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Unusual increased [beta]-globulins in an elderly patient.

CASE DESCRIPTION

A 90-year-old man who had a history of transurethral prostate resection for benign prostatic hyperplasia was admitted for left hip pain. A hip radiography examination revealed a large osteolytic lesion in the left greater trochanter, and a bone scintigraphy evaluation showed increased activity in the same area. Because of the patient's history, metastasis from prostate carcinoma was suspected, and bone biopsies were performed the same week. Ten days later, the patient was hospitalized in the emergency department after a fall that fractured the femoral neck. At admission, plasma sodium, chloride, and potassium concentrations were all within their respective reference intervals. The plasma concentration of total protein was 5.0 g/dL (50 g/L) [reference interval, 6.0-8.0 g/dL (60-80 g/L)], and that of albumin was 1.5 g/dL (15 g/L) [reference interval, 3.0-4.5 g/dL (30-45 g/L)]. The albumin-corrected calcium concentration was 10.8 mg/dL (2.69 mmol/L) [reference interval, 9.0-10.6 mg/dL (2.25-2.65 mmol/L)]. Hematology tests showed mild normocytic (mean corpuscular volume, 95.1 fL; reference interval, 80-97 fL]), aregenerative [reticulocyte count, 54 X [10.sup.3]/[micro]L (54 X [10.sup.9]/L); reference interval, 25-80 X [10.sup.3]/[micro]L (25-80 X [10.sup.9]/L) anemia, with a low hemoglobin concentration [9.9 g/dL (99 g/L); reference interval, 13.0-17.0 g/dL (130-170 g/L)] and leukopenia [2.5 X [10.sup.3]/[micro]L (2.5 X [10.sup.9]/L); reference interval, 4.0-10.0 X [10.sup.3]/[micro]L (4.0-10.0 X [10.sup.9]/L)]. The plasma creatinine value was 1.1 mg/dL(97/[micro]mol/L) [reference interval, 0.71-1.20 mg/dL (62-[10.sup.6] /[micro]mol/L)], and the urea nitrogen concentration was 35.0 mg/dL (12.5 mmol/L) [reference interval, 7.8-19.6 mg/dL (2.8-7.0 mmol/L)]. The urine protein value was 540 mg/24 h (reference interval, 0-150 mg/24 h). Urine protein electrophoresis (UPEP) [4] showed the presence of an unusual spike in the [beta] region (Fig. 1A). We measured an increased urine concentration of free [kappa] light chain [1.4 mg/dL (14 mg/L); reference interval, 0-0.2 mg/dL (0-2 mg/L)], along with a normal concentration of urine [[beta].sub.2]-microglobulin.

The patient underwent surgical repair of his left femur. In the meantime, our laboratory asked for serum to complete the investigation of the abnormal UPEP pattern. Investigations of the serum sample revealed a decreased albumin concentration [1.45 g/dL (14.5 g/L); reference interval, 3.0-4.5 g/dL (30-45 g/L)]. Immunoglobulin quantification revealed an increased IgG value [2950 mg/dL (29.5 g/L); reference interval, 700-1000 mg/dL (7-10 g/L)]. IgA and IgM were within their respective reference intervals. Serum protein electrophoresis (SPEP) showed an increased a1 region, along with decreased albumin and y regions. Moreover, we found an increased [beta] region (19.1 g/L, quantified from the electrophoresis trace and the total protein concentration), along with a loss of separation between the {1 and {2 regions because of a narrow spike (Fig. 1B). With a normal concentration of [beta]-globulins of approximately 300 mg/dL (3 g/L), we estimated the monoclonal protein concentration at approximately 1600 mg/dL (16 g/L). Serum immunofixation was performed with antibodies specific for heavy chains (G, A, M) and light chains ([kappa], [lambda]). A band was present in the IgG lane with a [beta] electrophoretic mobility, with no corresponding band for the light chain (Fig. 1C). Serum free light chains included a decreased free [kappa] chain [0.06 mg/dL (0.6 mg/L); reference interval, 0.33-1.94 mg/dL (3.3-19.4 mg/L)], a decreased free [lambda] chain [0.045 mg/dL (0.45 mg/L); reference interval, 0.57-2.63 mg/dL (5.7-26.3 mg/L)], and a normal [kappa]/[lambda] ratio of 1.33 (reference interval, 0.26-1.65). The results of IgG subclass quantification were as follows: IgG1, 768 mg/dL (7.68 g/L) [reference interval, 500-800 mg/dL (5-8 g/L)]; IgG2, <9.0 mg/dL (<0.09 g/L) [reference interval, 90-300 mg/dL (0.9-3 g/L)]; IgG3,23.0 mg/dL (0.23 g/L) [reference interval, 10-80 mg/dL (0.1-0.8 g/L)]; and IgG4, 1.0 mg/dL (0.01 g/L) [reference interval, 10-60 mg/dL (0.1-0.6 g/L)].

[FIGURE 1 OMITTED]

DISCUSSION

Abnormal SPEP and UPEP patterns representing potential monoclonal peaks have to be characterized with additional tests. It is necessary to differentiate analytical artifacts from the presence of a monoclonal gammopathy. Interferences can arise from the presence of radiopaque imaging agents or antibiotics. We verified the absence of fibrinogen (found with plasma tube or incomplete blood clotting) and hemoglobin in the patient's serum (no hemolysis in the sample), which are the most common confounding factors leading to apparent monoclonal peaks in the [beta]-globulin region of an SPEP analysis. Confirmation of a monoclonal protein can be reliably done only by immunofixation or immunotyping. Serum immunofixation electrophoresis revealed the presence of a monoclonal [gamma] band with no corresponding light chain (Fig. 1C), a finding consistent with the diagnosis of [gamma]-heavy chain disease ([gamma]-HCD).

HCDs are rare proliferative B-cell disorders characterized by the production of monoclonal proteins with incomplete heavychain components and without associated light chains (1). HCDs involving the 3 main immunoglobulin classes (IgA, IgM, IgG) have been described. The most frequent HCD is [alpha]-HCD, whereas [mu]w-HCD is very rare; [gamma]-HCD is of intermediate incidence. The median age at the time of [gamma]-HCD diagnosis is approximately 60 years. Fatigue, weakness, and lymphadenopathy are the most frequent initial symptoms, and hepato- and splenomegaly are the most common physical findings, which are often accompanied by anemia. The clinical course can range from an asymptomatic state to a rapid progression leading to death within a few weeks (2). [gamma]-HCD can also be associated with other lymphoplasma cell proliferative diseases or autoimmune disorders. Because of the strong clinical heterogeneity and the varying SPEP patterns with an inconstant presence of a monoclonal peak, [gamma]-HCD is thought to be an underdiagnosed disease.

At admission, the patient's hip pain and subsequent fractured femur neck suggested prostate cancer; however, the associated proteinuria and anemia also suggested a gammopathy. With only these clinical features, the [gamma]-HCD diagnosis relied on laboratory tests. In accordance with other reports, a monoclonal peak with a [beta] mobility was present after both SPEP and UPEP. This feature is the most common of [gamma]-HCD electrophoresis patterns (3), although this peak can be found in a-globulin or [gamma]-globulin regions.

[FIGURE 2 OMITTED]

The concentrations of serum free light chains were very low, supporting the diagnosis of [gamma]-HCD. The presence of polyclonal free [kappa] light chains in the patient's urine (Fig. 2, lane 11) seemed independent of [gamma]-HCD, because k light chains in the urine are due to both physiological degradation of polyclonal immunoglobulins and renal excretion of serum free [kappa] light chains (Fig. 2, lane 5). Furthermore, such urine free light chains cannot explain the total proteinuria we measured. To investigate which protein was mainly responsible for the monoclonal peak observed after UPEP (4), we performed co-immunofixation of both serum and urine proteins (Fig. 2). Using [gamma] antiserum, we found a monoclonal component in serum with the same [beta] mobility as that of urine, confirming the presence of the monoclonal IgG heavy chain without the corresponding light chain.

[gamma]-HCD proteins have a lower molecular weight than normal IgGs (4). Effectively, this feature is due not only to the absence of light chains but also to the presence of deletions in either the variable or the constant domain. These deletions can produce broad truncated regions (5) that can reach 50%-60% of the length of a typical heavy chain (4). Because of their small molecular size, these [gamma]-HCD monoclonal proteins are usually present in both serum and urine (6),as we observed in our case. To assess the molecular weight of the [gamma]-HCD protein, we purified it and subjected the fraction to SDS-PAGE (data not shown). A normal y heavy chain has a molecular weight of 51 kDa, and the size of the truncation of the patient's monoclonal [gamma] heavy chain was approximately 25%, because we estimated the protein's molecular weight to be between 37.5 and 40 kDa from the molecular weight markers and standard purified IgGs. The markedly increased IgG1 value and the very low concentrations of the 3 other subtypes probably identifies the M protein, although various explanations are possible for the discrepancy between the total-IgG measurement and the sum of the concentrations of the 4 IgG subtypes. First, nephelometric techniques can suffer from antigen excess (7) (also known as the high-dose hook effect), but an appropriate serial-dilution assay excluded this possibility. The observed underestimation of the monoclonal protein could then be explained by self-aggregation of truncated heavy chains. This self-aggregation is well recognized for IgM and IgA, but cases of covalent polymers of IgG have also been described (8). It is also possible that the antisera we used for typing the subclasses could have contained antibodies that recognized domains that were absent from the patient's truncated IgG, because these antisera were obtained from sheep that had been immunized against human IgG1, IgG2, IgG3, and IgG4.

Because an association of [gamma]-HCD with other hematologic malignancies, such as malignant lymphoma and other lymphoplasma cell proliferative disorders, has been described, our patient was referred to a hematologist-oncologist. Analysis of a bone marrow biopsy revealed a double population of B lymphocytes. The first clone was of small size, expressed IgM [kappa], and was negative for CD5, CD23, and CD10. These results are consistent with B-cell non-Hodgkin lymphoma. The second clone of B lymphocytes expressed an IgG without a corresponding light chain, which is consistent with our earlier findings. The pathology laboratory examined the femoral biopsy sample and reported the presence of undifferentiated cells of unknown origin. These cells were not of hematologic lineage, and a prostate origin was thus suspected. A computed tomography examination of the chest and pelvis revealed numerous nodules in the lungs, liver, and bones, suggesting metastatic dissemination. Unfortunately, the diagnosis of [gamma]-HCD had no impact on the clinical course and the treatment of the patient, because his health deteriorated rapidly and he died 3 weeks later.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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: No authors declared any potential conflicts of interest.

References

(1.) Franklin EC, Lowenstein J, Bigelow B, Meltzer M. Heavy chain disease--a new disorder of serum gamma-globulins: report of the first case. Am J Med 1964;37:332-50.

(2.) Kyle RA, Greipp PR, Banks PM. The diverse picture of gamma heavy-chain disease. Report of seven cases and review of literature. Mayo Clin Proc 1981;56:439-51.

(3.) Wahner-Roedler DL, Witzig TE, Loehrer LL, Kyle RA. Gamma-heavy chain disease: review of 23 cases. Medicine (Baltimore) 2003;82:236-50.

(4.) Lee MT, Parwani A, Humphrey R, Hamilton RG, Myers DI, Detrick B. Gamma heavy chain disease in a patient with diabetes and chronic renal insufficiency: diagnostic assessment of the heavy chain fragment. J Clin Lab Anal 2008;22: 146-50.

(5.) Stramignoni A, Carbonara A, Paolino W, Navone R, Coda R, Leonardo E. G1 heavy chain disease: clinicopathological, ultrastructural and immunochemical study of a new case. J Cancer Res Clin Oncol 1980;96:93-103.

(6.) Kambham N, Markowitz GS, Appel GB, Kleiner MJ, Aucouturier P, D'Agati VD. Heavy chain deposition disease: the disease spectrum. Am J Kidney Dis 1999;33:954-62.

(7.) Daval S, Tridon A, Mazeron N, Ristori JM, Evrard B. Risk of antigen excess in serum free light chain measurements. Clin Chem 2007;53:1985-6.

(8.) Yoo EM, Wims LA, Chan LA, Morrison SL. Human IgG2 can form covalent dimers. J Immunol 2003;170:3134-8.

QUESTIONS TO CONSIDER

1. What are the potential causes of increased [beta]-globulins?

2. What can explain the discrepancy between the presence of k light chains in the urine and the presence of a monoclonal IgG without light chains in the serum?

3. What investigations should be performed to characterize the protein responsible for the spike in the region of the UPEP results?

4. What can explain the observation that the sum of the 4 IgG subclasses [<800 mg/dL (<8 g/L)] was not equal to the total IgG concentration [2950 mg/dL (29.5 g/L)]?

POINTS TO REMEMBER

* [gamma]-HCD is an underdiagnosed and atypical lymphoproliferative process with high variability in the clinical picture; the laboratory often plays a central role in the diagnosis.

* [gamma]-HCD therapy depends on the underlying clinicopathologic features rather than on the presence of the monoclonal protein.

* The monoclonal IgG found in y-HCD presents no light chains, and the heavy chains are frequently truncated, yielding an abnormal protein with a low molecular weight.

* Electrophoresis and immunofixation of proteins from both serum and urine samples are necessary to establish a [gamma]-HCD diagnosis and should be performed in all cases of lymphoplasma cell proliferative disease, especially if proteinuria or an abnormal SPEP result are present.

Benoit Busser, [1] * Sylvain Millet, [2] Claude Eric Bulabois, [3] Patrice Faure, [1] and Jean Charles Renversez [1]

[1] Departement de Biochimie, Toxicologie, Pharmacologie, Pole de Biologie, CHU Grenoble, Grenoble, France; 2 Departement d'Hematologie, Onco-Genetique et Immunologie, Poole de Biologie, CHU Grenoble, Grenoble, France; 3 Unitede Soins Intensifs d'Hematologie, Poole de Cancerologie et d'Hematologie, CHU Grenoble, Grenoble, France.

* Address correspondence to this author at: Departement de Biochimie, Toxicologie, Pharmacologie, CHU Grenoble BP217, 38042 Grenoble Cedex 09, France. Fax 4-76-76-51-81; e-mail bbusser@chu-grenoble.fr.

Received May 12, 2010; accepted September 20, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.150425

[4] Nonstandard abbreviations: UPEP, urine protein electrophoresis; SPEP, serum protein electrophoresis; [gamma]-HCD, [gamma]-heavy chain disease.

Commentary

David F. Keren

Busser et al. have shared an unusual case featuring a broad, prominent increase in the [beta] region of a urine protein electrophoresis result. The immunofixation electrophoresis (IFE) result identifying the band in serum and urine as containing only y chains with no corresponding light chain secured the diagnosis of [gamma]-heavy chain disease.

The serum protein electrophoresis had been performed by capillary electrophoresis, a technique in which radiocontrast dyes and antibiotics can create spikes that mimic an M protein (1-5). Such spikes are typically quite discrete, however. Hemoglobin and fibrinogen are other common M protein mimics, but they have characteristic migrations that clinical laboratories usually recognize (6).

Some M proteins that migrate in the [beta] region of serum can be quite subtle and easily missed. Narayan et al. reported that in electrophoresis systems that produce crisp resolution of the [[beta].sub.1]- and [[beta].sub.2]-globulins, an increase in either of these fractions not explained by an obvious process, such as [beta]-[gamma] bridging, deserves an IFE evaluation (7). These investigators reported that of 36 samples with such an increase, 12 had an M protein by IFE. Similarly, Katzmann et al. found M proteins when they reflexed to IFE serum samples that had the following suspicious findings: "fuzzy" bands (54% M proteins), [beta] regions of 1.6-1.9 g/dL (16-19 g/L) (10% M proteins), and hypogammaglobulinemia (12% M proteins) (6).

An unusual phenomenon, "phantom light chain," rarely accounts for an apparent lack of a light chain in some cases of myeloma. This term, coined by Cejka and Kithier and originally described in a case of IgD [lambda] myeloma (8), has also been reported in a case of IgA [lambda] myeloma (9). The apparent lack of light chains by IFE may be due to heavy chains cloaking light chain determinants; however, by using other antisera, or a stronger concentration of the antisera, one can demonstrate the light chains (9).

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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: No authors declared any potential conflicts of interest.

References

(1.) Arranz-Pena ML, Gonzalez-Sagrado M, Olmos-Linares AM, Fernandez-Garcia N, Martin-Gil FJ. Interference of iodinated contrast media in serum capillary zone electrophoresis. Clin Chem 2000;46:736-7.

(2.) Blessum CR, Khatter N, Alter SC. Technique to remove interference caused by radio-opaque agents in clinical capillary zone electrophoresis. Clin Chem 1999;45:1313.

(3.) Bossuyt X, Mewis A, Blanckaert N. Interference of radio-opaque agents in clinical capillary zone electrophoresis. Clin Chem 1999;45:129-31.

(4.) Bossuyt X, Peetermans WE. Effect of piperacillin-tazobactam on clinical capillary zone electrophoresis of serum proteins. Clin Chem 2002;48:204-5.

(5.) Bossuyt X, Verhaegen J, Marien G, Blanckaert N. Effect of sulfamethoxazole on clinical capillary zone electrophoresis of serum proteins. Clin Chem 2003; 49:340-1.

(6.) Katzmann JA, Stankowski-Drengler TJ, Kyle RA, Karen SL, Snyder MR, Lust JA, Dispenzieri A. Specificity of serum and urine protein electrophoresis for the diagnosis of monoclonal gammopathies. Clin Chem 2010;56:1899 900.

(7.) Narayan S, Lujan MG, Baskin LB, Devaraj S, Rutherford C, Jialal I. Measurement of [[beta].sub.1]- and [[beta].sub.2]-globulins improves detection of M-spikes on high-resolution electrophoresis. Clin Chem 2003;49:676-8.

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

(9.) Su L, Keren DF, Warren JS. Failure of anti-lambda immunofixation reagent

David F. Keren *

Warde Medical Laboratory, Ann Arbor, MI.

* Address correspondence to the author at: Warde Medical Laboratory, 300 W. Textile Rd., Ann Arbor, MI 48108. Fax 734-214-0399; e-mail dfkeren@yahoo.com.

Received February 16, 2011; accepted February 24, 2011.

DOI: 10.1373/clinchem.2011.162669

Commentary

Geraldine P. Schechter *

Two monoclonal proteins--a urinary k light chain and a serum [gamma] heavy chain without an identifiable light chain--were discovered in this elderly patient. Absence of a light chain is usually due to truncation of the heavy chain, thereby preventing the binding of light chains.

The patient's presentation with anemia, leukopenia, hypoalbuminemia, and albuminuria raises the possibility of myeloma rather than metastatic prostate cancer; however, malignancies associated with monoclonal [gamma] heavy chains are usually lymphoproliferative disorders that exhibit adenopathy rather than plasma cell myeloma with lytic bone lesions (1).

Both M proteins are low in concentration, and these findings may simply reflect a diclonal gammopathy of unknown significance in an elderly individual with an unrelated neoplasm. Monoclonal gammopathy of unknown significance (MGUS) has been found in 7.5% of patients older than 80 years, and diclonal gammopathy occurred in 3% in a population-based study (2). Individuals with MGUS may have small amounts of urinary free light chains without a serum M protein. These uncommon MGUS clones of light chains have the potential to progress to light chain myeloma (3). In 9% to 17% of patients with monoclonal y heavy chains, there is no associated pathologic finding, and the truncated protein simply represents a rare type of MGUS (1).

Alternatively, this patient's pathologic fracture may be due to hyposecretory multiple myeloma derived from one or both clones. Rare patients with myeloma have exhibited truncated [gamma] chains. If so, the relationship of the 2 clones to the malignancy may be determined by immunohistochemical or flow cytometry analysis. A recent report described a myeloma patient in whom all of the malignant plasma cells contained a truncated [gamma] chain, whereas a subclone of 30% of the cells also contained light chain (4). Heavy [gamma] chains may also cause immunoglobulin deposition disease or amyloid fibrils (5, 6), which could explain the patient's albuminuria.

References

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

(2.) Kyle RA, Therneau TM, Rajkumar SV, Larson DR, Plevak MF, Offord JR, et al. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006;354:1362-9.

(3.) Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes the diagnosis of multiple myeloma in most patients. Blood 2009;113:5418-22.

(4.) Richter AG, Harding S, Huisson A, Drayson M, Pratt G. Multiple myeloma with monoclonal IgG3 heavy chains and free kappa light chains. Acta Haematol 2010;123:158-61.

(5.) Ronco P, Plaisir E, Aucouturier P. Monoclonal immunoglobulin light and heavy chain deposition diseases: molecular models of common renal diseases. Contrib Nephrol 2011;169:221-31.

(6.) Miyazaki D, Hazaki M, Gorio T, Kametani F, Tsuchiya A, Matsuda M, et al. AH amyloidosis associated with an immunoglobulin heavy chain variable region (VH1) fragment: a case report. Amyloid 2008;15:125-8.

Geraldine P. Schechter [1,2] *

[1] Hematology Section, Medical Service, Washington Veterans Affairs Medical Center, and 2 Department of Medicine, George Washington University, Washington, DC.

* Address correspondence to the author at: Veterans Affairs Medical Center, 50 Irving St. NW, Washington, DC 20422. Fax 202-518-4300; e-mail g.p.schechter@med.va.gov.

Received February 27, 2011; accepted March 10, 2011.

DOI: 10.1373/clinchem.2011.162677

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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: No authors declared any potential conflicts of interest.
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Title Annotation:Clinical Case Study
Author:Busser, Benoit; Millet, Sylvain; Bulabois, Claude Eric; Faure, Patrice; Renversez, Jean Charles
Publication:Clinical Chemistry
Date:Jul 1, 2011
Words:3661
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