Sensitivity of serum free light chain assays

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Chapter

6

SECTION 1 - Introduction

Sensitivity of serum free light chain assays

Contents

FLC 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 immunofixation electrophoresis.

6.1. Introduction

Comparison of sensitivity of serum and urine electrophoresis with serum free light chains
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 (summarised in Table 4.1) . 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 protein electrophoresis (SPE)

Normal serum protein electrophoresis gel and densitometry
Figure 6.2. Normal SPE. (Courtesy of JA Katzmann).
Serum scanning densitometry and serum immunofixation of a monoclonal protein
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).
Serum protein electrophoresis and serum free light chain measurments for light chain multiple myeloma
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.

Current algorithms for the identification, characterisation and quantification of monoclonal intact immunoglobulins rely upon serum protein electrophoresis (SPE) and serum immunofixation electrophoresis (sIFE). SPE 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 limitations of SPE are discussed in Chapter 32, which also introduces new immunoglobulin heavy chain/light chain immunoassays, for the detection and quantitation of monoclonal intact immunoglobulins.

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 other serum protein bands [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 the clinical details “possible multiple myeloma - please investigate”. This example highlights that screening with SPE alone would miss a significant proportion of monoclonal gammopathies. Since a urine sample is only available for <5 to 52% of paired serum samples, sFLC immunoassays offer a more reliable method to screen for monoclonal gammopathies, and 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) and is now recommended in international guidelines (Chapter 25).

6.3. Serum immunofixation electrophoresis

Free light chains in AL amyloidosis light chain multiple myeloma and light chain deposition disease
Figure 6.5. FLC concentrations in sera containing monoclonal FLCs that were “difficult to detect” by IFE.

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 [4], it was shown that all of 46 serum samples with low concentrations of monoclonal FLCs were correctly identified by sFLC immunoassay (Figure 6.5). 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 which were negative for monoclonal FLCs by sIFE, but the corresponding urine IFE (uIFE) was positive. In addition, one sample shown in Figure 6.5 was negative by sIFE, 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 concentration of the alternate (uninvolved) FLC and, thereby, enhances the sensitivity of the κ/λ ratio. The uninvolved FLC concentration, and hence the κ/λ ratio, are both important aspects of the diagnostic accuracy of sFLC immunoassays.

Figure 6.5 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 may prevent the formation of visible, narrow monoclonal bands during electrophoresis and cause an overestimation of FLC concentrations by immunoassay. This is further discussed in Section 4.2I.

6.4. Capillary zone electrophoresis (CZE)

Serum free light chains in patients with light chain multiple myeloma with monoclonal proteins that were undetectable by capillary zone electrophoresis
Figure 6.6. 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).

CZE is used in many clinical laboratories for serum protein separation, and is able to detect and quantify most monoclonal proteins. However, CZE fails to detect 5% of monoclonal proteins identified by sIFE [5][6][7]. These so-called “false negative” results encompass low-concentration and “hidden” monoclonal proteins (e.g. in the transferrin peak).

Marien et al. [8] 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. [7], and by others in monoclonal protein screening studies (Chapter 23).

Figure 6.6 indicates that several samples with FLC concentrations greater than 1,000 mg/L by immunoassay were not detected by CZE. This may be due to polymerisation of monoclonal FLCs, which may prevent the formation of visible, narrow monoclonal bands during electrophoresis and cause an overestimation of FLC concentrations by immunoassay (Section 4.2I).

6.5. Total κ and λ immunoassays

Insensitivity of total light chain assays for light chain multiple myeloma
Figure 6.7. 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. [8] 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 6.7). 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 protein electrophoresis

urine protein electrophoresis patterns with normal serum electrophoresis control
Figure 6.8. UPE examples. 1 Slight glomerular proteinuria; 2 Albumin only; 3 Normal serum control; 4 λ FLC; 5 Tubular proteinuria, uIFE negative; 6 uIFE identifies IgGκ plus κ FLC; 7 uIFE shows κ banding, no monoclonal protein detected.

Urinary FLC measurements are normally based upon electrophoretic tests (Figure 6.8). These may require samples to be concentrated up to 200-fold prior to analysis by UPE and scanning densitometry (combined with total protein measurements), with IFE to confirm and type any paraprotein bands. Urine electrophoresis can detect FLCs 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 plasma cell dyscrasias produce only small amounts of monoclonal FLCs, so little, if any, passes the absorptive surface of the renal proximal tubules. As a result, these patients may have undetectable levels of FLCs in the urine. Therefore, whilst urine electrophoresis is much more sensitive than SPE for the detection of monoclonal FLCs, sFLC immunoassays are usually preferable (Chapter 24). Although it is important to note that small amounts of monoclonal FLCs have occasionally been identified in the urine of some AL amyloidosis patients with normal sFLC ratios. This is further discussed in Section 24.10.


The methodology employed for UPE/uIFE varies considerably between laboratories, and has been shown to contribute to differences in reported results. Variations include:

  • Use of 24-hour vs random urine collections, with or without correction for creatinine excretion [9].
  • Use of concentrated versus unconcentrated urine for electrophoresis [10].
  • Variable screening algorithms (uIFE for all samples versus UPE followed by uIFE for abnormal samples) [10].
  • High analytical variability of different methods used for the determination of total protein concentration in the presence of monoclonal FLCs [11].


The visual interpretation of monoclonal bands by UPE/uIFE offers many challenges to even experienced users [12]. Some examples are listed below:

  • Polyclonal background FLC may produce high background staining and “ladder banding” which obscure monoclonal FLC bands and make scanning densitometry inaccurate.
  • Heavy proteinuria may obscure monoclonal FLC bands and makes quantitation of monoclonal protein bands by scanning densitometry inaccurate.
  • False positive results may be produced by a variety of proteins including β2-microglobulin, and lysozyme.
  • Non-specificity of antibodies used on IFE.

As an alternative to urine electrophoretic tests, FLC immunoassays can be used on urine samples. However, International guidelines state that “Measurement of urine FLC levels is not recommended” (see Chapter 25). Urinary FLC immunoassays do not solve any of the renal threshold, urine collection and urine measurement problems, and therefore, sFLC immunoassays are preferable to urine. This is further discussed in Chapter 24, which brings together all the arguments for the use of serum rather than urine for the measurement of FLCs.

Conclusions

Serum free light chain assay identifies monoclonal light chains in samples misclassified as negative by serum electrophoresis
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).

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 [13], details of which are presented in Chapter 25. sFLC assays are recommended for use in combination with serum electrophoresis to screen for pathological monoclonal plasma cell proliferative disorders other than AL amyloidosis, which also requires a 24-hour uIFE [13].


Test Questions
  1. Which is more sensitive, CZE or SPE?
  2. Do total serum light chain tests have a useful role in the laboratory?
  3. What is the origin of ladder banding in UPE?


Chapter 5 Back to Contents Page Section 2

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. 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
  6. 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
  7. 7.0 7.1 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
  8. 8.0 8.1 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
  9. Kaplan JS, Horowitz GL. Twenty-four-hour Bence-Jones protein determinations: can we ensure accuracy? Arch Pathol Lab Med 2011;135:1048-51 PMID: 21809998
  10. 10.0 10.1 Holding S, Spradbery D, Hoole R, Wilmot R, Shields ML, Levoguer AM, Dore PC. Use of serum free light chain analysis and urine protein electrophoresis for detection of monoclonal gammopathies. Clin Chem Lab Med 2011;49:83-8 PMID: 20961192
  11. Maisnar V, Tichy M, Stulik J, Vavrova J, Friedecky B, Palicka V et al. The problems of proteinuria measurement in urine with presence of Bence Jones protein. Clin Biochem 2011;44:403-5 PMID: 21291878
  12. Keren DF. Protein electrophoresis in clinical diagnosis. Arnold (Hodder Headline), 2003
  13. 13.0 13.1 Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009;23:215-24 PMID: 19020545

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