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Interference in protein electrophoresis

Last author update: 30 August 2023
Last staff update: 1 September 2023

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PubMed Search: Interference in protein electrophoresis

Ahsan Farooq, M.D.
Yusheng Zhu, Ph.D.
Page views in 2023 to date: 85
Cite this page: Farooq A, Zhu Y. Interference in protein electrophoresis. website. Accessed September 21st, 2023.
Definition / general
  • Interference causes a clinically significant difference in the assay result due to another component or property of the sample
  • For serum electrophoresis, it can be exogenous or endogenous
Essential features
  • Interference in serum electrophoresis can cause misinterpretation, leading to improper patient management
  • Immunofixation, immunosubtraction and clinical assessment can mitigate most of the interferences
Factors interfering with electrophoresis
  • Analytical interferences
    • Important causes of laboratory error
    • Clinically significant consequences, including over or undertreatment and misdiagnosis
Types of interferences

Endogenous Exogenous
  • Fibrinogen
  • Hemolysis
  • Antianimal antibodies
  • Polyclonal increases in IgG4: monoclonal gammopathy mimicry
  • Contrast dyes
  • Antifungals and antibiotics
  • Gelatin based plasma substitutes
  • Hydroxocobalamin
  • Monoclonal antibody therapies

  • Fibrinogen is not normally present in serum specimens if adequate preanalysis is performed
  • Fibrinogen may be present in serum
    • Disorders of coagulation
    • Anticoagulation therapy
    • When a plasma sample is erroneously provided instead of serum
  • When serum protein electrophoresis (SPEP) is performed on these samples, fibrinogen migrates to the β / γ region and it may be misinterpreted as a monoclonal immunoglobulin
  • Following immunofixation electrophoresis (IFE) the apparent monoclonal protein is absent, this combined with the characteristic localization of the band in the β / γ region should establish the identity of this band as fibrinogen
  • Although not routinely performed in diagnostic practice, IFE with antifibrinogen antibodies provides solid proof that the band is indeed fibrinogen
  • Commercial sources of antiserum used for IFE may contain antibodies with specificity towards proteins that are typically absent from serum, such as fibrinogen, which can produce clinically misleading results
  • For proper M protein analysis of these rare cases, either another blood sample should be obtained or the fibrinogen should be selectively eliminated prior to SPEP (Clin Chim Acta 2006;368:192)
  • See image 1
  • Reference: Clin Biochem 2018;51:72
  • The 2 main interference mechanisms are spectral interference from high concentrations of hemoglobin and direct release of analytes from red blood cells
  • Hemolysis can be broadly divided into either prephlebotomy (in vivo hemolysis) or during phlebotomy (in vitro) causes
  • In SPEP, hemoglobin and hemoglobin complexes that show up as discrete bands in the α2 and β regions may be misinterpreted as monoclonal proteins
  • This is easily avoided by identifying hemolyzed specimens prior to interpretation or by reflexing to IFE to confirm the presence of any abnormal bands
  • Reference: Clin Biochem 2018;51:72
Human antianimal antibodies (HAAAs)
  • HAAAs are typically formed after exposure to animal proteins (either through occupational exposure or medical therapy)
  • The most common HAAAs encountered are human mouse antibodies (HAMAs)
  • Disorders affected by HAMAs include cases of organ transplantation, cancer, inflammation, infection and kidney disease, etc. (Clin Chem 1999;45:942)
  • HAMAs interfere with serum IFE and may falsely appear as monoclonal proteins
  • When SPEP / IFE in isolation are not sufficient to drive decision making, clinical awareness of this issue is the best course of action
  • Reference: Clin Biochem 2018;51:72
IgG4 related disease (IgG4 RD)
  • IgG4 RD is a syndrome of unknown etiology that have lymphoplasmacytic infiltrate enriched with polyclonal IgG4 positive plasma cells, a variable degree of fibrosis and elevated serum concentrations of polyclonal IgG4 (N Engl J Med 2012;366:539)
  • The focal band detected by SPEP from patients with IgG4 RD can be confirmed as polyclonal by IFE or immunosubstraction in cases where the connecting light chains are both kappa and lambda
  • Rarely, significant kappa:lambda skewing can occur in the polyclonal IgG4 fraction; in such cases, clinical awareness and isoelectric focusing can be helpful to differentiate between polyclonal and monoclonal IgG
  • See image 2
  • Reference: Clin Biochem 2018;51:72
Contrast dyes
  • Contrasts dyes, used in imaging testing, interfere with capillary zone electrophoresis (CZE)
  • Migrate to α2 globulin fraction or less frequently the β2 globulin fraction
  • Reflex testing to IFE or immunotyping can confirm absence of a monoclonal component
  • Clinicians should be aware that imaging using contrast dyes should be done after blood collection
  • Reference: Clin Biochem 2018;51:72
Antifungals and antibiotics

Drugs Peak zones on CZE
5 fluorocytosine (5FC) Cathodal region of γ globulin fraction
Ceftriaxone Anodal region of the prealbumin fraction
Sulfamethoxazole Albumin peak
Piperacillin with tazobactam α2 β1 globulin region

  • These interferences are more likely to occur when blood samples are collected closer to the time of perfusion, especially in the context of renal failure and drug accumulation
  • Reflex testing to IFE or immunotyping can confirm the absence of a monoclonal component
  • See image 3
  • Reference: Clin Biochem 2018;51:72
Gelatin based plasma substitutes
  • Gelatin based plasma substitutes can increase the γ globulin fraction, with a polyclonal pattern shifted to the β2 globulin fraction, simulating a βγ block in CZE
  • Reflex testing to IFE or immunotyping can confirm absence of monoclonal component
  • SPEP is rarely performed in an emergency context of infusion of gelatin based substitutes; together with their short half life (~2.5 hours), the risk of this type of interference is uncommon
  • Reference: Clin Biochem 2018;51:72
  • Interference with SPEP concerning the α1 globulin fraction
  • Hydroxocobalamin is the standard therapy for cyanide poisoning
  • Interference will be much easier to detect on SPEP as hypoproteinemia is very frequently associated with severe burns
  • Reflex testing to IFE or immunotyping can confirm absence of a monoclonal component
  • Reference: Clin Biochem 2018;51:72
Monoclonal therapies
  • Many of the available monoclonal therapeutics can appear as monoclonal IgG kappa by IFE
  • Of the monoclonal drugs used for non-plasma cell dyscrasia based diseases, only rituximab and bevacizumab in therapeutic doses produced a visible M protein on SPEP / IFE
  • Drugs like siltuximab, daratumumab and elotuzumab (all IgG kappa monoclonal antibodies) can appear as a small M protein, most often in concentrations of no more than 1 g/L or 0.1 g/dL, when used in therapeutic doses (Clin Chem 2010;56:1897)
  • The main concern with the presence of these therapies is misclassification of disease response as having a very good partial response (VGPR) as opposed to a complete response
  • Practically applicable mitigation response to monoclonal therapy varies with subtype of clonal immunoglobulin produced by plasma cell neoplasm

IgG plasma cell neoplasm Plasma cell neoplasm other than IgG
  • When starting therapy, laboratory might not be able to separate therapeutic monoclonal bands and should report the detection and quantification of the M protein as standard with the additional information that the quantification might be influenced by interference from the therapeutic drug
  • When evaluating treatment response close to complete response (M protein < 2 g/L), laboratory can add a specific assay, e.g., the daratumumab specific immunofixation electrophoresis reflex assay (DIRA), to mitigate the potential comigration of the therapeutic drug with the malignant M protein
  • Another option that laboratories can use is to ask for a report from pharmacy indicating each instance when a patient receives monoclonal therapy susceptible to interfere with IFE
  • When starting therapy, laboratory should report the detection of the malignant M protein as standard with the supplementary information of a second M spike / M band at the expected electrophoretic position of the therapeutic drug
  • Therapeutic M protein should also be quantified at initial recognition and should under normal circumstances not exceed 1 g/L
  • As malignant M protein and the known therapeutic M protein are of different Ig subtype, the evaluation of treatment response should not be hindered by the phenomenon of comigration of the M proteins

  • Daratumumab specific immunofixation electrophoresis reflex assay (DIRA) using an antibody targeting daratumumab to alter the migration of daratumumab during electrophoresis should be utilized
  • Reference: Clin Biochem 2018;51:72
Monoclonal immunoglobulin rapid accurate molecular mass (miRAMM) assay
  • miRAMM assay uses affinity purification followed by microflow LC-ESI-Q-TOF MS to determine the exact mass of monoclonal immunoglobulin chains
  • This method is effectively immune to interferences including those from a monoclonal therapy because it will resolve each immunoglobulin peak by its unique mass and allow for it to be identified and differentiated (Methods 2015;81:56)
  • Reference: Clin Biochem 2018;51:72
Diagrams / tables

Contributed by Ahsan Farooq, M.D. and Yusheng Zhu, Ph.D.


Patient with IgG4 RD

Patient with IgG4 RD

Patient on 5 fluorocytosine

Patient on 5 fluorocytosine

Multiple myeloma patient on daratumumab

Multiple myeloma patient on daratumumab

Board review style question #1
Which of the following statements regarding electrophoreses after initiation of monoclonal therapy is true?

  1. Electrophoresis studies should be done before administration of monoclonal antibodies
  2. Immunofixation electrophoresis (IFE) will show a peak in the anodal part of the γ region with daratumumab
  3. Short time interval between serum protein electrophoresis (SPEP) and drug administration should decrease chances of interference
  4. SPEP showing a peak of 300 mg/dL will always mean a very good partial response (VGPR)
Board review style answer #1
A. Electrophoresis studies should be done before administration of monoclonal antibodies. Before each dose, the concentration of therapeutic monoclonal will be lowest and thus give less interference. Answer C is incorrect because with longer intervals between the dose and testing, interference will be less. Answer B is incorrect because daratumumab usually migrates to the cathodal part of the γ region. Answer D is incorrect because a value of < 1 g/dL can be due to therapeutic monoclonal therapy.

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Reference: Interference in protein electrophoresis
Board review style question #2

Serum electrophoresis of a 60 year old man identifies a peak. Immunofixation electrophoresis (IFE) is performed. Which most the following is most consistent with the findings?

  1. Patient has multiple myeloma
  2. Plasma sample of patient with macrocytic anemia
  3. Serum sample obtained immediately after monoclonal therapy
  4. Serum sample of patient with IgG related disease
Board review style answer #2
B. Plasma sample of patient with macrocytic anemia. Plasma contains fibrinogen; plasma sample was erogenously run instead of serum. Answer C is incorrect because IFE shows no monoclonal antibody. Answer D is incorrect because the band is in β / γ region. Answer A is incorrect because IFE shows no monoclonal antibody.

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Reference: Interference in protein electrophoresis
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