Serum Protein Electrophoresis and Immunofixation

Serum protein electrophoresis separates serum proteins by charge on agarose gel or by capillary zone electrophoresis at alkaline pH. It gives albumin, alpha-1, alpha-2, beta and gamma fractions, quantified by densitometry. A discrete band, or M-protein, indicates a monoclonal immunoglobulin made by a single plasma cell or B-cell clone. Immunofixation then identifies the heavy chain class (IgG, IgA, IgM, IgD, IgE) and light chain type (kappa or lambda), and is about 10 times more sensitive than electrophoresis for small bands.

The pair is the first line screen for plasma cell disorders and Waldenström macroglobulinaemia, and it also defines patterns in inflammation, liver disease, nephrotic syndrome and immunodeficiency. Serum free light chains are added to find light chain only disease, which gives no band.

Monoclonal gammopathy of undetermined significance (MGUS) is present in about 3% of people over 50 years and 5% over 70 years. It is about twice as frequent in people of African ancestry and is more common in men. Multiple myeloma follows MGUS at about 1% each year.

Indications. Unexplained anaemia, bone pain or fractures, hypercalcaemia, kidney impairment, recurrent infection, unexplained peripheral neuropathy, nephrotic range proteinuria in adults, raised ESR or plasma viscosity with rouleaux, high total globulin, low immunoglobulins, hyperviscosity symptoms, carpal tunnel syndrome with macroglossia or heart failure (amyloidosis), and follow up of a known paraprotein.

Specimen. Serum, not plasma, because fibrinogen in plasma gives an extra band. Fasting is not needed. Samples are stable for about 7 days at 2 to 8 °C.

Adult reference ranges are method specific, and local ranges take precedence.

Albumin. The largest fraction, made in the liver and a negative acute phase protein. Range: 35 to 50 g/L. Raised: dehydration. Lowered: inflammation, liver disease, nephrotic syndrome, protein losing enteropathy, malnutrition, burns, haemodilution.

A bisalbuminaemia (double albumin band) is an inherited variant of no clinical importance.

Alpha-1 globulins. Mainly alpha-1 antitrypsin and alpha-1 acid glycoprotein. Range: 1.0 to 4.0 g/L. Raised: acute inflammation, pregnancy, oestrogen therapy, hepatocellular carcinoma (AFP). Lowered: alpha-1 antitrypsin deficiency, severe liver disease, nephrotic syndrome.

A flat or absent alpha-1 band suggests alpha-1 antitrypsin deficiency, and needs a quantitative assay and phenotyping.

Alpha-2 globulins. Alpha-2 macroglobulin, haptoglobin and caeruloplasmin. Range: 5.0 to 9.0 g/L. Raised: acute inflammation, nephrotic syndrome (alpha-2 macroglobulin is retained while smaller proteins are lost), corticosteroid therapy. Lowered: intravascular and severe extravascular haemolysis (haptoglobin), severe liver disease.

An acute phase pattern combines low albumin with raised alpha-1 and alpha-2 bands. A nephrotic pattern combines very low albumin and gamma with a strongly raised alpha-2.

Beta globulins. Transferrin (beta-1), complement C3 and LDL (beta-2), and, in plasma, fibrinogen. Range: 6.0 to 11.0 g/L. Raised: iron deficiency (transferrin), pregnancy, oestrogen therapy, obstructive jaundice and hypercholesterolaemia (LDL), liver disease, and IgA paraprotein. Lowered: inflammation and malnutrition (transferrin), complement consumption.

A beta to gamma bridge, with fusion of the two zones, is characteristic of cirrhosis from raised polyclonal IgA. An extra band between beta and gamma indicates fibrinogen from a plasma sample, or a paraprotein.

Gamma globulins. Immunoglobulins, mainly IgG. Range: 6.0 to 16.0 g/L. Raised polyclonal (broad based): chronic infection (HIV, hepatitis B and C, tuberculosis, visceral leishmaniasis, malaria), autoimmune disease (systemic lupus erythematosus, Sjögren syndrome, rheumatoid arthritis, autoimmune hepatitis), cirrhosis, sarcoidosis, lymphoproliferative disease. Lowered: primary and secondary immunodeficiency, chronic lymphocytic leukaemia, nephrotic syndrome, protein losing enteropathy, light chain myeloma, immunosuppression.

A narrow based raised band is monoclonal until proven otherwise.

Monoclonal protein (M-protein). A discrete band, most often in the gamma region (IgG), sometimes in the beta region (IgA) or near the origin (IgM). Its concentration is measured by densitometry in g/L and followed over time.

Immunofixation. Serum is electrophoresed in lanes and fixed with antisera to IgG, IgA, IgM, kappa and lambda. A band that matches one heavy chain and one light chain confirms a monoclonal protein and gives its class. It is required whenever electrophoresis shows a band or a suspicious asymmetry, and in all cases where the clinical suspicion is high but the pattern is normal.

Monoclonal gammopathy classification.

• MGUS: serum M-protein under 30 g/L, clonal bone marrow plasma cells under 10%, and no end organ damage or amyloidosis. Non-IgM MGUS and IgM MGUS are separate entities, and light chain MGUS has an abnormal free light chain ratio with no heavy chain.
• Smouldering myeloma: M-protein of 30 g/L or more, or marrow plasma cells 10 to 60%, with no myeloma defining events.
• Multiple myeloma: clonal marrow plasma cells of 10% or more, or a biopsy proven plasmacytoma, with one or more myeloma defining events: calcium more than 0.25 mmol/L over the upper limit (or over 2.75 mmol/L), creatinine over 177 micromol/L or creatinine clearance under 40 mL/min, haemoglobin more than 20 g/L under the lower limit (or under 100 g/L), one or more lytic bone lesions, marrow plasma cells of 60% or more, an involved to uninvolved free light chain ratio of 100 or more, or more than one focal lesion on MRI.
• Waldenström macroglobulinaemia: an IgM M-protein of any size with lymphoplasmacytic marrow infiltration. Hyperviscosity occurs at IgM concentrations over about 30 to 40 g/L.
• AL amyloidosis: a small M-protein, often lambda light chain, with organ involvement. The band may be absent, and free light chains are needed.

Risk of progression in MGUS. Three factors raise risk: M-protein of 15 g/L or more, a non-IgG isotype, and an abnormal free light chain ratio (under 0.26 or over 1.65). Risk of progression at 20 years is about 5% with none, about 20% with one, about 37% with two and about 58% with three.

Response and progression in myeloma. A fall of at least 50% in M-protein is a partial response. Disappearance with negative immunofixation is a complete response. A rise of at least 25% from the lowest value, with an absolute increase of at least 5 g/L, is progression.

Free light chains. Reference: kappa 3.3 to 19.4 mg/L, lambda 5.7 to 26.3 mg/L, ratio 0.26 to 1.65 (ratio 0.37 to 3.1 in kidney impairment). Light chain only myeloma and AL amyloidosis show an abnormal ratio with no band.

Other patterns.

• Oligoclonal bands: after transplantation, in immune reconstitution, infection and autoimmune disease, and often transient.
• Biclonal gammopathy: two bands, in a small proportion of cases.
• Therapeutic monoclonal antibodies (daratumumab, isatuximab) appear as an IgG kappa band and can obscure a complete response.
• Cryoglobulin: a band that is lost when the sample is kept cold.

Critical values and findings needing immediate action.

• M-protein with calcium over 3.0 mmol/L, acute kidney injury (cast nephropathy), cord compression symptoms (back pain with leg weakness or sphincter change), or a high plasma cell count in blood.
• IgM paraprotein with headache, visual disturbance, bleeding or confusion, indicating hyperviscosity.
• A new M-protein with heart failure, nephrotic syndrome or neuropathy, indicating amyloidosis.
• Marked hypogammaglobulinaemia with severe or recurrent infection.

Interferences and pitfalls.

• A small band in the beta region can be masked by transferrin or C3, and a band in the alpha-2 or beta region may need immunofixation to be seen.
• IgD and IgE paraproteins and light chain only disease can be missed on electrophoresis because they give a small or absent band.
• Fibrinogen from incompletely clotted or plasma samples, haemolysis (haemoglobin to haptoglobin complex) and iodinated contrast agents produce false bands.
• A reduced gamma region may be the only clue to light chain myeloma.
• Densitometry of a band over a polyclonal background, or in the beta region, is imprecise, and serial values should come from the same method.
• A negative electrophoresis does not exclude a small M-protein, so immunofixation and free light chains are added when suspicion is high.
• Electrophoresis alone cannot separate benign from malignant disease.

Special groups.

• Children: gamma globulin is low at 3 to 6 months, and monoclonal bands are rare.
• Pregnancy: albumin falls, and alpha and beta fractions rise.
• Older adults: MGUS prevalence rises with age, and an M-protein needs interpretation against symptoms and investigations.
• People of African ancestry: MGUS is about twice as common and appears earlier.
• Transplant recipients and HIV: transient bands are common.
• Regions: visceral leishmaniasis, malaria, schistosomiasis, tuberculosis and chronic hepatitis give strong polyclonal gamma rises.

Follow up tests.

• Immunofixation, serum free light chains, quantitative immunoglobulins
• Urine protein electrophoresis with immunofixation, 24-hour urine protein
• Full blood count and film, calcium, creatinine and eGFR, albumin
• Beta-2 microglobulin, lactate dehydrogenase, C-reactive protein
• Bone marrow aspirate and trephine biopsy, flow cytometry, FISH for plasma cell abnormalities
• Skeletal imaging by low dose whole body CT, MRI or PET-CT
• Serum viscosity, cryoglobulins
• Amyloid assessment (tissue biopsy, NT-proBNP, troponin, echocardiography)
• Anti-MAG antibodies, MYD88 mutation testing