diag.of gammop Dr. A Sarrafnejad PhD Dep. Immunology School of public health TUMS

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2 laboratory diag.of gammop Dr. A Sarrafnejad PhD Dep. Immunology School of public health TUMS Sarrafnejad@tums.ac.ir

3 Arne Wilhelm Kaurin Tiselius Nobel Prize for Chemistry (1948)

4 Electrophoresis ٤ After 20 min, the current is turned off the proteins stained to make them visible The separated proteins appear as distinct bands. serum albumin moves closest to the positive electrode, why? Dr Gihan Gawish

5 + Fraction identification α-1 acidglycoprotein α-1 antitrypsin Haptoglobin TBG, Transcortin α-2 macroglobulin Ceruloplasmin Gammaglobulins C3 complement B Lipoprotein Hemopexin Transferrin

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7 Bis albuminemia Causes: genetic, lipoproteins, biliary pigments, antibiotics, products of con

8 Congenital deficiency of A1AT AT A1AT = 0.17g/L (normal range: g/l) Alb α1 α2 β1 β2 γ

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14 Monoclonal peak in Gamma Alb α1 α2 β1 β2 γ

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16 Oligoclonal pattern Alb α1 α2 β1 β2 γ

17 Monoclonal peak in beta Alb α1 α2 β1 β2 γ

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19 κ λ

20 + Capillary Za\ Electrophoresis in Minicap/Capillarys Anode + DETECTION OF PROTEINS EOF Protein migration INJECTION OF SERUM Electro migration Positive charges of the buffer solution Negative charges of capillarywall The Electro-Osmotic Flow (EOF) isa stronger force than the Electrical Field. As a result, all proteins are carriedtowards the cathodic end of the capillary.

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22 Electrophoretic System in + Minicap/Capillarys p y p p Capillarys Electrophoresis Principle Thermic bridge Temperature Controlled by Peltier device Detector Migration Deuterium lamp Capillary in thermo- conductive resin Cathode - High Voltage Anode +

23 INJECTION OF SAMPLE Current Analytical Chemistry Current Analytical Chemistry. 2005, 1

24 Clinical Manifestations Plasma Cell proliferation Pancytopenia, bone damage, constitutional symptoms, anorexia, cachexia, hypercalcemia Monoclonal protein production Renal failure, hyperviscosity, amyloidosis, hypoalbuminemia, neurologic symptoms Immunodeficiency i Infection, autoimmune phenomena

25 Lytic Bone Lesions in Myeloma Important for diagnosis Treatment of impending fracture

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27 What is important in this graph?

28 Abnormal peak in gamma The peak disappears in Ig G The peak disappears in Kappa Conclusion: Detection of monoclonal Ig G Kappa

29 IgA lambda

30 IgG lambda + free light chains lambda

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38 Protein Total = 14 mg/dl Urine Volume = 700 ml Protein.24hrs = 98 mg/dl

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40 Protein Total = 12 mg/dl Urine Volume = 1600 ml Protein.24hrs = 192 mg/dl

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42 Plasma Cells travel back to bone marrow Memory B cell Activated B cell Plasma Cell

43 Properties of Plasma Cells Proliferate Secrete Immunoglobulins Make space Ifl Influence bone turnover Secrete Inflammatory mediators

44 Initial Diagnostic Workup H&P CBC BUN/creat, lytes Calcium/albumin Quant Ig SPEP/immunofix Bone Marrow Biopsy 24-hour urine UPEP/immunofix Beta2-microglobulin Skeletal survey

45 MULTIPLE MYELOMA Hematologic findings at presentation Mayo Clin Proc 2003;78:21 Hgb < 12 73% Hgb < 8 7% ESR > 20 84% ESR > % WBC < % WBC < % Plts < 100K 5% Plts > 500K 2%

46 Multiple Myeloma: Incidence and Epidemiology i 1% of all malignancies 10% of hematological malignancies (2 nd most common) 3-4 per 100,000 population 16,000 new cases/yr; 11,000 deaths/yr Median age: 65 yo; 3%<40 yo M>F Risk: radiation exposure

47 Etiology Familial clustering African Americans Radiation Agriculture, Benzene, Radiation, i Sheet metal work Chronic inflammatory disorders

48 Multiple Myeloma: Indication for Treatment: CRAB Calcium (Hypercalcemia) Renal insufficiency Anemia Bone lesions

49 Non secretory myeloma no secretion of protein (relatively rare if serum and urine carefully studied for presence of M- protein) prognosis same as myeloma or longer

50 Amyloidosis Amyloid: homogenous, amorphous extracellular material y g, p with fibrillar structure; made of low MW proteins that precipitate in tissues

51 Figure I 123 labelled serum amyloid P scans in a 52-year-old woman, viewed g y y, posteriorly. Reduction of AL deposits in the liver and spleen after one year of chemotherapy can be seen. Serum κ FLCs reduced from 551mg/L to 52mg/L over the same period. (Courtesy of PN Hawkins).

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53 Figure 2.1. Henry Bence Jones (Courtesy of RA Kyle and The British Journal of Haematology.

54 Figure 6.1. Sensitivity of assays for serum FLC quantitation with error bars indicating the different claimed limits.

55 Table 5.2. Median values and ranges for free and total light chain concentrations and κ/λ ratios in the sera of 282 normal individuals.

56 LABORATORY DIAGNOSIS OF MULTIPLE MYELOMA Protein electrophoresis Screening Serum (SPEP) and Urine (UPEP) [random or 24 hour specimen) Confirmation Immunofixation Electrophoresis (IFE) Free light chains (FLC) with ratio Serum by nephelometry or turbidimetry Histopathology of bone marrow aspiration or biopsy Percent plasma cells

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58 Plasma cell leukemia loss of adhesion molecules that localize in marrow (CD56, VLA-5, MPC-1) greater than 20% %plasma cells in blood and > 2000/ul more frequent: younger age, hepatosplenomegaly, lymphadenopathy, few lytic lesions, small M-protein, poor prognosis

59 Non-myeloablative SCT Immuno suppression only Stem cells Manipulate the Immune response to Manipulate the Immune response to maximize Graft vs. Disease

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61 Light Chain 1% WS 1% M.M 43% MGUS 55% MGUS 689 M.M WS Light Chain Total

62 IgM/Lambda 2% Immunotyping Kappa Light Chain 1% Lambda Light Chain 1% IgM/Kappa 7% IgA/Lambda 7% G / K 391 IgA/Kappa 14% IgG/Kappa 47% G / L 177 A/ K 115 A / L 58 M / K M / L IgG/Lambda 21% K L.C 5 L L.C 5

63 Figure Diagnostic accuracy of different assays in AL amyloidosis. Electrophoretic test results in serum (A) and urine (B) were based on 430 patients studied at The Mayo Clinic. Serum FLCs (C) are from 262 patients at The UK National Amyloidosis Centre.

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