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Technical Briefs |
1 Dipartimento di Scienze Cliniche Luigi Sacco, Università degli Studi di Milano, Milan, Italy 2 Laboratorio di Biotecnologie e Tecnologie Biomediche, IRCCS Policlinico San Matteo, Dipartimento di Biochimica, Università degli Studi di Pavia, Pavia, Italy
aaddress correspondence to this author at: Dipartimento di Scienze Cliniche Luigi Sacco, Via G.B. Grassi 74, 20157 Milan, Italy; fax 39-02-3564-018, e-mail carlo.franzini{at}unimi.it
Capillary electrophoresis (CE) is an analytical technique for the separation of molecules on the basis of molecular size, electric charge, and hydrophobicity. Since automated clinical instruments have become available, the major clinical application of this technique has been the rapid and effective separation of serum proteins.
The detection of monoclonal protein components (MCs) is the main purpose of serum protein separation assays. Several reports have dealt with the feasibility of CE for the detection of MCs, and some pitfalls of this technique have also been described (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Immunosubtraction is used in conjunction with CE for typing any detected MC, for confirming the diagnosis of monoclonal gammopathies, and for identifying the heavy and light chains constituting the monoclonal immunoglobulin molecule. Different types of MCs (IgG, IgA, or IgM, with associated 
or 
light chains) and free light chains (either or ) have been reported to be detectable by CE, but there have been no observations concerning the behavior of free heavy chains in CE.
We report a case of 
-heavy chain disease (-HCD) detected in the course of routine serum assay by CE, characterized by immunosubtraction, and confirmed by further techniques.
The patient was a 91-year-old man who had a complete medical examination, supplemented with numerous laboratory tests, including serum protein electrophoresis, 
2 years before the admission, without any significant finding but mild anemia. At admission he presented with bilateral peripheral edema, no dyspnea, and no palpable lymph node enlargement. Abdominal echography showed an enlarged steatotic liver and enlarged spleen. Laboratory investigation showed normocytic anemia (blood hemoglobin, 77.0–99.0 g/L; mean corpuscular volume, 99.3–102.6 fl), increased serum lactate dehydrogenase, low serum total protein (41.0 g/L) and albumin (28.0 g/L), and low serum cholinesterase. Serum immunoglobulin concentrations (determined by nephelometry on the Beckman Coulter Immage analyzer) were as follows: IgG, 5.31 g/L (reference interval, 7.0–16.0 g/L); IgA, 0.27 g/L (reference interval, 0.70–4.0 g/L); IgM, 0.28 g/L (reference interval, 0.40–2.3 g/L); chains (total), 1.71 g/L (reference interval, 6.0–12.6 g/L); -chains (total), 0.59 g/L (reference interval, 3.40–7.07 g/L).
Serum protein CE was performed on a Beckman CZE Paragon 2000 system (Beckman Coulter) equipped with software release 1.6. On visual inspection, an anomaly in the beta zone and a markedly lowered -globulin zone were evident (Fig. 1A
). The anomalous peak in the beta zone was 3.5% of the total protein, corresponding to 1.4 g/L; the polyclonal -globulin fraction was 4.0% of the total protein, corresponding to 1.7 g/L. Immunosubtraction was performed with reagents (agarose-bound antibodies) and an instrument (CZE Paragon 2000) from Beckman Coulter. Pretreatment with anti- led to the disappearance of the extra peak in the beta zone and to a further decrease of the -globulin zone (Fig. 1B
), whereas pretreatment with immobilized anti-
(not shown), anti-µ (not shown), anti- (Fig. 1C
), and anti- (Fig. 1D
) did not induce any significant modification of the electrophoretic pattern with the exception of a further reduction of the polyclonal -globulin zone after treatment with anti- and anti- antibodies. Thus, the anomalous peak in the beta zone was classified as -type free heavy chain, leading to the presumptive diagnosis of -HCD.
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Serum and urine agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE) were performed with, respectively, Paragon SPE Gel and Paragon Twin IFE Gel reagents (both from Beckman Coulter). Serum AGE demonstrated a small MC migrating in the beta fraction, which was confirmed by IFE as a -type MC lacking any light chain; a low -globulin concentration was also evident. AGE and IFE of the patient’s urine (total protein concentration, 0.4 g/L) also showed the presence of a MC, which was reactive only with anti- antiserum.
These data were consistent with the presence of free heavy chain in the blood and urine of the patient. For a definitive confirmation, the patient’s serum was assayed by a immunoselection method. This method and its different variants (14)(15) are able to provide direct evidence for the occurrence of free heavy chains. An immunoselection assay was performed with in-house reagents and gel plates. Antibodies were from Dako (polyclonal rabbit anti-human light chain, light chains, and heavy chains). In brief, this technique has steps beginning with electrophoretic separation on agarose gel plates incorporating selected antibodies; only those proteins not recognized by the antibodies are left free to migrate. After migration, double diffusion with an appropriate antibody is performed in an orthogonal direction to detect reactive molecules not blocked by the antibodies incorporated in the gel. The assay was performed with plates incorporating anti- and anti- antibodies and with anti- as a diffusion reagent. The results demonstrated that the patient’s serum, but not the control, included a molecule reactive with the anti- antibody but not with any of the two anti-light chains antibodies, thereby providing evidence of the presence of free -type heavy chains.
Structural variants of immunoglobulins have been reported in association with neoplasms of lymphocytes or plasma cells (16)(17). Structural studies have shown that most -HCD proteins are dimers of truncated heavy chains devoid of light chains. They consist mainly of the Fc region with a molecular mass of the monomeric -chain of 27–49 kDa, thus accounting for its presence in urine. Several structural variants involving deletion of the CH1 domain, often with a portion of the amino terminus of the variable region, have been reported, indicating complex underlying genetic defects.
Considering the advanced age of our patient, the decision was made to avoid any invasive diagnostic procedure, although laboratory and clinical evidence was consistent with lymphoplasma cell proliferative disease. In a recent review summarizing the clinical data of 23 cases of -HCD (18), 16 were found to have an associated lymphoplasma cell proliferative disorder; in 3 other cases the lymphoplasma cell proliferative disorder was coupled to an autoimmune disorder, 3 others had an autoimmune disorder only, and 1 had no underlying disease. HCD may therefore present without any specific clinical picture and may be serendipitously discovered during routine electrophoretic examination of serum protein. Results in this report show that CE analysis coupled with immunosubtraction is able to detect and characterize low-concentration free heavy chains in serum.
References
- and µ-heavy chain diseases and related disorders. J Clin Pathol 1974;28(Suppl 6):65-71.
-Heavy chain disease. Review of 23 cases. Medicine 2003;82:236-50.[CrossRef][Medline]
[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
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  I. Infusino, P. Luraschi, M. Panteghini, and C. Franzini Pretreatment of serum with penicillamine: effects on capillary electrophoresis patterns and on immunonephelometric measurement of immunoglobulins. Clin. Chem., April 1, 2006; 52(4): 772 - 774. [Full Text] [PDF]
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G. Marien, G. Verhoef, and X. Bossuyt Detection of Heavy Chain Disease by Capillary Zone Electrophoresis Clin. Chem., July 1, 2005; 51(7): 1302 - 1303. [Full Text] [PDF]
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