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Proteins and electrophoresis

15 min

Skills you'll build2 skills
  • 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

At the bench: Review a separation pattern and its method controls

Try first

Try first

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

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Electrophoresis patterns

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 contains most of the immunoglobulins.1 The shape of a change is more informative 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. One clone makes identical molecules with the same charge and size, so they migrate together into a narrow peak. Its size and position do not identify 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

Workup of 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

Follow the bench demonstration

Sources for this step
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

Read each decision and its explanation.

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?

TestResultReference intervalFlag
Kappa free light chains68 mg/L High3.3–19.4 mg/L3.3–19.4High
Lambda free light chains14 mg/L5.7–26.3 mg/L5.7–26.3
Kappa/lambda ratio4.86 High0.26–1.650.26–1.65High
Creatinine0.8 mg/dL0.5–1.0 mg/dL0.5–1.0

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 is 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 identifies the heavy and light chains.

  6. Note the limitation of the size estimate. 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 falsely increase a densitometric estimate there.

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

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A serum protein electrophoresis on a 72-year-old woman gives this trace. Point to the abnormal fraction.

A serum protein electrophoresis trace. Dashed lines divide it into five fractions, left to right: albumin, alpha-1, alpha-2, beta and gamma. Albumin is the tallest peak. Alpha-1 and alpha-2 are low humps, and beta has two low humps. In the gamma region one peak rises to about three-quarters of the albumin height from a base about a tenth as wide as the region, and the rest of gamma lies close to the baseline.
  1. Albumin
  2. Alpha-1
  3. Alpha-2
  4. Beta
  5. Gamma
Hint
  1. Check each fraction in turn, from albumin to gamma.
  2. Look at how wide each rise is as well as how tall.
Show the answer

One narrow peak over a low gamma background

The gamma region contains one narrow peak. Its base spans about a tenth of the region, it rises to about three-quarters of the albumin height, and the rest of gamma lies low. A peak this narrow comes from identical molecules that migrate together, so the laboratory works it up as a possible monoclonal protein. Albumin, alpha-1, alpha-2 and beta keep their usual shapes.

Review Pattern recognition

TestResultReference intervalFlag
Kappa free light chains7.6 mg/L3.3–19.4 mg/L3.3–19.4
Lambda free light chains94 mg/L High5.7–26.3 mg/L5.7–26.3High
Kappa/lambda ratio0.08 Low0.26–1.650.26–1.65Low
Creatinine0.8 mg/dL0.5–1.0 mg/dL0.5–1.0

Specimen: H 4, L 10, I 1. Serum, gold top

Problem 2 of 3 · Hints available

A 72-year-old woman's serum trace shows one peak in mid-gamma. Its base spans about a tenth of the gamma region, and the rest of gamma lies low. Serum free light chains were run on the same specimen. What does the laboratory do next?

One clone makes identical molecules with the same charge and size, so they migrate together into a thin peak. The ratio of 7.6 ÷ 94 mg/L = 0.08 is below its interval with lambda in excess, and creatinine is within its interval. Either finding sends the specimen to immunofixation, which confirms the protein and identifies its heavy and light chains.

Many clones make immunoglobulins of many charges, which spread across the whole gamma region. This peak covers about a tenth of it.

The specimen line shows a gold-top serum tube, so fibrinogen does not explain the peak. Fibrinogen also runs near the beta-gamma boundary, and this peak is in mid-gamma.

Electrophoresis alone cannot confirm or type the band. Immunofixation confirms and types it before a monoclonal protein is reported.

Hint
  1. Ask how many different molecules it takes to make a peak this thin.
  2. Read the tube type in the specimen line before you consider fibrinogen.

Review Monoclonal protein studies

TestResultReference intervalFlag
Kappa free light chains14.2 mg/L3.3–19.4 mg/L3.3–19.4
Lambda free light chains11.8 mg/L5.7–26.3 mg/L5.7–26.3
Kappa/lambda ratio1.200.26–1.650.26–1.65
Creatinine0.9 mg/dL0.5–1.0 mg/dL0.5–1.0

Specimen: H 2, L 8, I 1. Serum, gold top, centrifuged 40 minutes after collection

Problem 3 of 3 · On your own

A 58-year-old woman has a serum protein electrophoresis and serum free light chains ordered together. No earlier electrophoresis is on file. Point to the fraction that determines the next step.

A serum protein electrophoresis trace. Dashed lines divide it into five fractions, left to right: albumin, alpha-1, alpha-2, beta and gamma. Albumin is the tallest peak. Alpha-1 and alpha-2 are low humps. Beta has two low humps, and a sharp peak about a quarter of the albumin height rises at its slow end. Gamma is a low hump that spans the whole region.
  1. Albumin
  2. Alpha-1
  3. Alpha-2
  4. Beta
  5. Gamma
Show the answer

One small narrow band at the slow end of beta

A small narrow band rises at the slow end of beta. The specimen is serum, so fibrinogen does not explain it. A restricted band alone sends the specimen to serum immunofixation. The normal free light-chain ratio of 1.20 does not change that. Densitometry reads a beta-region band less accurately because transferrin and complement migrate with it.

Review Pattern recognition

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • A serum protein electrophoresis is ordered on Perry Sites, 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 migrates to the same place and must 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 start a monoclonal protein workup because of a specimen problem.

The band would appear on the report without explanation, and a reader could mistake it for a monoclonal protein.

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

Review Ordering, identification, and acceptability

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 plasma specimen explained the band. Plasma contains fibrinogen, which forms a narrow band near the beta-gamma boundary. The serum specimen showed the patient's true pattern, a polyclonal rise with a beta-gamma bridge.

For reference

Sources checked