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Immunofixation 

  • Author: Anastasios Dimou, MD; Chief Editor: Eric B Staros, MD  more...
 
Updated: Dec 11, 2015
 

Reference Range

Immunofixation consists of an electrophoresis phase and a fixation phase.[1]

Serum or urine immunofixation negative for a monoclonal protein or a polyclonal pattern is considered to be normal. CSF immunofixation that does not reveal oligoclonal bands is also considered normal. A polyclonal immunoglobulin pattern in the serum or urine immunofixation is considered to be nonspecific.

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Interpretation

Immunofixation can either reveal a normal pattern or identify a monoclonal protein or a polyclonal immunoglobulin pattern.

A normal result includes a darker immunoglobulin G (IgG) lane, a lighter immunoglobulin A (IgA), an absent immunoglobulin M (IgM), and a denser kappa compared to lambda lane, with ratio of 2:1. In a normal result, the lanes are broad and there is a gradual and smooth reduction in the color density toward the edges of the lane with no narrow dense band with sharp borders identified within the lane.

In some cases, all the lanes are homogeneously darkened to the same degree. This pattern represents the presence of polyclonal immunoglobulin. Again, the lanes are broad and the transition to the lane borders is smooth. In this case, the IgM lane, which is normally absent, is broad with smooth borders.

When a narrow band with sharp borders can be identified, it implies the presence of a monoclonal protein (see image below).

On the left a serum protein electrophoresis (SPEP) On the left a serum protein electrophoresis (SPEP) of a patient with a monoclonal band in the gamma region is shown. Subsequent immunofixation with electrophoresis identified the monoclonal protein as an IgG antibody (paraprotein) with a lambda light chain (arrow).

Finally, when several bands with sharp borders are seen after immunofixation in cerebrospinal fluid (CSF) but not in serum, the result is reported as positive for oligoclonal bands.

The identification of a monoclonal immunoglobulin is helpful in the diagnosis of the following conditions[2] :

The identification of oligoclonal bands in CSF is helpful in the diagnosis of multiple sclerosis.[9]

In addition, immunofixation can be used to monitor therapy in plasma cell dyscrasias (ie, multiple myeloma and Waldenstrom macroglobulinemia). If the monoclonal protein level decreases or is undetectable after chemotherapy, it might indicate a response to treatment. On the contrary, a persistent monoclonal protein despite treatment is a sign of refractory disease.[10]

The presence of oligoclonal bands in CSF but not in the serum, given their intrathecal production, is helpful in the diagnosis of multiple sclerosis. Other inflammatory conditions of the CNS can present with oligoclonal bands in CSF resulting from intrathecal production of antibodies.[11] HIV-related encephalitis, neurosyphilis, Lyme meningoencephalitis, neurosarcoidosis, acute disseminated encephalomyelitis (ADEM), systemic lupus erythematosus (SLE), Sjögren syndrome, subacute sclerosing panencephalitis, CNS malignancy, neuromyelitis optica, and transverse myelitis can all reveal oligoclonal banding in CSF immunofixation, precluding the use of immunofixation for the detection of oligoclonal bands as a sole diagnostic technique in multiple sclerosis.

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Collection and Panels

The specimen can be serum, urine, or CSF.

Serum

Sample: Serum (whole blood or plasma is inappropriate, as fibrinogen may alter the results)

Tube: Red top (see image below)

Red top tube for the collection of serum used. Ima Red top tube for the collection of serum used. Image courtesy of Rush University.

Technique: Venipuncture

Sample size: 4 mL

Urine

Sample: 24-hour urine collection, concentrated

Collection in a container: A plastic collection container is used. The patient urinates each time into a clean dry vessel and then pours the urine in the plastic container, which is kept at a cold temperature (eg, in the refrigerator).

Technique: Collection starts early in the morning after the first morning urine has been voided and continues for the subsequent 24 hours, including the first morning urine of the following day.

Sample size: Every drop of urine within 24 hours is included in the sample.

Cerebrospinal fluid

Sample: CSF

Tube: CSF conical tube

Technique: Lumbar puncture

Sample size: 1 mL

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Background

Description

Immunofixation consists of an electrophoresis phase and a fixation phase.[1] In the former, the serum is applied to an agarose gel, and the negatively charged serum proteins move toward the cathode under the influence of an electric current. The speed of movement is dictated by their charge. During the fixation phase, antiserum containing anti-IgG, anti-IgA, anti-IgM, anti-light chain kappa, or anti-light chain lambda is inoculated with the serum proteins. If a monoclonal protein is present, a precipitant will form at this phase. Finally, the sample is washed to remove the proteins that do not precipitate and then stained, destained, and dried.

In most cases in which a monoclonal protein is present, it shows as a narrow band at the IgG lane in combination with a monoclonal band at the kappa or lambda lane. IgA or IgM bands are less common. Finally, isolated light chain bands, either kappa or lambda, represent more commonly pure kappa or lambda light chain gammopathies or, less commonly, immunoglobulin D (IgD) or immunoglobulin E (IgE) gammopathies. Given the dismal impact on prognosis in the latter scenario,[12] an additional assay for the identification of IgD or IgE must be performed. If the result is negative, it is safe to conclude that a pure kappa or lambda light chain is present.[10]

Indications/Applications

Primary screening for plasma cell dyscrasias: No studies have addressed the role of serum or urine immunofixation in screening of the general population for multiple myeloma and related disorders. Therefore, such an approach cannot be currently recommended.[9]

Surveillance among patients with MGUS or smoldering (asymptomatic) multiple myeloma: Immunofixation of the urine and serum should be included in a battery of tests after 2-3 months following diagnosis of smoldering myeloma, then every 4-6 months for 1 year, and, finally, every 6-12 months if the results are stable. Surveillance for multiple myeloma in patients with MGUS and favorable prognostic factors (ie, low levels of monoclonal protein and IgG type) should include monitoring at 6 months and then every 2-3 years thereafter. For patients with MGUS and high risk for progression to multiple myeloma, surveillance should be performed at 6 months and then yearly thereafter.[13, 14]

Diagnosis of plasma cell dyscrasias:[9] Immunofixation is clearly indicated upon clinical or laboratory evidence of a plasma cell dyscrasia for the diagnosis of multiple myeloma, Waldenstrom macroglobulinemia, or amyloid light-chain (AL) amyloidosis. Typically, the monoclonal protein is discovered, and workup then ensues. These conditions should be promptly diagnosed and treated. In particular, when serum and urine protein electrophoresis results are negative, immunofixation is indicated as a more sensitive test if the clinical suspicion remains high. In addition, when protein electrophoresis assays are positive for a monoclonal protein, serum and urine immunofixation are indicated for the appropriate identification of the immunoglobulin and the corresponding light chain. However, most patients with a monoclonal protein will be finally diagnosed with MGUS rather than any of the malignant plasma cell dyscrasias for which treatment is recommended.

Follow-up of multiple myeloma, Waldenstrom macroglobulinemia, and AL amyloidosis: Immunofixation of urine and serum samples should be performed in all patients with multiple myeloma, Waldenstrom macroglobulinemia, and AL amyloidosis every 3 months after the completion of treatment in order to evaluate for response or to document a relapse.[15]

Diagnosis of multiple sclerosis: Immunofixation in CSF for the detection of oligoclonal bands is indicated every time there is suspicion for multiple sclerosis. However, the presence of oligoclonal bands does not necessarily confirm the diagnosis, as other conditions can present with oligoclonal bands in the CSF.

Considerations

A negative urine or serum immunofixation result does not always rule out a plasma cell dyscrasia, as a nonsecretory or oligosecretory multiple myeloma can present with a negative immunofixation finding in both the urine and the serum. A serum free light chain ratio is indicated in the case of a negative immunofixation result when the suspicion for multiple myeloma is still high.[10, 16]

A 24-hour concentrated urine sample is appropriate for urine immunofixation. This sample is often difficult to collect, and suboptimal collection might alter the result. A concomitant measurement of the creatinine in the 24-hour urine sample for correction of sampling errors has been proposed.[17]

Detection of a paraprotein by immunofixation is possible in lymphomas.[14]

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Contributor Information and Disclosures
Author

Anastasios Dimou, MD Resident Physician, Department of Internal Medicine, Albert Einstein Medical Center

Disclosure: Nothing to disclose.

Coauthor(s)

John C Leighton, Jr, MD, FACP Clinical Associate Professor, Jefferson Medical College of Thomas Jefferson University; Chief, Division of Medical Oncology, Jeanes Hospital; Associate Director, Associate Chief, Divisions of Hematology/Medical Oncology, Albert Einstein Cancer Center; Associate Member Kimmel Cancer Center, Thomas Jefferson Hospital

John C Leighton, Jr, MD, FACP is a member of the following medical societies: American College of Physicians, American Medical Association, American Society of Hematology, American Society of Clinical Oncology

Disclosure: Nothing to disclose.

Chief Editor

Eric B Staros, MD Associate Professor of Pathology, St Louis University School of Medicine; Director of Clinical Laboratories, Director of Cytopathology, Department of Pathology, St Louis University Hospital

Eric B Staros, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology

Disclosure: Nothing to disclose.

References
  1. Cawley LP, Minard BJ, Tourtellotte WW, et al. Immunofixation electrophoretic techniques applied to identification of proteins in serum and cerebrospinal fluid. Clin Chem. 1976. 22:1262-8.

  2. Guido Tricot. Multiple Myeloma. Ronald Hoffman, Leslie E. Silberstein, Sanford J. Shattil, Bruce Furie, Philip McGlave. Hematology: Basic Principles and Practice. 5th. Philadelphia, PA, USA: Elsevier Churchill Livingstone; 2008. chapter 87.

  3. Monoclonal Gammopathy of Uncertain Origin.

  4. Multiple Myeloma.

  5. Beaumont-Epinette MP, Moreau C, Besnard S, Latute F, Collet N, Sebillot M, et al. Heavy/light chain specific immunoglobulin ratios provides no additional information than serum proteins electrophoresis and immunofixation for the diagnosis and the follow-up of intact immunoglobulin multiple myeloma patients. Pathol Biol (Paris). 2015 Aug 26. [Medline].

  6. Yang G, Geng C, Chen W. Clinical characteristics of a group of patients with multiple myeloma who had two different λ light chains by immunofixation electrophoresis: A retrospective study from a single center. Exp Ther Med. 2015 May. 9 (5):1895-1900. [Medline].

  7. Waldenstrom macroglobulinemia.

  8. Immunoglobulin-related amyloidosis.

  9. http://emedicine.medscape.com/article/1146199-overview.

  10. Dimopoulos M, Kyle R, Fermand JP, et al. Consensus recommendations for standard investigative workup: report of the International Myeloma Workshop Consensus Panel. Blood. 2011. 117:4701-5.

  11. Cavuoti D, Baskin L, Jialal I. Detection of oligoclonal bands in cerebrospinal fluid by immunofixation electrophoresis. Am J Clin Pathol. 1998. 109:585-8.

  12. Morris C, Drake M, Apperley J, et al. Efficacy and outcome of autologous transplantation in rare myelomas. Haematologica. 2010. 95:2126-33.

  13. Kyle RA, Durie BG, Rajkumar SV, et al. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management. Leukemia. 2010. 24:1121-7.

  14. Bird J, Behrens J, Westin J, et al. UK Myeloma Forum (UKMF) and Nordic Myeloma Study Group (NMSG): guidelines for the investigation of newly detected M-proteins and the management of monoclonal gammopathy of undetermined significance (MGUS). Br J Haematol. 2009. 147:22-42.

  15. NCCN practice guidelines.

  16. Sasson SC, McGill K, Wienholt L, Carr A, Brown DA, Kelleher AD, et al. Comparison of the Freelite serum free light chain (SFLC) assay with serum and urine electrophoresis/immunofixation and the N Latex FLC assay. Pathology. 2015 Oct. 47 (6):564-9. [Medline].

  17. Kaplan JS, Horowitz GL. Twenty-four-hour Bence-Jones protein determinations: can we ensure accuracy?. Arch Pathol Lab Med. 2011. 135:1048-51.

 
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On the left a serum protein electrophoresis (SPEP) of a patient with a monoclonal band in the gamma region is shown. Subsequent immunofixation with electrophoresis identified the monoclonal protein as an IgG antibody (paraprotein) with a lambda light chain (arrow).
Red top tube for the collection of serum used. Image courtesy of Rush University.
 
 
 
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