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Proteins and electrophoresis

15 min

  • Tell a broad polyclonal gamma increase from a narrow monoclonal peak on electrophoresis
  • Add immunofixation and free light chains to electrophoresis for a suspected M-protein

Read the full reference

Try first

Try first

Two serum electrophoresis traces both show more protein than usual in the gamma region. Which feature tells a monoclonal increase from a polyclonal one?

The next section explains it.

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Read the shape of the trace

At the alkaline pH used for serum protein electrophoresis, the proteins separate into albumin, alpha-1, alpha-2, beta and gamma fractions. The gamma region holds most of the immunoglobulins.1 The shape of a change says more than its size.

PatternWhat the trace shows
Acute inflammationAlpha-1 and alpha-2 rise, albumin may fall
Polyclonal increaseBroad rise across the gamma region
Monoclonal proteinNarrow, restricted peak in gamma, beta or occasionally alpha
Nephrotic lossLow albumin with a prominent alpha-2 region
CirrhosisLow albumin with a broad beta-gamma bridge

A broad gamma rise comes from many clones, as in chronic inflammation, infection, liver disease and autoimmune disease. A narrow peak comes from one clone. Its size and position do not name the disorder.1,2

Serum is the preferred specimen. Fibrinogen in plasma can form a restricted band near the beta-gamma boundary that looks like a monoclonal protein.1

Work up a suspected monoclonal protein

College of American Pathologists guidance sets the first laboratory steps:2

  1. Run serum protein electrophoresis and serum free light chains.
  2. When either is abnormal, run serum immunofixation or a validated equivalent.
  3. When amyloid light-chain amyloidosis is suspected, run all of these together with urine immunofixation. The monoclonal protein there can be very small.

Immunofixation applies antisera to heavy and light chains in parallel lanes. A band that reacts with one heavy-chain and one light-chain reagent is typed. It detects small proteins that electrophoresis misses. A light-chain band with no IgG, IgA or IgM partner is tested for IgD and IgE before it is reported as light chain only.2

The free light-chain assay measures unbound kappa and lambda chains and their ratio. Kidney impairment and inflammation raise both chains, so the method's own interval and kidney rules apply.3 Densitometry estimates the size of a measurable peak, and proteins that migrate with it in the beta region make that estimate less accurate.2

References
  1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
  2. Keren DF, Bocsi G, Billman BL, et al. Laboratory detection and initial diagnosis of monoclonal gammopathies. Arch Pathol Lab Med. 2022;146(5):575-590. doi:10.5858/arpa.2020-0794-CP
  3. Mayo Clinic Laboratories. Immunoglobulin free light chains, serum. Accessed September 27, 2026. https://www.mayocliniclabs.com/test-catalog/overview/608250

Watch one

A serum protein electrophoresis and serum free light chains are ordered on a 67-year-old woman with anemia and back pain. The trace shows a small, narrow band in the beta region. The gamma region looks normal.

What does the laboratory do next, and what does it report?

TestResultPreviousReference intervalFlag
Kappa free light chains68 mg/L3.3–19.4 mg/LHigh
Lambda free light chains14 mg/L5.7–26.3 mg/L
Kappa/lambda ratio4.860.26–1.65High
Creatinine0.8 mg/dL0.5–1.0 mg/dL

Specimen: H 3, L 12, I 1. Serum, gold top

  1. Check the specimen type. It is serum, so fibrinogen does not explain the band.

    Fibrinogen in a plasma specimen can form a band that mimics a monoclonal protein.

  2. Read the shape. The band is narrow, so a monoclonal protein is suspected even though it sits in the beta region.

    A restricted band means one clone, wherever it migrates.

  3. Check kidney function. Creatinine is 0.8 mg/dL, within its interval.

    Kidney impairment raises both light chains and shifts the ratio, so kidney function comes before the ratio.

  4. Read the ratio: 68 ÷ 14 mg/L = 4.86, above the interval, with kappa in excess.

    An abnormal ratio points to excess production of one light chain.

  5. Add serum immunofixation. The band reacts with anti-IgA and anti-kappa.

    Either abnormal screening result sends the specimen to immunofixation, which names the heavy and light chains.

  6. Note the limit on size. A beta-region peak measured by densitometry may read high, so serial measurements stay on the same method.

    Transferrin and complement migrate in the beta region and inflate a densitometric estimate there.

Report an IgA kappa monoclonal protein typed by immunofixation, with a kappa/lambda ratio of 4.86.

Your turn

Problem 1 of 3

A serum electrophoresis trace shows a broad gamma-region increase. Which description best fits?

Incorrect. A monoclonal protein forms a narrow, restricted peak or band. An increase spread across the whole gamma region comes from many clones.

Incorrect. An acute inflammatory pattern raises the alpha-1 and alpha-2 fractions. The gamma region holds the immunoglobulins.

Correct. Many immunoglobulin clones contribute to a broad gamma increase, which is common in chronic inflammation, infection, liver disease, and autoimmune disease.

Hint
  1. Picture the shape of the increase before its size.
  2. Ask how many clones it takes to widen the whole gamma region.

Review Pattern recognition

Problem 2 of 3

Serum electrophoresis shows a narrow restricted peak. Which test helps identify its immunoglobulin heavy- and light-chain type?

Incorrect. The free light-chain assay measures unbound kappa and lambda chains and calculates their ratio. It gives no heavy-chain identity for the restricted band.

Incorrect. Densitometry estimates the M-protein concentration. Naming the heavy and light chains in the band takes specific antisera.

Correct. Antisera to heavy and light chains are applied in parallel lanes, and a band that reacts with one heavy-chain and one light-chain reagent is typed. Immunosubtraction is an equivalent step on validated capillary systems.

Hint
  1. The question asks for the heavy chain and the light chain.
  2. Ask which test applies antisera to the band itself.

Review Monoclonal protein studies

Problem 3 of 3

Amyloid light-chain amyloidosis is suspected in a 59-year-old man. His serum protein electrophoresis shows no restricted peak. What does the laboratory workup call for?

Densitometry measures a peak that is already seen. No peak is present to measure.

Electrophoresis misses small monoclonal proteins and free light chains. In amyloidosis the protein is often too small to see on the trace.

Ruled out a monoclonal protein from one electrophoresis

Serum electrophoresis can miss small monoclonal proteins and free light chains. Immunofixation is more sensitive, and the free light-chain ratio detects light-chain disease. Excluding a monoclonal process on a negative electrophoresis can miss a small M-protein, especially when amyloid light-chain (AL) amyloidosis is suspected.

A suspected amyloidosis calls for all of these together with the electrophoresis. Immunofixation finds small proteins, and the light-chain ratio finds light-chain disease.

A repeat of the same test has the same blind spot. The workup adds the more sensitive tests now.

Review Monoclonal protein studies

Use it

  • A serum protein electrophoresis is ordered on Walter Brandt, 61, who has long-standing liver disease.
  • The tube received is a lithium heparin plasma tube.
  • The trace shows a narrow band between the beta and gamma regions, a low albumin and a broad gamma rise.
  • No earlier electrophoresis is on file.
Decision 1 of 3

What is the most likely source of the narrow band?

Plasma keeps its fibrinogen, which migrates near the beta-gamma boundary as a restricted band. The specimen type explains it.

A restricted band in serum would raise that question. In plasma, fibrinogen sits in the same place and has to be excluded first.

C-reactive protein forms a band on some systems only when it is very high. The plasma tube gives a simpler explanation.

Review Pattern recognition

Decision 2 of 3

What do you do with this trace?

The band may be fibrinogen. Reporting it would send the patient into a monoclonal workup on a specimen problem.

The band would stand on the report unexplained, and a reader could take it for a monoclonal protein.

The wrong specimen type caused the band, so a serum tube answers the question. Record the reason on the order.

Review Monoclonal protein studies

Decision 3 of 3

The serum trace has no narrow band. Albumin is low, and a broad gamma rise runs into the beta region. Which pattern fits?

Low albumin with a broad beta-gamma bridge is the cirrhotic pattern. Many clones raise the immunoglobulins, and IgA migrating toward beta fills the gap.

A monoclonal protein is narrow. A broad rise that bridges two regions comes from many clones.

Called a broad gamma increase monoclonal

A monoclonal protein forms a narrow, restricted peak, and a broad increase across the gamma region reflects many clones. Calling a broad increase monoclonal can report an M-protein that is not there and send the specimen into an unneeded monoclonal workup.

Nephrotic loss leaves a prominent alpha-2 region and often a low gamma. This trace has a raised gamma.

Review Pattern recognition

The clue that settled this case is the specimen type. A plasma tube carries fibrinogen, which forms a narrow band near the beta-gamma boundary. The serum specimen showed the patient's real pattern, a polyclonal rise with a beta-gamma bridge.

Keep

Sources checked