Immunofixation Compared with Immunoelectrophoresis for the Routine Characterization of Paraprotein Disorders

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1 Immunofixation Compared with Immunoelectrophoresis for the Routine Characterization of Paraprotein Disorders STEPHEN K. ERRD, M.D., PH.D., KTHERINE H. CHEN, PH.D., ND HSSN KHYM-BSHI, PH.D. The authors prospectively compared immunofixation electrophoresis (IE) with Immunoelectrophoresis (IEP) for the evaluation of paraproteins in 32 patient specimens. They used a simple modification of their existing agarose protein electrophoresis system for IE. Thirteen specimens (40%) required IE for paraprotein identification, 18 (56%) were definitive by either method, and 1 (3%) did not show a paraprotein by either method. Low titer and/or avidity with anti-lambda antibody from three of seven suppliers prevented or complicated interpretation of IEP patterns with two of four known lambda paraprotein-containing specimens but had no such effect with IE. In view of these and various additional technical considerations, the authors suggest IE for the routine characterization of paraproteins in the serum or urine. (Key words: Immunoelectrophoresis; Immunofixation; Serum protein electrophoresis; ammopathy; Paraprotein disorders; alse negative reactions) m J Clin Pathol 1987; 88: IMMUNOELECTROPHORESIS (IEP) is considered the reference method for the identification of paraproteins in serum or urine. 1,3 With this method, the specimen is electrophoresed and then allowed to diffuse through the matrix against appropriate antibodies, each applied in a well parallel and adjacent to the electrophoresis lane. The identification of a given paraprotein is accomplished by the recognition of characteristic precipitin band abnormalities. 2 ',23 Immunofixation electrophoresis (IE) was more recently introduced as a method for typing paraproteins. 4,20 s with IEP, the specimen must first be electrophoresed. In contrast to IEP, however, antibody is subsequently applied either directly or by way of an absorbent carrier onto the surface of the electrophoresed lane. fter a brief incubation period, e.g., one hour, a complex precipitates and is thereby "fixed" in the matrix when the specificity of the antibody and the paraprotein heavy or light chain type agree. The matrix is then washed to allow uncomplexed proteins to diffuse away freely. fter staining, paraproteins are identified by visualizing discrete bands that correspond in position precisely to those seen by serum or urine protein electrophoresis (SPE, UPE). Received ugust 11, 1986; received revised manuscript and accepted for publication November 25, ddress reprint requests to Dr. Khayam-Bashi: Clinical Laboratories, Bldg. 100, San rancisco eneral Hospital, 1001 Potrero venue, San rancisco, California Department of Laboratory Medicine, University of California, San rancisco, at the Clinical Laboratories, San rancisco eneral Hospital Medical Center, San rancisco, California There is considerable confusion regarding the appropriate application of IEP and IE for routinely investigating paraprotein disorders. reater sensitivity and resolution and ease of interpretation are characteristic of IP 4,16,18,20,27,29 Th ese factors may facilitate a more successful and/or complete identification of paraproteins for difficult patterns, such as with multiclonal disorders 7 ' 24 or faint monoclonal bands. 418,20,29 or these and related reasons, some have recommended IE as the primary method for characterizing paraprotein abnormalities. 20,27 However, others have suggested that IEP should remain the routine method of choice, to be augmented by IE only when necessary. 10 ' 24 inally, a major recent review on IE failed to make any recommendations in this regard. 9 To clarify this confusion, we prospectively assayed 32 patient specimens by IEP and IE, using a simple modification of a conventional SPE agarose film system for the latter. We also independently compared the influence of antibody supplier on test performance and the ease of execution by our technologists for the two methods. Methods Patient Specimens Serum and urine specimens were routinely submitted to the biochemistry laboratory for immunoelectrophoretic evaluation, based on the clinical suspicion of a paraprotein disorder. Of these, specimens were included in our study if a paraprotein abnormality was visually suggested by the presence of an abnormal band in the serum or urine protein electrophoretogram. We evaluated 28 serum and 4 urine specimens by the two methods. Urine specimens were concentrated up to 200-fold by using a disposable ultrafiltration concentrator with an exclusion limit of 15,000 daltons (Minicom- B15, micon Corporation, Danvers, M). Urine pro- 198 on 18 ugust 2018

2 Vol. 88 No. 2 IMMUNO1TION OR PRPROTEIN DISORDERS 199 tein was quantified by a Coomassie dye binding assay. 2 Serum protein was assayed using the biuret reagent by SMC 26 (Technicon Instruments Corporation, Tarrytown, NY). Serum and Urine Protein Electrophoresis SPE and UPE were performed on agarose gel (Corning Medical, Palo lto, C) according to the manufacturer's instructions, with the exception of a buffer that contained 50 mmol/l sodium barbital and 1.8 mmol/l calcium lactate, ph 8.6. Serum and urine specimens were either used immediately or stored at 4 C for up to 48 hours before electrophoresis. els were stained for protein with mido Black and scanned on a densitometer (Helena, Beaumont, T) for relative quantification of protein fractions. serum specimens were reconstituted from lyophilized preparations (merican Dade, Miami, L). Immunoelectrophoresis and Immunofixation Electrophoresis or both IEP and IE, agarose film (Corning) was used as the support matrix. When necessary, IgM paraprotein specimens were reassayed after reduction with mercaptoethanol. 15 IEP was otherwise performed by established procedures. 21,23 or the prospective evaluation of patient specimens, antibodies with specificities directed against,, M, kappa, and lambda chain determinants were obtained either from ICL Scientific (ountain Valley, C) or tlantic ntibodies (Scarborough, ME). Results were classified as definitive if the analysis clearly identified the abnormal band or bands shown by the prior SPE or UPE. IE was performed by applying a 2.5-cm-long by 0.75-cm-wide strip of antibody-impregnated filter paper centered over the abnormal band on the electrophoresed lane in agarose films (Corning). fter completion of the prospective patient specimen comparison, filter paper strips of 1.25 cm length and 0.75 cm width were used in the same fashion for the IE assay of 22 additional patient specimens. Serum and urine specimens were either diluted or concentrated, respectively, to approximately 5 g/l before electrophoresis for IE. Other details of the procedure were as described elsewhere. 7 In a separate evaluation, antibodies from the two sources noted above and from five additional commercial suppliers (Corning Medical, Palo lto, C; CalBiochem-Behring Diagnostics, La Jolla, C; Meloy Laboratories, Tarrytown, NY; Kallestad Laboratories, ustin, T; Tago Inc., Burlingame, C) were compared for the two methods, using selected patient specimens. or the antilambda antibody from these seven sources, three were from burro, two from rabbit, and two from goat. Results Prospective Patient Specimen Comparison IE was much more successful than IEP in identifying paraprotein abnormalities among serially submitted specimens. Of 32 total specimens, 31 were definitively characterized by IE, as compared with only 18 so characterized by IEP. Therefore, 13 specimens, representing 40% of the total, required IE for definitive paraprotein identification. One specimen did not show a paraprotein by either IE or IEP. Situations that precluded identification of paraprotein abnormalities by IEP included a high polyclonal background in nine, a biclonal process in two, and interfering globulins in two. representative comparative analysis for one of the specimens with a high polyclonal background is shown in igure 1. The SPE shows an abnormal band migrating at the cathodal end of the gamma region superimposed over a background of polyclonal gamma globulins (ig. \, top). Definitive identification of this band as a monoclonal Ig-lambda is clearly shown by IE (ig. I ) but not by IEP (ig. IB). One of two patients with a biclonal gammopathy had IgMkappa and IgM-lambda components in her serum and has been described elsewhere. 7 The other was a patient with simultaneous Ig-lambda and Ig-kappa circulating paraproteins. nother patient's serum specimen with a marked abnormal gamma band by SPE showed an oval area of precipitation around the origin of each lane with IEP, preventing the formation of identifying precipitin arcs. lthough some background polyclonal fixation staining of this band was evident by IE, the identification was definitive by this method, based on the much more marked staining by anti- and antikappa. urine specimen from this patient behaved similarly. Comparison of ntibody Supplier The influence of antibody supplier using reagent from seven different sources was evaluated for the two methods. Significant intersupplier antilambda antibody variability prevented interpretation of IEP patterns with two of four known lambda paraprotein-containing specimens but had no such effect on IE. This is shown for one of these two specimens in igure 2. The prominent gamma-migrating paraprotein band shown for the patient 1 specimen by SPE (ig. 2, top) is readily identified with any of the four antilambda antibodies shown, labeled, E,, and (ig. 2). lthough a prozone effect is suggested for the E,, and lanes by the clear area in the center of their respective bands, the lambda identity is nonetheless clearly established, particularly when each is compared with the negative companion on 18 ugust 2018

3 200 ERRD, CHEN, ND KHYM-BSHI.J.C.P. ugust 1987 nti: IEP Specimen: SPE IE nti: M K M K K ^s^* Patient Patient K Patient. I. 1. High polyclonal background specimen compared by IE and IEP. The anode is on the left.. The SPE (top) shows an abnormal band at the cathodal end of the gamma region superimposed over a diffuse background of polyclonal globulins. IE shows staining of this band with anti- and antilambda but not with antibodies to, M, or kappa. B. The IEP pattern of arcs does not permit identification of the abnormal gamma band. The patient specimen arcs for, kappa, and lambda are not significantly different than those for the normal control specimen. B. antikappa antibody pattern (not shown). In contrast, igure 2B shows absence of IEP arc formation with this specimen by the antilambda antibody, despite dilution of the specimen up to eightfold. Similar IEP behavior with this specimen was observed for the E and antilambda antibodies (not shown). This IEP problem did not correlate with the antibody species of origin. or comparison, a readily interpretable pattern of IEP arcs formed with the same antilambda antibody is shown in the lower half of igure 2B, using one of the two prominent lambda paraprotein specimens for which antilambda antibody arc formation was not problematic. Other Technical Considerations Of the three IgM paraprotein-containing specimens among those compared in our study, two required reduction with mercaptoethanol for identification by IEP but not IE. or each of 22 additional patient specimens that were submitted after the completion of our prospective comparison, an abnormal SPE or UPE band was clearly identified by IE alone with the use of antibody application filter paper strips of the shortened 1.25 cm length (see "Methods"). lthough the hands-on technologist time was comparable for the two procedures, IE was preferred because of the greater ease of antibody application and shorter turnaround times. Discussion Paraprotein disorders are associated with a wide spectrum of diseases, such as lymphoma, myeloma, amyloidosis, and macroglobulinemia. 13 The diagnosis of paraprotein abnormalities by definition depends upon the laboratory identification of one or more abnormal monoclonal immunoglobulins in the serum and/or urine. Laboratory documentation of paraproteins may be important even in the absence of an apparent underlying disorder, because a significant proportion of such patients may ultimately express one of the diseases typi- on 18 ugust 2018

4 Vol. 88 No. 2 IMMUNOITION OR PRPROTEIN DISORDERS 201 SPE Patient 1 Supplier: E nti: nti Supplier: IEP Specimen Dilution : 8x P 4x T 1 1 2x E N undil T undil 2x 4x 8x P T 1 3 E N T. I. 2. Supplier dependence of antilambda antibody patterns. The anode is on the left.. prominent abnormal band in the gamma region is shown by SPE for the specimen for patient 1 (lop). IE staining of this band is seen with anti- antibody and with antilambda antibody from four different suppliers, labeled, E,, and. B. IEP with the antilambda antibody shows no visible arc formation for the patient 1 specimen, despite dilution (top), but good arc formation with the specimen from patient 3 (bottom). Both specimens show good arc formation with the antilambda antibody from supplier (center). B. cally associated with paraproteinemia. 12 The measurement of paraprotein levels may additionally be useful for estimating prognosis and monitoring therapy. 58 Our prospective comparison of IEP and IE, using a simple modification of an agarose SPE system for the latter, suggests that IE as such is preferable over IEP for the routine evaluation of serum and/or urine monoclonal disorders. The significant proportion, i.e., 40%, of serially encountered specimens that could be characterized as to paraprotein type by IE but not by IEP is particularly compelling in this regard. Specimen characteristics that made IE necessary for definitive analysis in our study included a high polyclonal background, faint paraprotein bands, and biclonal gammopathy. lthough the preferential utility of IE for specimens of this type has been described previously, 4, ",25 we are aware of only one other study in which the prevalence of such specimens was prospectively evaluated. 10 The interference in IEP of background polyclonal globulins, which has been referred to by others as the "umbrella effect," 17,25 was the most prevalent cause of inconclusive IEP analysis in our study (see ig. 1). The IE behavior of another specimen was consistent with that recently described 22 and was attributed to nonspecific cryoglobulin precipitation. One final specimen was negative by both methods, most likely reflecting the presence of a nonimmunoglobulin band in the serum protein electrophoretogram. To our knowledge, an objective prospective assessment of the relative utility of IE and IEP for routine paraprotein evaluation has not previously been done. Keshgegian and Peiffer 10 evaluated 97 specimens but limited their comparison of these two methods to only the 33 samples with uncertain results by IEP. The proportion of their total specimens represented by this subset, i.e., 34%, is similar to that observed by us. Whichter and associates 28 found IE superior to agarose UPE for the detection of Bence Jones protein in 12 urine speci- on 18 ugust 2018

5 202 ERRD, CHEN, ND KHYM-BSHI J.C.P. -ugust 1987 mens, but no comparison was made with IEP. Other studies that have compared IE and IEP have relied upon a selection of stored specimens, thereby potentially biasing the distribution of definitive evaluations by either method. It was equally significant that we observed intersupplier variability of antilambda antibody that markedly influenced interpretability with IEP but not with IE. The weak intensity of IEP precipitin arcs and the clear zone within immunofixed bands by IE is consistent with a prozone effect due to low antibody titer. However, some of the uninterpretable IEP patterns with antilambda antibody failed to improve upon specimen dilution (ig. 2B), suggesting low antibody avidity. This problem was observed with two successive lots of antilambda antibody from one of the three problem suppliers, although analysis of repeated lots from other suppliers was not done. Nonetheless, the observed IEP patterns among two of four lambda paraprotein-containing patient specimens suggests that this problem may not infrequently complicate lambda component identification by IEP. In addition, light chain identification by IEP for two of three IgM paraprotein-containing specimens required repeat analysis after sample reduction with mercaptoethanol. 15 This procedure was unnecessary with IE, as has been described by others. 6 " 20 lthough a variety of IE kits are now commercially available, we used a simplified convenient method based on an inexpensive modification of our existing SPE agarose film system. s originally suggested by Cawley and associates, 4 we found the use of filter paper strips for antibody application to be particularly convenient. However, we were able to achieve good results without having to blot the whole agarose film with filter paper after antibody application, as has been recommended by some. 4-9 ' ' 25 somewhat greater reagent cost for antibody was associated with IE compared with IEP because of the larger volumes required for wetting the filter paper strips. However, this additional cost was largely offset by fewer repeat IE assays necessitated by ambiguous patterns. In addition, we have recently reduced antibody consumption by one-half by shortening the filter paper strip length from 2.5 to 1.25 cm without any compromise in performance. lthough this modification stresses the importance of careful placement of the antibody-impregnated filter paper strip, it also emphasizes the interaction between the technologist and the laboratory pathologist during the immunochemical evaluation, which we believe to be desirable. In summary, we prospectively determined that 40% of our patient specimens required IE for paraprotein characterization. This in conjunction with significant supplier-dependent antilambda antibody performance with IEP and greater ease of performance and interpretation of immunofixation lead us to recommend IE as the preferred method for routinely investigating paraprotein abnormalities. cknowledgments. The authors appreciate the assistance and participation of the biochemistry laboratory technologists who performed the immunoelectrophoretic analyses. They also thank Helen Chong and Sally Wykkel for providing them with selected patient specimens. References 1. Bentwich Z, Bianco N, Jager L, et al (IUIS/WHO working group): Use and abuse of laboratory tests in clinical immunology: Critical considerations of eight widely used diagnostic procedures. 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6 Vol. 88 No. 2 IMMUNOITION OR PRPROTEIN DISORDERS 203 tion, in the detection of an occult myeloma paraprotein. Clin Chem 1982;28: Ritchie R, Smith R: Immunofixation. I. eneral principles and application to agarose gel electrophoresis. Clin Chem 1976; 22: Ritchie R, Smith R: Immunofixation. III. pplication to the study of monoclonal proteins. Clin Chem 1976; 22: Ritzmann SE, Lawrence M: Qualitative Immunoelectrophoresis, Serum protein abnormalities. Edited by SE Ritzmann, and JC Daniels. Boston, Little Brown and Co., 1975, pp Schultz L, ink LM, IliffM: Immunofixation of cryoglobulins: Pitfalls and solutions. Clin Chem 1985; 31: Stites DP: Clinical laboratory methods for detection of antigens and antibodies, Basic and clinical immunology, fifth edition. Edited by DP Stites, JD Stobo, HH udenberg, JV Wells. Los ltos, Lange, 1984, pp Sun T: Immunofixation electrophoresis, Physiology of immunoglobulins: Diagnostic and clinical aspects. Edited by SE Ritzmann. New York, R Liss, 1982, pp Sun T, Lien YY, Degnan T: Study of gammopathies with immunofixation electrophoresis. m J Clin Pathol 1979;72: Technicon, Inc: Product labeling, vols 1 and 2. Technical Publication No. U3-0306B41, 1981, Tarrytown, New York. 27. Whicher JT: Immunofixation on cellulose acetate is more efficient than immunoelectrophoresis for detection of paraproteins. Clin Chem 1983; 29: Whicher JT, Hawkins L, Higginson J: Clinical applications of immunofixation: more sensitive technique for the detection of Bence Jones protein. J Clin Pathol 1980; 33: Williams TL, Weisenburger DD, narra DJ: Cell surface and serum monoclonal immunoglobulin in Burkitt's Lymphoma. rch Pathol Lab Med 1985; 109:389. on 18 ugust 2018