Serum Protein Electrophoresis with Immunofixation Dr.Ajay Phadke Centre Head SRL Diagnostics-Dr.Avinash Phadke’s Lab

What is electrophoresis? Electrophoresis is a method of separating proteins based on their physical properties. Proteins can be separated using a buffered solid medium (agarose electrophoresis ) or using only the liquid phase (capillary electrophoresis) The net charge (positive or negative) and the size and shape of the protein commonly are used in differentiating various serum proteins. A negatively charged particle usually travels to the positively charged electrode(Gel EP) In Capillary EP ? Negatively charged particle travels to the negatively charged electrode(Cathode) WHY?

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

The complementary positively charged ions in the surrounding buffer are free to move under the electromotive force, and they carry with them molecules of the solvent water. This buffer flow is termed electro-osmosis or endosmosis, which also carries the proteins with it to some extent by mechanical flow, not by charge. The actual distance traveled by a particular protein migrating in an electrical field is determined by the elec- tromotive force (a feature of the protein itself and the pH) and the electro- osmotic force (a function primarily of the support medium). When the electro-osmotic force is greater than the electrophoretic force acting on weakly anionic proteins (e.g., γ-globulins), those proteins move from the application point toward the cathode, even though their charge is slightly negative.

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

When do doctors ask for SPE? Unexplained anemia / weakness / fatigue / ↑ ESR Unexplained renal insufficiency Heavy proteinuria in patient >40yrs Bence Jones proteinuria Hypercalcaemia Hypergammaglobulinemia Immunoglobulin deficiency Peripheral neuropathy (5% will have MGUS) Recurrent infections Unexplained bone pain / pathologic fracture / lytic lesion-

1.Elderly patient with suspicion for MM i.e bone pain, lytic lesion 2. fever for >1 month 3. ESR increase, persistent anemia, fatigue 4. CRP high 5.Heavy proteinuria in adults 6.persistent increase in calcium 7.Peripheral neuropathy since a percentage have MGUS

Capillarys 2 Minicap

Carousel 28 positions

Easy access to consumables: reagent cups, waste & reagents containers Reagent Compartment Easy access to consumables: reagent cups, waste & reagents containers (2 buffer vials on board)

Fraction identification Albumin Beta-1 Alpha-1 Alpha-2 Beta-2 Gamma a-1 acidglycoprotein a-1 antitrypsin TBG, Transcortin Gammaglobulins C3 complement B Lipoprotein Haptoglobin a-2 macroglobulin Ceruloplasmin Hemopexin Transferrin

Normal Alb a1 a2 b1 b2 g Gaussian aspect in gamma & No increase or additional deformation/peak in gamma, beta 1, beta 2 and alpha 2 Alb a1 a2 b1 b2 g

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

In diabetes mellitus, patients with Type 2-2 haptoglobin have shown higher risk for vascular complications, perhaps owing to different ability to clear hemoglobin, thus leading to altered iron handling and heightened oxidative load Serum haptoglobin rises in response to stress, infection, acute inflam- mation, or tissue necrosis,

Fibrinogen levels become elevated with the other acute phase reactants, occasionally to over 1.0 g/L. In such instances, the erythrocyte sedimentation rate (ESR) is also markedly elevated owing to fibrinogen content directly. Fibrinogen levels also rise with pregnancy and use of contraceptive medications. Low levels generally indicate extensive activation of coagulation with consumption of fibrinogen.

Congenital deficiency of A1AT A1AT = 0.17g/L (normal range: 0.97 – 1.93 g/L) Alb a1 a2 b1 b2 g

Because of the rapid dynamics of its synthesis and clearance, prealbumin is considered to be a better early indicator of change in nutritional status Presence of a distinct band of prealbumin is used only as a landmark to confirm that the specimen was likely CSF. A protein band frequently appears in the pre-albumin position of of serum from patients who have had heparin therapy. In the circulation, heparin activates and releases lipoprotein lipase activity, which attacks triglycerides in lipoprotein fractions.

Albumin Albumin concentrations are vital to the understanding and interpretation of calcium and magnesium levels because these ions are bound to albumin, and so decreases in albumin are directly responsible for depression of their concentrations

C3 (and also C4) concentration is a convenient marker for assessing disease activity in rheumatic disorders such as lupus erythematosus and rheumatoid arthritis. C4 is not appreciated on serum protein electrophoresis because its concentration is normally only about one-fifth that of C3. Both C3 and C4 are now easily quantitated by nephelometry for monitoring rheumatic disease activity

Haptoglobin phenotypes in alpha 2 zone Phenotype I - I Phenotype I - II Phenotype II - II

Monoclonal peak in Gamma Alb a1 a2 b1 b2 g

Monoclonal peak in beta Alb a1 a2 b1 b2 g

Oligoclonal pattern Alb a1 a2 b1 b2 g

Immunofixation & immunotyping Principle: Apply the patients sample on gel. Separate the sample. Add antibody . If positive = on washing this sample remains because of large size of complex. Immunotyping : similar principle. Automated, not labour intensive. BASIC DIFFERENCE: way how sample is processed. WE MIX sample with antibody before processing.Complex is made EVEN BEFORE SEPERATION TAKES PLACE. Then injected into capillary. Monoclonal complex will MIGRATE SLOWLY and will NOT form a peak. THEREFORE, in IF you are looking for the band to be PRESENT. While in IT you are looking for it to be ABSENT! IFE is Very labour intensive

IEP: serum applied to aggel in wells. EP. Antisera added IEP: serum applied to aggel in wells. EP . Antisera added. 24 hr incubation. ARCS formed IFE:Sample on solid matrix IT: NO GEL. Migration in buffered medium. Mono-specific antisera. REDUCTION technique. Antisera binds to Immunoglobulin. Heavy, large molecule created. Pulled OUT of viewing area. If PEAK DETECTEd, just click on immunotyping after selecting dilution. Hypgogamma : Ig<0.8g/L(1/10) Std :Ig 0.8- 2.0 g/L(1/20) Hypergamma: Ig >2.0g/L(1/40)

Monoclonal peak or polyclonal increase in gamma? Pointedpeak Rounded top Narrow basement Large basement Monoclonal peak Polyclonalincrease IT IT Complete substractionwith the antiserumagainst a heavychain and partial substractionwith the antiseraagainst kappa and lambda Complete substraction of the peakwith one antiserumagainst a heavychain and a light chain

Normal sample Zoom

A ELP G ~ 80% IgG ~ 15% IgA K L M 2/3 Kappa 1/3 Lambda ~ 5% IgM

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

IgG lambda

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IgG kappa

Fusion between beta 1& 2

IgA lambda

IgG lambda + free light chains lambda

When interpreting IT, always consider: « If removingsomething, whatisremaining? » In eachwindow, removing one specific class of IgG highlights what is happening with the residual immunoglobulins that remain after substraction

When removing the IgM, the whole peak disappears When removing the Kappa → a slight peak in the residual Lambda remains When removing the Lambda → a peak in the residual Kappa remains Ig M Kappa + Ig M Lambda There are 2 monoclonal peaks, one Kappa and one Lambda. Both disappear when removing the IgM…

Protein 6

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IgM kappa

IgM Kappa+ IgM Lambda + oligoclonal profile in IgG

IgG IgM Before BME treatment

Treatment with Beta Mercaptoethanol (BME) to depolymerize a monoclonal Ig : * Prepare a 1% BME reducing solution : 1) 90µL H2O + 10µL BME 2) 10µL 1/10 diluted BME + 90µL Fluidil (ref 4587) * 100µL of 1% BME reducing solution + 300µL serum * Incubate 10 mn at room temperature Analyze immediately the treated sample in Capillarys or Minicap without any delay  To separate co-migrating Igs of different types (Ig M vs Ig G)

IgG kappa + IgM Lambda IgM L IgG K After BME treatment

One or Two Ig G kappa?

Smoothing 0 Change of the smoothing option 2 IgG kappa Smoothing 2

Polyclonal increase or monoclonal Ig in gamma? Zoom

Polyclonal increase or monoclonal Ig in gamma?

Protein 6

Polyclonal increase of Ig G

Oligoclonal profile Oligoclonal: Presence of multiple faint peaks or distorsions This profile is observed in: Autoimmune diseases (Rheumatoid arthritis, Gougerot Sjögren syndrome, Lupus erythematosus) Infectious (viral, bacterial, parasitic) diseases Autoimmune reactions (Patients with transplants or patients under immuno- suppressive treatments) Oligoclonal profile is linked with a dysregulation of immune system

Oligoclonal profile on Hydragel IF

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Oligoclonal profile in Ig G (with major Ig G lambda to recontrol)

Hints and tips for IT interpretation Examine carefully all IT curves without a zoom to verify the correct overlapping on albumin and the zone of interest between ELP and antisera curves Verify that the correct sample dilution has been used Compare the residual heavy and light chains after substraction and their position to verify additional presence of other monoclonal Ig If there is no correspondence between heavy and light chains, complete the test with an immunofixation to check for free light chains and/or IgD, IgE

Once a monoclonal gammopathy is identified by serum protein electrophoresis, multiple myeloma must be differentiated from other causes of this type of gammopathy. (Waldenström’smacroglobulinemia, smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, plasma cell leukemia, heavy chain disease)

sizeof the M-protein spike sizeof the M-protein spike. Although this spike is usually greater than 3 g per dL in patients with multiple myeloma, up to one fifth of patients with this tumor may have an M-protein spike of less than 1 g per dL.10Hypogammaglobulinemia on serum protein electrophoresis occurs in about 10 percent of patients with multiple myeloma who do not have a serum M-protein spike.11 Most of these patients have a large amount of Bence Jones protein (monoclonal free kappa or lambda chain) in their urine.11 Thus, the size of the M-protein spike is not helpful in excluding multiple myeloma. If multiple myeloma still is considered clinically in a patient who does not have an M-protein spike on serum protein electrophoresis, urine protein electrophoresis should be performed.

With the addition of sodium sulfate, sodium sulfite, ammonium sulfate, or methanol, the globulins tend to precipitate, leaving albumin in solution. By measuring total protein in the original serum and protein in the precipitate or the supernatant, values for albumin and globulin can be derived.

1.Elderly patient with suspicion for MM i.e bone pain, lytic lesion 2. fever for >1 month 3. ESR increase, persistent anemia, fatigue 4. CRP high 5.Heavy proteinuria in adults 6.persistent increase in calcium 7.Peripheral neuropathy since a percentage have MGUS

When does one advise urine EP The following conditions (to list a few) warrant urine protein electrophoresis: 1) monoclonal protein in serum is >1.5 g/dL, 2) monoclonal free light chains are detected in serum, 3) hypogammaglobulinemia is present in serum; 4) serum electrophoresis shows nephrotic pattern. “In the context of screening, the serum FLC assay in combination with serum protein electrophoresis (PEL) and immunofixation yields high sensitivity, and negates the need for 24-h urine studies for diagnoses other than light chain amyloidosis (AL).” • “...once diagnosis of a plasma cell disorder is made, 24-h urine studies are required for all patients.” • “For AL screening, however, the urine IFE should still be done in addition to the serum tests including the serum FLC.” • “The FLC assay cannot replace the 24-h urine protein electrophoresis for monitoring myeloma patients with measurable urinary M proteins”.

What history is important? What would you report?

Increase in alpha1, alpha 2 Advise renal profile,UPE and IT

65 year old patient. Weakness, What do you see on the graph. What will you advise?

50 year old female What is your impression? What would you advise?

30 Year old Female. What is your opinion? What history will you take

Hb: 8.0 RDW: 20.3 Retic N Ferritin : 3.0 Normal range(4.6-204) B 12 : 254

What is important in this graph?