Katzmann, Jerry A.

AbstractBACKGROUNDThe use of electrophoresis to monitor monoclonal immunoglobulins migrating in the β fraction may be difficult because of their comigration with transferrin and complement proteins.METHODSImmunoassays specific for IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ heavy/light chain (HLC) were validated for use in the clinical laboratory. We assessed sample stability, inter- and intraassay variability, linearity, accuracy, and reference intervals for all 6 assays. We tested accuracy by verifying that the sum of the concentrations for the HLC-pairs accounted for the total immunoglobulins in each of 129 healthy sera, and that the HLC-pair ratios (rHLCs) were outside the reference interval in 97% of 518 diagnostic multiple myeloma (MM) samples.RESULTSWe assessed diagnostic samples and posttreatment sera in 32 IgG and 30 IgA patients for HLC concentrations, rHLC, and total immunoglobulins and compared these nephelometry results with serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). In sample sets from patients with IgG MM, the sensitivity of SPEP was almost the same as for rHLC, and no additional advantage was conferred by running HLC assays. In pre- and posttreatment samples from patients with IgA MM, the SPEP, rHLC, and IFE identified clonality in 28%, 56%, and 61%, respectively. In addition, when M-spikes were quantifiable, the concentration of the involved HLC was linearly related to that of the SPEP M-spike, with a slope near 1.CONCLUSIONSThe use of IgA HLC assays for monitoring β-migrating IgA monoclonal proteins can substitute for the combination of SPEP, IFE, and total IgA quantification.

The serum immunoglobulin free light chain (FLC) assay quantitates free kappa (κ) and lambda (λ) light chains. FLC elevations in patients with diffuse large B‐cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and chronic lymphocytic leukemia (CLL) are associated with an inferior survival. These increases in FLC can be monoclonal (as in myeloma) or polyclonal. The goal was to estimate the frequency of these elevations within distinct types of B‐cell and T‐cell non‐Hodgkin lymphoma (NHL) and whether the FLC measurements are associated with event‐free survival (EFS). We studied serum for FLC abnormalities using normal laboratory reference ranges to define an elevated κ or λ FLC. Elevations were further classified as polyclonal or monoclonal. Four hundred ninety‐two patients were studied: 453 B‐cell and 34 T‐cell NHL patients. Twenty‐nine % (142/453) of patients had an elevated FLC of which 10% were monoclonal elevations. Within B‐cell NHL, FLC abnormalities were most common in lymphoplasmacytic (79%), mantle cell (68%), and lymphomas of mucosa associated lymphoid tissue (31%); they were least common in follicular (15%). The hazard ratio (HR) for EFS in all patients was 1.41 (95% CI; 1.11–1.81); in all B‐cell NHL the HR was 1.44 (95% CI 1.11–1.96); in all T‐cell NHL the HR was 1.17 (95% CI 0.55–2.49). FLC abnormalities predicted an inferior OS (HR = 2.75, 95% CI: 1.93–3.90, P < 0.0001). The serum FLC assay is useful for prognosis in both B‐cell and T‐cell types of NHL. In B‐cell NHL further discrimination between a monoclonal and polyclonal elevation may be helpful and should be analyzed in prospective clinical trials. Am. J. Hematol. 89:1116–1120, 2014. © 2014 Wiley Periodicals, Inc.

AbstractBACKGROUNDMeasurement of IgG subclasses is a useful tool for investigation of humoral immune deficiency in the presence of total IgG within reference intervals and IgG4-related disease. Nephelometry has been the method of choice for quantification. We describe an LC-MS/MS method that can multiplex all 4 subclasses along with total IgG by use of either IgG subclass-specific peptide stable isotope–labeled internal standards or a surrogate digest standard for quantification and does not rely on antigen/antibody reactions.METHODSWe combined serum with labeled internal peptide standards and intact purified horse IgG. Samples were denatured, reduced, alkylated, and digested. We analyzed the digested serum by LC-MS/MS for IgG subclasses 1–4 and total IgG.RESULTSWe assayed 112 patient sera by LC-MS/MS and immunonephelometry. The mean of the slopes and R2 values for IgG1, IgG2, IgG3, IgG4, and IgG were 1.18 and 0.93, respectively. Interassay imprecision for the LC-MS/MS method was &lt;15% for total IgG and subclasses and was slightly improved by use of a calibrator peptide from an exogenous horse IgG. Summed total IgG correlated with the measured total IgG within 10%. Reference intervals and analytical measuring range were all similar to our previous validation data for the immunonephelometry assays.CONCLUSIONSTotal IgG and IgG subclasses 1, 2, 3, and 4 can be quantified by LC-MS/MS with performance comparable to nephelometry.