Risk of Antigen Excess in Serum Free Light Chain Measurements

Measurement of immunoglobulin κ and λ free light chains (FLC) has improved the diagnostic evaluation and monitoring of monoclonal gammopathies (1), particularly light chain multiple myeloma(2), nonsecretory multiple myeloma, plasmacytoma, systemic amyloid light chain (AL) amyloidosis, heavy chain myeloma(3), and monoclonal gammopathy of undetermined significance(4). FLC measurement methods, however, are affected by analytical problems inherent to nephelometric techniques. A 76-year-old woman was admitted for evaluation of bone pain and hypercalcemia. The pains were located in the left inguinal fold. Examination showed no lymphadenopathy or hepatosplenomegaly. X-rays of the skeleton showed 2 voluminous gaps in the pubic branches. Laboratory examinations showed hypercalcemia of 2.91 mmol/L and renal insufficiency. Complete blood cell count showed a macrocytic anemia with a hemoglobin concentration of 97 g/L, rouleaux formation, and circulating plasmacytes. Bone marrow contained 85% dystrophic plasmacytes. Serum total protein was 68 g/L. Protein electrophoresis showed hypogammaglobulinemia of 2.2 g/L [reference interval (RI) 7–11 g/L] with a moderate increase of the α-2 fraction (12.2 g/L, RI, 6–9 g/L). Immunofixation electrophoresis of blood and urine showed a major band of monoclonal κ FLC migrating in the α-2 region. Serum FLC measurement showed a major increase in the κ FLC (Table 11 ). We arrived at a diagnosis of light chain κ multiple myeloma. In our laboratory, serum κ and λ FLC measurements are carried out with a nephelometric technique on the BNprospec automat (Dade Behring) using the Freelite reagents (The Binding Site) according to the manufacturer’s recommendations. A 1st FLC measurement is recommended on a sample diluted at 1:100. If the result is lower than the low end of the assay range, the machine automatically starts the assay again by diluting at 1:20 and then at 1:5. Likewise, if the concentration in FLC is higher than the high value of the range, the analyzer automatically starts serial dilutions and continues to a dilution of 1:32 000. Initially, the κ chains were <0.294 mg/L [RI, 3.3–19.4 mg/L (5)] and λ chains were <0.405 mg/L [RI, 5.7–26.3 mg/L(5)]. In view of this unusual result (double negativity), a 1:32 000 dilution was made manually and the κ-chain concentrations were then >60 100 mg/L. Measurement at a dilution of 1:2000 was then carried out for the λ chains, and the result was <0.41 mg/L (Table 11 ). Serum immunofixation confirmed the result, with a strong κ monoclonal band. Thirty-five days later the κ chains were 112 mg/L and λ chains <0.405 mg/L. Given the patient’s history, however, a measurement at a dilution of 1:32 000 was made manually for κ FLC, and the concentration obtained was 12 600 mg/L (Table 11 ). In this patient, we repeatedly observed that very high FLC concentrations yielded paradoxically low results. This situation is consistent with antigen excess. The “limitations” section of the product insert from the company does alert the user to the possibility of this phenomenon, but to our knowledge this report is the 1st to demonstrate that the problem of antigen excess in this assay is not merely theoretical but can actually occur clinically. The main risk is that a value within the RI, or one that is substantially underestimated, will be reported instead of the true value. To eradicate antigen excess, any suspect sample must be tested again at a higher dilution. Serum FLC measurements should always be interpreted together with other laboratory test results and clinical findings. This protocol requires a laboratory capable of performing serum protein electrophoresis, immunofixation, serum FLC measurement, and measurements of IgG, -A, and -M. Dissociation of these techniques among various laboratories can cause errors in interpretation. In conclusion, we have illustrated that antigen excess can cause falsely low serum FLC results with nephelometric techniques. Clinically suspicious results should be repeated after sample dilution. Serum FLC measurements according to the dilutions. Serum FLC measurements reported to the medical record after manual dilution. Serum FLC measurements according to the dilutions. Serum FLC measurements reported to the medical record after manual dilution. These authors contributed equally to this work. Grant/funding support: None declared. Financial disclosures: None declared.