Sensitivity of serum free light chain assays

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6.1. Introduction

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

Many factors need to be considered when deciding upon the most appropriate methods for measuring free light chains (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 their important features are discussed in other chapters of Wikilite. This chapter compares 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 multiple myeloma (MM) and usually involves 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 protein is shown in Figure 6.3.

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

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 urine samples, but these are only available for 15-40% of the patients in most laboratories. Serum FLC (sFLC) assays would avoid the need for testing urine in all 9 patients presented (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).

Murray et al. ^[4] analysed the relationship between monoclonal protein quantification by SPE and immunonephelometry. Measurement of 2,095 IgG monoclonal proteins demonstrated nonlinearity of SPE at high monoclonal IgG concentrations. They concluded that changes in plasma cell populations may not be accurately reflected by changes in monoclonal protein values determined by SPE scanning densitometry and that clinicians should be aware of these limitations when quantifying IgG by SPE.

Urine protein electrophoresis (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” caused by polyclonal FLCs prevent attainment of the ideal sensitivity. Furthermore, some patients with LCMM produce only small amounts of FLCs, so little, if any, passes the absorptive surface of the renal proximal tubules. These patients may, therefore, have no detectable levels of urine FLCs (uFLCs).

6.3. Serum immunofixation electrophoresis (sIFE)

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 uFLCs by immunoassay.

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

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).

sIFE 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. In a study from The Mayo Clinic ^[5], 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 sFIE and urine IFE (uIFE) and sFLC immunoassays.

The high clinical 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 highlights the poor correlation that exists 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 electrophoresis. 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 light chain deposition disease (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 patients with AL amyloidosis (Chapter 15). Some reports have suggested that sFLC analysis is not as sensitive as IFE for detecting some monoclonal gammopathies ^[6][7]. This is true, since there are many patients who produce monoclonal intact immunoglobulins alone, which 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 proteins. However, compared with IFE, CZE fails to detect monoclonal proteins in 5% of positive samples ^[8][9][10]. These so-called “false negative” results encompass low-concentration and “hidden” monoclonal proteins (e.g. in the transferrin peak).

Marien et al. ^[11] compared the sensitivity of sFLC assays and CZE for the detection of low concentration monoclonal immunoglobulins. Frozen sera from 55 patients, previously shown by IFE to contain monoclonal proteins, but which were 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 monoclonal gammopathy of undetermined significance (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. ^[10], and by 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 sensitive 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

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.

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.

The sensitivity of sFLC assays and total κ and λ assays were compared in a study by Marien et al. ^[11] Sixteen serum samples from patients with LCMM were investigated (samples were 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 (Figure 6.8). This compared with only 5 of the 16 samples by total κ and λ assays, and one λ patient was misclassified as κ. Total κ and λ assays do not have a role in the clinical laboratory.

6.6. Urine tests for free light chains (FLCs)

There are many methods for detecting urine FLCs, and some of these 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. ^[12] 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.

Table 6.1. Sensitivity of different analytical techniques for FLC detection in 33 patients with MM ^[13]. (Tκ+λ = total κ + λ concentrations; TP = total protein).

Herzog and Hoffman ^[13] 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 as assessed by IFE. Five of the patients had LCMM while 28 patients had intact monoclonal immunoglobulins in the serum with additional uFLC excretion. Fifteen 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 uIFE and positive results were the basis for sample selection.

Van Hoeven et al. ^[14] compared the sensitivity of three tests for FLCs (sFLC, uFLC und UPE) with reference to uIFE in 98 urine samples. sFLC assays were more sensitive than UPE for the detection of monoclonal FLCs. The authors also showed that the sensitivity of uFLC was greater than UPE (75% versus 44%) in samples with an abnormal uIFE. The difference between uFLC and UPE was even more pronounced for positive uIFE samples (89% versus 52%).

Fulton et al. ^[15] compared sFLC with uIFE when screening for monoclonal gammopathies. In 314 samples from 142 patients the sensitivity of the sFLC ratio and uIFE was 91% and 81%, respectively, with a specificity of 100% and 99%. 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. ^[16] 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).

Snyder et al. ^[17] assessed whether measuring uFLCs and/or urine total light chains was useful in addition to UPE for monitoring patients with a urine monoclonal protein. Using 336 uIFE-positive urine samples (that were also SPE positive), they showed that the diagnostic sensitivities of uFLC and urine total light chain measurement were 80% and 70%, respectively. In samples that were SPE negative, diagnostic sensitivities decreased substantially. They concluded that uFLC and urine total light chain assays are not useful in addition to SPE in diagnostic testing. Urine total light chain values were in closer agreement with serum monoclonal protein values measured by SPE and provided a quality check on measurements of urinary monoclonal protein values in disease monitoring.

Le Bricon et al. ^[18] analysed the sensitivity of different urine assays for Bence Jones proteins in 20 patients with MM. The urine samples contained monoclonal intact immunoglobulins and/or monoclonal FLCs. Comparisons were made between assays for total protein (Pyrogallol Red), sodium dodecyl sulfate-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. Six of the 20 patients were abnormal by the total protein test, 11 by SDS-AGE and 16 by FLC immunoassays. Details of the 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 these were present in low concentrations. There was a reasonable correlation between the quantitative results for the different methods. Herzum et al. also compared FLC immunoassays with other urine tests for FLCs ^[19]. 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, which measure peptide bonds and are considered to be good general assays for proteins, had the best correlation with FLC immunoassays. This provides supporting evidence for the accuracy of FLC tests.

Lueck et al. ^[20] assessed the utility of antibodies specific for FLCs in uIFE using the Sebia electrophoresis system. They found that antibodies against total light chains detected FLC bands more effectively than FLC-specific antibodies and concluded that such antibodies were of little clinical use. However, no comparison was made in this study of the latex-enhanced FLC nephelometric immunoassays on the urine samples.

Table 6.2. Comparison of urine tests in 6 patients who were negative for monoclonal urine proteins by SDS-AGE but were abnormal by FLC ^[18]. There was good overall correlation, but sample 6 probably contained albumin.

6.7. Discordant serum and urine FLC 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, IIMM: intact immunoglobulin multiple myeloma. 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. ^[21] using sFLC immunoassays. Part of the explanation is that uFLCs and sFLCs are frequently polymerised, which leads to high results by immunoassay (Chapter 4). Also, protein dye tests underestimate the concentrations of FLCs to a variable extent.

Discordant serum and urine FLC results are also caused by inaccuracies in 24-h urine testing. Siegel et al. ^[22] evaluated 623 24-hour urine samples that had unexpectedly increased creatinine clearance (CrCl) for 24-hour protein, uIFE and UPE monoclonal proteins. Unexpectedly, abnormally increased CrCl was found in 19% of the samples, which was accompanied by increased urinary M-protein and total protein, but with no sFLC increase in the corresponding serum samples. Thus, results requiring 24-hour urine collections were highly susceptible to error, whereas sFLC analysis was unaffected by these errors.

Although not quantitative, IFE is considered to be the “gold standard” for identifying monoclonal uFLCs. 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 to be 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 majority of the staining intensity (80% or more) is due to the secondary antibody 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 ^[23], 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 is usually clinically insignificant (Chapter 19.4, Figure 19.5) although rarely patients may have AL amyloidosis ^[24] (Chapter 15).

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

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

Table 6.3. Comparison of serum and urine tests for identifying 110 patients with AL amyloidosis ^[23]. Urine tests provided no additional diagnostic benefit.

Conclusions

Since normal sFLC concentrations are considerably lower than the detection limit of all serum electrophoretic methods, some samples will always be misidentified as negative using these techniques. The numbers of patients and types of diseases missed by serum electrophoretic tests are shown diagrammatically in Figure 6.9. Although reports vary in their claims for the levels of sensitivity achieved with electrophoretic tests, the benefit of the increased sensitivity of sFLC measurements is clearly evident. Hence, sFLC measurement has now been included in the international guidelines of the International Myeloma Working Group ^[26], details of which are presented in Chapter 25.

References