DOI 10.1515/cclm-2013-0714 Clin Chem Lab Med 2014; 52(5): 665–669
Erin Kaleta, Robert Kyle, Raynell Clark and Jerry Katzmann*
Analysis of patients with γ-heavy chain disease by the heavy/light chain and free light chain assays Abstract Background: The objective of this study was to evaluate the performance of the heavy/light chain and free light chain immunoassays in patients with heavy chain disease, and to assess the ability of the heavy/light chain assay to measure and confirm the abnormal, truncated heavy chain. Methods: Frozen serum samples from 15 γ-heavy chain disease patients were tested for IgGκ, IgGλ, total IgG, free light chains, and M-spike concentrations. Results: The (Gκ+Gλ)/IgGtotal ratio for these 15 patients ranged from 0.02 to 0.80. The 10 patients with IgG concentrations above 1 g/dL all had ratios below 0.3 indicating that a substantial portion of IgG was not quantitated by the Gκ and Gλ reagents. The average M-spike was 1.61 g/dL and the average calculated abnormal γ-chain concentration was 2.94 g/dL. Additionally, free light chain analysis revealed the presence of monoclonal free κ light chain in three of the 15 patients. Conclusions: This study demonstrates utility of a nephelometric assay to identify truncated immunoglobulin heavy chains in γ-HCD and that 20% of these patients also have monoclonal free light chain. Keywords: free light chain assay; heavy chain disease; heavy/light chain assay; nephelometry. *Corresponding author: Jerry Katzmann, PhD, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic Hilton 210, 200 1st St SW, Rochester, MN 55905, USA, Fax: +1 507 2664088, E-mail: [email protected]. Erin Kaleta and Raynell Clark: Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Robert Kyle: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA
Introduction Immunoglobulin molecules are tetra peptides containing two identical heavy chains and two identical light chains that are tightly bound together. Heavy chain diseases
(HCD) are rare lymphoplasmacytic proliferative disorders that are characterized by the production of a monoclonal immunoglobulin that is a truncated heavy chain which lacks corresponding light chains. These diseases include α-, γ-, and μ-HCD [1–3]. α-HCD is the most common of these disorders and usually presents as extranodal marginal zone lymphoma of mucosa-associated lymph node tissue (MALT) caused by chronic antigenic stimulation with intestinal organisms. This variety of HCD is treated by managing exposure to intestinal pathogens. μ-HCD is extremely rare, with only 33 reported cases in the literature as of 2005, and is commonly associated with lymphoproliferative malignancy [1]. γ-HCD has more diverse clinical features, and can present as disseminated lymphoproliferative disease, localized proliferative disease, or no apparent proliferative disease, which typically has an underlying autoimmune disorder. The median age of onset is 60 years, with symptoms involving anemia, fatigue, lymphadenopathy, splenomegaly and hepatomegaly [4, 5]. The testing strategy that is typically followed for any monoclonal gammopathy includes serum and/or urine protein electrophoresis, followed by immunofixation electrophoresis (IFE) [6]. The results with HCD samples demonstrate a monoclonal heavy chain with no corresponding light chain. In addition to the lack of light chain, heavy chains are typically truncated, further reducing the molecular weight [2], and the truncated heavy chain is often so small that serum concentrations are low due to renal clearance [7]. Due to these low serum concentrations, the definitive absence of light chain may be difficult to establish. In addition, some intact monoclonal immunoglobulins have poor reactivity with light chain anti-sera and show only faint light chain on IFE, blurring the distinction between poorly reacting light chain and absent light chain. Thus, identification of HCD can be difficult. The HevyliteTM immunoassay targets the unique junctional epitope between immunoglobulin heavy chain and light chain combination, and therefore the monoclonal protein in HCD will not be recognized by heavy/light chain reagents [8]. For these studies, we have focused on γ-HCD, measuring intact Gκ, Gλ, total IgG, and calculated the (Gκ+Gλ)/IgGtotal ratio in order to quantitate the portion of IgG that is derived from the monoclonal plasma cell clone.
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666 Kaleta et al.: Heavy/light chain and free light chain assays with HCD
Materials and methods Stored residual serum samples from 15 patients with previously diagnosed γ-HCD were identified by querying the dysproteinemia database, which contains data on all patients with plasma cell proliferative disorders seen at Mayo Clinic in Rochester, MN. The search was filtered for γ-HCD patients with a detectable serum M-spike. Serum samples that had been stored between 1978 and 2003 were retrieved from frozen storage (−20 °C). The database inquiries and use of stored sera were approved by the Mayo Clinic Institutional Review Board. Samples were analyzed by serum protein electrophoresis (SPEP) and immunofixation (IFE) to confirm the diagnosis of HCD. SPEP was performed on the Helena SPIFE SPE system (Helena Laboratories, Beaumont, TX, USA), stained with Acid Blue and visualized on the Helena SPIFE 3000 system. IFE was performed on Sebia HYDRAGEL 9IF kits with testing performed on the Sebia HYDRASys System (Sebia, Norcross, GA, USA). Nephelometry was performed to measure total immunoglobulins with a Siemens BNII and Siemens IgG antisera (Siemens, Tarrytown, NY, USA). Heavy/light chain measurements were performed on the BNII with immunoglobulin heavy/light chain reagents (Hevylite™, The Binding Site, Birmingham, UK). To establish expected results for the (Gκ+Gλ)/IgG ratio and the Gκ/Gλ ratio, we analyzed sera from 275 normal donors for Gκ, Gλ, and IgG concentrations. The 95% reference range for total IgG was 0.77–1.59 g/dL, for the ratio of IgGκ:IgGλ was 0.71–3.23 and for the ratio of (Gκ+Gλ)/IgGtotal is 0.80–1.12. The free light chain assay was performed on the BNII with the Free Light Chain assay (Freelite™, The Binding Site, San Diego, CA, USA).
Results SPEP was performed to identify the migration of the truncated immunoglobulin fragments and to quantitate the amount of monoclonal protein. Nine of the proteins migrated in the β-fraction and six migrated in the γ-fraction (Table 1). The M-spikes ranged from 0.37 to 4.17 g/dL with an average of 1.61 g/dL. All samples had a discrete γ-heavy chain band by IFE with an absence of a corresponding band in the anti-κ or anti-λ lanes (Figure 1). The total IgG, IgGκ, and IgGλ was determined (Table 1). On average, the sum of the IgGκ+IgGλ accounted for only 18% of the total IgG indicating that 82% of the IgG did not have an associated light chain. The distribu tions of the individual ratios of (Gκ+Gλ)/IgGtotal) are shown in Table 1. The ability of the heavy/light r eagents to account for the total IgG is significantly reduced and must be quantitating only the polyclonal IgG in the patient’s serum. In support of this, the ratios of the IgGκ to IgGλ for each patient fell within the 95% normal reference range (0.71–3.23). The IgG that is not recognized by the heavy/light reagents [IgG–(Gκ+Gλ)] is an indirect measure of the monoclonal heavy chain fragment concentration. The calculated heavy chain fragment concentrations ranged from
0.15 to 8.86 g/dL and averaged 2.94 g/dL compared to the average M-spike of 1.61 g/dL (Table 1). The relationship between the M-spike concentration and the calculated monoclonal IgG fragment is shown in Figure 2. These patients demonstrated a two-fold difference between M-spike and nephelometry. Greater differences between the two methods were seen with γ- and β-migrating protein at higher protein concentrations > 3 g/dL. The low concentration monoclonal proteins migrating in the β region are not always resolved from normal β-proteins, increasing the effective M-spike values which mask the difference between the two measures. In addition to heavy/light chain analysis, all patient sera were assessed for free light chain κ- and λ-concentrations and FLC κ/λ ratio (Table 2). In our patient set, 5/15 had abnormally elevated FLC κ/λ ratios, two patients of which were only slightly elevated, potentially due to renal dysfunction as a consequence of the monoclonal gammopathy. The remaining three patients have elevations clearly indicating monoclonal κ-FLC. Retrospective history review of urine protein electrophoresis and IFE indicated that one patient (Patient 10) showed evidence of a monoclonal κ-protein which had a different mobility than the IgG band.
Discussion The lack of recognition by the heavy/light chain reagents was consistent with the presence of γ-HCD and was in agreement with electrophoresis and immunofixation findings. The identification of γ-HCD by IFE relies on an IgG band and a lack of corresponding κ- or λ-reactivity. The heavy/light chain assay offers additional confirmation for difficult to interpret serum protein immunofixation results by providing numerical results to be paired with traditional electrophoresis and immunofixation findings. In this study we selected for HCD patients with a large enough abnormality to present with an M-spike, and the heavy/light chain assay results indicated γ-HCD in all 15 patients. With each patient the Gκ+Gλ did not account for the total IgG and the Gκ/Gλ ratio was within normal limits, indicating that the remaining intact immunoglobulins were polyclonal protein. It is possible that quantitative assays using anti-Fc and anti-Fab might also provide information regarding a discrepancy between Fc and Fab quantitation. The various interactions of anti-Fab with HCD γ-chains, however, are unknown. The lack of agreement between M-spike quantitation and IgG quantitation was an unexpected result. In
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Kaleta et al.: Heavy/light chain and free light chain assays with HCD 667
Table 1 Heavy/light chain immunoglobulin analysis in patients with γ-HCD. Patient ID
IgG Quant, g/dL
Gκ+Gλ, g/dL
Gκ+Gλ/IgG
Calculated clonal heavy chain, g/dL [IgG–(Gκ+Gλ)]
M-spike, g/dL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Reference range Average
0.46a 0.73a 1.08 1.65a 2.25a 2.85a 4.05a 6.46a 8.99a 1.94a 2.03a 3.09a 3.15a 4.08a 5.38a 0.77–1.59 –
0.29 0.58 0.24 0.33 0.59 0.29 0.08 0.13 0.18 0.16 0.06 0.46 0.90 0.20 0.48 –
0.63a 0.79a 0.22a 0.20a 0.26a 0.10a 0.02a 0.02a 0.02a 0.08a 0.03a 0.15a 0.03a 0.05a 0.09a 0.80–1.12 0.18
0.17 0.15 0.84 1.32 1.67 2.55 3.96 6.34 8.86 1.79 1.97 2.64 3.05 3.88 4.90 – 2.94
0.37 βb 0.50 βb 0.58 βb 1.12 βb 1.60 β 2.46 β 1.75 β 2.28 β 4.17 β 0.82 γ 0.52 γ 1.84 γ 1.81 γ 1.84 γ 2.55 γ – 1.61
a Denotes a result outside of the reference range; bPatients 1–4 have monoclonal proteins that are not completely resolved from the β-fraction, and the M-spike value contains co-migrating β-proteins.
all cases of γ-migrating monoclonal protein and in those with β-migration with nephelometric concentrations > 3 g/dL, the M-spike quantitation by electrophoresis was nearly two-fold lower than the calculated value from nephelometry. The M-spike quantitation is derived from the amount of dye bound by the protein and should be an indirect measure of mass. The nephelometric assay correlates light scatter with a standard curve that is based upon intact immunoglobulin in order to convert light scatter of molecules to mass. Although the monoclonal heavy chain fragment has lost some antigenic sites and anti-γ
anti-α
anti-µ
anti-κ
anti-λ
IgG-(GK+GL) with Nephelometr,y g/dL
SPEP
light scatter, it is being converted based on an IgG standard that is anywhere from six- to 10-fold larger and results in an overestimation due to these differences. An additional complication relates to the differences in reactivity of intact polyclonal Ig compared to monoclonal Ig [9]. Due to these differences in quantitation, as well as the inclusion of polyclonal Ig by nephelometry, the International Myeloma Working Group guidelines recommend monitoring monoclonal gammopathies using densitometry-based methods in electrophoresis as opposed to nephelometric values [10, 11]. Analysis of the FLC ratio showed significant elevation in three patients, indicating the presence of κ-FLC.
Figure 1 Immunofixation electrophoresis of serum from Patient 6 with γ-HCD. The (Gκ+Gλ)/IgGtotal ratio for this patient was 0.10. The arrow indicates heavy-chain reactivity with lack of corresponding light chain reactivity.
Measuring heavy chain protein: M-spike vs Nephelometry
10
Beta migration
9
Gamma migration
8
y = 2.095x-0.443
7 6 5 4 3 2 1 0
0
1
2 3 M-spike, g/dL
4
5
Figure 2 Comparison of electrophoretic M-spike versus nephelometric quantitation of monoclonal heavy chain protein.
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668 Kaleta et al.: Heavy/light chain and free light chain assays with HCD Table 2 Free light chain analysis in patients with γ-HCD. Patient ID
Gκ+Gλ/IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Reference range
0.63a 0.79a 0.22a 0.20a 0.26a 0.10a 0.02a 0.02a 0.02a 0.08a 0.03a 0.15a 0.03a 0.05a 0.09a 0.80–1.12
κ-FLC, mg/dL
λ-FLC, mg/dL
0.76 1.03 1.21 1.54 1.55 0.79 0.61 0.84 84.8a 1.07 3.89a 2.32a 136a 0.29 1.97a 0.26–1.65
0.47 1.62 1.19 3.06a 3.41a 1.46 1.37 0.92 378a 1.04 1.15 3.09a 91.1a 0.95 3.87a 0.33–1.94
0.62 1.57 0.99 1.99 2.20 1.85 2.23 1.09 4.46a 0.98 0.30a 1.33 0.67 3.29a 1.96 0.57–2.63
κ/λ Ratio
Denotes a result outside of the reference range.
a
Presumably, both heavy chain and light chain were produced early within the clonal expansion, and a subsequent mutation resulted in a truncated form of heavy chain which was unable to form intact immunoglobulin. In some clones, light chain synthesis may have been maintained and both the truncated heavy chain and light chain were produced and secreted as separate molecules. In this study, only κ-FLC elevations were seen, but this does not exclude the possibility of γ-HCD with λ-FLC, and may be a result of a small sample population which was inherent with this low prevalence disease.
Conclusions This study demonstrates that heavy/light chain analysis is a diagnostic tool that can aid in the characterization of γ-HCD, a disease that may be difficult to diagnose with
laboratory findings which rely on the absence of detectable protein. Patients with γ-HCD demonstrated an expectedly low (Gκ+Gλ)/IgGtotal ratio, indicating that the monoclonal protein identified was a heavy chain variant with little to no intact light chain. In addition, at least three of 15 γ-HCD patients also secreted monoclonal κ-FLC. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received August 30, 2013; accepted November 14, 2013; previously published online December 11, 2013
References 1. Wahner-Roedler DL, Kyle RA. Heavy chain diseases. Best Pract Res Clin Haematol 2005;18:729–46. 2. Wahner-Roedler DL, Witzig TE, Loehrer LL, Kyle RA. Gamma-heavy chain disease: review of 23 cases. Medicine (Baltimore) 2003;82:236–50. 3. Bieliauskas S, Tubbs RR, Bacon CM, Eshoa C, Foucar K, Gibson SE, et al. Gamma heavy-chain disease: defining the spectrum of associated lymphoproliferative disorders through analysis of 13 cases. Am J Surg Pathol 2012;36:534–43.
4. 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. 5. 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. 6. Katzmann JA, Kyle RA, Benson J, Larson DR, Snyder MR, Lust JA, et al. Screening panels for detection of monoclonal gammopathies. Clin Chem 2009;55:1517–22.
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Kaleta et al.: Heavy/light chain and free light chain assays with HCD 669 7. 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. 8. 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;55:1646–55. 9. Murray DL, Ryu E, Snyder MR, Katzmann JA. Quantitation of serum monoclonal proteins: relationship between agarose
gel electrophoresis and immunonephelometry. Clin Chem 2009;55:1523–9. 10. Kyle RA, Child JA, Anderson K, Barlogie B, Bataille R, Bensinger W, et al. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003;121:749–57. 11. Durie BG, Harousseau J-L, Miguel JS, Bladé J, Barlogie B, Anderson K, et al. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467–73.
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Erin Kaleta, Robert Kyle, Raynell Clark and Jerry Katzmann*
Analysis of patients with γ-heavy chain disease by the heavy/light chain and free light chain assays Abstract Background: The objective of this study was to evaluate the performance of the heavy/light chain and free light chain immunoassays in patients with heavy chain disease, and to assess the ability of the heavy/light chain assay to measure and confirm the abnormal, truncated heavy chain. Methods: Frozen serum samples from 15 γ-heavy chain disease patients were tested for IgGκ, IgGλ, total IgG, free light chains, and M-spike concentrations. Results: The (Gκ+Gλ)/IgGtotal ratio for these 15 patients ranged from 0.02 to 0.80. The 10 patients with IgG concentrations above 1 g/dL all had ratios below 0.3 indicating that a substantial portion of IgG was not quantitated by the Gκ and Gλ reagents. The average M-spike was 1.61 g/dL and the average calculated abnormal γ-chain concentration was 2.94 g/dL. Additionally, free light chain analysis revealed the presence of monoclonal free κ light chain in three of the 15 patients. Conclusions: This study demonstrates utility of a nephelometric assay to identify truncated immunoglobulin heavy chains in γ-HCD and that 20% of these patients also have monoclonal free light chain. Keywords: free light chain assay; heavy chain disease; heavy/light chain assay; nephelometry. *Corresponding author: Jerry Katzmann, PhD, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic Hilton 210, 200 1st St SW, Rochester, MN 55905, USA, Fax: +1 507 2664088, E-mail: [email protected]. Erin Kaleta and Raynell Clark: Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Robert Kyle: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA
Introduction Immunoglobulin molecules are tetra peptides containing two identical heavy chains and two identical light chains that are tightly bound together. Heavy chain diseases
(HCD) are rare lymphoplasmacytic proliferative disorders that are characterized by the production of a monoclonal immunoglobulin that is a truncated heavy chain which lacks corresponding light chains. These diseases include α-, γ-, and μ-HCD [1–3]. α-HCD is the most common of these disorders and usually presents as extranodal marginal zone lymphoma of mucosa-associated lymph node tissue (MALT) caused by chronic antigenic stimulation with intestinal organisms. This variety of HCD is treated by managing exposure to intestinal pathogens. μ-HCD is extremely rare, with only 33 reported cases in the literature as of 2005, and is commonly associated with lymphoproliferative malignancy [1]. γ-HCD has more diverse clinical features, and can present as disseminated lymphoproliferative disease, localized proliferative disease, or no apparent proliferative disease, which typically has an underlying autoimmune disorder. The median age of onset is 60 years, with symptoms involving anemia, fatigue, lymphadenopathy, splenomegaly and hepatomegaly [4, 5]. The testing strategy that is typically followed for any monoclonal gammopathy includes serum and/or urine protein electrophoresis, followed by immunofixation electrophoresis (IFE) [6]. The results with HCD samples demonstrate a monoclonal heavy chain with no corresponding light chain. In addition to the lack of light chain, heavy chains are typically truncated, further reducing the molecular weight [2], and the truncated heavy chain is often so small that serum concentrations are low due to renal clearance [7]. Due to these low serum concentrations, the definitive absence of light chain may be difficult to establish. In addition, some intact monoclonal immunoglobulins have poor reactivity with light chain anti-sera and show only faint light chain on IFE, blurring the distinction between poorly reacting light chain and absent light chain. Thus, identification of HCD can be difficult. The HevyliteTM immunoassay targets the unique junctional epitope between immunoglobulin heavy chain and light chain combination, and therefore the monoclonal protein in HCD will not be recognized by heavy/light chain reagents [8]. For these studies, we have focused on γ-HCD, measuring intact Gκ, Gλ, total IgG, and calculated the (Gκ+Gλ)/IgGtotal ratio in order to quantitate the portion of IgG that is derived from the monoclonal plasma cell clone.
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666 Kaleta et al.: Heavy/light chain and free light chain assays with HCD
Materials and methods Stored residual serum samples from 15 patients with previously diagnosed γ-HCD were identified by querying the dysproteinemia database, which contains data on all patients with plasma cell proliferative disorders seen at Mayo Clinic in Rochester, MN. The search was filtered for γ-HCD patients with a detectable serum M-spike. Serum samples that had been stored between 1978 and 2003 were retrieved from frozen storage (−20 °C). The database inquiries and use of stored sera were approved by the Mayo Clinic Institutional Review Board. Samples were analyzed by serum protein electrophoresis (SPEP) and immunofixation (IFE) to confirm the diagnosis of HCD. SPEP was performed on the Helena SPIFE SPE system (Helena Laboratories, Beaumont, TX, USA), stained with Acid Blue and visualized on the Helena SPIFE 3000 system. IFE was performed on Sebia HYDRAGEL 9IF kits with testing performed on the Sebia HYDRASys System (Sebia, Norcross, GA, USA). Nephelometry was performed to measure total immunoglobulins with a Siemens BNII and Siemens IgG antisera (Siemens, Tarrytown, NY, USA). Heavy/light chain measurements were performed on the BNII with immunoglobulin heavy/light chain reagents (Hevylite™, The Binding Site, Birmingham, UK). To establish expected results for the (Gκ+Gλ)/IgG ratio and the Gκ/Gλ ratio, we analyzed sera from 275 normal donors for Gκ, Gλ, and IgG concentrations. The 95% reference range for total IgG was 0.77–1.59 g/dL, for the ratio of IgGκ:IgGλ was 0.71–3.23 and for the ratio of (Gκ+Gλ)/IgGtotal is 0.80–1.12. The free light chain assay was performed on the BNII with the Free Light Chain assay (Freelite™, The Binding Site, San Diego, CA, USA).
Results SPEP was performed to identify the migration of the truncated immunoglobulin fragments and to quantitate the amount of monoclonal protein. Nine of the proteins migrated in the β-fraction and six migrated in the γ-fraction (Table 1). The M-spikes ranged from 0.37 to 4.17 g/dL with an average of 1.61 g/dL. All samples had a discrete γ-heavy chain band by IFE with an absence of a corresponding band in the anti-κ or anti-λ lanes (Figure 1). The total IgG, IgGκ, and IgGλ was determined (Table 1). On average, the sum of the IgGκ+IgGλ accounted for only 18% of the total IgG indicating that 82% of the IgG did not have an associated light chain. The distribu tions of the individual ratios of (Gκ+Gλ)/IgGtotal) are shown in Table 1. The ability of the heavy/light r eagents to account for the total IgG is significantly reduced and must be quantitating only the polyclonal IgG in the patient’s serum. In support of this, the ratios of the IgGκ to IgGλ for each patient fell within the 95% normal reference range (0.71–3.23). The IgG that is not recognized by the heavy/light reagents [IgG–(Gκ+Gλ)] is an indirect measure of the monoclonal heavy chain fragment concentration. The calculated heavy chain fragment concentrations ranged from
0.15 to 8.86 g/dL and averaged 2.94 g/dL compared to the average M-spike of 1.61 g/dL (Table 1). The relationship between the M-spike concentration and the calculated monoclonal IgG fragment is shown in Figure 2. These patients demonstrated a two-fold difference between M-spike and nephelometry. Greater differences between the two methods were seen with γ- and β-migrating protein at higher protein concentrations > 3 g/dL. The low concentration monoclonal proteins migrating in the β region are not always resolved from normal β-proteins, increasing the effective M-spike values which mask the difference between the two measures. In addition to heavy/light chain analysis, all patient sera were assessed for free light chain κ- and λ-concentrations and FLC κ/λ ratio (Table 2). In our patient set, 5/15 had abnormally elevated FLC κ/λ ratios, two patients of which were only slightly elevated, potentially due to renal dysfunction as a consequence of the monoclonal gammopathy. The remaining three patients have elevations clearly indicating monoclonal κ-FLC. Retrospective history review of urine protein electrophoresis and IFE indicated that one patient (Patient 10) showed evidence of a monoclonal κ-protein which had a different mobility than the IgG band.
Discussion The lack of recognition by the heavy/light chain reagents was consistent with the presence of γ-HCD and was in agreement with electrophoresis and immunofixation findings. The identification of γ-HCD by IFE relies on an IgG band and a lack of corresponding κ- or λ-reactivity. The heavy/light chain assay offers additional confirmation for difficult to interpret serum protein immunofixation results by providing numerical results to be paired with traditional electrophoresis and immunofixation findings. In this study we selected for HCD patients with a large enough abnormality to present with an M-spike, and the heavy/light chain assay results indicated γ-HCD in all 15 patients. With each patient the Gκ+Gλ did not account for the total IgG and the Gκ/Gλ ratio was within normal limits, indicating that the remaining intact immunoglobulins were polyclonal protein. It is possible that quantitative assays using anti-Fc and anti-Fab might also provide information regarding a discrepancy between Fc and Fab quantitation. The various interactions of anti-Fab with HCD γ-chains, however, are unknown. The lack of agreement between M-spike quantitation and IgG quantitation was an unexpected result. In
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Kaleta et al.: Heavy/light chain and free light chain assays with HCD 667
Table 1 Heavy/light chain immunoglobulin analysis in patients with γ-HCD. Patient ID
IgG Quant, g/dL
Gκ+Gλ, g/dL
Gκ+Gλ/IgG
Calculated clonal heavy chain, g/dL [IgG–(Gκ+Gλ)]
M-spike, g/dL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Reference range Average
0.46a 0.73a 1.08 1.65a 2.25a 2.85a 4.05a 6.46a 8.99a 1.94a 2.03a 3.09a 3.15a 4.08a 5.38a 0.77–1.59 –
0.29 0.58 0.24 0.33 0.59 0.29 0.08 0.13 0.18 0.16 0.06 0.46 0.90 0.20 0.48 –
0.63a 0.79a 0.22a 0.20a 0.26a 0.10a 0.02a 0.02a 0.02a 0.08a 0.03a 0.15a 0.03a 0.05a 0.09a 0.80–1.12 0.18
0.17 0.15 0.84 1.32 1.67 2.55 3.96 6.34 8.86 1.79 1.97 2.64 3.05 3.88 4.90 – 2.94
0.37 βb 0.50 βb 0.58 βb 1.12 βb 1.60 β 2.46 β 1.75 β 2.28 β 4.17 β 0.82 γ 0.52 γ 1.84 γ 1.81 γ 1.84 γ 2.55 γ – 1.61
a Denotes a result outside of the reference range; bPatients 1–4 have monoclonal proteins that are not completely resolved from the β-fraction, and the M-spike value contains co-migrating β-proteins.
all cases of γ-migrating monoclonal protein and in those with β-migration with nephelometric concentrations > 3 g/dL, the M-spike quantitation by electrophoresis was nearly two-fold lower than the calculated value from nephelometry. The M-spike quantitation is derived from the amount of dye bound by the protein and should be an indirect measure of mass. The nephelometric assay correlates light scatter with a standard curve that is based upon intact immunoglobulin in order to convert light scatter of molecules to mass. Although the monoclonal heavy chain fragment has lost some antigenic sites and anti-γ
anti-α
anti-µ
anti-κ
anti-λ
IgG-(GK+GL) with Nephelometr,y g/dL
SPEP
light scatter, it is being converted based on an IgG standard that is anywhere from six- to 10-fold larger and results in an overestimation due to these differences. An additional complication relates to the differences in reactivity of intact polyclonal Ig compared to monoclonal Ig [9]. Due to these differences in quantitation, as well as the inclusion of polyclonal Ig by nephelometry, the International Myeloma Working Group guidelines recommend monitoring monoclonal gammopathies using densitometry-based methods in electrophoresis as opposed to nephelometric values [10, 11]. Analysis of the FLC ratio showed significant elevation in three patients, indicating the presence of κ-FLC.
Figure 1 Immunofixation electrophoresis of serum from Patient 6 with γ-HCD. The (Gκ+Gλ)/IgGtotal ratio for this patient was 0.10. The arrow indicates heavy-chain reactivity with lack of corresponding light chain reactivity.
Measuring heavy chain protein: M-spike vs Nephelometry
10
Beta migration
9
Gamma migration
8
y = 2.095x-0.443
7 6 5 4 3 2 1 0
0
1
2 3 M-spike, g/dL
4
5
Figure 2 Comparison of electrophoretic M-spike versus nephelometric quantitation of monoclonal heavy chain protein.
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668 Kaleta et al.: Heavy/light chain and free light chain assays with HCD Table 2 Free light chain analysis in patients with γ-HCD. Patient ID
Gκ+Gλ/IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Reference range
0.63a 0.79a 0.22a 0.20a 0.26a 0.10a 0.02a 0.02a 0.02a 0.08a 0.03a 0.15a 0.03a 0.05a 0.09a 0.80–1.12
κ-FLC, mg/dL
λ-FLC, mg/dL
0.76 1.03 1.21 1.54 1.55 0.79 0.61 0.84 84.8a 1.07 3.89a 2.32a 136a 0.29 1.97a 0.26–1.65
0.47 1.62 1.19 3.06a 3.41a 1.46 1.37 0.92 378a 1.04 1.15 3.09a 91.1a 0.95 3.87a 0.33–1.94
0.62 1.57 0.99 1.99 2.20 1.85 2.23 1.09 4.46a 0.98 0.30a 1.33 0.67 3.29a 1.96 0.57–2.63
κ/λ Ratio
Denotes a result outside of the reference range.
a
Presumably, both heavy chain and light chain were produced early within the clonal expansion, and a subsequent mutation resulted in a truncated form of heavy chain which was unable to form intact immunoglobulin. In some clones, light chain synthesis may have been maintained and both the truncated heavy chain and light chain were produced and secreted as separate molecules. In this study, only κ-FLC elevations were seen, but this does not exclude the possibility of γ-HCD with λ-FLC, and may be a result of a small sample population which was inherent with this low prevalence disease.
Conclusions This study demonstrates that heavy/light chain analysis is a diagnostic tool that can aid in the characterization of γ-HCD, a disease that may be difficult to diagnose with
laboratory findings which rely on the absence of detectable protein. Patients with γ-HCD demonstrated an expectedly low (Gκ+Gλ)/IgGtotal ratio, indicating that the monoclonal protein identified was a heavy chain variant with little to no intact light chain. In addition, at least three of 15 γ-HCD patients also secreted monoclonal κ-FLC. Conflict of interest statement Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Received August 30, 2013; accepted November 14, 2013; previously published online December 11, 2013
References 1. Wahner-Roedler DL, Kyle RA. Heavy chain diseases. Best Pract Res Clin Haematol 2005;18:729–46. 2. Wahner-Roedler DL, Witzig TE, Loehrer LL, Kyle RA. Gamma-heavy chain disease: review of 23 cases. Medicine (Baltimore) 2003;82:236–50. 3. Bieliauskas S, Tubbs RR, Bacon CM, Eshoa C, Foucar K, Gibson SE, et al. Gamma heavy-chain disease: defining the spectrum of associated lymphoproliferative disorders through analysis of 13 cases. Am J Surg Pathol 2012;36:534–43.
4. 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. 5. 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. 6. Katzmann JA, Kyle RA, Benson J, Larson DR, Snyder MR, Lust JA, et al. Screening panels for detection of monoclonal gammopathies. Clin Chem 2009;55:1517–22.
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Kaleta et al.: Heavy/light chain and free light chain assays with HCD 669 7. 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. 8. 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;55:1646–55. 9. Murray DL, Ryu E, Snyder MR, Katzmann JA. Quantitation of serum monoclonal proteins: relationship between agarose
gel electrophoresis and immunonephelometry. Clin Chem 2009;55:1523–9. 10. Kyle RA, Child JA, Anderson K, Barlogie B, Bataille R, Bensinger W, et al. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003;121:749–57. 11. Durie BG, Harousseau J-L, Miguel JS, Bladé J, Barlogie B, Anderson K, et al. International uniform response criteria for multiple myeloma. Leukemia 2006;20:1467–73.
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