitors of the coagulation process.

Activated PC, together with co-factor PS, normally inactivates factor Va and factor VIIIa while antithrombin, the major serine protease inhibitor of coagulation, inactivates thrombin.

9. B - schistocyte 10. LT had three of four of the clinical manifestations that suggest an inherited form of thrombophilia: first episode of thrombosis under 45 years of age, recurrent thrombosis, and a family history of thrombosis. It is suggested that if an individual have any of these manifestations that they be worked up for thrombophilia.

11. Primary disorder 12. The most common defects that occur with thrombophilia should be tested. This includes activated protein C resistance (if positive, a molecular test for factor V Leiden), prothrombin 20210 molecular test, and tests for protein S, protein C, and antithrombin.

13. It is probable that any one of these may be abnormal. About 75 percent of thrombophilic families with hereditary defects have a defect in one of these proteins.

14. The following are principles of the tests mentioned in #12: a. APCR: An APTT is performed with and without APC. The test with APC should be prolonged compared with the test without APC. The APC inactivates factor Va if it is a normal molecule. b. Factor V Leiden and prothrombin 20210: There are molecular tests for these mutations using PCR technology. c. Protein S: There may be a quantitative or qualitative defect. For both defects a functional assay may be used. In this assay patient platelet poor plasma is mixed with PS deficient plasma, APC, factor Va, and calcium. The clotting time is proportional to the PS activity. The clotting time is interpreted from a reference curve using various dilutions of PS activity. Immunologic methods will not detect qualitative deficiencies of PS, but they will detect quantitative deficiencies. d. Protein C: Quantitative analyses can be determined by EIA or ELISA. Functional assays use an activator of PC in a chromogenic or clot-based assay. In the chromogenic assay, APC is measured by its enzymatic activity on a chromogenic substrate. The clotting assay is based on the ability of APC to prolong the APTT via inactivation of factor Va and factor VIIIa. e. Antithrombin: Can be measured by EIA, ELISA, radial immunodiffusion, or microlatex particle immunologic assay. Functional assays use thrombin or factor Xa as targets for inhibition by AT and utilize either clotting of fibrinogen or cleavage of a synthetic chromogenic substrate as the endpoint of activity. The AT activity in the presence of heparin can also be determined.

VIII. HEMATOLOGY/HEMOSTASIS PROCEDURES 1. Preanalytical, analytical, and postanalytical 2. Universal precautions are precautions defined by the Centers for Disease Control and Prevention (CDC) to minimize the risk of biohazard exposure to health care workers. The original guidelines are updated by the CDC periodically. Within a facility, the term standard precautions is used to define the facility’s policies regarding universal precautions.

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3. Impedance and optical light scattering 4. Electrical impedance: When a blood cell with poor conductivity passes through an electrical field, there is increased resistance. The number of pulses and the amplitude of the pulse indicate the number of cells and their volume, respectively.

Optical light scattering: As a single blood cell passes through a beam of light the light is scattered. The degree of forward scatter is related to cell size while the degree of side scatter is related to cell complexity or granularity.

5. Hemoglobin electrophoresis: a. Principle: A hemolysate of packed RBCs is applied to a cellulose acetate plate. The plate is placed in an electrophoretic chamber with an alkaline buffer. The buffer permits the flow of electrons from the cathode to the anode in the chamber. It alters the charge of the hemoglobin molecules based on their amino acid composition and allows the hemoglobins to migrate and separate based on the strength of their negative charge. The plate is then stained and evaluated with a densitometer to determine the amount of hemoglobin present in each band. b. Clinical significance: This procedure allows detection of abnormal hemoglobins such as HbS or HbC. It also is valuable in thalassemias to evaluate the quantities of normal hemoglobins (HbA, HbA2, and HbF). c. Sources of error: The pH of the buffer may be too high or too low; inadequate time for the migration of the hemoglobin sample; wrong interpretation of the electrophoretic pattern.

6. 7.9 * 109/L 7. This is the beginning of a trend. There may be a problem with the instrument or reagents. Both should be checked to find the problem. The calibration of the instrument should be checked; reagents should be checked for expiration date and storage conditions.

8. PNH is due to an acquired defect in the blood cells’ membranes. There is a lack of a glycolipid structure that serves as an anchor for some proteins. The net result is cells that are excessively sensitive to complement. PCH is an acquired immune hemolytic anemia in which a biphasic antibody, Donath-Landsteiner (DL), reacts with erythrocytes causing cell lysis. The antibody is specific for the P-antigen.

Tests: The test for PCH is the Donath-Landsteiner test for detecting the presence of these antibodies. If DL antibodies are present, the test results show hemolysis. The screening test for PNH is the sugar-water test. Blood is added to a sugar-water solution. Normal erythrocytes will not hemolyze whereas the erythrocytes in a patient with PNH will hemolyze. The acidified serum test (Ham test) is suggested if the screening test is positive. In this test, hemolysis occurs when acidified serum and patient cells are incubated together in PNH. 9. Proficiency testing is an external quality control program designed to assure accuracy of test systems by comparing test results of externally provided samples to that of other laboratories.

Competency testing refers to the competency of personnel performing the test rather than the test system. CLIA requires that laboratories assess the competence of laboratory personnel to perform tests twice during the first year of employment and annually thereafter. The mechanism is not defined by CLIA. Thus, there may be a variety of methods used.

10. a. If the cell lacks surface immunoglobulins or diagnostic surface antigens b. When the abnormal phenotype is only present in a portion of the population/specimens c. Presence of an abnormal phenotype that is not specific for a single subtype of lymphoid malignancy

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11. D - submetacentric 12. B - heating to 95°C 13. When a laser beam interacts with a particle, the light that proceeds in a forward direction is measured as forward scatter and indicates the particle size.

Side Scatter is the detection of scattered light at a 90° angle. This indicates the particle’s internal complexity and granularity.

14. No, specimens from a red top tube cannot be used for immunophenotyping. This procedure utilizes the flow cytometry technology, which needs a suspension of cells. Red top tubes have no anticoagulant, therefore the cells will form a clot.

15. DNA sequencing or a DNA probe is recommended to determine the nucleotide sequence of abnormal hemoglobin. Abnormal nucleotide sequences code for the alteration in amino acid order that create abnormal hemoglobin.

16. Southern Blot analysis isolates DNA, cleaves it at specific nucleotide sequences, and separates these portions by size using gel electrophoresis. Complementary probes are used to detect the sequence of interest.

Fluorescence In Situ Hyrbridization (FISH) uses the whole chromosome and hybridizes a fluorescent labeled complementary probe. Samples are visualized by microscopy.

17. This question utilizes the student’s comprehension of the molecular analysis techniques and requires their evaluation and synthesis abilities. There is no one correct answer to this question.

18. T helper absolute count = Absolute lymphocyte count X %CD4/100.

CD4 are T helper cells and CD8 are T suppressor cells; therefore, the student should only calculate the absolute value for CD4 cells. Absolute CD4 values 6200/uL are indicative of AIDS. This 26-year-old male patient’s T helper absolute count is 0.385 * 109/L or 385/uL. This value is considered normal.

19. The sugar-water screening test may be helpful.

20. If PNH were present, the test would show hemolysis of the patient’s RBCs. 21. Blood is added to a 10 percent sugar-water solution which promotes the attachment of complement to the complement sensitive RBCs of patients with PNH. Since PNH RBCs are extremely sensitive to complement lysis, the cells hemolyze.

22. The acidified serum test (Ham test) should be ordered to confirm PNH since the sugar-water test is not specific for PNH. 23. Patients who have hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) may also have a positive Ham test. However, in patients with HEMPAS, the patient’s erythrocytes are not lysed by the patient’s own serum, and the sugar-water test is negative.

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