1. Understanding Your Blood Work
MYELOMA CANADA CONFERENCE
Understanding Your Blood Work
Donna E. Reece, M.D.
Princess Margaret Hospital
23 October 2010

2. What’s in your blood? Formed elements made in the bone marrow
Red blood cells: carry oxygen to tissues
Measured by hemoglobin, hematocrit (packed cell volume), number of RBCs
White blood cells (leukocytes):several types; fight infection
Neutrophils  first line of defense
Lymphocytes  invovled in more complicated immune reactions
Platelets: clot the blood

3. Complete Blood Count (CBC) Determination
Courtesy of Dr. A. Xenocostas

4. Normal Blood Counts Normal Hgb (Hemoglobin) Normal WBC (Leukocytes)
Men
Women
Normal WBC (Leukocytes)
Total
Differential
Neutrophils (Polys, ANC)
Lymphocytes
Eosinophils
Monocytes
Basophils
Normal platelets
Fatigue possible if <
Transfusion if < 80
Risk of infection if neutrophils <1.0
Risk of bleeding if <10

5. Example of CBC Results

6. Manual White Blood Cell Differential
Courtesy of Dr. A. Xenocostas

7. Normal Peripheral Blood Film
Lazarchick, J. ASH Image Bank 2001;2001:100185

8. Example of CBC Results

9. What’s in your blood? Sugars (glucose) Fats
Minerals (potassium, sodium, calcium, zinc)
Breakdown products of metabolism (carbon dioxide, other cellular wastes)
Creatinine
Uric acid

10. What’s in your blood? Proteins
Carrier proteins (for hormones, nutrients, vitamins)
Albumin: good protein made by the liver
Beta 2 microglobulin
Enzymes
Antibodies/immunoglobulins
Fight invaders like germs by adhering to them and neutralizing them (very specific)

11. Automatic Chemistry Analyzer
Courtesy of Dr. A. Xenocostas

12. Bone Marrow Aspirate
Courtesy of Dr. A. Xenocostas

13. Bone Marrow Aspirate
Courtesy of Dr. A. Xenocostas

14. Bone Marrow Findings in Myeloma Bone Marrow Aspirate
Normal bone marrow makes all types of blood cells (RBCs, WBCs and platelets)
Normal bone marrow has less than 5 % plasma cells
Plasma cells make antibodies
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.

15. Bone Marrow Aspirate in Multiple Myeloma
Lazarchick, J. ASH Image Bank 2001;2001:100185

16. Antibodies/Immunoglobulins
Plasma cells make antibodies (immunoglobulins, Igs) which consist of 2 heavy chains and 2 light chains
One type of antibody is made to bind with one foreign substance (virus, bacteria, etc.)

17. Patterns of Antibody Production

18. Free Light Chain Production
KAPPA
LAMBDA
** Cells producing Ig molecules or antibodies are plasma cells found in the BM.
** Normal plasma cell content in the BM is approximately 1% but it can rise to over 90% in MM. BM may contain 5-10% plasma cells in chronic infections and autoimmune diseases and this is associated as well with hypergammaglobulinemia and corresponding increases in polyclonal FLCs concentrations
** Ig molecules or antibodies produced by plasma cells consist of 2 identical heavy chains, and 2 identical light chains synthesized separately and being brought together prior the secretion of the intact Ig.
** There is approximately 40% excess FLC production over heavy chains synthesis to allow proper conformation of the intact Ig molecules.
** Light chains are of 2 types: Kappa et Lambda
** There are twice as many k producing plasma cells as lambda and, k FLCs are normally monomeric molecules of app. 25Kda while lambda tends to be dimeric with a MW of about 50KDa.
** Production of FLC in normal individuals reach app. 500mg/day (from BM and LN cells)
Plasma cells produce twice as many kappa as lambda light chains, but the median K/L ratio is 0.6 (meaning more lambda than kappa in the serum). This seeming paradox is explained by the slower clearance of lambda from the serum, such that its half life is somewhat longer and hence it has higher concentrations.
FLC-producing
plasma cells
FLC-producing
plasma cells

19. Types of Multiple Myeloma

20. Multiple Myeloma
Diagnosis based on finding over 10% plasma cells (antibody forming cells) in bone marrow
In most cases, these excess plasma cells make an excess of one kind of antibody (monoclonal antibody)
Symptomatic myeloma characterized by “CRAB”
Anemia (low hemoglobin*)
Bone lesions (found on xrays)
Kidney damage (elevated creatinine*)
Elevated blood calcium*
*Blood tests

21. Blood Tests in Myeloma To measure the burden/amount of myeloma
Measurements of the product of the myeloma cells
(Monoclonal antibody level in blood and/or urine)
To measure extent of myeloma damage
CBC: complete blood count (includes Hgb, WBC, platelets)
Creatinine (kidney function)
Calcium (comes from bone destruction)
To assess prognosis
Beta 2-microglobulin (high is unfavourable)
Albumin level (high is favourable)

22. Criteria for Symptomatic (Active)Myeloma
Bone marrow plasma cells > 10%
Evidence of end organ damage
Calcium > 2.8 mmol/L Hypercalcemia
Hgb < 100 g/L (or 20 g/L below normal)Anemia
Creatinine > 176 umol/L Renal dysfunction
Lytic bone lesions
Myeloma without symptoms (“smouldering myeloma) does not need treatment
The disease may smoulder for months or years without causing problems

23. Findings at Diagnosis in Symptomatic Myeloma
• Anemia or low hemoglobin: 80%
• Renal (kidney) dysfunction: 20%
• Hypercalcemia: 25%
• Bone destruction: 70%
• Hyperviscosity: <5%
Investigation of a patient with suspected myeloma should include the following:
Full blood count
Serum urea, electrolytes and creatinine
Serum calcium
Serum albumin
Serum uric acid
Electrophoresis of serum and concentrated urine followed by immunofixation to confirm and type any paraprotein present. Immunofixation is also indicated in patients in whom there is a strong suspicion of myeloma but routine electrophoresis is negative
Quantification of serum paraprotein
Quantification of urinary light chain excretion; either calculated on a random urine sample in relation to the urine creatinine or measured directly on a 24-h urine collection
Quantification of non-isotypic serum immunoglobulins
Creatinine clearance, measured or calculated
Plasma viscosity
Standard X-rays of the skeleton including lateral and antero-posterior (AP) cervical, thoracic and lumbar spine, skull, chest, pelvis, humeri and femora (radionuclide bone scanning is not usually helpful)
Magnetic Resonance Imaging (MRI) is an essential investigation for patients with suspected spinal cord compression
Computerized tomography (CT) scanning is not routinely indicated in assessment but is helpful for imaging extramedullary disease
Bone marrow aspirate
Trephine biopsy and clonality studies (e.g. light chain restriction) are helpful in selected patients
b2-microglobulin (in the presence of normal renal function), lactate dehydrogenase (LDH) and C-reactive protein are prognostic markers
A marrow sample should be sent for cytogenetic studies whenever possible. Although routine cytogenetic studies rarely provide helpful diagnostic or prognostic information, interphase cytogenetics using fluorescence in situ hybridization (FISH) analysis will identify abnormalities in most patients, the most frequent being translocations involving 14q and deletions of chromosome 13. Complete or partial deletion of chromosome 13 is a powerful adverse prognostic factor (Desikan et al, 2000; Konigsberg et al, 2000; Zojer et al, 2000; Facon et al, 2001). Cytogenetic information may therefore be useful in guiding decisions about treatment.
The diagnosis is usually confirmed by demonstration of a paraprotein in serum or urine and/or lytic lesions on X-ray, together with over 10% plasma cells in bone marrow (Greipp, 1992). Other conditions in which a paraprotein may be present include monoclonal gammopathy of
undetermined significance (MGUS), AL amyloidosis, B-cell non-Hodgkin's lymphoma (includingWaldenstrom's macroglobulinaemia), chronic lymphocytic leukaemia and connective tissue disorders. Currently accepted criteria for distinguishing myeloma from MGUS are shown in Table II. Clear distinction may require careful observation over time (3±6 months). In patients in whom the investigations fulfil the diagnostic criteria of myeloma rather than MGUS, but in whom the disease is asymptomatic and stable over the period of observation, the disease has variously been termed equivocal, indolent or smouldering myeloma (Greipp, 1992; Malpas, 1998). Distinguishing between each of these terms is of no practical value as the main issue is whether or not treatment is required. A new staging system and clinical classification of MGUS and myeloma is currently in preparation by an international working group. It is anticipated that the recommendations will be available by 2002 and will be incorporated in the first revision of these guidelines.

24. How do we measure antibodies?
Protein electrophoresis
Can be done on the blood (serum) or urine
Tells if there is a monoclonal antibody (M-spike, M protein)
Requires interpretation

25. How do we measure antibodies?
Quantitative immunoglobulins
Gives the total amounts of IgG, IgA and IgM
Concentration in g/L
Normal total IgG
Normal total IgA
Normal total IgM
Does not indicate if there is a monoclonal antibody present
Automated

26. How do we measure antibodies?
Serum free light chain assay (Freelite)
Measures free kappa and lambda light chains in the blood
Can detect tiny levels, before light chains spill into the urine

27. Serum Protein Electrophoresis
Lazarchick, J. ASH Image Bank 2001;2001:100185

28. Protein Electrophoresis--Densitometer
Courtesy of Dr. A. Xenocostas

29. Serum Protein Electrophoresis (SPEP)
Normal SPEP
Multiple Myeloma
Densitometer can measure M spike in g/L
Kyle RA and Rajkumar SV. Cecil Textbook of Medicine, 22nd Edition, 2004

30. Serial Quantitative Immunoglobulins and
M spike from Serum Protein Electrophoresis
Induction
with dex
2005
2006
After ASCT
2007
Progression

31. Electrophoresis/Immunofixation
Multiple Myeloma – Immunofixation
Electrophoresis/Immunofixation
Uses of Immunofixation:
- Identify monoclonal protein
- More sensitive than EP
** SPEP is a simple manual/semi-automated method, Inexpensive and this method is considered quantitative because the bands can be scanned and measured by densitometry
** However, on the other hand, the sensitivity of SPEP for FLC detection is quite low between mg/L.
SPEP is – for FLCs in all patients with NSMM, majority of patients with AL amyloidosis, and many patients with LCMM. It can even be – in a portion of patients with IIgMM.
** Since this method cannot detect mildly elevated levels of protein, it becomes difficult with SPEP to reach an early diagnosis. Indeed, the tumor burden is already quite significant when SPEP turns +.
** Here are 2 examples of SPEP gels. 1 normal and the other abnormal showing a monoclonal protein spike in the gamma region.

33. 24-Hour Urine Protein Results with Electrophoresis

34. Serial 24-Hour Urine Protein Results

35. Serum Free Light Chain Assay (sFLC)
Hidden surface
Antibody
target
Free Lambda
Free Kappa
Antibody target
Intact Ig
Previously hidden
surface
** sFLC assay is an immunoassay with an exquisite specificity using sheep polyclonal antibodies to sequester light chains epitopes. This exceptional specificity is occurring mainly for 2 reasons:
1) Epitopes are only found in the constant region of the light chains
2) Those epitopes are hidden when lambda and kappa light chains are bound to heavy chains and consequently can only be recognized when light chains are free.
** sFLC assay is now available and can be performed on various nephelometric or turbidimetric platforms.
** Now, let’s talk about Ig structure and how the sFLC assay is working.
** As previously mentioned, intact Ig are composed of 2 identical heavy chains and 2 identical light chains, each containing variable and constant domains.
** Variable domain of light chain and heavy chain together form the antigen-binding site, therefore both chains contribute to the antigen-binding specificity of the Ig molecule
Normal Ranges1
Serum
Free Kappa mg/L
Free Lambda mg/L
Kappa/Lambda:
1Katzmann et al. Clin Chem. 2002;48:1437–1444.

36. Serum Free Light Chain Assay (sFLC) -Advantages-
Increased sensitivity
Electrophoresis can detect g/L
Immunofixation can detect g/L
Serum FLC assay can detect < g/L
Serum is easier to work with than urine
Serum FLCs have a short half-life compared to intact monoclonal immunoglobulins
can detect changes in myeloma cell growth or regrowth faster
A number of B-cell and plasma cell disorders are associated with the presence of circulating immunoglobulin molecules, or parts of immunoglobulin molecules. The majority are associated with a clonal expansion of a monoclonal plasma cell population, some are associated with mature B-lymphocyte neoplasms, and some with both (lymphoplasmacytic lymphomas). Missing from this slide is light chain deposition disease which constitutes less than 1% of all monoclonal gammopathies.

37. Free Light Chain Escape
IgA paraprotein
Serum lambda FLC
Bortezomib
Robson, E. Unpublished data

38. Increasing M protein and
Monitoring Myeloma
ResearchTrial
Bortezomib + Vorinostat
Increasing M protein and
IgG levels
Anemia
New skeletal findings
Courtesy of Dr. A. Xenocostas

39. Myeloma Lab Tests Key Points
Need to determine the best test for monitoring the activity of the myeloma cells
Serum M spike (Intact IgG or Ig A)
Quantitative IgA level in about 5-8% of patients
Urine M spike (kappa or lambda light chain)
Freelite in serum (kappa or lambda)
The “trend” is important
Some variability in results is inherent in the tests
Results have to be interpreted in context of overall status of the patient