Sensitivity of serum free light chain assays

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Chapter

6

SECTION 1 - Immunoglobulin free light chains and their analysis

Sensitivity of serum free light chain assays
Summary: Free light chain in immunoassays:-
  1. Are inherently much more sensitive than electrophoretic tests (Figure 6.1).
  2. Identify additional patients in all diseases associated with monoclonal gammopathies.
  3. Provide quantitative κ/λ ratios compared with qualitative IFE.

Contents

6.1. Introduction

Figure 6.1 Sensitivity of assays for serum FLC quantitation with error bars indicating the different claimed limits.

There are many factors that need to be taken into account when deciding upon the most appropriate methods for measuring monoclonal FLCs. Requirements for the assays include sensitivity, accuracy, speed, cost, reliability, hands-on time, etc. A number of publications have compared different FLC assays and many of the important features are discussed in other chapters. This chapter is concerned only with a comparison of the sensitivity of routine laboratory assays for FLC detection in a clinical setting (Figure 6.1) - arguably the most important issue [1][2].

6.2. Serum and urine protein electrophoresis (SPE and UPE)

Figure 6.2 Normal SPE. (Courtesy of JA Katzmann).
Figure 6.3 SPE and IFE of a serum IgGλ monoclonal protein. Also identified is a small amount of polyclonal IgG with its associated κ and λ staining, and virtually no IgA or IgM. (Courtesy of JA Katzmann).

SPE is the standard screening method for MM and is usually based upon scanning agarose electrophoretic gels after the serum proteins have been separated, fixed and stained. A normal SPE is shown in Figure 6.2 and a monoclonal immunoglobulin is shown in Figure 6.3.

The sensitivity of SPE for FLC detection is between 500mg/L and 2,000mg/L depending upon whether or not the monoclonal protein migrates alongside beta proteins [3]. SPE is negative for FLCs in all patients with NSMM, the majority of patients with AL amyloidosis and many patients with LCMM and other plasma cell dyscrasias (Chapter 8 , 9 and 15) .

Figure 6.4 SPE in 9 patients with LCMM and one normal sample compared with the concentrations of sFLCs (mg/L). Some samples appear relatively normal by SPE but sFLC concentrations are grossly abnormal in all samples.
Figure 6.5 UPE in 9 patients with LCMM and one normal serum sample compared with measurements of urine FLCs by immunoassay.

Examples of sera containing medium to high concentrations of FLCs are shown in Figure 6.4. Most show the typical electrophoretic abnormalities of monoclonal bands and hypogammaglobulinaemia. Sample No 2, however, appears normal yet the λ FLC concentration is elevated 50-fold. This is typical of many serum samples that are sent to the laboratory with a diagnosis of, “possible multiple myeloma - please investigate”. Current practice would require testing a urine sample, but this is typically only available for 15-40% of the patients. sFLC assays would avoid the need for testing urine in all of the nine patients shown (Chapter 24). Screening sera by SPE and sFLCs is a simple and sensitive strategy for identifying new patients with monoclonal gammopathies and avoids the need for urine tests (Chapter 23).

UPE is much more sensitive than SPE since urine can be concentrated many times (Figure 6.5). Thus, FLCs in urine can be detected at <10-20mg/L, although most laboratories claim a detection limit in the region of 40-50mg/L. In practice, high concentrations of background proteins and “ladder banding” of polyclonal FLCs prevent attainment of the ideal sensitivity. Furthermore, some patients with LCMM produce only small amounts of FLCs so that little, if any, passes the absorptive surface of the renal proximal tubules. These patients may, therefore, have no detectable levels of uFLCs.

6.3. Serum immunofixation electrophoresis (IFE)

Serum IFE is approximately 10-fold more sensitive for FLC detection (150-500mg/L) than SPE but still considerably less sensitive than FLC immunoassays. One particular disadvantage of IFE is that it cannot be used to quantify monoclonal immunoglobulins because of the presence of the precipitating antibody. IFE is also rather laborious to perform and visual interpretation may be difficult. A typical result on a sample containing a substantial amount of IgGλ monoclonal protein, is shown in Figure 6.3.

Figure 6.6 FLC concentrations in sera containing monoclonal FLCs that were “difficult to detect” by IFE.

In a study from The Mayo Clinic [4], it was shown that all of 46 serum samples with low concentrations of monoclonal FLCs were correctly identified by FLC immunoassay (Figure 6.6). IFE was less sensitive and detected FLCs in some of the sera only after multiple assays and at different sample dilutions. The study included serum samples from patients in whom IFE was only positive for uFLCs. In addition, one sample shown in Figure 6.6 was negative by serum and urine IFE and sFLC immunoassays.

The high sensitivity of FLC assays is dependent upon assessing the individual FLC concentrations and the κ/λ ratios. Tumour suppression of the normal plasma cells in the bone marrow reduces the concentrations of the alternate FLC and, thereby, enhances the sensitivity of the κ/λ ratio. The alternate FLC concentrations and hence the κ/λ ratios, are important aspects of the diagnostic accuracy of sFLC immunoassays.

Figure 6.6 shows that there is a poor correlation between sFLC concentrations and the detection of monoclonal proteins by IFE. This may be due to polymerisation of the FLCs, which prevents the formation of visible, narrow, monoclonal bands during SPE. This has been observed in patients with NSMM (Chapter 9) and probably occurs in most MM sera. However, rare serum samples from patients with AL amyloidosis and LCDD may be negative by sFLC assays but positive by IFE. This indicates that sFLC assays and IFE should be used as complementary diagnostic tests in occasional patients with AL amyloidosis (Chapter 15) .

Figure 6.7 Serum κ and λ FLC concentrations in 20 control samples and 21 samples that were normal by CZE but had monoclonal proteins by IFE. (Courtesy of X Bossuyt).

Some reports have suggested that sFLC analysis is not as sensitive as IFE for detecting some monoclonal gammopathies [5][6]. This is true, since there are many patients who produce monoclonal intact immunoglobulins alone. These patients, of course, cannot be detected by sFLC analysis. Such reports indicate some confusion by the authors about the specificity of the assays rather than their sensitivity.

6.4. Capillary zone electrophoresis (CZE)

CZE is used in many clinical laboratories for serum protein separation and is able to detect most monoclonal immunoglobulins. However, when compared with IFE, CZE fails to detect monoclonal proteins in 5% of positive samples [7][8][9]. These so called “false negative” results encompass low-concentration and “hidden” monoclonal proteins (e.g. in the transferrin peak).

Marien et al.[10], compared the sensitivity of sFLC assays and CZE for the detection of low concentration monoclonal immunoglobulins. Frozen sera from 55 patients, previously shown to contain monoclonal proteins by IFE, but negative by CZE, were assessed by immunoassays for FLCs. They included both intact immunoglobulin and FLC monoclonal proteins.

The results showed that all 21 samples from patients with LCMM had abnormal FLC results (Figure 6.7). In addition, 13 out of 33 samples from patients with intact monoclonal immunoglobulins had abnormal FLC ratios (not shown). These patients had MGUS and B-cell derived malignant diseases. FLC immunoassays were, therefore, more reliable than CZE for detecting LCMM and identified many abnormal samples in patients from other disease groups. Similar findings were reported from Bakshi et al [9]., and others in monoclonal protein screening studies (Chapter 23).

Figure 6.7 indicates that several samples with FLC concentrations greater than 1,000 mg/L were not detected by CZE. Under ideal conditions the sensitivity of this technique may be better but it is still substantially less than IFE. However, CZE does at least provide a quantitative measure of the monoclonal proteins whereas IFE does not.

6.5. Total κ and λ immunoassays

Unfortunately, immunoassays for total κ and λ are sometimes used to try and identify patients with LCMM. This is in spite of warnings by The College of American Pathologists and others, that the method is not sensitive enough for routine clinical use [1]. Indeed, samples containing many grams of FLCs may be completely missed using this technique.

Figure 6.8 Comparison of total serum light chains (A) and sFLC κ/λ ratios (B) for identifying patients with κ and λ LCMM. κ patients: black squares; λ patients: blue triangles. The red triangle is a misclassified sample. Normal range limits are shown.

The sensitivity of sFLC assays and total κ and λ assays were compared in a study by Marien et al.[10] 16 serum samples from patients with LCMM were investigated (samples from the CZE study described above). Total κ and λ concentrations were measured using Beckman-Coulter reagents on the Immage nephelometer and sFLC concentrations were measured by Freelite assays from The Binding Site. All samples were abnormal by FLC assays. This compared with only 5 of the 16 samples by total κ and λ assays, and one λ patient was misclassified as κ (Figure 6.8). Total κ and λ assays do not have a role in the clinical laboratory.

6.6. Urine tests for free light chains

There are many methods for detecting urine FLCs, some of which are outlined in Chapter 4. Serum immunoassays are preferable because urine is a poor fluid for assessing FLC concentrations (Chapter 3). As shown by Nowrousian et al.[11], significant amounts of sFLCs are necessary to cause FLC proteinuria in patients with MM. In that study, median monoclonal serum κ and λ concentrations were 113 mg/L and 278mg/L respectively, before Bence Jones proteinuria occured. Further clinical comparisons of serum and urine sensitivity are described in Chapter 24.

Analytical technique
Normal ranges Sensitivity
Serum FLC κ/λ ratio κ/λ >0.26 to <1.65 33 out of 33
Urine FLC κ/λ ratio κ/λ >2.04 to <10.37 29 out of 33
Urine total κ/λ ratio + levels κ/λ >1 to <5.2 & Tκ + λ<10mg/L 29 out of 32
Urine albumin/total protein albumin<0.3 & TP <300mg/L 14 out of 31
Urine total protein total protein <100mg/L 26 out of 32

Table 6.1. Sensitivity of different analytical techniques for FLC detection in 33 patients with MM (Herzog). (Tκ+λ = total κ + λ concentrations; TP = total protein).

Herzog and Hoffman compared the sensitivity of 3 different urine protein tests (Table 6.1) with serum and urine FLC immunoassays in 33 patients with MM who had Bence Jones proteinuria by IFE. Five of the patients had LCMM while 28 patients had intact monoclonal immunoglobulins in the serum with additional uFLC excretion. 15 of the latter patients also excreted intact monoclonal immunoglobulins into the urine. Results showed that sFLC κ/λ ratios provided the most sensitive analysis and better than uFLC κ/λ ratios (Table 6.1). Total protein tests for uFLCs were poor. The reference method for this study was urine IFE and positive results were the basis for sample selection. Other studies have shown that in a clinical setting, sFLC tests are considerably more sensitive than uIFE for identifying patients with residual disease (Chapters 8, 9, 10, 11, 12 and 24).

A comparison of uIFE and uFLC immunoassays for Bence Jones protein detection was made by Viedma et al [12]. While FLC immunoassays correctly identified many of the positive and negative samples, high background polyclonal FLCs (arising from renal damage) obscured correct interpretation of κ/λ ratios. Hence, uIFE was more accurate for monoclonal protein identification. Similar results have been observed by Katzmann et al (personal communication).

Le Bricon et al.[13], analysed the sensitivity of different urine assays for Bence Jones proteins in 20 patients with MM. The urines contained monoclonal intact immunoglobulins and/or monoclonal FLCs. Comparisons were made between assays for total protein (Pyrogallol Red), SDS-agarose gel electrophoresis (SDS-AGE: sensitivity 50mg/L in unconcentrated urine) and FLC immunoassays (sensitivity <1mg/L). The results confirmed the superior sensitivity of the FLC immunoassays. 6 of the 20 patients were abnormal by the total protein test, 11 by SDS-agarose gel electrophoresis and 16 by FLC immunoassays. 6 samples that were only abnormal by FLC immunoassays are shown in Table 6.2. The immunoassays were particularly helpful for identifying monoclonal FLCs when they were present in low concentrations. There was a reasonable correlation between the quantitative results for the different methods. Herzum et al.[14], also compared FLC immunoassays with other urine tests for FLCs. The FLC assays showed good precision (<10%), linearity and correlation between different instruments (Hitachi and BNII). However, the assays overestimated the amounts of Bence Jones protein present in urine samples. 37 samples were compared using the following methods: benzethonium chloride, Biuret, modified Biuret and FLC immunoassays. Measurements using immunoassays produced the highest results in 26 of the samples. The Biuret methods had the best correlation with the FLC immunoassays. The Biuret method measures peptide bonds, and is considered to be a good general assay method for proteins. The good correlation with the FLC test results provides supporting evidence for their accuracy.

Lueck et al.[15], assessed the utility of antibodies specific for FLCs in urine IFE using the Sebia electrophoresis system. They found that antibodies against total light chains detected FLC bands more effectively than the FLC specific antibodies and concluded that such antibodies were of little clinical use. However, there was no comparison with the latex-enhanced FLC nephelometric immunoassays on the urine samples.

Serum M-
protein		 Urine protein
(mg/L)		 Urine free light chains Urine SDS-
AGE
κ & λ(mg/L) κ/λ ratio
IgG kappa 50 92:10 9.2 Negative
IgA kappa 40 28:6 4.7 Negative
IgA lambda 50 7:94 0.07 Negative
IgA lambda 960 66:749 0.09 +/- polyclonal
IgD lambda 100 3:21 0.14 Negative
FLC 1,990 60:21 0.29 +/- polyclonal

Table 6.2. Comparison of urine tests in 6 patients who were negative for monoclonal urine proteins by SDS-agarose gel electrophoresis but were abnormal by FLC [13]. There was good overall correlation, but sample 6 probably contained albumin.

6.7. Discordant serum and urine free light chain results

Figure 6.9 κ/λ logarithmic plot of sFLCs showing samples that would be misidentified as negative using SPE and serum IFE. (High pIgG: polyclonal hypergammaglobulinaemia. For the data on the patient groups see the relevant chapters).

Quantitation of monoclonal FLCs by electrophoretic methods often produces lower results than FLC immunoassays. H. Zitterberg (Goteborg University, Sweden: Personal communication) reviewed the laboratory records from 22 newly diagnosed LCMM patients who presented over a 2-year period. By quantitative electrophoresis, none had more than 3g/L of sFLCs and 19 were below 1g/L. This is in contrast to 50% that were over 3g/L in the study by Bradwell et al.[16], using sFLC immunoassays. Part of the explanation is that urine and serum FLCs are frequently polymerised and this leads to high results by immunoassay (Chapter 4). Also, protein dye tests underestimate the concentrations of FLCs to a variable extent.

Although not quantitative, IFE is considered to be the “gold standard” for identifying monoclonal urine FLCs. Nevertheless, the detection of minor monoclonal bands by this method can be misleading. There are many reasons:-

  1. Precipitating antibodies against one or other FLC may not be completely specific and may cross-react with other urine proteins. This can produce a false positive band with the appearance of a monoclonal FLC.
  2. A narrow protein precipitate band can occur at the sample application site on the gel and appear like a monoclonal band.
  3. Restricted “ladder banding” in concentrated samples can give the appearance of monoclonality.
  4. Heavy proteinuria containing polyclonal FLCs may produce confusing background staining which obscures correct interpretation of monoclonal FLC bands.
  5. An inadequate antibody to one of the FLCs (usually λ) gives the impression that only the alternate FLC is being excreted. Although there may be a broad band, it can suggest monoclonality. This is a fairly frequent occurrence.
  6. After transplantation, oligoclonal bands are produced for many months. These may produce confusing results in urine samples and appear monoclonal. sFLC ratios, on the other hand, may be normal.
  7. IFE is not quantitative so it is difficult to compare one result with another. The majoriy of the staining intensity is due to the second antibody (80% or more), so it is not possible to assess the amounts of monoclonal protein present.

Even when a monoclonal uFLC band is visually convincing, it should be evaluated alongside other clinical and laboratory data because it may be inconsequential. As discussed elsewhere, clinically significant monoclonal uFLCs are exceptional when sFLCs are normal. In a study of 110 patients with AL amyloidosis [17], all but one had serum monoclonal proteins and the remaining patient was negative by all tests including urine analysis (Table 6.3). Isolated, minimal uFLC excretion has usually been considered as clinically insignificant, an opinion that is further supported by this data (Chapter 19.4, Figure 19.6).

Urine IFE may correctly identify monoclonal FLCs when serum analysis is normal, under the following unusual circumstances:-

  1. When there is damage to isolated nephrons allowing leakage of monoclonal FLCs into urine. Serum levels, in contrast, may not be raised sufficiently to produce abnormal κ/λ ratios as defined by the normal range (Chapter 5).
  2. Intact immunoglobulin molecules that enter the proximal tubules may become absorbed and partially metabolised by tubular cells. FLCs and other immunoglobulin fragments could subsequently be released and enter the urine.
  3. When monoclonal FLCs are truncated and rapidly cleared. The short serum half-life prevents accumulation in serum but significant levels could be present in urine. This must be a very rare occurrence since it has not been observed in any study to date.
  4. Abnormal amino-acid sequences could change the shape of the “hidden” epitopes on the FLC constant regions. This might prevent detection by antisera specific for FLCs. However, IFE antisera against the whole FLC structures would detect some remaining, undistorted epitopes and produce positive results [18]. This is speculative and has not been observed to date.
  5. When the serum and urine samples are collected at different times, results may be discordant. Normally, serum samples are collected at the clinic but the urine is either collected earlier or is subsequently sent in by post. If the samples are separated by a significant time, even a few days, results may be quite different because of the short serum half-life of FLCs (Chapter 10).

In spite of the possibility that uIFE is more sensitive for monoclonal FLCs under some circumstances, serum assays provide a reliable basis for assessing true FLC production by the tumour. Detailed physiological, technical and clinical reasons for using sFLC assays rather than urine assays are discussed in Chapter 24.

Test
Sensitivity
Serum FLC κ/λ ratio 91%
Serum IFE 69%
Urine IFE 83%
FLC κ/λ ratio and urine IFE 91%
FLC κ/λ ratio and serum IFE 99%
Serum IFE and Urine IFE 95%
All three tests 99%

Table 6.3. Comparison of serum and urine tests for identifying 110 patients with AL amyloidosis [17]. Urine tests provided no additional diagnostic benefit.

Conclusions

Since normal sFLC concentrations are considerably below the detection limit of all electrophoretic methods, some patients will always be missed using these techniques. The numbers of patients and the types of diseases missed by serum electrophoretic tests are shown diagrammatically in Figure 6.9. Reports vary in their claims for the achieved levels of sensitivity for electrophoretic tests, but the figure clearly indicates the benefits of the increased sensitivity of sFLC measurements. Further details are given in later chapters.

Test Questions
  1. What is the origin of ladder banding in UPE?
  2. Which is more sensitive, CZE or SPE?
  3. Do total serum light chain tests have a useful role in the laboratory?
  4. Under what circumstances is uIFE unreliable?
  5. Which is more specific for monoclonal proteins - urine FLC immunoassays or uIFE?


Chapter 5 Back to Contents Page Chapter 7

References

  1. ↑ 1.0 1.1 Kyle RA. Sequence of testing for monoclonal gammopathies. Arch Pathol Lab Med 1999; 123: 114 – 8 PMID: 10050783
  2. ↑ Guinan JE, Kenny DF, Gatenby PA. Detection and typing of paraproteins: comparison of different methods in a routine diagnostic laboratory. Pathology 1989; 21: 35 – 41 PMID: 2762044
  3. ↑ Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47: 673 – 80 PMID: 11274017
  4. ↑ Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, Kyle RA. 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 PMID: 12194920
  5. ↑ Jaskowski TD, Litwin CM, Hill HR. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis. Clin Vaccine Immunol 2006; 13: 277 – 80 PMID: 16467338
  6. ↑ Mehta J, Stein R, Vickrey E, Resseguie W, Singhal S. Significance of serum free light chain estimation with detectable serum monoclonal protein on immunofixation electrophoresis. Blood 2006; 108: 5048a
  7. ↑ Katzmann JA, Clark R, Sanders E, Landers JP, Kyle RA. Prospective study of serum protein capillary zone electrophoresis and immunotyping of monoclonal proteins by immunosubtraction. Am J Clin Pathol 1998; 110: 503 – 9 PMID: 9763037
  8. ↑ Bossuyt X, Marien G. False-negative results in detection of monoclonal proteins by capillary zone electrophoresis: a prospective study. Clin Chem 2001; 47: 1477 – 9 PMID: 11468244
  9. ↑ 9.0 9.1 Bakshi NA, Gulbranson R, Garstka D, Bradwell AR, Keren DF. Serum free light chain (FLC) measurement can aid capillary zone electrophoresis in detecting subtle FLC-producing M proteins. Am J Clin Pathol 2005; 124: 214 – 8 PMID: 16040291
  10. ↑ 10.0 10.1 Marien G, Oris E, Bradwell AR, Blanckaert N, Bossuyt X. Detection of monoclonal proteins in sera by capillary zone electrophoresis and free light chain measurements. Clin Chem 2002; 48: 1600 – 1 PMID: 12194945
  11. ↑ Nowrousian MR, Brandhorst D, Sammet C, Kellert M, Daniels R, Schuett P, Poser M, Mueller S, Ebeling P, Welt A, Bradwell AR, Buttkereit U, Opalka B, Flasshove M, Moritz T, Seeber S. Serum Free Light Chain Analysis and Urine Immunofixation Electrophoresis in Patients with Multiple Myeloma. Clin Cancer Res. 2005 Dec 15;11(24 Pt 1):8706-14. PMID: 16361557
  12. ↑ Viedma JA, Garrigos N, Morales S. Comparison of the Sensitivity of 2 Automated Immunoassays with Immunofixation Electrophoresis for Detecting Bence Jones Proteins. Clin Chem. 2005 Aug;51(8):1505-7 PMID: 16040842
  13. ↑ 13.0 13.1 Le Bricon T, Bengoufa D, Benlakehal M, Bousquet B, Erlich D. Urinary free light chain analysis by the Freelite immunoassay: a preliminary study in multiple myeloma. Clin Biochem 2002; 35: 565 – 7 PMID: 12493586
  14. ↑ Herzum I, Heinz R, Bruder-Burzlaff B, Renz H, Wahl HG. Reliability of the new freelite assay for quantification of free light chains in urine. Clin Chem 2004; 50: C38a
  15. ↑ Lueck N, Agrawal YP. Lack of utility of free light chain-specific antibodies in the urine immunofixation test. Clin Chem 2006; 52: 906 – 7 PMID: 16638965
  16. ↑ 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 PMID: 12583950
  17. ↑ 17.0 17.1 Katzmann JA, Abraham RS, Dispenzieri A, Lust JA, Kyle RA. Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice. Clin Chem 2005; 51: 878 – 81 PMID: 15774572
  18. ↑ Coriu D, Weaver K, Schell M, Eulitz M, Murphy CL, Weiss DT, Solomon A. A molecular basis for nonsecretory myeloma. Blood 2004; 104: 829 – 31 PMID: 15090444
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