6/17/2013

Serum Protein Electrophoresis

Karina Rodriguez-Capote MD, PhD, FCACB,

Clinical Chemist, DynaLIFEDx

2013 Laboratory Medicine Symposium

Objectives • Describe the electrophoresis procedure used to separate serum proteins and to identify a monoclonal protein • Indications for serum protein electrophoresis (SPE) • Describe how immunofixation electrophoresis (IFE) is used to identify the heavy and light chain of a monoclonal protein • Interpretation: common patterns & pitfalls • Describe the diagnostic criteria used to identify patients with monoclonal gammopathy

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Electrophoresis: the proteins are separated according to their electrical charges on agarose gel using both the electrophoretic and electroendosmotic forces present in the system.

Staining : After the proteins are separated, the plate is placed in a solution to stain the protein bands. The staining intensity is related to protein concentration

After dehydration in methanol, the plate background is then made transparent by treatment with a clearing solution.

Drying Interpretation

Additional testing: IFE, FLC, IGQ

Plate Soaking Time...... 20 minutes Electrophoresis Time...... 15 minutes Stain Time...... 6 minutes Destain Time in 5% Acetic Acid...... 3 times/10 minutes Dehydration Time in Methanol...... 2 times/2 minutes Clearing ...... 5-10 minutes. Drying ...... 20 minutes

Typical gels: 28 patients and 2 controls

Interpretation of the results is done case by case We evaluate the need of further testing such as: immunofixation (IFE) serum free light chain analysis immunoglobulin quantification A1AT quantification and phenotyping

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Normal SPE pattern

Alpha 1 region: α1antitrypsin, α1acid glycoprotein, alpha- 1 anti- chymotrypsin, α1-Fetoprotein , SBG, TBG

Alpha 2 Region:

GC-globulin (vitD binding), 2 macroglobulin, haptoglobin, HDL, ceruloplasmin

Beta Region: Hemopexin, transferrin, β-lipoprotein, LDL, C3, C4

Gamma Region:

albumin Immunoglobulins , CRP, LDH, fibrinogen,

+ - Location of some of the more commonly known proteins Liver Proteins Lymphoid Proteins

Am Fam Physician. 2005; 71(1):105-112.

Interpretation ► Results on normal individuals will involve age and gender-related variations and day-to-day biologic variations.

► Different protein patterns are found in many conditions:

• pregnancy • rheumatic disease • chronic liver disease • protein-loss disorders • systemic lupus erythematous • nephrotic syndrome • rheumatoid arthritis • genetic deficiencies • inflammatory response • malnutrition

SPE is not necessary to diagnose or monitor these conditions.

Am Fam Physician. 2005 Jan 1;71(1):105-112.

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When should SPE be ordered?

Guidelines recommend the use of SPE in two main contexts: screening and monitoring of monoclonal gammopathies Multiple Myeloma, Waldenström’s Macroglobulinemia, primary amyloidosis or other related disorders

1. Canadian Coalition for Quality in Laboratory Medicine 2. BJH 2005 Guidelines 4. CAP 1999 Guidelines 3. NCCN 2008 Guidelines 5. Am Fam Physician. 2005 71(1):105-112

What is a monoclonal gammopathy? ? ► Monoclonal gammopathy is a disorder caused by proliferation of a single clone of plasma cells, which in turn produce a single immunoglobulin. ► Levels of other normal immunoglobulins eventually decrease, referred to as immune paresis. ► While this is most often associated with multiple myeloma, it is quite frequently an unexpected discovery, and may be related to a number of conditions.

Monoclonal protein = Paraprotein = M-protein/band/spike = Monoclonal gammopathy

1. BJH 2005 Guidelines CAP 1999 Guidelines 2. NCCN 2008 Guidelines Am Fam Physician. 2005 71(1):105-112.

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INCIDENCE: Estimated New Cases for Multiple Myeloma by Sex and Province, Canada, 2011

Canada NL PE NS NB QC ON MB SK AB BC

Males 1,300 15 10 40 30 350 510 40 35 110 150

Females 1,000 10 5 30 25 270 420 30 25 90 110

Totals 2,300 25 15 70 55 620 930 70 60 200 260

Estimated Age-Standardized Incidence Rates for Multiple Myeloma by Sex and Province, Canada, 2011 (Cases per 100,000)

Canada NL PE NS NB QC ON MB SK AB BC

Males 6 4 10 6 6 7 6 6 5 6 5

Females 4 3 3 4 4 4 4 4 3 4 3

Canadian Cancer Society, Provincial/Territorial Cancer Registries Public Health Agency of Canada, 2011

Myeloma is more common in older persons

Incidence 8 per 100,000 in African descents 4 per 100,000 in Caucasians 1-2 per 100,000 in Hispanic and Asian groups

Canadian Cancer Society, Provincial/Territorial Cancer Registries Public Health Agency of Canada, 2011

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Indications for requesting SPE

Based on clinical findings: ► Suspected Multiple Myeloma, Waldenström’s Macroglobulinemia, primary amyloidosis or other related disorders ► Unexplained bone pain or fracture ► Recurrent infections ► Unexplained peripheral neuropathy (not able to be attributed to another condition, e.g. type 2 diabetes, chemotherapy)

Based on radiological findings: ► Lytic lesions in bone ► Unexplained osteopaenia (as not all patients with MM will have osteolytic lesions)

1. BJH 2005 Guidelines 3. CAP 1999 Guidelines 2. NCCN 2008 Guidelines 4. Am Fam Physician. 2005 71(1):105-112.

Indications based on laboratory findings:

► High (or low) total serum globulin or immunoglobulin ► Extremely high percentage of lymphocytes ► Incidental finding of an increased total protein level ► Unexplained anaemia (multiple myeloma is a recognised cause of non- iron deficiency anaemia) or other persisting cytopaenias for which there is no other explanation ► Unexplained high ESR (>50) with a normal CRP ► Unexplained hypercalcaemia or renal impairment ► Red cell rouleaux formations noted on the peripheral blood smear ► Unexplained high urine protein with relatively low or normal urine albumin ► Presence of urine free light chains (Bence-Jones proteinuria)

1. BJH 2005 Guidelines CAP 1999 Guidelines 2. NCCN 2008 Guidelines Am Fam Physician. 2005 71(1):105-112.

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Red flags for potential diagnosis of MM in patients with back pain

► Age > 50 years ► Back pain that is worse in supine position ► Pain that is worse at night or awakens patient from sleep ► Pain with a band-like distribution around the body ► Pain that is not relieved with conventional methods (i.e., rest, nonsteroidal anti-inflammatory drugs) ► Associated constitutional symptoms (fever, weight loss, dehydration) ► Progressive neurologic deficit in lower extremities

1. Best Tests, July 2011 2. CAP 1999 Guidelines 3. Am Fam Physician. 2005 71(1):105-112

Immunofixation ► When SPE identifies a monoclonal gammopathy, the laboratory will automatically perform immunofixation (i.e. reflex test) to further determine the exact type of monoclonal protein.  Heavy chain subtypes: ─ common: IgG, IgA or IgM ─ rare: IgD or IgE  Light chain subtypes: kappa or lambda (κ or λ). ► In a minority of cases only light chains (without heavy chains) are produced.  Light chain-only monoclonal gammopathy are often barely visible in serum but may show as large amounts of monoclonal light chains excreted in the urine; hence the need to consider urine testing when clearly suspecting a monoclonal gammopathy

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Potential False Alarms ► Fibrinogen (peak -)

► Hemoglobin-haptoglobin  Hemolysis (2) ► Transferrin  Iron deficient anemia () ► High levels of lipids (hyperlipidemia)  ()

► Nephrotic Syndrome  (2 or , ↓ Albumin and gamma globulins) ► Complement C3: peak β region

► Hyperthyroidism (TBG  2)

► Pregnancy (Alpha fetoprotein  1) ► Oligoclonal Bands (infection, autoimmune diseases) ► Radiographic agents may produce an uninterpretable pattern. ► Some drugs can affect protein electrophoresis results: corticosteroids, cholesterol-lowering drugs (statins), oral contraceptive use, steroid use, chlorpromazine (Thorazine), neomycin, penicillin.

1. BJH 2005 Guidelines 3. CAP 1999 Guidelines 2. NCCN 2008 Guidelines 4. Am Fam Physician. 2005 71(1):105-112

How can the ordering physician help the lab (and the patient)… ? ►Appropriate test utilization – CRP is the most sensitive indicator of an acute phase reaction (inflammation, trauma, infection) – A1AT quantification with reflex phenotyping are the recommendations for alpha1- antitrypsin deficiency

►A diagnostic comment can be so helpful… – peripheral neuropathy – osteoporosis Any of these raises the pre-test – back pain probability and may influence our – unexplained anemia decision to add an IFE – R/O myeloma – unexplained renal failure

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Summary

► SPE and IFE are used to identify a monoclonal protein in the serum of patients with Multiple Myeloma and MGUS. ► Patients with Multiple Myeloma and MGUS are followed by measuring the concentration of the monoclonal protein using SPE. ► Although abnormal protein levels may be found in many conditions (such as kidney disease, chronic liver disease, systemic lupus erythematous, rheumatoid arthritis), serum protein electrophoresis is usually not done to diagnose or monitor these conditions. ► CRP is the most sensitive indicator of an acute phase reaction (inflammation, trauma, infection)

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