Urine Protein Electrophoresis and Bence Jones Protein

Urine protein electrophoresis (UPEP) separates urinary proteins on agarose gel or by capillary zone electrophoresis into albumin, alpha-1, alpha-2, beta and gamma regions. Urine immunofixation (UIFE) then identifies the type of any monoclonal band by reacting it with antisera against IgG, IgA, IgM, kappa and lambda.

Bence Jones protein is the free monoclonal light chain (kappa or lambda) excreted in urine, which was originally detected by heat coagulation and is now identified by electrophoresis and immunofixation. A timed 24-hour collection allows measurement of total protein and the monoclonal band in g/24 hours.

The test is used with serum protein electrophoresis, immunofixation and serum free light chains to diagnose and monitor plasma cell disorders: myeloma, light chain amyloidosis, Waldenström macroglobulinaemia and monoclonal gammopathy. It also characterises proteinuria as glomerular, tubular or overflow.

Multiple myeloma has an incidence of about 5 to 7 per 100 000 people a year, and about 50 to 70% of patients excrete Bence Jones protein. Light chain cast nephropathy ("myeloma kidney") occurs in 20 to 40% of patients at presentation. Light chain amyloidosis affects about 1 per 100 000 people a year, and 70 to 80% have a monoclonal protein in urine.

Indications. Suspected myeloma (bone pain, anaemia, hypercalcaemia, kidney failure, recurrent infection, raised ESR), unexplained proteinuria with a negative dipstick, nephrotic syndrome of unknown cause, suspected amyloidosis, light chain deposition disease, peripheral neuropathy with a paraprotein, monitoring of light chain myeloma, and follow up of monoclonal gammopathy.

Specimen. A 24-hour urine collection in a container without preservative (record the total volume), or a random early morning sample. A concentrated aliquot is needed because the protein concentration is often low. Refrigerate during collection. A matching serum sample is sent with the request.

Normal urine. Total protein under 150 mg/24 hours (under 0.15 g/24 hours). The pattern is mainly albumin with small amounts of alpha-1, alpha-2 and beta globulins (including Tamm-Horsfall protein). No monoclonal band.

Patterns.

• Glomerular proteinuria: mainly albumin and transferrin (selective in minimal change disease, non-selective with larger proteins in other glomerulonephritis).
• Tubular proteinuria: small proteins (alpha-1 microglobulin, beta-2 microglobulin, retinol binding protein) in the alpha and beta regions, with albumin under 50% of the total. Seen in Fanconi syndrome, interstitial nephritis, heavy metal and drug toxicity.
• Overflow proteinuria: a sharp band in the gamma, beta or alpha region from monoclonal light chains (Bence Jones), haemoglobin or myoglobin.
• Mixed patterns occur in advanced kidney disease.

Bence Jones protein.

• A monoclonal band in the urine, with a free kappa or lambda light chain on immunofixation (without a matching heavy chain), is Bence Jones protein.
• Lambda is more often associated with amyloidosis, and kappa with light chain deposition disease. Kappa to lambda ratio in myeloma is about 2 to 1.
• Quantity over 1 g/24 hours with kidney failure suggests cast nephropathy, and over 200 mg/24 hours defines measurable disease in light chain myeloma.
• Urine M-protein over 200 mg/24 hours or serum M-protein over 10 g/L, with features of end organ damage, supports active myeloma.

Monitoring. Fall of 90% or more in urine M-protein (or under 200 mg/24 hours) defines a very good partial response, and absent protein by immunofixation with a normal free light chain ratio defines complete response. A rise of 25% (and at least 200 mg/24 hours) defines progression. Serum free light chains are now preferred for monitoring, and urine is used when serum is not informative.

Causes of a monoclonal band in urine.

• Multiple myeloma (including light chain only myeloma, where serum may be normal).
• AL amyloidosis and light chain deposition disease.
• Waldenström macroglobulinaemia and other B-cell lymphoma.
• Monoclonal gammopathy of undetermined significance (small amounts, under 0.5 g/24 hours), smouldering myeloma, and plasmacytoma.
• Rarely, chronic lymphocytic leukaemia and cryoglobulinaemia.

Critical values and findings needing immediate action.

• Bence Jones protein with acute kidney injury, hypercalcaemia, bone pain or anaemia: urgent haematology and nephrology review, fluids and avoidance of nephrotoxins and contrast.
• Nephrotic syndrome with a monoclonal band: amyloidosis or light chain deposition disease, and biopsy.
• Cord compression symptoms, hyperviscosity (visual change, bleeding, confusion) or severe hypercalcaemia in a patient with a paraprotein.
• Rapid fall in creatinine clearance with a positive result.

Interferences and pitfalls.

• The urine dipstick detects albumin and misses light chains, so a negative dipstick does not exclude Bence Jones proteinuria. Sulfosalicylic acid testing detects all proteins.
• Poorly concentrated urine can miss small bands, and concentration before electrophoresis is needed.
• Unlike serum, urine electrophoresis can show non-specific bands from contamination, bacteria or contrast media. Radiographic contrast gives false bands.
• A polyclonal increase in light chains (kidney failure, inflammation) does not mean a plasma cell disorder.
• Free light chains in serum are more sensitive than urine electrophoresis for detecting light chain disease, and both are done at diagnosis.
• Incomplete 24-hour collections lead to under quantification.
• Heavy albuminuria can obscure small bands.
• Interpretation needs an experienced reader, and immunofixation is required to confirm any band.

Special groups.

• Older adults: monoclonal gammopathy of undetermined significance affects over 3% of those over 50 years and 5% of those over 70 years, and most never progress (about 1% a year).
• Patients with kidney failure of unknown cause: myeloma is found in 1 to 2% of patients over 50 years with unexplained kidney failure, and testing is essential.
• Children: very rare, and Bence Jones protein is unusual.
• Pregnancy: no major change.
• Regions: late presentation with kidney failure is common, and tuberculosis and HIV can cause polyclonal gammopathy that confuses interpretation.

Follow up tests. Serum protein electrophoresis with immunofixation, serum free light chains with ratio, quantitative immunoglobulins, calcium, creatinine and eGFR, full blood count, bone marrow aspirate and trephine, flow cytometry, skeletal survey or low dose whole body CT, beta-2 microglobulin, albumin, LDH, FISH cytogenetics, kidney biopsy with Congo red staining, and NT-proBNP and troponin in suspected amyloidosis.