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

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

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

Normal Blood Counts Normal Hgb (Hemoglobin) Normal WBC (Leukocytes) Men 140-160 Women 210-140 Normal WBC (Leukocytes) Total 4.0-10.0 Differential Neutrophils (Polys, ANC) Lymphocytes Eosinophils Monocytes Basophils Normal platelets 150-350 Fatigue possible if < 100-110 Transfusion if < 80 Risk of infection if neutrophils <1.0 Risk of bleeding if <10

Example of CBC Results

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

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

Example of CBC Results

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

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)

Automatic Chemistry Analyzer Courtesy of Dr. A. Xenocostas

Bone Marrow Aspirate Courtesy of Dr. A. Xenocostas

Bone Marrow Aspirate Courtesy of Dr. A. Xenocostas

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.

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

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.)

Patterns of Antibody Production

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

Types of Multiple Myeloma

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

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)

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

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.

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

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

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

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

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

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

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

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 500-2000mg/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.

24-Hour Urine Protein Results with Electrophoresis

Serial 24-Hour Urine Protein Results

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 3.3-19.6 mg/L Free Lambda 5.6-26.3mg/L Kappa/Lambda: 0.26-1.65 1Katzmann et al. Clin Chem. 2002;48:1437–1444.

Serum Free Light Chain Assay (sFLC) -Advantages- Increased sensitivity Electrophoresis can detect 0.5- 2.0 g/L Immunofixation can detect 0.15-0.5 g/L Serum FLC assay can detect < 0.005 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.

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

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

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