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Restricted IgG-Kappa and Free Alpha-Heavy-Chain Bands in an Asymptomatic 62-Year-Old Man.

CASE

A 62-year-old Virginia man in good health was found on routine testing to have a decreased platelet count of 124k/[micro]L (reference interval: 150-450k/[micro]L). There was no evidence of bruising or bleeding, and there was no family history of thrombocytopenia. His weight was stable, and he had no complaints of pain and no gastrointestinal, musculoskeletal, or hematological symptoms. He denied fatigue, fevers, chills, and night sweats. Past medical history included hypothyroidism and chronic obstructive pulmonary disease. Physical examination was unremarkable.

Other laboratory test results included hemoglobin of 13.8 g/dL (reference interval: 14.0-18.0 g/dL) and mean red-cell volume of 98.1 fL (reference interval: 83.0-95.0 [micro]L). Plasma potassium was 4.1 mmol/L (reference interval: 3.5-4.5 mmol/L), calcium 8.8 mg/dL (reference interval: 8.8-10.5 mg/dL), albumin 4.23 g/dL (reference interval: 3.4-5.0 g/dL), and alkaline phosphatase 61 U/L (reference interval: 40-150 U/L). Review of a peripheral blood smear found no obvious evidence of pseudothrombocytopenia. Serum ferritin was 53 ng/mL (reference interval: 20-275 ng/mL) and iron saturation was 18% (reference interval: 16%-48%).

Serum protein electrophoresis (Fig. 1A) revealed 2 restricted bands in the y region, identified by immunofixation (IFE) (3) electrophoresis (Fig. 1B) as IgG [kappa] proteins. The combined concentration of these 2 bands was <1.0 g/dL as estimated by densitometry of the stained serum protein electrophoresis gel. IFE (Fig. 1B) also revealed an [alpha]-heavy chain band in the [alpha]-2 region with no corresponding light-chain band. Immunoglobulin quantification results were as follows: IgA 107 mg/dL (reference interval: 60-263 mg/dL), IgG 1376 mg/dL (reference interval: 694-1618 mg/dL), and IgM 36 mg/dL (reference interval: 60-263 mg/dL). Serum free [kappa] light chain was 3.01 mg/dL (reference interval: 0.33-1.94 mg/ dL), and serum free [lambda] light chain was 1.85 mg/dL (reference interval: 0.57-2.63 mg/dL). The [kappa]: [lambda] free-light-chain ratio was 1.6 (reference interval: 0.26 -1.65). Urine IFE showed no monoclonal immunoglobulins.

DISCUSSION

To evaluate asymptomatic mild thrombocytopenia, the first step in the laboratory is to rule out pseudothrombocytopenia by repeating the complete blood count and reviewing the peripheral blood smear. If platelet clumping is observed, the platelet count can be repeated with blood collected in a sodium citrate tube to avoid clumping that is sometimes seen in EDTA collection tubes. The most common pathological causes for asymptomatic thrombocytopenia in outpatients include immune-mediated thrombocytopenia, liver disease, and infections with HIV and hepatitis C virus. Specific laboratory tests are available for all these except immune-mediated thrombocytopenia, which is largely a diagnosis of exclusion (1). Immune thrombocytopenia has been reported to be present in patients with multiple myeloma or monoclonal gammopathy of undetermined significance (2).

In this patient, serum protein electrophoresis revealed 2 restricted bands, which were identified by IFE as 2 IgG [kappa] monoclonal bands. Two IgG bands may, in theory, represent 2 clones of plasma cells. Commonly, however, when the light chains are identical for the 2 IgG bands, as

was the case in this patient, a finding of2 bands is considered likely to represent a single gene product with differential posttranslational processing or formation of dimers. Taken together with the patient's lack of symptoms, the low (<1.0 g/dL) concentration of the monoclonal protein and a normal ratio of free [kappa] to free [lambda] light chains, the IgG [kappa] finding suggested a diagnosis of monoclonal gammopathy of undetermined significance.

The unexpected IFE finding was of an a-heavy chain in the [alpha]-2 region, with no corresponding light chain. This finding was observed only because the presence of the [gamma]-regions bands on protein electrophoresis led to the IFE study. The [alpha]-heavy-chain band was not visualized by protein electrophoresis because it comigrated with proteins in the [alpha]-2 band of the gel. The unexpected finding led to concern for a diagnosis of heavy-chain disease.

Normal immunoglobulin molecules include 2 or more pairs of heavy chains and light chains, which are connected by disulfide bonds. In heavy-chain disease, the constant-1 domain of the immunoglobulin heavy-chain molecule, which is responsible for light-chain binding, is truncated. This results in the production of abnormal heavy chains without corresponding light chains and thus no light chain band in the electrophoretic position of the heavy chain, as was seen in this patient. The truncated heavy chains may be small enough to be cleared by the kidneys, and in some patients with heavy-chain disease the urine contains a heavy-chain peak with no monoclonal light chain. This is analogous to patients with light-chain disease, whose urine often has a light-chain concentration that is higher in urine than in serum. This patient, however, had no monoclonal a chains detected in the urine.

Cases of heavy-chain disease may involve [gamma], [alpha], or [mu] heavy chains. When an abnormal monoclonal band is identified, it is usually a broad band found in the [alpha]-2 or [beta]-globulin region. Diagnosis of heavy-chain disease requires the detection of a monoclonal heavy chain without associated light chains. The presence or absence of corresponding light chains is typically assessed by serum or urine IFE (3, 4).

The clinical findings in this patient were not suggestive of [alpha]-heavy-chain disease ([alpha]-HCD). [alpha]-HCD, also called immunoproliferative small intestinal disease, is associated classically with severe gastrointestinal symptoms, including abdominal pain and severe malabsorption manifested by chronic diarrhea and weight loss; it classically occurs in young adults (ages 20s to 30s) living in the Mediterranean region or Middle East, with a slight male dominance (5). The patient had none of these features. Common abnormal clinical chemistry findings in patients with [alpha]-heavy-chain disease include hypokalemia, hypocalcemia, hypomagnesemia, hypoalbuminemia, and increased serum alkaline phosphatase (owing to intestinal enzyme), none of which were seen in this patient. Moreover, [alpha]-HCD is rare, with fewer than 500 cases reported worldwide (6).

In IFE, which revealed the a-heavy-chain band, individual immunoglobulins are precipitated by antisera. With its lower detection limit, it is more diagnostically sensitive than protein electrophoresis. Because the immunoprecipitation relies on interaction of antibody molecules with antigen, interpretation of the IFE can be complicated by several factors:

1) Under- or overdilution of serum prevents recognition of light chains. Antigen-excess, or a "prozone" effect, may lead to a false-negative result. When a large amount of monoclonal protein is present, the immune complexes formed are small and are washed away during the washing step, leading to an absence of a band on the gel. An overdiluted sample, however, is also problematic because it may lead to a false-negative result. According to the protocol in our institution, for patients with a total immunoglobulin [less than or equal to] 2000 mg/dL, as in this patient, a 3-fold dilution of serum was used in both light-chain tracks, leaving open the possibility that a different dilution might have revealed a light-chain partner of the [alpha]-heavy chain.

2) Differing analytical performance of heavy- and light-chain reagents may lead to failure to identify light chains. Differing analytical detection limits for heavy and light chains can lead to either false-negative or false-positive results for heavy-chain disease. Moreover, some antisera reagents, especially anti-IgM and anti-IgA reagents, have cross-reactivities with other proteins. Use of a different antisera reagent is an approach to evaluate results that suggest heavy-chain disease.

3) Masking of the corresponding light-chain epitopes by bound heavy chain can prevent binding of anti-light-chain antisera to the light chains and thus prevent visualization of a light chain that is present. One approach to addressing this possibility is to treat the serum with a sulfhydryl reducing agent to make the epitopes accessible to antisera used in IFE (7). Capillary zone electrophoresis with immunosubtraction can also be tried. Another approach is to use recently developed immunoassays capable of individually quantifying isotype-specific heavy/light chain pairs (Hevylite, Binding Site). In a series of 15 patients with [gamma]-heavy-chain disease, the ratio of (G[kappa] + G[lambda]) to total IgG was <0.8 in all cases (8).

Serum from the patient presented here was analyzed by mass spectrometry (MASS-FIX) (9). The serum underwent 5 unique nanobody enrichments directed against the heavy-chain constant domains of (a) IgG, (b) IgA, and (c) IgM or against the light-chain constant domains of (d) [kappa] and (e) [lambda] immunoglobulins. Mass analysis of each of the 5 enrichments was performed by MALDITOF-MS in the positive-ion mode as described elsewhere (10). Mass spectra were generated from each enrichment and overlaid for analysis. The light-chain [LC] +2 region was examined for the presence of M-proteins (Fig. 2).

The [kappa], IgG, IgA, and [lambda] immunopurifications each showed a single sharp peak. The masses of the light-chain peaks from the IgG-enriched preparation and from the [kappa]-enriched preparation were identical, indicating the presence of an IgG [kappa] M-protein. Similarly, the masses of the IgA peak and of the [lambda] peak were identical, indicating the presence of an IgA [lambda] M-protein. Thus, the patient had a single IgG [kappa] M-protein and an intact IgA [lambda] protein. The heavy-chain regions (not shown) were inspected for the presence of a truncated heavy chain as expected in heavy-chain disease; none was found, thus providing further evidence that the patient did not have [alpha]-HCD.

FOLLOW-UP

The patient has remained free of symptoms, including gastrointestinal symptoms, in the year since the original IFE electrophoresis was done. The prognosis of patients with 2 monoclonal proteins is reportedly similar to that of patients with monoclonal gammopathies, with progression to multiple myeloma occurring in about 1% of patients each year (10).

QUESTIONS TO CONSIDER

1.What tests are appropriate to evaluate asymptomatic mild thrombocytopenia?

2. What are the expected clinical and laboratory findings in alpha-heavy-chain disease?

3. What is the likely explanation of the IFE finding of a free alpha-heavy chain in this patient?

4. What other laboratory tests would you perform to characterize the alpha-heavy-chain band found on IFE?

POINTS TO REMEMBER

* IFE results are best interpreted in the context of clinical findings.

* Alpha-heavy chain disease is rare and is usually associated with severe gastrointestinal symptoms.

* MALDI-TOF-MS analysis is an alternative to IFE for analytically sensitive detection and characterization of monoclonal immunoglobulins.

* Heavy-chain-only results often are artifacts that require additional testing to reveal the light-chain component; additional tests that may be used include sample pretreatment with a reducing agent before IFE, IFE with different antilight-chain antisera (or the same antisera with a different dilution of serum), capillary zone electrophoresis with immunosubtraction, IgA Hevylite testing, and MALDI-TOF-MS.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: D.L. Murray, W02014150170, W02015154052.

References

(1.) George JN, Arnold DM. Approach to the adult with unexplained thrombocytopenia. http://www.uptodate.com/contents/approach-to-the-adult-with-unexplained-thrombo cytopenia (Accessed October 2016).

(2.) Shimanovsky A, Alvarez Argote J, Murali S, Dasanu CA. Autoimmune manifestations in patients with multiple myeloma and monoclonal gammopathy of undetermined significance. BBA Clin 2016;6:12-8.

(3.) Bianchi G, Anderson KC, Harris NL, Sohani AR. The heavy chain diseases: clinical and pathologicfeatures. Oncology (Williston Park) 2014;28:45-53.

(4.) Wahner-Roedler DL, Kyle RA. Heavy chain diseases. Best Pract Res Clin Haematol 2005;18:729-46.

(5.) Rajkumar SV. The heavy chain diseases. https://www.uptodate.com/contents/theheavy-chain-diseases? (Accessed October 2016).

(6.) Orphanet. Prevalence and incidence of rare diseases: bibliographic data. http://www. orpha.net/orphacom/cahiers/docs/GB/Prevalence_of_rare_diseases_by_alphabetical_ list.pdf (Accessed November 2016).

(7.) Cejka J, Kithier K. IgD myeloma protein with "unreactive" light chain determinants. Clin Chem 1979;25:1495-8.

(8.) Kaleta E, Kyle R, Clark R, Katzmann J. Analysis of patients with gamma-heavy chain disease by the heavy/light chain and free light chain assays. Clin Chem Lab Med 2014;52:665-9.

(9.) Mills JR, Kohlhagen MC, Dasari S, Vanderboom PM, Kyle RA, Katzmann JA, et al. Comprehensive assessment of M-proteins using nanobody enrichment coupled to MALDI-TOF mass spectrometry. Clin Chem 2016;62:1334-44.

(10.) Mullikin TC, Rajkumar SV, Dispenzieri A, Buadi FK, Lacy MQ, Lin Y, et al. Clinical characteristics and outcomes in biclonal gammopathies. Am J Hematol 2016;91: 473-5.

Min Yu, [1] * David E. Bruns, [1] Jerry A. Katzmann, [2] Lawrence M. Silverman, [1] and David L. Murray [2]

[1] Division of Laboratory Medicine, Department of Pathology, University of Virginia, School of Medicine and Health System, Charlottesville, VA; [2] Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

* Address correspondence to this author at: Division of Laboratory Medicine, Department of Pathology, University of Virginia School of Medicine and Health System, PO Box 800168, Charlottesville, VA22908. Fax434-924-2107; e-mail my7m@virginia.edu.

Received November 4,2016; accepted February 13,2017.

DOI: 10.1373/clinchem.2016.269050

[C] 2017 American Association for Clinical Chemistry

[3] Nonstandard abbreviations: IFE, immunofixation electrophoresis; a-HCD, alpha-heavy chain disease.

Caption: Fig. 1. Serum protein electrophoresis (A). IFE electrophoresis (B). In panel A, serum proteins were separated by electrophoresis in agarose and stained with Amido black. The patient's sample is in lane 10. In panel B, separated immunoglobulins in the patient's serum were visualized by use of IFE with antisera against human immunoglobulins G, A, and M, and against human kappa and lambda light chains in lanes marked G, A, M, K, and L, respectively. In the lane marked ELP, electrophoretically separated proteins were stained with acid violet.

Caption: Fig. 2. Mass distributions of light chains in patient serum. Enriched preparations of immunoglobulins were made by use of nanobodies against IgG, IgA, IgM, and kappa and lambda light chains, as indicated. The images are from the region of the mass spectrum with 2+ charges per light chain. The masses of the light-chain peaks from IgG-enriched and from kappa-enriched preparations are identical, indicating the presence of an IgG-kappa M-protein. Similarly, the masses of the IgA peak and of the lambda peak are identical, indicating the presence of an IgA lambda M-protein.
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Title Annotation:Clinical Case Study
Author:Yu, Min; Bruns, David E.; Katzmann, Jerry A.; Silverman, Lawrence M.; Murray, David L.
Publication:Clinical Chemistry
Date:Feb 1, 2018
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