Dr. Andrea Ciapini With the partecipation of: Prof. Bruno Milanesi Prof. Barbara Mroczko - Dr. Fangyin Zeng - Dr. Maurizio Cortesi Dr. Edmondo Adorisio - E.B.C. Guadalupe Valenzo Valencia
Electrophoresis Atlas of Serum Proteins, Serum Immunofixation, Urine Proteins and Cryoglobulins
E L E CTROPHOR E SIS A TL A S
INTRODUCTION
At the same time the latter must realize the potential, but also the limits and possible causes for error, which the laboratory study methods present.
For many years, perhaps too many, the passion for studying plasma proteins has run constantly through my professional interests. There has been discussion for decades on protein separation methods in the elctrophoretic field, buffers, supports, stains, grade of resolution, focusing, sensitivity, precision and accuracy. Sometimes in an exasperated attempt at “perfection”, these methods have been attributed with potential they don’t have due to constitutive limits. But the fact remains that, as Robert F. Ritchie states (Clin chem Lab Med 2001, 39(11) 1045-1053) [1]: “Serum proteins have a unique feature that few test have: they are interrelated. In other words, an effect on one will change others. Serum proteins analysis is, in fact, one multi-use test, not a series of independent test. As a result, measuring only one protein, e.g. haptoglobin to identify hemolysis, decreases diagnostic ability tremendously.” It follows that in order to fully use the diagnostic potential provided by the analysis of plasma protein, close cooperation between the laboratory operator and the clinic is indispensable as well as the fact that both must have sufficient knowledge in the reciprocal fields of interest. Our intention is to alert the laboratory worker to the interpretive complexity, due to the numerous interactions and the possible overlapping of disorders, and the risk, except on rare occasions, of drawing safe diagnostic conclusions without clinical input.
Electrophoresis
Of course, nor can we be so simplistic as to attribute only the task of detecting the presence of monoclonal components to the study of proteins separated in electrophoresis! Based on these presuppositions this atlas is born, the intention of which is simply to highlight through images and brief comments the potential of electrophoresis which, if “interpreted visibly” the same way as radiographic and cardiographic reports, provides the laboratory worker with the opportunity to give the clinic useful comparisons on the patients’ protein balances without abusing the task of the clinic itself. What was previously stated falls into the spirit of recommendations of the Institute of Medicine (1992) which states a guideline is: “Any type of document which has the objective of indicating which diagnostic-therapeutic options must be adopted in specific clinical circumstances” Even the attached comment, for an exhaustive report on an electrophoresis separation, possesses the same value as other reports which are part of Laboratory Medicine, as indicated by:
I
2) Qualitative assessment 3) Assessment of monoclonality (the most appropriate of the three purposes) A. Burlina 1992 Many readers will find the definition of “Semi-quantitative Electrophoresis” to be inappropriate, feeling that they should trust exclusively in Nefelomet“Il Laboratorio Medico (o Clinico) è il Laboratorio per l’esame biologico, microbiologico, ric/Turbidimetric dosages to assess the concentration of the specific proteins. immunologico, immunoematologico, ematologico, biofisico, citologico, anatomo“The Pathology Laboratory (orchimico, Clinic) is the Laboratory for biological, patologico ed altro di materiali derivati dal corpo umano al fine di fornire informazioni per la They are perfectly correct! microbiological, immunological, chemical, immunohaematologic, haematodiagnosi, prevenzione e trattamento di malattie oppure per la valutazione della salute di esseri umanianatomical-pathological “ logic, biophysical, cytologic, exams and other maWhat the authors intend by “Semi-quantitative Electrophoresis” is simply terials from the human body in order to provide information for diagnosis, the consideration that a “good electrophoretic system, automated and under “…e che può fornire un servizio di consultazione comprendente tutti gli aspetti delle ricerche incluso dei risultati e il suggerimento di appropriati statistic control” prevention and treatment dioflaboratorio, diseases or l’interpretazione for assessment of human health” approfondimenti diagnostici “ “...and which can provide a consulting service including all the aspects can provide, over time, statistically comparable and homogeneous data. Internationathe l Stanresults dard ISO and 15189:suggestions 2003(E) punto 3.8of appropriate of laboratory research, including This situation allows the ponderal electrophoretic data to be accepted as indiagnostic analysis” dicative of normal, increased or decreased values with a high level of statistic Inoltre: International Standard ISO 15189:2003(E) point 3.8 [2] probability. “Il laboratorio, oltre che la responsabilità di accurate risposte, ha quella di assicurarsi quanto Other services: più possibile che esse siano interpretate ed applicate nel miglior interesse del paziente. “ To the specific methods the task of calculating the “true” ponderality. The “The laboratory, in addition to being responsible for providing accurate is further highlighted by the following sub“Azioni specialistiche nella selezione ed interpretazione dei test fanno parte del servizioabove del responses, must ensure asLaboratorio. much as“possible that they are interpreted and apchapters: plied in the best interest ofC6.3 theAnnex patient. “ C Ethics in laboratory medicine a) Electrophoretic system, “automated and under statistical control”: “Specialist actions in International selection Standard and interpretation of the tests are part of ISO 15189:2003(E) the service which the Laboratory provides. “ Queste definizioni dell’ “International Standard ISO” sostentano la definizione di appropriatezza Appropriateness:: adapt and suitable Appropriateness elettroforetico : C6.3 Annex C Ethics dell’esame in laboratory medicine [3] International Standard“appropriato ISO 15189:2003(E) è il test in cui il risultato fornisce una risposta al quesito clinico e mette in grado di una decisione o intraprendere un’azione “ These definitions of theprendere “International Standard ISO” sustain the definition ….appropriate is the test able to give a replay to a of appropriateness of the CP electrophoretic exam; Price 2000 clinical problem and permit to keep a decision or to “appropriate is the test able to give a replay to a clinical problem and undertake an action. “appropriato è tutto ciò che può essere benefico per il paziente” permit to keep a decision or to undertake an action.” CP Price 2000 “in una medicina sempre più complessa, occorre evitare che alcuni pazienti non ottengano CP Price 2000 l’intervento di cui hanno bisogno ed altri abbiamo l’intervento di cui non necessitano” Test “appropriate is anything that can be a benefit for the patient” RH Brook 1994 “in a more and more complex world of medicine, situations must be Clinic query La elettroforesi delle siero-urine proteine è un test che ha come finalità : avoided where some patients do not obtain the treatment which they need 1) Valutazione semiquantitativa Report and others undergo treatments which they did not need” result Exhaustive 2) Valutazione qualitativa RH Brook 1994 3) Valutazione delle monoclonalità (la più appropriata delle tre finalità) The purpose of a sero-urine protein electrophoresis test is: Effective diagnosis Response to the problem 1) Semi-quantitative assessment
Electrophoresis
II
The meaning of effective diagnosis
Disease?
Pathology note
Diagnosis
EBLM
Theatment
EBM
Positive result
D. Giavarina 2004
Personal gold standard
The purposes of effective diagnostics in EFT
Monoclonality research
Qualitative : chemical-physical modifications care of
C3\Hpt-Hb\C.M.
Electrophoresis
Quantitative assessment
Global gold standard
Quantitative: hetero/homozygous conditions care of
Personal gold standard
Alb.\AAT\Trf\ C3\Hpt.
Independent gold standard
Separate gold standard
Qualitative assessment
A. Burlina 1992
Individual increase in Knowledge: continuous training.
III
Independent gold standard
How should quality control be implemented in ETF ?
Global gold standard Personal gold standard
Independent gold standard
• Small and simple things for big results!
Separate gold standard
Quality control
Aimed first and foremost at the best possible evidence. Example: The execution of the urinary proteins must go through the agarose support and a sensitive colouring.
The reference values must be researched by each laboratory and by their own test population.
Separate gold standard Global gold standard Personal gold standard
Independent gold standard
The reference values of the manufacturer versus the laboratory
Separate gold standard
Behaviour uprooted from preconceptions and focused on the complete picture requires cooperation between laboratory technicians and physicians to respond to the following questions : a) Is the test accurate and reproducible? Will it be executed and interpreted correctly? b) What is the pre-test predictability? c) Will the post-test probabilities modify treatment of the disease? d) Will the significance of the response and the report be positively assessed by the physician?
Electrophoresis
EP Areas
Manufacturer X Densitometer A values in %
Manufacturer X Densitometer B values in %
Values of the laboratory in % n = 7700
Albumin
56 / 61 / 66
60 / 65.5 / 71
56 / 61 / 66
Gamma
10 / 14 / 18
8.5 / 12.2 / 16
11 / 15.5 / 20
Annotations on the reference values: The A and B densitometers are made by the same manufacturer and both instruments read the same support in agarose. It is helpful to consider, in light of the values expressed by the manufacturer on comparison with those prepared in the laboratory, the need for each laboratory to estimate their own reference values.
IV
Fig. 1 = Ideal or pathognomonic test with 100% sensitivity and clinical specificity and with absolute diagnostic power. [4] Fig. 2 = Pointless test with 50% clinical sensitivity and specificity (like flipping a coin) Fig. 3 = Ordinary test which has a certain area of overlapping, sensitivity and clinical specificity is intermediate as is the diagnostic power. [5]
Quality control
Precision: Monthly check on the same sample repeated at last 21 times.
Function of the Tonks test A = healthy subjects B = sick subjects C = overlapping of the codes of the two previous areas and its width delimits sector 2 which includes false positives and false negatives.
The Tonks test suggests the max value of CV% within which the data remain significant.. significant
Albumin Alpha 1 Alpha 2 Beta 1 Beta 2 Gamma
CV % Max = 2 CV % Max = 6 CV % Max = 5 CV % Max = 4 CV % Max = 4 CV % Max = 6
The systematic, weekly use of the Tonks test contracts and keeps the C area under control.
Percentage of clinical errors applying the Acland Lipton test to the Tonks data
Tonks DB: Clin Chem 9:217 1963 Albumin: Tonks Max.= 2%
The diagnostic significance of a test
Albumin: Normality range = 56-66 % With a value of 54% what are the probabilities of becoming normal for reproducibility error?
Probability = 1.5 %
Electrophoresis
V
Percentage of clinical errors applying the Acland Lipton test to the Tonks data
Percentage of clinical errors applying the Acland Lipton test to the lowest Tonks data
Gamma: Tonks Max.= 6 %
Gamma: Normality range = 11-20 % What probability does a value of 13% have of becoming normal for reproducibility?
Gamma: Normality range = 11-20 % What probability does a value of 13% have of becoming normal for reproducibility? Probability = 5 %
Tonks Gamma = 4 % Probability = 2.5 %
b) The electrophoretic data as approximate significantly of normality, increase, decrease • Materials and methods: • selection of 20 serums for alignment of the ETF albumins to the nephelometric ones • 12 serums with polyclonal IgA with concentrations between100 and 600 mg/dl • 100 serums (age between 18 and 75 years, normal panels and with various pathologies, 55 women and 45 men) • total proteins dosage with biuret method and white sample. • Behring nephelometer for dosage of the specific proteins of the 100 serums • system for Microgel electrophoresis in agarose and colouring support Acid Blue • Visual inspection (2 expert operators) and proteins assessment with adjectivations corresponding to numbers from 1 to 5. • Kolmogorov-Smirnov [6] test and parallelism test for the study of the populations of electrophoretic data (adjectivations translated in numbers from 1 to 5) and nephelometric data (g/L). • In the graphics the green curves are relative to electrophoretic data while the red curves are relative to the nephelometric data.
Conclusions on Tonks data
Albumin Alpha 1 Alpha 2 Beta 1 Beta 2 Gamma
Electrophoresis
CV % Max = 2 CV % Max = 6 CV % Max = 5 CV % Max = 4 CV % Max = 4 CV % Max = 6
Each reduction of the Tonks values will lead to a minor probability that the data can go from Normal to Pathological and viceversa: see following slide.
VI
Dinamica ETF fino a 3.5 g/L
Livello di significatività >0.05 Popolazioni con stessa distribuzione
ELECTROPHORETIC ALFA 2 NEPHELOMETRIC ALFA 2 + HPT
ELECTROPHORETIC ALBUMINNEPHELOMETRIC ALBUMIN
MEASUREMENTS IN
Albumine ETF allineate con Nef. Dinamica ETF fino a 49 g/L Albumine ETF allineate con Nef. Livello di significatività >0.05 ETF dynamic up to 49 g/L Test di parallelismo positivo Dinamica ETF fino a 49 g/L
Popolazioni con stessa distribuzione Level of significance >0.05 Livello di significatività >0.05 Population with the same distribution Popolazioni con stessa distribuzione
F calcolato < 3,96 F statistico Parallelism test positive Test di parallelismo positivo calculated F <3.96F F calcolato < 3,96 F statistico statistic
ELECTROPHORETIC ALPHA 1 NEPHELOMETRIC ALFA 1 AT
ETF dynamic up to 3.8 g/L Parallelism Dinamica ETF fino a 3.8 g/L test
Level of significance >0.05 Population with the same distribution
Livello di significatività >0.05 Popolazioni con stessa distribuzione
positive calculated F <3.96F Test di parallelismo statistic
positivo F calcolato < 3,96 F statistico
MEASUREMENTS IN
Dinamica ETF fino a 4.9 g/L
Dinamica ETF fino a 3.5 g/L ETF dynamic up to 3.5 g/L Dinamica ETF fino a 3.5 g/L
Electrophoresis
MEASUREMENTS IN
ELECTROPHORETIC BETA 1 NEPHELOMETRIC TRF
MEASUREMENTS IN
Level of significance >0.05 Livello di significatività >0.05 Population with the same distribution Popolazioni con stessa distribuzione Livello di significatività >0.05 Popolazioni con stessa distribuzione
Test di parallelismo positivo F calcolato < 3,96 F statistico
ETF dynamic up to 4.9 g/L
Positive parallelism
Test test di parallelismo positivo calculated F <3.96F F calcolato < 3,96 F statistico Test di parallelismo positivo statistic F calcolato < 3,96 F statistico
VII
Positive parallelism test Level of significance >0.05 di parallelismo positivo LivelloPopulation di significatività >0.05 calculatedTest F <3.96F with the same distribution statistic F calcolato < 3,96 F statistico
Popolazioni con stessa distribuzione
Dinamica ETF fino a 4.9 g/L
Test di parallelismo positivo F calcolato < 3,96 F statistico
Livello di significatività >0.05 Popolazioni con stessa distribuzione
The average difference between the values found with the two methods for the immunoglobulins oscillates between 2 and 3 g/L less for Electrophoresis.
ELECTROPHORETIC BETA 2 NEPHELOMETRIC C3
This difference can be attributed to two factors: 1) The different affinity between antibodies and heat for the Ig classes (obvious statement not to be commented given the stoichiometry between colouring and amino acids). 2) The percentage of IgA, electrophoretics, which are not calculated because they migrate under the C3 and the Tf.
MEASUREMENTS IN
For point number 2 a study was conducted by means of the Cubic Spline (third degree equation) applied to the graphic points which draw the gamma area.
Dinamica ETF fino a 1.7 g/L ETF dynamic up to 1.7 g/L
Livello di significatività >0.05 Popolazioni con stessa distribuzione Level of significance >0.05 Population with the same distribution
Positive parallelism test calculated F <3.96F statistic
Test di parallelismo positivoSince the Cubic Spline [7] [83] is a polynominal regression equation it was F calcolato < 3,96 F statistico possible to request, based on the slope of the gamma curve toward C3 and Tf, the tendency to regress to the base line of the graphic if the zero point were the minimum between C3 and gamma and then between Tf and gamma.
ELECTROPHORETIC IMMUNOGLOBULINS IgG +IgA + IgM NEPHELOMETRIC
The first graphic shows how a percentage of Ig migrate under C3 and up to the beginning of Tf.
MEASUREMENTS IN
ETF dynamic up to 26 g/L Level of significance >0.05 Population with the same distribution
Dinamica ETF fino
Livello di significatività >0.05 Popolazioni con stessa distribuzione
Electrophoresis
Positive parallelism test calculated F <3.96F a 26 g/L statistic
Test di parallelismo positivo F calcolato < 3,96 F statistico VIII
La differenza media tra i valori trovati con i due metodi per le immunoglobuline oscilla tra 2 e 3 g/L in meno per la Elettroforesi. Questa differenza è da imputare a due fattori:
The second graphic shows how the hypogammaglobulinemic sample must be reconsidered in light of the large percentage of Ig which migrate under C3 and well beyond the densitometric minimum.
Total amount not calculated by the densitometer in mg/dl
Amount not calculated under C3 in mg/dl
Amount not calculated under Tf in mg/dl
600
184
99
85
500
136
73
63
400
108
58
50
300
80
43
37
200
50
27
23
100
20
11
9
The above data shows how an electrophoretic system “under control” (automated and under statistic) can provide semi-quantitative data intended to determine a correlation congruent with the nephelometric data. This means that such a “system under control” can be an excellent indicator for definition of normal specific proteins, increased or decreased leaving the nephelometry/turbidimetry the task of confirmation and the “true” weighted dosage. In order for the following three “Assessments” to be realised 1) Semi-quantitative assessment: hetero\homozygous conditions care of Alb.\AAT\Trf\ C3\Hpt. 2) Qualitative assessment chemical-physical modifications care of C3\Hpt-Hb\M.C. 3) Assessment of the monoclonalities (the most appropriate of the three finalities) (burlina 1992) it is fundamental that each laboratory performs an upgrade of their Electrophoretic system in light of the recommendations expressed in the context of the Evidence Based Medicine (EBM) and the Evidence Based Laboratory Medicine (EBLM): Evidence Based Medicine (EBM):
The third graphic shows how a significant percentage of Ig migrate under C3 and Tf.
The use of the Cubic Spline has allowed the following table to be prepared of Ig not calculated by the densitometer due to the incorrect position of the minimum placed all the way on the bottom.
Electrophoresis
Tenor of IgA in mg/dl
IX
“the conscientious, explicit and judicious use of current best evidence in making decisions about the care or individual patients”. This definition by Sackett et al specifically includes the laboratory as well in that it is an integral part of the physician’s decisional process. EBM has also been defined as a process of personal updating throughout one’s entire life. From this description it follows that medicine is a field in continuous discovery, evolution and change, therefore it is important to ensure that the practice is based on the best available evidence and that there is an opportunity to adopt new procedure for which the benefit has been demonstrated. Evidence Based Laboratory Medicine (EBLM): The practice of EBLM requires the integration of individual clinical experience with the best laboratory evidence derived from systematic research. Evidence Based Laboratory Medicine (EBLM) and Gold Standard [8]: Introducing the principles of Evidence Based Medicine in the laboratory means implementing a continuous process of identification of those tests which, on one side can offer the highest diagnostic efficiency and reliability (Gold Standard) and, on the other side, can cause lower risks and less inconveniences for the patient as well as saving work, time and money. Gold Standard The “Gold Standard” concept is a concept in continuous development which cannot and must not know the Enthalpic phase. It is a concept of a continuous search for the “best truth (method) available” for a specifically aimed and efficient support work for the true final users of this action: the patients! The current Gold Standard in the Electrophoretic field is represented by the Agarose support. Who attests to this (some organisms and conventions in chronological order): 1. College of American Pathologists Arch. Pathol. Lab. Med. Vol. 123 page 126 February 1999 [9] 2. 36th national SIBioC Conference in Padua 2004
Electrophoresis
3. XV Edition of “Proteins from the Laboratory to the Clinic” CEFAR 2006 4. Am J Clin Pathol 2008; 129:451-458 “Performance Comparison of Capillary and Agarose Gel Electrophoresis for the Identification and Characterization of Monoclonal Immunoglobulins” [10] 5. Clin Chem Lab Med 2009; 47: 1021-1022 2009 [11] If meta-analysis is a “structured and systematic integration of information derived from different studies on a given problem... the analytical procedure for serum-urine proteins on agarose is probably capable of achieving objectives useful for the clinic for the purpose of improving the diagnostic approach and/or therapeutic intervention. The use of the best available method does not complete the task of the laboratory. The approach to electrophoretic methods, being a diagnostic procedure, must consider a descriptive and an interpretive phase [12]: a) visual inspection of protein patterns (descriptive phase prepared in molecular terms with indication of absence and presence of supernumerary protein, most adjectival increase or reduction, specific individual proteins. At the descriptive phase, the laboratory must undertake identification of M.C. and the search for the B.J. protein. The laboratory may also proceed with quantification of specific proteins to guarantee visual observation) b) the “elecrophoretic semeiotic” phase (interpretive phase prepared in relation to physiopathological panels and clinical associations; the acquisition of clinical news is desirable. c) exhaustive reporting a) visual inspection of protein patterns formidable methodology to identify proteinic modification, individual or together, with respect to the physiological panel: Guidelines for Clinical and Laboratory Evaluation of Patients with Monoclonal Gammopathies [13] David F. Keren, MD; Raymond Alexanian, MD; James A. Goeken, MD; Peter D. Gorevic, MD; Robert A Kyle, MD; Russell H. Tomar, MD
X
“Serum and urine electrophoresis …….. the gel should be examined directly by the interpreter.” seguenti è possibile osservare la“Serum caducità della (Arch PatholNelle Labfigure Med. 1999;123:106–107) andlettura urinedensitometrica electro- senza l’intervento della interpretazione visiva: phoresis …….. the gel should be examined“Serum directly by the interpreter.” and urine electrophoresis …….. the gel should be examined directly by the interpreter.” In the following figures the caducity of the densitometric reading withNelle figure seguenti è possibile osservare la caducità out the intervention of visual interpretation [12]: della lettura densitometrica senza l’intervento della interpretazione visiva:
La piccola banda omogenea in ETF (freccia) non viene rilevata dalla lettura The small homogeneous densitometrica.band in ETF (arrow) is not detected by the den-
Guidelines: clinical associations
Prealbumin
Nephrosis
Albumin
Non existent
Alpha 1 antitrypsin
Oestrogenic effect, inflammation, necrosis, heteroplasia, hepatopathies
Alpha 2 macroglobulin
Nephrosis, hepatopathies, diabetes, III tri. preg., infancy, senility
Aptoglobin
Necrosis, heteroplasia, inflammation
Transferrin
Iron def. anaemia, hepatitis, pregn.
C3
Obstruction, biliary, non recent inflammation
Gammaglobulin
Hepatic and biliary cirrhosis, alcoholism, chronic infections, chronic hepatitis, auto-immune diseases
La piccola banda omogenea in ETF (freccia) non viene rilevata dalla lettura
sitometric reading. densitometrica.
Guidelines: clinical associations
La migrazione ETF mette in evidenza due bande omogenee delle quali la più catodica risulta non esistente nel grafico (Frecce)
Prealbumin
Inflammations, heteroplasia, hepatopathies
Albumin
Hepatopathies, nephropathies, cachexies, dispersing enteropathies, heteroplasia
Alpha 1 antitrypsin
Genetic defect
Alpha 2 macroglobulin
Fibrinolysis, rheumatoid arthritis, eclampsia
Aptoglobin
Congenital defect, hepatopathies
Transferrin
Hepatopathies, nephropathies, chronic infections
The ETF migration shows two homogeneous bands of which the C3 Auto-immune diseases, hepatopathies b) fase di “semeiotica Lala migrazione ETFelettroforetica” mette in evidenza due bande omogenee delle quali la più catodica more cathodic is non existent in the graphic (Arrows) Gammaglobulin Congenital/infant acquired immune deficiencies risulta non esistente nel grafico (Frecce)sullo stato dell’ assetto proteico. che fornisce, attraverso ad “archetipi cognitivi”, conclusioni b) the “elecrophoretic semeiotic” phase c) exhaustive reporting b) la conclusions fase di “semeiotica elettroforetica” which provides on the state of the protein structure through able to present the clinic with an exhaustive response to the clinical query in order to undertake the appropriate steps for action on the patient. “cognitive archetypes”. che fornisce, attraverso ad “archetipi cognitivi”, conclusioni sullo stato dell’ assetto proteico.
Electrophoresis
XI
Rarely, however, are we aware of the evidence which the use of a laboratory test can modify, for a given patient or group of patients, the clinical behaviour with respect to diagnosis or therapy. For this reason the laboratory must: • provide homogeneity to the results • improve effectiveness of the methods • provide data on new possible methodologies The diagnostic process is structured on four main reference models: Laboratory task 1) Analytic identification of the panel Physician task 1) Recognition of the panel 2) Physiopathological reasoning 3) Probabilistic diagnosis All of this must take place in complete observance of the individual competences in order to realise the Gold Standard concept of globalism: a) personnel: Individual growth in knowledge and continuous training b) independent: Aimed first and foremost at the best possible evidence. c) separate: Behaviour uprooted from preconceptions and focused on the to the following questions: a) Is the test accurate and reproducible? Will it be executed and interpreted correctly?
Regarding this the report must suggest outcomes, whether the test is needed only once (1) or whether the test must be repeated in a follow-up (2): A1 A A1 A
Clinical needs Necessità clinica Clinical needs
tests tes Necessità clinica
tes tests
Decision/Action Decisione/Azione Decision/Action Decisione/Azione
Intervention Intervent
Intervent Intervention
Outcome Outcom
Outcome Outcom
B B2 B B2
Clinical needs Necessità clinica Clinical needs Necessità clinica
tests test
Intervention Intervento
Intervention Intervento
tests test
Outcome Da Price CP, modificato.
• • •
c) Will the post-test probabilities modify treatment of the disease? d) Will the significance of the response and the report be positively assessed by the physician? The essential purpose of the preparation of this atlas text is to provide the reader with an evident and exhaustive iconographic material of the individual specific proteins and immuno-fixative panels, which can be highlighted with electrophoretic method on agarose support. A. Ciapini
Outcome
By CP Price, modified. Da Price CP, modificato. Global Gold Standard In the laboratory different types of evidence are accepted: the data of analytical performances. the internal and external quality control data. the data relative to the specificity and sensitivity of the tests.
Electrophoresis
XII
PREALBUMIN
ALBUMIN
ALPHA 1 ANTITRYPSIN ALPHA 2 MACROGLOBULIN RBP TRANSFERRIN Î&#x2019;2 MICROGROBLUIN
FREE K OR L
POLICLONAL IG CISTATIN C LYSOZYME
The graphic representation of an electrophoretic migration and the position of the specific proteins.
Urine protein patterns on agarose and acid violet colouring Position of the individual plasmatic proteins Separation by net electrical charge [28]
ICONOGRAPHIC ATLAS OF SERUM AND URINE PROTEINS ON AGAROSE GEL
PRE-ALBUMIN ALBUMIN ALFA LIPOPROTEINS
ANTICHIMOTRYPSIN Gc GLOBULINS CERULOPLASMIN EMOPESSIN COMPLEMENT FRACTION C4
The specific sieric proteins most frequently identifiable upon visual inspection [17] [29] [30]
HETEROZYGOSITY OF ALPHA 1 ANTI-TRYPSIN INTER ALPHA-TRYPSIN INHIBITOR ALPHA 2 MACROGLOBULIN ALPHA 2 MACROGLOBULIN APTOGLOBULIN INSOLUBLE COLD GLOBULIN β LIPOPROTEIN TRANSFERRIN
COMPLEMENT FRACTION C3 FIBRINOGEN OR FDP
SERUM PROTEIN PATTERNS OF INDIVIDUAL SPECIFIC PROTEINS AND COMIGRANT METABOLITES
PROTEIN Pre-albumin/Transthyretin
Taxonomy Eukaryotes Bacteria Organisms Homo Sapiens Helix pomatia Gallus gallus Streptococcus Brassica napus Finegoldia magna
Clinical significance: with its high content of tryptophan, its short half-life (2 days), low concentration, prealbumin is a thin and quick indicator of inflammatory reaction, denutrition (jointly with PCR), decrease of hepatic parenchyma. Different genetic variations, of the more than 50 identified, are amyloidogenic. â&#x2013;˛ treatment with corticosteroids, use of anabolic steroids, Hodgkins disease, alcoholism, acromegaly â&#x2013;źAPR, hepatopathy, nephropathies, hereditary amyloidosis, hyperestrogenism, thyrotoxicosis, intravenous administering of liquids
The prealbuminic molecule connected to triiodothyronine
Electrophoresis
7
PROTEIN Pre-albumin/Transthyretin
Track No. 21
Aι Aι α1 α2 β1 β2 γ
pre
Aι Aι α1 α2 β1 β2 γ
pre
ID 12008
The colourings normally in use such as: Acid blue, amide black and red Ponceau S show a low index of affinity for this protein [17] [28]. An excellent densitometer is able to highlight even small quantities of Prealbumin which connect tot he aforementioned anionic colourings. This is perhaps the only case in which the instrument can replace the visual observation of ETF.
Electrophoresis
8
PROTEIN Pre-albumin/Transthyretin
Track No. 3
Aι Aι α1 α2 β1 β2 γ
pre
ID 23009
Electrophoresis
9
PROTEIN Pre-albumin/Transthyretin
The use of colourings with greater affinity index, for example acid violet, allows us to highlight the transthyretin.
ID 30010
Sample with normal Transthyretin
Electrophoresis
ID 30011
Sample with decreased Transthyretin
10
PROTEIN Albumin
Taxonomy Eukaryotes Bacteria Organisms Homo Sapiens Helix pomatia Gallus gallus Streptococcus Brassica napus Finegoldia magna
Clinical significance: historically a general indicator of the protein state of the organism, its reliability is compromised by: • long half-life (19 days) • decreased synthesis during most inflammatory processes At today: prognostic value, in particular in chronic patients ▲ haemoconcentration ▼ APR, hepatopathy, nephrosic syndrome, malnutrition, pregnancy, premature newborns, genetic analbuminaemia
Electrophoresis
11
PROTEIN Albumin
Track No. 8
Track No. 9
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 45012
On the left subject with reduction of Albumin On the right the sample with normal Albumin
Electrophoresis
12
PROTEIN Albumin
Track No. 8
Track No. 9
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00313
Hypoalbuminemia from clinostatism. Sample on the left: Patients hospitalised and bedridden show hypoalbumins for redistribution of albumin in the extravascular spaces.
Electrophoresis
13
PROTEIN Albumin, analbuminemic condition
Track No. 12
Aι α1 α2 β1 γ ID 00311
Electrophoresis
14
PROTEIN Albumin
Track No. 21
Track No. 20
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 90015
On the right the sample with normal albumin carrying drugs
Electrophoresis
15
PROTEIN Albumin
Track No. 1
Track No. 2
Aι α1 α2 β1 γ
Aι α1 α2 β1 β2 γ
ID 80016
On the left the only event which determines an increase of Albumin: dehydration
Electrophoresis
16
PROTEIN Albumin
Track No. 9
Track No. 10
Aι α1 α2 β1 γ
Aι α1 α2 β1 γ
ID 70017
On the left an example of overload of the Albumin transport function with appearance of “quick” Albumin for anodic enlarging caused by the presence of bilirubin (jaundiced sample). In the beta-anodic area a small “arc” is also noted made up of bilirubin.
Electrophoresis
17
PROTEIN Albumin
Track No. 12
Track No. 13
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 60018
On the right a sample with fast heterozygote alloalbuminemia (autosomic codominant transmission).
Electrophoresis
18
PROTEIN Albumin
Track No. 2
Track No. 3
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 50019
On the right a sample with slow homozygote alloalbuminemia (autosomic codominant transmission).
Electrophoresis
19
PROTEIN Albumin
Track No. 17
Track No. 18
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 76020 004520
On the left a sample with slow heterozygote alloalbuminemia (autosomic codominant transmission).
Electrophoresis
20
PROTEIN Albumin
Track No. 7
Track No. 8
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 12021
bisalbuminemia (always fast) from ascertained pancreatitis or use of drugs.
Electrophoresis
21
PROTEIN Albumin
Track No. 12
Track No. 13
Aι α1 α2 β1 γ
Aι α1 α2 β1 β2 γ
ID 22022
bisalbuminemia (always fast) from drugs.
Electrophoresis
22
PROTEIN Alpha lipoprotein (APO A)
Taxonomy Eukaryotes Bacteria Virus Archea Organisms Homo Sapiens Mus musculus Bos taurus Rattus nor vegicus Escherichia coli Gallus gallus Torpedo californica
Electrophoresis
Electrophoresis of lipoproteins is a simple method, useful for studying dyslipidemia. Their differentiation based on the electrophoretic mobility is the basis of the Fredrickson classification. Alpha lipoproteins or HDL (High Density Lipoproteins): are the fastest fraction. They represent the majority of the lipoproteins and migrate into the position between Albumin and alpha 1 antitrypsin.
23
THE MONOCLONAL COMPONENTS Their position in ETF migration
After stimulation Ag assists with a rapid expansion of the clone; this is accompanied by a morphologic change and by a small lymphocyte a mature plasma cell is reached equipped with Golgi apparatus and endoplasmic reticulum. During the subsequent cellular divisions the V region is expressed with constance while the H region shifts from D to M to G to A.
From Monoclonal Gammopathies Aguzzi/Bienveniu/Jones/Whicher
Electrophoresis
115
THE MONOCLONAL COMPONENTS Their percentage frequency in relation to the position assumed in ETF migration The study was conducted on 1654 samples with presence of MC. In the gamma area, as can be observed, 88% of the MC migrate.
A. Ciapini; Proteins from the laboratory to the Clinic; Desenzano del Garda 13-14 January 2006
Electrophoresis
116
THE MONOCLONAL COMPONENTS Alpha 1 area
Track No. 7
IFE shows the presence of a monoclonal component of the free lambda type.
Track No. 8
Aι α1 α2 β1 β2 γ ID 00117
In the sample on the left the alpha 1 area is in a more anodic position with respect to the same protein in the sample on the right.
Aι α1 α2 β1 β2 γ
Electrophoresis
117
THE MONOCLONAL COMPONENTS Alpha 2 area
Track No. 19
Track No. 18
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00118
Normal sample on the left Sample on the right with presence of MC IgA K in alpha 2 anodic area.
Electrophoresis
118
THE MONOCLONAL COMPONENTS Alpha 2 area
Track No. 7
Track No. 8
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00119
Normal sample on the left Sample on the right with presence of MC IgA λ in alpha 2 inter-area - transferrin.
Electrophoresis
119
THE MONOCLONAL COMPONENTS Alpha 2 area
Track No. 8
Track No. 9
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00120
Sample on the right with alpha 2 increase and decrease of aptoglobin Sample on the left with presence of MC IgA K in alpha 2 inter-area - transferrin.
Electrophoresis
120
THE MONOCLONAL COMPONENTS The Gamma area Inter Alpha 2 - Transferrin Track No. 23
Track No. 22
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00121
Normal sample on the right Sample on the left with presence of homogeneous ETF band (IgA ʎ) in central Alfa2 - Transferrin position
Electrophoresis
121
THE MONOCLONAL COMPONENTS The Transferrinic area
Track No. 2
Track No. 3
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00122
Normal sample on the right with absence of abnormalities referring to the presence of homogeneous bands. Sample on the left with slower transferrinic area and presence of homogeneous band, referring to monoclonal free lambda in the inter-space Tf-C3. Reduction of albumin, C3 and hypogammaglobulinemia.
Electrophoresis
122
THE MONOCLONAL COMPONENTS The Transferrinic area
Track No. 26
Track No. 25
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00123
Normal sample on the right with increased transferrin, but with the absence of abnormalities referring to the presence of homogeneous bands. Sample on the left with anodically widened transferrinic area with hazy morphological profile due to presence of monoclonal light chains λ.
Electrophoresis
123
THE MONOCLONAL COMPONENTS The Transferrinic area
Track No. 8
Track No. 9
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00124
Sample on the left with presence of Gc (specific group component) Sample on right with increased transferrin and intense colouring, more cathodic position due to presence of Monoclonal IgA K component with similar net electrical charge.
Electrophoresis
124
THE MONOCLONAL COMPONENTS The (Gamma) Inter Transferrin-C3 area
Track No. 19
Track No. 18
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00125
Normal sample on the left Sample on the right with widened transferrin and hazy on the cathodic margin due to the presence of K type monoclonal free chains.
Electrophoresis
125
THE MONOCLONAL COMPONENTS The (Gamma) Inter Transferrin-C3 area
Track No. 3
Track No. 4
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00126
Normal sample on the left Sample on the right with the presence of homogeneous ETF band (IgA K) in anodic position with respect to C3 which was physically moved and retromigrated in the cathodic direction, by spherical encumbrance bound to the presence of the MC.
Electrophoresis
126
THE MONOCLONAL COMPONENTS The (Gamma) Inter Transferrin-C3 area
Track No. 3
Track No. 4
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00127
Normal sample on the left Sample on the right with presence of ETF homogeneous band (IgA λ) in anodic position with respect to C3 which appear weakly on the side facing the cathode and simultaneous presence of Beta 2 lipoprotein leaning against the anodic side MC.
Electrophoresis
127
THE MONOCLONAL COMPONENTS The C3 area
Track No. 19
Track No. 18
Aι α1 α2 β1 β2 γ
Aι α1 α2 β1 β2 γ
ID 00128
Normal sample on the left Sample on the right with presence of ETF homogeneous band (IgA λ) in anodic position with respect to C3 which appears reduced, hazy and asymmetric-anodic
Electrophoresis
128
IMMUNOFIXATIONS
[7] [15] [25] [50] [51] [52]
ATLAS negative sieric immunofixations
Negative IFE pattern for presence of MC
SPE
IgG
ID 200195
Electrophoresis
IgA
IgM
魏 位
SPE
IgG
IgA
ID 200195
195
IgM
魏 位
ATLAS negative serum immunofixations
Negative patterns for presence of MC. The IFE on the left shows a reduction of the polyclonal heterogeneity for class G and subclass I. Class A and M are normal. The IFE on the right shows a medium reduction of polyclonalism G (still in the reference environment) and pushed by A.
SPE
IgG
ID 200196
Electrophoresis
IgA
IgM
魏 位
SPE
IgG
IgA
ID 200196
196
IgM
魏 位
ATLAS serum immunofixations: class G different sieric samples
SPE
IgG
IgA
IgM
κ λ
ID 200197
Presence of 1 IgG class MC and large size λ type. Depressed polyclonal.
Electrophoresis
SPE
IgG
IgA
IgM
κ λ
ID 200197
SPE
IgG
IgA
IgM
κ λ
ID 200197
Presence of 1 IgG class MC and medium size K type. Absent polyclonal.
197
Presence of 1 IgG class MC and medium size λ type. Absent polyclonal.
ATLAS sieric immunofixations: class G
Presence of 2 IgG class MC and type K; small concentration cathodic, while anodic, in transferrinic position, has a medium concentration.
SPE
IgG
IgA
IgM
魏 位
ID 200198
Electrophoresis
198
ATLAS sieric immunofixations: class G different sieric samples
SPE
IgG
IgA
IgM
κ λ
ID 200199
Presence of 2 IgG class MC and K type. Good conservation of the polyclonals.
Electrophoresis
SPE
IgG
IgA
IgM
κ λ
ID 200199
SPE
IgG
IgA
IgM
κ λ
ID 200199
Presence of 2 IgG class MC and K type. discrete conservation of the G polyclonals and absence of A and M.
199
Presence of 2 IgG class MC and K type. Low conservation of the G polyclonals.
ATLAS sieric immunofixations: class G different sieric sample
SPE
IgG
IgA
IgM
κ λ
ID 200200
Presence of 1 very small IgG class MC and type λ. The high concentration of the K polyclonals can mislead in attribution of the type of light chain.
Electrophoresis
SPE
IgG
IgA
IgM
κ λ
ID 200200
SPE
IgG
IgA
IgM
κ λ
ID 200200
Presence of IgG class MC and λ type.
200
Presence of 2 IgG class MC and K type. Strong depletion of the polyclonals.
ATLAS sieric immunofixations: class G
Presence of 2 IgG class MC and type lambda; small concentration cathodic, while anodic has a lower concentration. Note the strong reduction of the polyclonal heterogeneity of the immunofixated IgA (more anodic with respect to the anodic MC) which simulates a compatible band with a “monoclonal” morphology.
SPE
IgG
IgA
IgM
κ λ
ID 200201
Electrophoresis
201
SPE
ATLAS
ATLAS
sieric immunofixations: class A
sieric immunofixations: class A
IgG
IgA
IgM
κ λ
SPE
ID 200202
IgA
IgM
κ λ
The sample has an A class MC and λ type. Note the different reactivity of the antiserum (affinity and avidity) compared with the class and type.
Presence of 3 IgA class MC and lambda type, the presence of a lambda type MC is noted confirmed with antiserum anti free light chains (positive Bence Jones). For class A it is useful to disaggregate the sample and verify the number of secernent clones.
Electrophoresis
IgG
ID 100202
202
SPE
ATLAS
ATLAS
sieric immunofixations: class A
sieric immunofixations: class M
IgG
IgA
IgM
κ λ
SPE IgG
ID 200203
The sample has 2 MC in gamma- C3 position of IgA class and λ type;
Electrophoresis
IgA
IgM
κ λ
ID 100203
The sample has 2 MC in central gamma position of IgM class and respectively type k e λ.
203
ATLAS sieric immunofixations: class M
The sample has an IgM class MC and K type.
SPE
IgG
IgA
IgM
魏 位
ID 200204
Electrophoresis
204
SECOND ANNEX
Appendice seconda Prima pagina il Titolo: ok quello esistente Inserire questa diciture e figura
a) Le proteine urinarie in elettroforesi b) La concentrazione dei liquidi biologici
THE ETF URINARY BLOCK AND THE CONCENTRATION OF BIOLOGICAL LIQUIDS IN THE PRACTICE OF CLINICAL CHEMISTRY LABORATORIES AFFERENT ARTERIOLE
DISTAL TUBULE
GLOMERULE
Il glomerulo
THE NEPHRON: KIDNEY FUNCTIONAL UNIT
(CAPILLARY BED) AFFERENT ARTERIOLE
BOWMAN’S CAPSULE
AFFERENT ARTERIOLE
DISTAL CONVOLUTED TUBULE
PROXIMAL CONVOLUTED TUBULE
BOWMAN’S CAPSULE
COLLECTING DUCT
PROXIMAL TUBE
HENLE’S LOOP
STRAIGHT VESSELS
DESCENDING LOOPS OF HENLE
COLLECTING DUCT
HENLE’S LOOP
Il glomerulo
a) The urinary proteins in electrophoresis b) The concentration of the biological liquids
The renal apparatus
The glomerule
The tubule
Seconda pagina Titolo: L’apparato renale
Electrophoresis
Il tubulo
287
ASCENDING LOOPS OF HENLE
VESCICLE AND THE EXTERNAL ENVIROMENT
Il tubulo
To concentrate or not to concentrate? And if yes, how? This is the problem [28]!
Electrophoresis of the urinary proteins that Tidstrom, at first, conducted on paper in 1963, madefollowing notable progressions in the use of new substrates that the technology furnished to the laboratory technician: from the paper it was passed to the starch gel, to the agar, to the cellulose acetate, to the acrylamide, and to the agarose, and finally with the capillary method
INTRODUCTION The relevance of the study of proteinuria is demonstrated by the great mass of existing literature on the subject. The association of the proteinura with abnormality of the kidney has been clearly demonstrated by Richard Bright around 150 years ago. The role of the kidney in the storage and metabolism of the plasma-proteins has been studied many times in the past, but it is in the last two decades that the subject has been the focus of numerous studies. The interest in the study of proteinuria is derived by the frequency of appearance of this sign, present in almost all kidney diseases, by the development of biochemical methods for the qualitative and quantitative study of urinary proteins and a more precise knowledge of the physiological processes of glomerular filtration and tubular re-absorption of the proteins in normal and pathological conditions. We think it useful to recall that the presence of proteinuria does not necessarily imply the existence of a renal pathology (even normal subjects have “physiological” proteinuria) and instead are the qualified electrophoretic studies to define a proteinuria as “pathological”, highlighting the presence of plasma originating proteins in the urine that under normal conditions do not exceed the glomerular barrier; of proteins present only when the tubule does not carry out its function of re-absorption; of abnormal plasma-tic proteins that pass in the urine; of proteins secreted by the kidney and urinary tract.
Electrophoresis
Table 1 Principle EP techniques used in the clinic, for the analysis of the proteinic block of the various biological liquids
288
Substrate
Research worker
Year
Filter paper
Konig Cremer and Tiselius Durrum Grassman Wunderly*
1937 1950 1950-1955 1950-1954 1954
Agar gel
Gordon Grabar e Williams Wieme
1949 1953 1956-1959
Starch gel
Smithies
1955
Cellulose acetate
Kohn
1957
Beta 2 microglobulin
Polyacrylamide gel
Raymond and Weintraub Davis and Ornstein
1959 1964
Immunoglobulins
Acrylamide-agarose gel
Uriel
1966
Enzym
Agarose gel
Laurell Johansson
1965 1972
PROTEINS OF RENAL ORIGIN around 40 \ 30 %
Capillary
Protein binding the retinol
Urine mucoid around 50 mg \ 24 hours
2001
Urokinase Secretory IgA
In the normal subject, the daily urinary elimination of protein consists of around 150 mg. Table 2 lists the main proteins of normal urine.
Tubular The proteins of plasmatic origin constitute 60 % 70 % and of these, about 15 mg are attributable to albumin, while other proteins or polypeptides are present in very small quantities or in traces; suffice it to say that with ultrasensitive techniques such as bi-dimensional electrophoresis, 500/600 spots are highlighted. The protein prevalent in the urine from renal endogen production is the urine mucoid or Tamm-Horsfall protein. It is a protein with a very elevated PM (7,000,000 D) but with the possibility of aggregates that can reach 23,000,000 or more. It is a polymeric protein with a high glycine content (30%), in which monomers have a PM of 80,000 D, with P.I. of 3.5; it is the essential matrix of the cylinders and seems to be secreted in the urine to the Henle ansa level and by distal twisted tubule. Its biological meaning is unknown, and it is hypothesized that it has an antibacterial and antiviral role. The urokinase, with its capacity to lyse the fibrin can have a role in removing coagulates at the capillary level, or, according to other AA, that of acting enzymatically on the cylinders.
Table 2 Total 24 hour proteinuria = 130 mg ( 80 \ 240 ) PROTEINS OF PLASMATIC ORIGIN around 60 \ 70 % Albumin around 15 mg \ 24 hours ( 2.0 - 30.0 ) Caeruloplasmin Alpha 1 acid glycoprotein Alpha 1 antitrypsin Alpha 1 micro-protein Emopessina Transferrin Aptoglobin
Electrophoresis
289
The Iga in the urine, which increase to around 1 mg/24 hours, are of the dimeric secretorial type, equal to those present in other secretions; the secretory component would be synthesized by the tubules; the function is an antibody type. The quantity and composition in urine protein depend on the balance between the transglomerular passing and the tubular re-absorption. Considering that the kidney receives around 25 kg of proteins daily, of which only nearly a gram crosses the glomerular barrier, then encountering a tubular re-absorption of around 90%, it includes the extreme efficiency of nephron in controlling this aspect of the protein homeostasis. To clinically frame the proteinurias, it is necessary to consider the physiopathological mechanisms that they determine and carry out at a glomerular and tubular level. Kidney anatomy, function and hydrodynamics The anatomy
FIBROUS CAPSULES CORTICAL SUBSTANCE RENAL PAPILLAE MEDULLARY SUBSTANCE CALYX RENAL PELVIS
URETER
URINES
The function
KIDNEY 25% OF THE TOTAL AMOUNT OF BLOOD EJECT FROM THE LEFT VENTRICLE AT EACH CARDIAC CYCLE IS DISTRIBUTED THROUGH THE ARTERIES TO THE KIDNEY FILTRATION
EACH KIDNEY CONTAINS ABOUT ONE MILLION MICROSCOPIC UNITS – NEURONS – THAT FORM URINE
IN RENAL CORPUSCLE THE FORMATION OF URINE BEGINS WITH THE BLOOD FILTRATION
EACH AFFERENT ARTERIOLE LEADS TO A BALL OF CAPILLARIES – THE GLOMERULUS – AROUND THIS BALL IS THE CLOSED END – CAPSULE OF BOWMAN
OF A LONG AND TORTUOUS TUBULE COMPOSED OF VARIOUS PARTS CAPSULE OF FIBROUS TISSUE
FIRST CONVOLUTED TUBULE SECOND CONVOLUTED TUBULE
ARTERY AND RENAL VEIN
PY
PELVIS
ID RAM
DESCEND AND ASCENDENT BRANCHES AND OF HENLE’S LOOP CALYX
Electrophoresis
LARGE AFFERENT ARTERY
THE BLOOD THAT LEAVES THE CAPILLARY BALL FLOW EFFERENT ARTERIOLE WHICH DIVIDES TO FORM A 2° NETWORK – CAPILLARY – AROUND THE TUBULES OF THE NEPHRON OWN. FINALLY THESE CAPILLARIES CONVERGE INTO A VEIN
NARROW EFFERENT ARTERY
EVERY MINUTE ABOUT 1200ML OF BLOOD ENTERS THE GLOMERULAR CAPILLARY SKEIN CONTAINING 540ML CELLS 660 ML PLASMA
EVERY MINUTE 1080ML OF CONCENTRATED BLOOD COME OUT 540ML CELLS 540 ML PLASMA
THIS BLOOD IS UNDER PRESSURE
(ALMOST TWICE OF OTHER SYSTEMATIC CAPILLARIES)
THIS PRESSURE TENDS TO BRING OUT WATER AND SALTS THROUGH THE VESSEL WALL
IT IS OPPOSED 1) TO THE PLASMA PROTEIN OSMOTIC TENSION THAT TENDS TO ATTRACT WATER ABD SALTS IN THE VESSELS AND 2) THE BACK PRESSURE IN THE CAPSULE OF BOWMAN
IN TUBULES IN THE FORMATION OF URINE IS COMPLETE BY REABSORPTION ESSENTIAL SUBSTANCES IN THE BLOOD STREAM OF ESSENTIAL SUBSTANCES COMING FROM THE FILTRATE AND SECRETION AND SYNTHESIS OF OTHER WASTE SUBSTANCES.
RENAL CORPUSCLE
THIS GIVES RISE TO AN EFFECTIVE FILTRATION OR A PUSHING FORCE OF
THE FIGURES REPRESENT THE VOLUME OF BLOOD THAT ENTERS AND LEAVES THE GLOMERULI OF TWO KIDNEYS IN HUMANS
TUBULAR COLLECTORS EMPTYING THE URINE FORMED IN THE PELVIS
URETER
290
ABOUT 120 ML PER MINUTE ARE FILTRED: PLASMA, SALTS. GLUCOSE AND OTHER SMALL MOLECULES ARE FILTRED: CELLS AND PLASMA PROTEINS ARE TOO LARGE TO PASS THROUGH THE CAPILLARY MEMBRANES AND CAPSULES IN RENAL TUBE.
100-150 LITERS OF FILTRATE DILUTED IN THIS WAY EVERY DAY FROM THE GLOMERULI OF TWO KIDNEYS. THIS FILTRATE CONTAINS GLUCOSE, SALT, UREA, URIC ACID, POTASSIUM, PHOSPHATES, SULFATES, ETC. IN THE SAME PROPORTION OF THE BLOOD PLASMA.
The glomerular function THE RENAL ARTERY DIVIDES INTO INTERLOBULAR ARTERIES SPLIT IN ARCUATE ARTERIES
Four main factors intervene in the determination of the glomerular filtration of the proteins: 1) The selective permeability: molecular weight and form
GIVE RISE TO STRAIGHT ARTERIES
2) The presence of anionic structure with the function of electrostatic repulsion 3) The plasmatic concentration
FROM WHICH DERIVE THE AFFERENT ARTERIOLES
4) The local hemodynamic conditions Reynolds Number = d . v . ρ / η
EACH AFFERENT ARTERIOLE IS DIVIDED INTO ABOUT 50 CAPILLARIES
ABOUT 120 ML/MIN ARE FILTERED
The physiological hydrodynamic The nephron consists of two parts: the glomerule and the tubule
WATER AND SALTS TEND TO LEAVE BUT ARE OPPOSED TO THE BACK PRESSURE OF THE PROTEINS AND THE CAPSULE
GLOMERULE
cells
cells
TUBE
The tubular function The mechanisms of re-absorption: All proteins with a molecular weight less that 40.00 D do not encounter, almost, difficulty in being filtered at the glomerular level and they are over 90% reabsorbed in the proximal tubule.
Electrophoresis
291
The purpose of study of the urinary electrophoretic proteinic block 1) M.C. research 2) “Semi-quantitative” observations of the different zones to frame the renal damage and classify the proteinuria and to address level II analysis. The pre-analytical phase: the container and its importance Experimental plan Collection of 15 urines (sodium azide 0.1%) with micro-molecular proteinuria Dosage K and L for nephelometric path Values between 23 and 765 mg/L. Urine mixed and divided into two parts. One part in glass and the other in plastic not for food. Storage time 24 hours. The results
TUBULAR MECHANISMS OF ABSORPTION OF PEPTIDES AND PROTEINS PEPTIDASE TUBULAR LUMEN
PEPTIDES
PROTEINS BRUSH BORDER ABSORPTION
APICAL VESICLE VACUOLE
PROXIMAL TUBULAR CELL
DEFECATION
AMINO ACIDS
CARRIER
TELOLYSOSOME
PRIMARY LYSOSOME METABOLISM
BASOLATERAL MEMBRANE CLEAVAGE PRODUCTS
While the peptides are split by enzymes of the orletto brush to amino acids which then can be absorbed through Na+- carrier dependents, the proteins are absorbed in the lumen of the vesicles and split in the heterolysosomes through fusion with primary lysosomes. The products of splitting are returned to the organism through the basolateral membrane. The residuals that cannot be digested are secreted in the lumen for exocytosis, with simultaneous secretion of lysosomial enzymes.
IF BJ positive
IF BJ negative
Glass
15
0
Plastic
13
2
Urine Nephelometric Evaluation stored in glass mg/L
Urine Nephelometric Evaluation stored in plastic mg/L
Urine neg. 1
37
About 5
Urine neg. 2
39
About 2
The metabolism and catabolism of the tubule: TUBULE
PERITUBULAR CAPILLARY
GLUCOSE
GLUCOSE
NaC1
NaC1
AMINOACIDS
AMINOACIDS
C VITAMIN
C VITAMIN
TUBULE
PERITUBULAR CAPILLARY
UREA, PHOSPHATE
UREA, PHOSPHATE
TUBULE
PERITUBULAR CAPILLARY
The pre-analytical phase: the collection of the urine TUBULE
For the research and classification of the proteinuria:
PERITUBULAR CAPILLARY
collect the second urination in the morning (G.P. Merlini)
CREATININE SULPHATES
UREA, PHOSPHATE UREA, PHOSPHATE
In confirmation of what was said, a study was conducted on carriers of proteinuria plasma by BJ over-flow, in order to assess the maximum excretion of free light monoclonal chains in urine collected with the timing:
URIC, URIC URIC
Electrophoresis
URIC
292
Clinical aspects From the semeiological viewpoint, the proteinuria may be: • Transitory • Intermittent • Persistent Transitory Proteinuria
COLLECTION INTERVALS: HOURS
Consists of the exhibition of occasional proteinuria with pathological values, usually moderate, that do not repeat over time in subsequent checks and has a benign type character.
Ciapini, M. Tani, B. Milanesi The Clinical Pathologist 3/2006
For the research, classification and evaluation of the excreted proteinic mass, collect the urines of the 24/hours with sodium azide 0.1%: Long term outcome of MGUS: R. A. Kyle and S. V. Rajkumar British Journal of Haematology, 134, 573589 2006 “A 24h urine specimen should be collected so that the amount of M protein can be measured. This provides a measure of the patient’s tumour mass and is useful in monitoring the course of the disease. “
With the end of functional proteinuria, meaning the proteinuria that, independently from pathological kidney states, accompanies several clinical conditions characterized by states of fever, to intense and prolonged exercise, to conditions of physical and emotional stress such as surgical interventions or the exposure to cold. This proteinuria is defined as transitory type and resolves together at the recovery of the physical condition that, altering it, was not the origin. Intermittent Proteinurias These proteinurias are characterized, as their definition indicates, by the appearance of pathological proteins over time intervals and in episodes in which the proteinuria re-enters into the range of physiological values.
EFFLUENT BLOOD FROM THE KIDNEY
These proteinurias may have completely random and irregular courses, or show a regular recurrence such as in orthostatic type proteinuria, in addition to normal values in the samples taken in the morning, following a period of supine rest, which presents pathological values in serotin samples, after the common orthostatic activity.
PROXIMAL
EFFLUENT BLOOD FROM THE GLOMERULUS
AND DISTAL TUBULES REABSORB
The intermittent proteinurias are of glomerular type and, although usually quantitatively small, they deserve attention because they can be indexes of incipient renal suffering.
DISCARDED MATERIALS ARE ELIMINATED WITH THE URINE EXCRETORY DUCTS
Electrophoresis
293
Study methods of the proteinurias
Data la entità spesso moderata di queste proteinurie torna utile dire e fare presGiven the often moderate entity of these proteinurias, it is useful to say and to make present the need for a good analytical quality of determination methods, for both the screening phase as well as the dosage, especially regarding the analytical sensitivity and the limitation of imprecision, in order to avoid false positive due to excessive analytical variability. The verification of finding an intermittent proteinuria requires the doctor to deepen the investigations in order to be able to resolve the potential doubt of a renal lesion that, as we have said, may be the cause. The typology of the intermittent proteinuria is often a prerogative of the child and adolescent age where it is fundamental to exclude the possibility of silent streptococcus infections. Persistent Proteinurias The persistent proteinurias can be distinguished in the pre-renal, renal and post-renal forms. The pre-renal proteinurias are the proteinurias from “overflow”, which are not the expression of renal disease, but proteins physiologically filtered by glomerule that are present in an increased concentration in the plasma for various pathological contingencies, exceeding the normal threshold of tubular re-absorption. Paradigmatic expression of this type of proteinuria and the Bence Jones proteinuria. The renal proteinurias are those already described in the etiopathogenetic classification, or: glomerulary, tubulary and mixed. The post-renal proteinurias are due to lesions, which often refer to an inflammatory nature, of the tissues of the kidney pathways such as: renal pelvis, ureter, bladder, prostate and genital organs. Sometimes these proteinurias have a composition which allows the identification of the presence of specific proteins such as the presence of enzymes or the Tamm-Horsfal protein.
Electrophoresis
The study methods of the proteinurias can be distinguished in quantitative (Table 3) and qualitative (Table 4) methods. Table 3: QUANTITATIVE METHODS
Table 4: QUALITATIVE METHODS
Dosage of the total proteinuria
Electrophore: On cellulose acetate
Dosage of single specific proteins:
On agarose gel
Albumin
On polyacrylamide gel
Transferrin
On polyacrylamide gel SDS (SDS PAGE)
IgG immunoglobulins
On Proteinuria SDS Hydragel agarose
Beta 2 microglobulin
Bi-dimensional electrophoresis
Alpha 1 microglobulin
Capillary
Protein binding the retinol
Chromatography: On Sephadex column
Determination of the CLEARANCES proteinic
HPLC
Enzymuria:
Immunofixation
Alanina aminopeptidasi ( AAP ) N Acetil glucosaminidasi ( NAG )
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If the technique used is electrophoretic with ample resolution power,
Technical aspects Determination methods of the total proteinuria
an increase in mass and sensitive dye, it is possible to overcome the pitfall
The more reliable techniques are those of Bradford\SDS and Pyrogallol.
of highlighting micro-proteins that in addition to being present in modest
The detection stripes for urine allow, in an approximate way, to follow the loss trend of albumin and do not give satisfactory results in the monitoring of other proteins; in the end, they are linked closely to the interpretation by the provider.
quantities have, also, the prerogative to migrate under those having greater molecular weight, especially in mixed type proteinurias; in these methodological situations, it is possible to have frameworks of proteinurias such as those in fig. 1 [28]:
As far as research of free light chains and micro-proteins, both nephelometric techniques as well as those mentioned demonstrate their limits, whether for the low molecular weights at stake, whether for low concentrations of the levels of normality (micro-proteins), and finally whether for the difficulty to correlate the responses of the calibrators on the proteins sought.
Figure 1 PREALBUMIN
Type arrangement of proteinuria: Qualitative Methods ALBUMIN
The technique most often used is electrophoresis. The true parameter limiting this technique is represented by the degree of sensitivity needed to reached.
ALPHA 1 ANTITRYPSIN
There are two possible solutions to the problem:
ALPHA 2 MACROGLOBULIN
Increase of the proteinic molecular mass by means of:
RBP
1) Concentration 2) Multiple deposits with reduction of the resolution
TRANSFERRIN
3) Immunofixative techniques
Î&#x2019;2 MICROGROBLUIN
Increase of the sensitivity of the detection marker by means of: 1) High sensitivity dyes
FREE K OR L
2) immunoenzymatic systems The level of attainable sensitivity with these systems varies between 1 and 15 mg\l. The recommended techniques are electrophoretic with ample resolution power, that in polyacrylamide in gradient of concentration and that being in agarose with pre-treatment of the sample with SDS.
Electrophoresis
POLICLONAL IG CISTATIN C LYSOZYME
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The use of agarose SDS, in which the separation is the only prerogative of the protein PM, allows an easy and obvious interpretation: fig. 2/3/4 Figure 2 Figure 3 Tubular proteins β2 MICRO LYSOZYME RBP MONOMERS DIMERS
ALPHA 1 MICRO ALBUMIN
Electrophoresis
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Figure 4
Table 5
Glomerular proteins
1) Proteinurias of plasmatic derivation a) Renal Glomerular b) Renal Tubular c) Pre-renal
ALBUMIN TRANSFERRIN
2) Proteinurias of tissular derivation a) Nephrogenetic
3) Post-renal proteinurias
PHENOTYPES ALPHA 2 MACRO
A first distinction is based on the origin of the proteins found at the urinary level and provides for three large categories: The only drawback in the use of this method of protein separation is linked to its inability to distinguish between monoclonal and polyclonal proteins; this is because the proteins of the same species have the same molecular weight and migrate in a homogeneous band.
Figure 5 NORMAL
TUBULAR DAMAGE
PLASMA CONCENTRATION
The sensitivity of the method normally reaches 5 mg/L.
FILTRATION LOAD
Pathogenetic classification of the proteinurias The complexity of the mechanisms underlying the determinism of proteinuria is reflected in the complexity of the interpretative diagnostic process of the proteinuric frameworks found in clinical practice.
RESORPTION
On the basis of the probable origin of urinary proteins, various classifications have been proposed among which we show that proposed by Boylan: table 5
Electrophoresis
GLOMERURAL DAMAGE
DAILY ELIMINATION mg AS AN EXAMPLE TWO PROTEIC SPECIES WERE CONSIDERED: ALBUMIN (STRIPED AREAS OF THE FIGURE) AND BETA 2 MICROGLOBULIN (WHITE AREAS OF THE FIGURES)
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Figure 6
In a more exhaustive way we propose another pathogenetic classification of the proteinurias: Table 6 and fig. 6/7: PATHOGENETIC CLASSIFICATION OF THE PROTEINURIAS 1) PLASMATIC PROTEINURIAS a) Renal Glomerular
TYPE
Selective glomerular proteinuria
Glomerular proteinuria non selective
CAUSE
Alterations of basal membrane load for loss of negative charges
Alterations of the glomerular barrier structure for an increase and/or enlargement of the pores
PROTEINIC CLASSES
Albumin Transferrin
Albumin Transferrin Immunoglobulins
MOLECULAR WEIGHTS
68.000 \ 77.000 D
68.000 \ 500.000 D
EXAMPLES OF CORRELATED PATHOLOGIES
Minimal change Nephropathy Focal glomerular sclerosis Glomerule membranous
Proliferative Glomerulonephritis Advanced diabetes Amyloidosis
Electrophoresis
LYSOZYME
β2 MICRO
RBP MONOMERS DIMERS
ALPHA 1 MICRO
ALBUMIN
PHENOTYPES ALPHA 2 MACRO
Sample 7 = physiological proteinuria with presence of albumin and transferrin Sample 6 = selective glomerular proteinuria with presence of increased albumin nd transferrin
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SIXTH ANNEX The Bence Jones protein
CONNECTING TUBULE
DISTAL CONVOLUTED TUBULE PROXIMALE CONVOLUTED TUBULE
CORTICAL NEPHR 85%
RENAL CORPUSCLE WITH THE BOWMAN’S CAPSULE AND THE GLOMERULUS
JUXTAMIDOLLARY NEPHR 15% CORTICAL CORTICAL COLLECTOR DUCT
MORPHOLOGICAL DIFFERENCES POSITION GLOMERULI SIZE HENLE LOOP LENGHT FUNCTIONAL DIFFEENCES GLOMERULAR FILTRATION RATE TUBULAR TRANSPORT CONTENT OF RENIN
PROXIMAL TUBULE STRAIGHT DISTAL TUBULE STRAIGHT
OUTER MEDULLARY
THIN DESCENDING TRACT THIN ASCENDING TRACT
a) Its relation with renal functionality b) The SIBioC guidelines c) The BJ and the contrastograph
INNER MEDULLARY
The role of the Cortical nephrons and Iuxtamidollari
Electrophoresis
OUTER MEDULLARY COLLECTOR DUCT
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INNER MEDULLARY COLLECTOR DUCT
The Bence Jones proteinuria: guidelines and contrastographs
In the following two months, the patient became emaciated, weak, and was tormented by the pain. He died on January 1, 1846, in full possession of his mental faculties. Dr. MacIntyre successively published (1850), post-mortem of the patient, both the examination as well as the description of the urine sent to Dr. Bence. Unfortunately for him, Henry Bence Jones had already published the results of the urinary tests of the patient in two articles of sole authorship, one of which was in The Lancet, in 1847. He considered the protein to be a “deutoxide hydrated of albumin”. His comment follows: “I do not need to comment on the importance of seeking for this oxide of egg albumin in other cases of mollities ossium”. The reputation of Bence Jones was guaranteed to posterity, while the contributions of colleagues were delivered to the footnotes of history. For all it may seem an injustice in relation to his colleague, William MacIntyre, but Henry Bence Jones accomplished much more in his career. He published another 40 articles and became wealthy and famous for his studies on his clinical practice, didactics and original observations; he received a scholarship from the Royal Society, at the tender age of 33 years. Florence Nightingale, once described him as “the best chemist doctor in London.” Surprisingly, there was no mention of the Bence Jones protein in his obituary and the eponymous (and the hyphen in his name) was not used until his death.
History: Even if FLC “immunoglobulins” are synonymous with proteins of Bence Jones, history could be more generous with the other researchers involved in their discovery. “Last Friday, October 1845, doctor William MacIntyre, doctor at the Western St. Marylebone Hospital, Marylebone, London, left his room on Harley Street. He was called to see Mr. Thomas Alexander McBean, 45 years old, a fruit vendor with all respect, that had strong bone pain and fractures. He was placed under the care of his general practitioner, Doctor Thomas Watson, for several months. After an examination of the patient, William MacIntyre observed the presence of edema. Considering the possibility of nephrosis, he tested the urine for its albumin content. With great wonder he checked that when heating the urine, the albuminous protein precipitated and dissolved again at 75 °C“ Both Dr. MacIntyre and Dr. Watson sent the urine samples to the chemical pathologist at the St. George Hospital, to the attention of Dr. Bence. A note from Dr. Watson accompanied the urine sent: .. “Dear Dr. Bence Jones, The test tube contains urine with very high specific weight: when boiled it becomes very opaque in addition of nitric acid, it is effervescent, takes on a reddish colour, and becomes quite clear, but when it cools it takes on the texture and appearance as seen. Heating re-liquefies. What is it ? “
Electrophoresis
382
Definition: The Bence Jones protein is a protein belonging to the class of globulins. Its molecular weight is 20 kDa [7] [8] [28] [91] [92] [93]. It is in fact the light chain of an antibody, detached from the heavy chain. The light chains called “Bence Jones protein” are different from the“usual” light chains that the B-lymphocytes of the body normally produce: • the Bence Jones are free and monoclonal, while the normal light chains are linked to the heavy chain corresponding to forming the antibody. • the Bence Jones are monoclonal, i.e. all genetically equal, while the normal light chains are polyclonal, that is to say, all with the subtle differences between them. LThe Bence Jones protein can be detected in the blood, in the urine or in both biological liquids of the same patient:
Electrophoresis
Several rare diseases may have Bence Jones protein production as a symptom; among these must be mentioned the macroglobulinemia of Wal-
383
2) Tubular re-absorption
denström; among the others, there are proteins of Bence Jones in blood or urine of patients with multiple myeloma, renal impairment, anemia. The Bence Jones can be produced by neoplastic plasma cells. They can be of the kappa or lambda type. The light chains can be both immunoglobulinic fragments and homogeneous immunoglobulins. They can be found in the urine by their small dimensions, following a simple renal clearance. In other words, the Bence Jones protein can be defined as Monoclonal Components, or “Clones of Lymphocyte B in expansion”
3) Tubular secretion EFFERENT ARTERIOLE
PERITUBULAR CAPILLARY DISTAL TUBULE
GLOMERULUS
AFFERENT ARTERIOLE CAPSULE OF BOWMAN
FILTRATION REABSORBTION SECRETION EXCRETION
LOOP OF HENLE TO THE RENAL VEIN
COLLECTOR DUCT
PERITUBULAR CAPILLARY
EFFERENT ARTERIOLE
TO THE RENAL VEIN
GLOMERULUS AFFERENT ARTERIOLE CAPSULE OF BOWMAN
The quantity of any substance present in the urine (excreted load) is the result of the following algorithm:
Crystal of the Bence Jones protein in X-ray Crystallography The kidney and the Bence Jones protein:
EXCRETED LOAD
One of the primary functions of the kidneys is to remove unnecessary substances from the blood by passing them into the urine, and to retain the necessary substances.
FILTERED LOAD
SECRETED LOAD
Now if we consider the molecular weight of a Bence Jones Protein, we
Formation of the urine derives from three processes: 1) Glomerular filtration
Electrophoresis
REABSORBED LOAD
must consider that the glomerule
384
The water is reabsorbed by osmosis. Active transport:
DISTAL CONVOLUTED TUBULE
MACULA DENSA
Primary - if coupled directly to an energy source (hydrolysis of ATP) pump
EFFERENT ARTERIOLE
JUXTAGLOMERULAR CELLS AFFERENT ARTERIOLA
EXTRAGLOMERULAR MESANGIUM VASCULAR POLE
ATPasi Na + / K+. Secondary = if coupled indirectly to an energy source.
FENESTRATED ENDOTHELIUM
PODOCYTES
FILTRATION TUBULAR CELLS
PERITUBULAR CAPILLARY
GLOMERULAR LOOPS
LUMEN
TO THE RENAL VEIN
PATERIAL SHEET OF THE GLOMERULAR CAPSULE
PARACELLULAR WAY TRANSCELLULAR WAY
UERINAY POLE
ACTIVE TRANSPORT DIFFUSION BLOOD
PROXIMAL CONVOLUTED TUBULE
is not capable to stop it because it is normal to find the presence of Albumin in the urine (P.M. 55,000 D), in slight quantities. At this point the tubular function intervenes:
SOLUTES
OSMOSIS
EXCRETION
RESORPTION
EFFERENT ARTERIOLE TO THE RENAL VEIN
PERITUBULAR CAPILLARY
RESORPTION Na+ (PRIMARY ACTIVE TRANSPORT) TUBULAR CELLS
TUBULE
FILTERED AMOUNT
CAPSULE OF BOWMAN GLOMERULUS
REASBSORBED AMOUNT
SECRETED AMOUNT
LUMEN TO THE BLADDER AND TO THE EXTERNAL ENVIROMENT
EXCRETED SOLUTE AMOUNT
BLOOD
AFFERENT ARTERIOLE
that provides for the re-absorbtion of the solutes, which it subdivides into active and passive mechanisms:
Electrophoresis
FILTRATION
385
OSMOSIS INTERSTITIAL FLUID BASAL MEMBRANE
1) Na+ ENTERS ALONG ITS ELECTROCHEMICAL GRADIENT 2) Na+ IS CATIVELY PUMPED THROUGH THE BASO-LATERAL MEMBRANE OF THE Na+ K+ PUMP