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Risk of antigen excess in serum free light chain measurements.

To the Editor:

Measurement of immunoglobulin [kappa] and [lambda] 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 [alpha]-2 fraction (12.2 g/L, RI, 6-9 g/L). Immunofixation electrophoresis of blood and urine showed a major band of monoclonal [kappa] FLC migrating in the [alpha]-2 region. Serum FLC measurement showed a major increase in the [kappa] FLC (Table 1). We arrived at a diagnosis of light chain [kappa] multiple myeloma.

In our laboratory, serum [kappa] and [lambda] 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 [kappa] chains were <0.294 mg/L [RI, 3.3-19.4 mg/L (5)] and k chains were <0.405 mg/L [RI, 5.726.3 mg/L (5)]. In view of this unusual result (double negativity), a 1:32 000 dilution was made manually and the [kappa]-chain concentrations were then >60100 mg/L. Measurement at a dilution of 1:2000 was then carried out for the [lambda] chains, and the result was <0.41 mg/L (Table 1). Serum immunofixation confirmed the result, with a strong [kappa] monoclonal band. Thirty-five days later the [kappa] chains were 112 mg/L and [lambda] chains <0.405 mg/L. Given the patient's history, however, a measurement at a dilution of 1:32 000 was made manually for [kappa] FLC, and the concentration obtained was 12 600 mg/L (Table 1).

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.

Grant/funding support: None declared. Financial disclosures: None declared.

DOI: 10.1373/clinchem.2007.093377

References

(1.) Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001;47:673-80.

(2.) Bradwell AR, Carr-Smith HD, Mead GP, Harvey TC, Drayson MT. Serum test for assessment of patients with Bence Jones myeloma. Lancet 2003;361:489-91.

(3.) Hill PG, Forsyth JM, Rai B, Mayne S. Serum free light chains: an alternative to the urine Bence Jones proteins screening test for monoclonal gammopathies. Clin Chem 2006;52: 1743-8.

(4.) Rajkumar SV, Kyle RA, Therneau TM, Melton LJ 3rd, Bradwell AR, Clark RJ, et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood 2005;106: 812-7.

(5.) 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;48:1437-44.

Sandrine Daval [1] [[dagger]] Arlette Tridon [1,2] [[dagger]] Nicole Mazeron [1] Jean-Michel Ristori [2,3] Bertrand Evrard [1,2] *

[1] Centre Hospitalier Universitaire Clermont-Ferrand Department of Immunology Hotel-Dieu Hospital Clermont-Ferrand, France

[2] University Clermontl Faculty of Medicine-Pharmacy Clermont-Ferrand, France

[3] Centre Hospitalier Universitaire Clermont-Ferrand Department of Rheumatology G Montpied Hospital Clermont-Ferrand, France

[[dagger]] These authors contributed equally to this work.

* Address correspondence to this author at: Department of Immunology, Hotel-Dieu, Blvd. Leon Malfreyt, F-63058 Clermont-Ferrand, France. Fax 33-4-73750-637; e-mail bevrard@chu-dermontferrand.fr.
Table 1. Serum FLC measurements according to the dilutions.

 Dilution [kappa], [lambda],
 mg/L mg/L

 1/100 <5.88 <8.09
Day 0 1/20 <1.17 <1.62
 1/5 <0.294 <0.405
 1/32 000 (a) >60 100
 1/2000 (a) <0.410
Day 35 1/100 112 <8.09
(1 course of chemotherapy) 1/20 <1.62
 1/5 <0.405
 1/32 000 (a) 12600
 1/2000 (a) <0.410

(a) Serum FLC measurements reported to the medical record
after manual dilution.
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Title Annotation:Letters
Author:Daval, Sandrine; Tridon, Arlette; Mazeron, Nicole; Ristori, Jean-Michel; Evrard, Bertrand
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Nov 1, 2007
Words:1017
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