Serum Free Light Chain in Lymphoproliferative Disorders
Serum Free Light Chain in Lymphoproliferative Disorders
The sFLC assay is a sensitive, latex-enhanced immunonephelometric test that quantitates free κ and λ FLC in serum and urine samples. The test is based on the presence of polyclonal antibodies in the reagent (Freelite, The Binding Site Ltd, Birmingham, UK) that react noncompetitively with Bence Jones proteins individually. The assay showed high sensitivity for κ FLC antibodies reacting with κ-labeled cells up to a dilution of 1:16,000 and no reactivity against polyclonal IgG, monoclonal IgA and IgM κ, and monoclonal λ FLC-coated cells at a dilution less than 1:2. Similar results were obtained with λ antibodies reacting with λ-labeled cells and polyclonal and monoclonal immunoglobulins as well as κ FLC-coated cells. At the same time, the detection limit for these assays is at least 20-fold and 50-fold lower than IFE and PEL, respectively. The studies mentioned in this review used the Freelite serum FLC assay from The Binding Site Ltd (Birmingham, UK) and was adapted to the BNII nephelometer (Dade Behring, Deerfield, IL).
In healthy individuals, sFLC concentrations depend on the balance between production by plasma cells and renal clearance. They are cleared rapidly with a serum half-life of 2 to 4 hours through the renal glomeruli and then metabolized in the proximal tubules of the nephrons. Normally little protein escapes to the urine, and sFLC concentrations have to increase manifold before the absorption mechanisms are overwhelmed. Katzmann et al defined the normal range for κ and λ FLC concentrations and ratio in healthy subjects. Fresh serum samples from 127 healthy subjects (age range, 21–62 years) and frozen serum samples from 155 donors (age range, 51–90 years) were collected from a serum bank in Olmsted Country, MN. The 95% reference intervals for κ and λ FLC were 3.3 to 19.4 mg/L and 5.7 to 26.3 mg/L, respectively. The 95% reference interval for the κ/λ ratio was 0.3 to 1.2. However, a 5% false-positive rate is considered a high number in diagnosing monoclonal FLC diseases; therefore, the reference range was expanded to 100% (0.26–1.65). The sensitivity dropped from 98% to 97%, and the specificity increased from 95% to 100%. The positive and negative predictive values were 100% and 99%, respectively. Any patient with a κ/λ ratio greater than 1.65 or less than 0.26 is considered to have excess κ or λ FLC, respectively. The 100% confidence interval used reduces the likelihood that polyclonal activation of B cells will cause an abnormal ratio and therefore the test must be interpreted in the context of a clinical situation and repeated at a later date. κ and λ sFLC concentrations may be abnormal because of a number of conditions including immune suppression, immune stimulation, reduced renal clearance, or monoclonal plasma cell proliferative disorders. Serum samples from patients with either polyclonal hyper-gammaglobulinemia or renal impairment often have elevated κ and λ FLC because of increased synthesis or reduced renal clearance; however, the sFLCr usually remains normal in these situations. However, Hill et al reported falsely elevated sFLCr in 14% of patients with polyclonal increase in Igs, with concurrently abnormal glomerular filtration rate, thus suggesting that these conditions can cause false-positive results for sFLCr. Nevertheless, a significantly abnormal κ/λ sFLCr should only be caused by a plasmaproliferative or lymphoproliferative disorder that secretes excess FLC and disturbs the normal balance between κ and λ secretion.
Although the test has major advances, it also has some limitations. First, there can be significant lot-to-lot variation (20% coefficient of variation [CV]) between batches of polyclonal FLC antiserum according to the manufacturer. Moreover, Tate et al showed that CV can change up to 29% and 45% for κ FLC and λ FLC, respectively, on repeated measurements. Their recommendation is to be cautious when following up a patient with serial sFLC when different reagent lots are used, because the κ/λ ratio can be doubled in a patient with a stable disease. This issue is mainly present in large multi-institutional trials, where serious consideration should be given for running samples at a centralized testing facility that performs lot-to-lot comparisons. Second, in the absence of additional offline dilutions, κ FLC, and to a lesser extent, λ FLC results may be lower than the real values. In addition, Tate et al showed that FLC might not dilute in a linear fashion, especially the κ type. False high results, by as much as 10-fold, may occur because of the polymerization of light chains. Finally, changes in amino acid sequence of the light chain cause a change in the FLC epitope configuration, which make them unrecognizable by the FLC reagents. Nakano and Nagata developed an FLC κ and λ enzyme-linked immunosorbent assay (range, 7.8–500 μg/L), and showed that there is cross reactivity of FLC κ and λ at 100 mg/L with intact IgGs of 0.11% and 0.12%, respectively. Moreover, the cross-reactivity between κ and λ FLCs is minimal at less than 0.015%. The greater the specificity, the better is one's ability to quantitate κ and λ FLC in the presence of a large excess of serum IgG, IgA, and IgM. This distinction is important, because in healthy individuals and in most patients with myeloma, most of the circulating light chain is bound to heavy chains, thus making less specific reagents a near surrogate for circulating heavy chain measurement.
Free Light Chain Assay
The sFLC assay is a sensitive, latex-enhanced immunonephelometric test that quantitates free κ and λ FLC in serum and urine samples. The test is based on the presence of polyclonal antibodies in the reagent (Freelite, The Binding Site Ltd, Birmingham, UK) that react noncompetitively with Bence Jones proteins individually. The assay showed high sensitivity for κ FLC antibodies reacting with κ-labeled cells up to a dilution of 1:16,000 and no reactivity against polyclonal IgG, monoclonal IgA and IgM κ, and monoclonal λ FLC-coated cells at a dilution less than 1:2. Similar results were obtained with λ antibodies reacting with λ-labeled cells and polyclonal and monoclonal immunoglobulins as well as κ FLC-coated cells. At the same time, the detection limit for these assays is at least 20-fold and 50-fold lower than IFE and PEL, respectively. The studies mentioned in this review used the Freelite serum FLC assay from The Binding Site Ltd (Birmingham, UK) and was adapted to the BNII nephelometer (Dade Behring, Deerfield, IL).
In healthy individuals, sFLC concentrations depend on the balance between production by plasma cells and renal clearance. They are cleared rapidly with a serum half-life of 2 to 4 hours through the renal glomeruli and then metabolized in the proximal tubules of the nephrons. Normally little protein escapes to the urine, and sFLC concentrations have to increase manifold before the absorption mechanisms are overwhelmed. Katzmann et al defined the normal range for κ and λ FLC concentrations and ratio in healthy subjects. Fresh serum samples from 127 healthy subjects (age range, 21–62 years) and frozen serum samples from 155 donors (age range, 51–90 years) were collected from a serum bank in Olmsted Country, MN. The 95% reference intervals for κ and λ FLC were 3.3 to 19.4 mg/L and 5.7 to 26.3 mg/L, respectively. The 95% reference interval for the κ/λ ratio was 0.3 to 1.2. However, a 5% false-positive rate is considered a high number in diagnosing monoclonal FLC diseases; therefore, the reference range was expanded to 100% (0.26–1.65). The sensitivity dropped from 98% to 97%, and the specificity increased from 95% to 100%. The positive and negative predictive values were 100% and 99%, respectively. Any patient with a κ/λ ratio greater than 1.65 or less than 0.26 is considered to have excess κ or λ FLC, respectively. The 100% confidence interval used reduces the likelihood that polyclonal activation of B cells will cause an abnormal ratio and therefore the test must be interpreted in the context of a clinical situation and repeated at a later date. κ and λ sFLC concentrations may be abnormal because of a number of conditions including immune suppression, immune stimulation, reduced renal clearance, or monoclonal plasma cell proliferative disorders. Serum samples from patients with either polyclonal hyper-gammaglobulinemia or renal impairment often have elevated κ and λ FLC because of increased synthesis or reduced renal clearance; however, the sFLCr usually remains normal in these situations. However, Hill et al reported falsely elevated sFLCr in 14% of patients with polyclonal increase in Igs, with concurrently abnormal glomerular filtration rate, thus suggesting that these conditions can cause false-positive results for sFLCr. Nevertheless, a significantly abnormal κ/λ sFLCr should only be caused by a plasmaproliferative or lymphoproliferative disorder that secretes excess FLC and disturbs the normal balance between κ and λ secretion.
Although the test has major advances, it also has some limitations. First, there can be significant lot-to-lot variation (20% coefficient of variation [CV]) between batches of polyclonal FLC antiserum according to the manufacturer. Moreover, Tate et al showed that CV can change up to 29% and 45% for κ FLC and λ FLC, respectively, on repeated measurements. Their recommendation is to be cautious when following up a patient with serial sFLC when different reagent lots are used, because the κ/λ ratio can be doubled in a patient with a stable disease. This issue is mainly present in large multi-institutional trials, where serious consideration should be given for running samples at a centralized testing facility that performs lot-to-lot comparisons. Second, in the absence of additional offline dilutions, κ FLC, and to a lesser extent, λ FLC results may be lower than the real values. In addition, Tate et al showed that FLC might not dilute in a linear fashion, especially the κ type. False high results, by as much as 10-fold, may occur because of the polymerization of light chains. Finally, changes in amino acid sequence of the light chain cause a change in the FLC epitope configuration, which make them unrecognizable by the FLC reagents. Nakano and Nagata developed an FLC κ and λ enzyme-linked immunosorbent assay (range, 7.8–500 μg/L), and showed that there is cross reactivity of FLC κ and λ at 100 mg/L with intact IgGs of 0.11% and 0.12%, respectively. Moreover, the cross-reactivity between κ and λ FLCs is minimal at less than 0.015%. The greater the specificity, the better is one's ability to quantitate κ and λ FLC in the presence of a large excess of serum IgG, IgA, and IgM. This distinction is important, because in healthy individuals and in most patients with myeloma, most of the circulating light chain is bound to heavy chains, thus making less specific reagents a near surrogate for circulating heavy chain measurement.
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