1. Diagnosis of Paraprotein Diseases CLS 404 Immunology Protein Abnormalities

2. Objectives Discuss the use of the following laboratory tests in the diagnosis of paraprotein diseases:  Protein level determinations  Immunoglobulin level determinations  Electrophoresis  Bone marrow differential

3. Objectives Distinguish paraprotein diseases from these non-paraprotein conditions:  Acute inflammation  Nephrosis  Cirrhosis  Infection

4. Protein Measurements Total serum protein  Elevated levels  May be detected before the patient exhibits symptoms  Also found in non-paraprotein diseases Additional tests required to distinguish between diseases

5. Immunoglobulin Levels Serum immunoglobulin levels  Detects increased quantities of a specific immunoglobulin class  Patient’s serum is mixed with antibodies to IgG, IgM or IgA and the formation of antigen-antibody complexes is measured  Cannot distinguish between monoclonal and polyclonal increase Serum protein electrophoresis  Demonstrates the monoclonal immunoglobulin (M protein)

6. Serum Protein Electrophoresis Abbreviated SPE Separation of proteins according to size and electrical charge Anode Patient serum Cathode (+ electrode) (- electrode) Application point

7. Protein Fractions IgG, IgA, IgM, IgD, IgE and C-reactive protein Transferrin, Complement, beta- Lipoprotein alpha-2-macroglobulin, Haptoglobin, Ceruloplasmin alpha-1-antitrypsin, alpha-1-glycoprotein, alpha-1-lipoprotein

8. Serum Protein Electrophoresis Will differentiate a monoclonal gammopathy from other causes of increased protein levels Will not detect an increase in light chains, as these are cleared from circulation too quickly  See slide on Bence Jones proteins

9. Electrophoresis Pattern of Normal Individual anode cathode

10. Electrophoresis Pattern of Monoclonal Gammopathy M protein spike Note the percentage of the gamma globulin fraction has doubled from the norm.

11. Electrophoresis Pattern of Polyclonal Gammopathy Polyclonal gammopathy is typically seen in infections. Note the % of the gamma globulin fraction is similar to that seen in monoclonal gammopathy, but the band is wider, reflecting the diversity of antibodies produced.

12. Electrophoresis Pattern of Acute Inflammation

13. Electrophoresis Pattern of Cirrhosis The pattern in cirrhosis shows a “bridging” of the beta and gamma globulin fractions.

14. Electrophoresis Pattern of Nephrosis

15. Immunoelectrophoresis (IEP) Semi-quantitative test for determining specific heavy chain and light chain components in a monoclonal gammopathy First, serum or urine proteins are separated by electrophoresis, usually on an agarose gel. Antibodies specific for heavy and light chains are added to the gel. The antibodies diffuse through the gel.

16. IEP If the antibody encounters its specific Ig chain, a precipitate forms. The gel is stained in order to visualize the precipitates. The amount of Ig present is indicated by the thickness and shape of the precipitate.

17. Immunofixation Electrophoresis Abbreviated IFE  More sensitive than IEP  More expensive than IEP As in IEP, the proteins are separated by electrophoresis. Ig specific antibody is applied directly to the gel, rather than relying on diffusion. The gel is stained to reveal antigen-antibody complexes. Dark bands form when monoclonal antibodies are present; light diffuse bands indicate polyclonal antibodies.

18. IFE –Example of an IgG monoclonal antibody with kappa light chains = Serum application point Anti-Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda Total protein

19. Bence Jones Proteins Light Ig chains found in the urine of multiple myeloma patients NOT detected by routine urine dipstick test Heat Precipitation – non-specific test  Bence Jones proteins remain in solution at room temperature  Precipitate out of solution at 56 o C  Dissolve again at 100 o C IEP and IFE – specific test for identification of particular light chain

20. Stains Light chain deposits in tissue, as seen in amyloidosis, can be detected by stains:  Congo red show these as apple green fibers under a polarizing microscope  Antibodies to the light chain tagged with fluorescent dyes or other chemicals

21. Immunofluorescence Tissue that is suspected of having light chain deposits (pink dots in the demonstration) is fixed to a slide. Fluorescently labeled antibody specific for kappa or lambda light chain is added to the slide. Antibody combines with antigen, and fluorescence can be detected microscopically.

22. Bone Marrow Differential Used to confirm a diagnosis of Multiple Myeloma, Waldenström's macroglobulinemia, or MGUS. An aspirate of bone marrow is obtained via a large bore needle inserted into the iliac crest of the hip.

23. Bone Marrow Differential Normal marrow typically shows less than 5% plasma cells. In Multiple Myeloma, plasma cells will increase to 10% - 30% or more of all marrow cells. In MGUS, plasma cells comprise less than 10% of marrow constituents.

24. Plasma Cells in Bone Marrow of Multiple Myeloma Patient The differential on this marrow revealed that over 80% of the cells in the marrow were plasma cells.

25. Plasma Cells in Bone Marrow of Multiple Myeloma Patient This magnification of the previous slide shows abnormal and very immature plasma cells (prominent nucleoli at arrows)

26. Peripheral Blood Plasma cells may be seen in Multiple Myeloma Plasmacytoid lymphocytes may be seen in Waldenström's Macroglobulinemia Red cells may exhibit a “stack of coins” appearance.  Called rouleaux  Caused by excess of serum proteins

27. Radiology X-rays demonstrate lesions in bones throughout the body

28. Diagnosis of paraprotein disease includes: Detection of high protein levels Confirming the high protein is due to gamma globulins Determining the presence of the M protein & classifying the Ig present through electrophoresis Bone marrow biopsy to verify abnormal numbers of plasma cells X-rays to visualize lytic bone lesions

29. This completes the presentation on the diagnosis of paraprotein disease. You are ready for the self assessment quiz!