Screening studies using serum free light chain analysis

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23.1. Introduction

Whether or not to measure serum FLCs at the time of the initial diagnostic request for, “possible myeloma - please investigate,” is an important issue. By tradition, SPE and/or serum IFE tests have been performed first, sometimes alongside IgG, IgA and IgM measurements. SPE can detect intact immunoglobulin monoclonal proteins in the 1-5g/L range (Chapter 6) which is more than adequate to identify all IIMM patients. Some centres screen with IFE because it is ten times more sensitive. However, occasional monoclonal FLCs are missed and IFE is non-quantitative. Furthermore, many low-level MGUS proteins are detected that are unlikely to progress to malignancy, provided sFLC levels are also normal (Chapter 19) ^[1]. A few laboratories measure total serum κ and λ in the initial screen but this is inadequate (Chapter 6).

Because of these deficiencies, UPE has been performed alongside serum tests, but many patients with NSMM, AL amyloidosis and other FLC-associated disorders are still missed. Furthermore, only a minority of patients have accompanying urine samples (typically 5-25% in the UK). It is, therefore, logical to test for sFLCs on receipt of the first blood sample. The results will also provide baseline values for subsequent disease monitoring.

Hence, a strategy of using SPE/IFE combined with sFLC analysis, “up-front,” allows identification of all clinically significant monoclonal gammopathies. It also allows risk assessment for disease progression in MM, MGUS, ASMM, plasmacytomas etc., and appropriate clinical decisions for monitoring patients.

This chapter discusses the current and potential screening options for identifying monoclonal proteins. As a general rule, intact monoclonal immunoglobulins can be identified using serum electrophoretic tests while monoclonal light chain diseases should be identified using sFLC assays. The combination of the 2 procedures produces good diagnostic accuracy and urine FLC analysis is not required at initial clinical presentation (Chapter 24).

23.2. Diagnostic protocols for monoclonal gammopathies

Figure 23.1 κ/λ ratios in 55 abnormal sera from a CZE and FLC screening study of 1,003 patients ^[2]. Sera with intact monoclonal immunoglobulins by CZE are shown in black or blue and samples containing only abnormal FLC κ/λ ratios are shown in yellow. Numbers against large symbols refer to additional patients with confirmed monoclonal gammopathy identified by FLC tests (Table 23.2).

Figure 23.2 FLCs in 925 sera screened at Derby, UK. Normal results are the FLC levels in samples with normal SPE. (Courtesy of P Hill).

The accuracy of different diagnostic protocols used for identifying monoclonal gammopathies are shown in Table 23.1. The basis of the numerical analysis is as follows:-

1. SPE identifies all patients with IIMM. By definition, they have at least 10g/L of monoclonal protein which is much greater than the sensitivity of the assays (2-5g/L). The test fails to identify approximately 30% of LCMM patients (the other 70% have sFLC bands or hypogammaglobulinaemia) and all NSMM (Chapter 9). Approximately 50% of AL amyloidosis patients are abnormal by SPE (Chapter 15) . Many intact immunoglobulin MGUS individuals are also identified.

2. The combination of SPE and sIFE is more sensitive but fails to identify a few patients with LCMM and all patients with NSMM. 70% of patients with AL amyloidosis are identified but those producing FLCs only are frequently missed. Many more MGUS individuals are identified but monoclonal proteins of less than 2-5g/L are of little clinical consequence if sFLCs are normal (Chapter 19).

3+4. The addition of UPE/uIFE to either of the above protocols identifies the remaining 30% of patients with LCMM and many patients with AL amyloidosis. Few additional MGUS patients are identified because they rarely produce sufficient monoclonal FLCs to exceed the renal threshold and enter the urine (Chapter 19). Also, since serum tests for FLCs are clinically more useful than UPE tests, these protocols are illogical (Chapter 24).

5. sFLC analysis identifies FLC monoclonal gammopathies but cannot be used alone. It is, by definition, a test for FLCs and not for intact monoclonal immunoglobulins. 30-60% of patients with MGUS, 10% with ASMM and 5% with IIMM do not have excess FLC production (see Chapters 19, 14 and 11).

Table 23.1. Approximate diagnostic sensitivity of tests for monoclonal gammopathies. (See relevant clinical chapters for details)

6. A sensitive protocol is to use SPE to identify all the intact monoclonal immunoglobulins and sFLC immunoassays to identify all the monoclonal FLCs. Approximately 20% of NSMM patients will be missed with this strategy but are not detected by any other serum or urine tests (Chapter 9). Such patients are nonproducers or non-excretors and may have mutations in the light chain DNA causing protein shape distortion. Nearly all patients with AL amyloidosis and LCDD are identified (Chapter 15 and 17). Some additional MGUS individuals are identified who produce only monoclonal sFLCs. Their clinical importance is not yet known but FLC MGUS is probably the precursor of LCMM and AL amyloidosis and may have a relatively poor outcome (Chapter 15). Patients with renal impairment are also identified (Chapter 20).

7. The addition of serum IFE to protocol 6 is not likely to identify any more patients with MM. It identifies approximately 2-10% additional AL amyloidosis patients (Chapter 15). It also identifies some MGUS individuals with minor monoclonal intact immunoglobulins who are unlikely to progress to overt disease, if there is no accompanying abnormal FLC κ/λ ratio (Chapter 19). The extra cost, the inconvenience of performing the test and problems interpreting the clinical relevance of minor MGUS bands may not be justified in all clinical laboratories.

In the unlikely event that SPE/IFE and serum FLC tests are normal but the clinical picture still suggests a plasma cell disorder, urinalysis for FLCs is required. However, caution should be exercised when considering the clinical importance of minor urine FLC bands when serum FLC tests are normal (see below and Chapter 24). After a monoclonal protein has been identified, IFE is required to characterise the heavy chain type. The FLC type can be identified by serum κ/λ ratios or from the IFE gels.

Table 23.2. Clinical and laboratory data in 16 patients (confirmed monoclonal lymphoproliferative diseases) that were normal by CZE but abnormal by serum FLCs ^[2]. NI: Not indicated, B-CLL: B-cell chronic lymphocytic leukaemia, SLL: B-cell small lymphocytic lymphoma, LPL: lymphoplasmacytic lymphoma, MZL: marginal zone lymphoma. Case numbers refer to the patients also shown in Figure 23.1.

23.3. Screening studies using serum free light chain analysis

Figure 23.3 Test results on 370 urines screened for monoclonal proteins at a UK Hospital. Of 925 sera sent for screening, 370 (40%) had accompanying urine samples of which 126 were suspicious by UPE but were negative by urine IFE. (Courtesy of P Hill).

Figure 23.4 Test results on 971 sera screened for monoclonal proteins at Nuneaton, UK ^[3]. Patient samples in the normal range have been excluded.

Eight screening studies have evaluated the use of sFLC assays, alongside other tests, as part of the initial diagnostic screen for monoclonal gammopathies. These are described below:-

1. Bakshi and colleagues ^[2] used a combination of CZE and sFLC analysis. 1,003 consecutive unknown samples were studied. 39 contained monoclonal proteins by CZE and 33 had abnormal κ/λ ratios to give a total of 55 abnormal sera.

16 samples (11 κ and 5 λ) were abnormal for monoclonal proteins only by sFLC analysis (Figure 23.1 and Table 23.2). Subsequent IFE showed that 5 of the 16 samples were abnormal, although some had barely visible bands, but 11 samples were clearly only abnormal by FLC assays. Of the 39 samples that were positive by CZE, 17 were associated with abnormal FLC κ/λ ratios. In every case, the FLC test results concurred with the light chain type of the intact monoclonal immunoglobulin.

9 of the extra monoclonal gammopathy patients identified from abnormal κ/λ ratios were eventually shown to have plasma cell dyscrasias: 3 with MM, 4 with B-CLL/small cell lymphoma, 1 aplastic anaemia and 1 lymphoma. 7 samples were not associated with significant diseases and were classified as FLC MGUS only (Chapter 19).

Addition of the FLC analysis to the CZE test in this screening trial increased the yield of lymphocyte and plasma cell proliferative diseases by 56%. The authors noted that the study emphasised the utility of sFLC testing for patients with monoclonal proteins when CZE was essentially non-diagnostic.

2. Hill et al. ^[4], studied 923 consecutive serum samples referred to a UK District General Hospital. 71 had abnormal sFLC ratios (Figure 23.2). 8 new B-cell dyscrasias were detected among 43 patients with negative SPE but abnormal sFLC κ/λ ratios. 2 patients with κ/λ ratios of 50 and 193 had κFLC LCMM; 5 patients had FLC MGUS and one a malignant lymphoma (but with no monoclonal band). 35 patients with negative SPE had abnormal κ/λ ratios but no monoclonal diseases. This false positive rate for a ratio of >1.65 was associated with polyclonal increases in immunoglobulins and renal impairment and was higher than previously noted. Such minor abnormalities need to be considered in the context of reference ranges for hospitalised patients and renal diseases (Chapter 5 and Chapter 20).

In this study, comparison was made between the utility of serum and urine tests for monoclonal FLCs. 370 (40%) of the serum samples were accompanied by urine samples. Of these, 141 (38%) had suspicious UPE tests and were further analysed by urine IFE. Only 15 of these urine samples (4% of the total tested) had confirmed monoclonal FLC indicating that 126 tests were performed unnecessarily (Figure 23.3). In 11 of the samples there was a corresponding serum FLC abnormality but 4 were normal. One of the 4 samples was associated with a serum IgGλ of 7g/L that was identified by IFE but had been missed on the initial SPE. The remaining 3 samples had urine FLC concentrations of approximately 50mg/L. One normalised as the patient recovered from illness, another was subsequently interpreted as polyclonal urine FLCs and the 3rd patient had persisting FLC proteinuria but was asymptomatic and had no evidence of a B-cell disorder from bone marrow biopsy. Hence, none of the “urine only” monoclonal FLCs had clinically active disease.

On the basis of these results the hospital practice was changed to a policy of no urine tests when screening for monoclonal gammopathies. This allowed all patients to have proper assessments for monoclonal FLCs, improved operational efficiencies and increased costs by less than £5 per patient. The cost increase is largely due to testing all patients for FLCs whereas previously urines were available in only 40% of cases.

3. Augustson et al. ^[5], studied 217 consecutive samples referred to a UK district general hospital. They found an extra 8 monoclonal gammopathies by abnormal sFLC κ/λ ratios. 3 of these patients had LCMM that were missed by the routine SPE tests and resulted in serious diagnostic delays. Subsequently, the study was increased to 971 patients ^[3] and results were similar to other screening studies (Figure 23.4).

4. Abadie et al. ^[6], reported results on 312 consecutive samples from Seattle hospitals in the USA. Serum FLC tests identified a higher percentage of true positive and true negative samples and a lower percentage of false positive and false negative samples than SPE. The two assays in combination offered the most reliable results (Figure 23.5).

5. Foray and Chapuis-Cellier ^[7] studied 75 patients with FLC monoclonal gammopathies. They observed 6 patients who were normal by urine tests but had abnormal sFLC results. They concluded that the assays should be used whenever monoclonal FLC diseases were suspected.

Figure 23.5 Combined assay performance of SPE and sFLC assays for identifying monoclonal gammopathies in a screening study ^[6]. PPV: Positive predictive value, NPV: Negative predictive value.

6. Katzmann et al. ^[8], in a large retrospective study, made a direct comparison of the relationship between the diagnostic sensitivity of serum FLC analysis and urine studies. 428 patients with monoclonal urine proteins were studied, a 10 times larger cohort than any other study (for details see Chapter 24). The clinical diagnoses had been established in all patients. The authors concluded that by adding sFLC analysis to serum IFE, urine screening tests were no longer necessary. These results addressed the uncertainties that had been discussed by Katzmann ^[9] in an earlier editorial in the Journal of Clinical Chemistry. Furthermore, the data supported the diagnostic sensitivity of sFLC assays that Katzmann ^[10] had previously reported for assessing patients with known monoclonal gammopathies (see audit below).

7. Beetham et al. ^[11], in the UK, prospectively investigated 932 consecutive patients by SPE and sFLCs plus serum or urine IFE where appropriate. 449 had serum studies only (because no urine samples were available) of which 53 had monoclonal proteins but importantly, no significant diseases were missed. 5 new cases of serum monoclonal FLCs only were detected but unfortunately no information was provided regarding their clinical relevance. The authors indicated that the results supported other published studies in that SPE, sIFE and sFLC analysis could replace urine studies for detecting monoclonal gammopathies. The other 483 samples were investigated in the context of a paired urine study (Chapter 24.6).

8. Holding et al. ^[12], screened 753 consecutive serum samples using CZE and sFLCs. There was 100% serum sensitivity for monoclonal positive urine samples and extra plasma cell diseases were detected using sFLC assays. At the time of the report, full clinical analysis was not available.

The results from the 8 studies described above are consistent. Adding sFLC assays to the SPE/IFE tests for routine screening, (in the absence of urine studies) increased the tumour pick-up rate by approximately one patient per 100 samples tested. Some of the additional patients identified had significant changes to therapy resulting from their earlier diagnoses. It is of interest that many of the patients identifed with this strategy had B-cell non-Hodgkin lymphoma or chronic lymphocytic leukaemia (Chapter 18). The diagnostic value of sFLC testing at the initial screening stage has recently been reviewed ^[13][14][15].

Table 23.3. Audit of serum FLC requests at the Mayo Clinic for the year 2003. Sensitivity for detection by FLC analysis was available for some of the diseases.

23.4. Audit of serum free light chain usage

Katzmann et al. ^[10] performed an audit of sFLC analysis for the year 2003, on patients attending The Mayo Clinic (Table 23.3). Of the 1,020 patients, 88% had plasma cell disorders. All 120 patients who had no plasma cell disorder had normal κ/λ ratios, in spite of a complex variety of different diseases. Thus, there were no false positive results in this study. The diagnostic sensitivity for AL amyloidosis by serum κ/λ ratios was 91% compared with 69% for serum IFE and 83% for urine IFE. Serum κ/λ ratios plus serum IFE produced a sensitivity of 99% which was not improved by adding urine IFE (Table 15.1). For 5 NSMM patients at clinical presentation, all had abnormal κ/λ ratios (100%). 5 of the 6 NSMM patients who had normal serum FLCs had achieved complete clinical remission after PBSCT and had normal bone marrow plasma cell content. The conclusion of the study was that the performance of the FLC assays, in a prospective analysis, matched the results from published retrospective validation studies.

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