PDF
ePub
Citation
Print
Abstract
Background
Determination of monoclonal gammopathy through conventional protein electrophoresis is sometimes difficult because of the presence of large proteins such as haptoglobin and transferrin, which may obscure the results. Ambiguity in an electrophoresis band can give rise to confusion or difficulty in interpretation. The heavy chain/light chain assay (HLC assay) using Hevylite antibody (The Binding Site, UK) has recently been developed for the accurate measurement of monoclonal proteins. We compared the immunotyping (IT) profiles to the immunoglobulin (Ig) heavy/light chain measurements obtained using the HLC assay and observed the ratios between intact Ig kappa and lambda.
Methods
We collected 35 and 28 sera from patients with suspicious and definitive monoclonal protein, respectively. Then we performed serum protein electrophoresis (SPEP) and IT by Capillarys2 (Sebia, USA). Monoclonal protein production was investigated using Freelite antibody (The Binding Site) and specific Ig(G, A)κ and Ig(G, A)λ Hevylite antibodies. The results were analyzed using PASW 18.0 for Windows (IBM, USA).
Results
Direct measurement of Ig heavy/light chains showed discordant IT results for 12 (34.2%) of 35 patients' sera with suspicious SPEP pattern and identical IT results for 28 patients' sera with definitive monoclonal peak in the SPEP results. Overall, the results of the HLC assay and IT showed good agreement (κ=0.718, P=0.000 by cross-tabulation Gamma, Kappa analysis).
Conclusions
The results of direct measurement of serum Ig heavy chain/light chain pairs were comparable to those of IT and were helpful for determination of monoclonality in the case of ambiguous electrophoresis results. Measurement of the heavy chain/light chain pair ratio also allowed precise quantification of the monoclonal Igs with ambiguous electrophoresis patterns and identification or discrimination of clonality.
Figures and Tables
Fig. 1
Schematic diagram and study plan. This study comprised2 phases: in phase I, the laboratory utility of direct measurement of serum immunoglobulin heavy/light chain pairs(the HLC assay) was determined. In phase II, the HLC assay was validated by testing samples showing a definitive monoclonal peak in SPEP and immunotyping.
Abbreviations: SPEP, serum protein electrophoresis; MM, multiple myeloma; HLC assay, heavy chain/light chain assay.
Fig. 2
Linearity results of Hevylite Ig'κ and Ig'λ assays performed using a mixture of normal pooled sera with high concentration controls (simulation test by serial dilution). (A) IgGκ assay, (B) IgGλ assay, (C) IgAκ assay, (D) IgAλ assay.
Fig. 3
Serial analysis of sera from an IgGκ multiple myeloma patient; a comparison of serum protein electrophoresis (SPEP), total immunoglobulin G (IgG), monoclonal IgG from SPEP densitometry, and HevyliteTM IgG κ:λ ratio.
Abbreviations: IgG, immunoglobulin G; κ, kappa; λ, lambda; TCD, thalidomide plus cyclophosphamide plus dexamethasone treatment; auto-PBSCT, autologous peripheral blood stem cell transplantation; Vel, velcade (bortezomib); IT, immunotyping; PR, partial response; CR, complete response; NR, normal range.
Table 2
Characteristics of the clinical specimen based on serum protein electrophoresis (SPEP) patterns
*Statistical differences in HLC-IgGκ, HLC-IgGκ/IgGλ ratios, and serum immunotyping results between the suspicious electrophoresis group and the definitive monoclonal peak group were evaluated (P<0.05); †Reference range for the HLC assay: IgGκ, 3.84-12.07 g/L; IgGλ, 1.91-6.74 g/L; IgGκ/IgGλ, 1.12-3.21 g/L; IgAκ, 0.57-2.08 g/L; IgAλ, 0.44-2.04 g/L; IgAκ/IgAλ, 0.78-1.94 g/L.
Abbreviations: Ig, immunoglobulin; sFLC assay, serum free light chain assay; FLCκ, free light chain-kappa; FLCλ, free light chain-lambda; HLC assay, heavy/lightchain assay.
Table 3
Samples showing ambiguous serum protein electrophoresis (SPEP) patterns (12 of 35), which also showed discordance in the immunotyping results and HLC ratios
*In the case of only numerical data without Ig'κ or FLCκ labeling, this indicates that the result of HLC assay or FLC assay was within the reference range (IgGκ/IgGλ, 1.12-3.21, IgAκ/IgAλ, 0.78-1.94; FLCκ/λ, 0.26-1.65); †Described as per the response criteria given by the International Myeloma Working Group.
Abbreviations: SPEP, serum protein electrophoresis; HLC ratios, heavy/lightchain assay κ:λ ratios; sFLC assay, serum free light chain assay; FLCκ, free light chain-kappa; MM, multiple myeloma; SD, stable disease; PR, partial response; VGPR, very good partial response.
References
1. Hortin GL. Amino acids, peptides, and proteins. In : Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 5th ed. Missouri: ELSEVIER Saunders;2012. p. 509–563.
2. McPherson RA, Massey HD. Laboratory evaluation of immunoglobulin function and humoral immunity. In : McPherson RA, Pincus MR, editors. Henry's clinical diagnosis and management by laboratory methods. 22th ed. Philadelphia: ELSEVIER Saunders;2011. p. 899–913.
3. Legg A, Harding S, Hughes RG, Levoguer AM, Bradwell AR. Serum free light chain and Hevylite analyses in the diagnosis, monitoring and prognosis of B cell disorders. Klin biochem metab. 2010; 18:56–61.
4. Yang Z, Harrison K, Park YA, Chaffin CH, Thigpen B, Easley PL, et al. Performance of the Sebia CAPILLARYS 2 for detection and immunotyping of serum monoclonal paraproteins. Am J Clin Pathol. 2007; 128:293–299.
5. McCudden CR, Mathews SP, Hainsworth SA, Chapman JF, Hammett-Stabler CA, Willis MS, et al. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins. Am J Clin Pathol. 2008; 129:451–458.
6. Bradwell AR, Harding SJ, Fourrier NJ, Wallis GL, Drayson MT, Carr-Smith HD, et al. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios. Clin Chem. 2009; 55:1646–1655.
7. Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, et al. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem. 2002; 48:1437–1444.
8. Lee JH, Bang SM. Epidemiological change of multiple myeloma in Korea. Korean J Hematol. 2006; 41:225–234.
9. Kang SY, Suh JT, Lee HJ, Yoon HJ, Lee WI. Establishment of serum reference range for free light chains and its clinical usefulness in multiple myeloma. Korean J Lab Med. 2004; 24:273–278.
10. Keren DF. Heavy/Light-chain analysis of monoclonal gammopathies. Clin Chem. 2009; 55:1606–1608.
11. Tovar N, Fernández de Larrea C, Elena M, Cibeira MT, Aróstegui JI, Rosiñol L, et al. Prognostic impact of serum immunoglobulin heavy/light chain ratio in patients with multiple myeloma in complete remission after autologous stem-cell transplantation. Biol Blood Marrow Transplant. 2012; 18:1076–1079.
XML Download