Serum versus urine tests for free light chains

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24.1. Introduction

The purpose of this chapter is to bring together all the arguments for the use of serum rather than urine for the measurement of FLCs. Most of the issues have been covered in some detail in preceding chapters, but they are scattered throughout the book rather than being focussed into a single coherent discussion. Given that a minority of individuals continue to favour urine over serum measurements; this chapter aims to persuade them otherwise. An analogy with diabetes mellitus is helpful: 40 years ago, all patients were monitored using urine glucose tests, whereas now they are monitored using blood glucose due to its overwhelming clinical advantages. As glucose and FLCs are handled in a similar manner by the kidneys, similar benefits accrue from serum over urine, for FLC analysis.

“If free light chains are in the urine they are always in the serum first.”

24.2. Renal threshold for FLC excretion

Figure 24.1 Serum and urine FLCs in 4 patients with light chain multiple myeloma (LCMM) or primary AL-amyloidosis. Serum tests (blue) were useful even when urine FLC excretion (pink) was minimal in patients 3 and 4. (Courtesy of M-A Alyanakian).

Figure 24.2 Amounts of sFLCs required to produce light chain proteinuria for κ and λ myelomas. Median, 95% and ranges are shown. (Courtesy of MR Nowrousian).

As described in Chapter 3, sFLCs are primarily cleared through the renal glomeruli and then metabolised in the proximal tubules of the nephrons. Only when the tubular absorptive capacity is exceeded are significant amounts of FLCs seen in the urine as “overflow proteinuria”. Since normal production is about 500mg/day and the renal absorptive capacity is 10-30g/day, production must increase many times before urine contains significant amounts of FLCs ^[1].

The clinical effect of renal tubular absorption on urine FLC (uFLC) concentrations is shown in Figure 24.1. Serum and urine FLC concentrations are compared in 4 patients undergoing treatment ^[2]. Patients 1 and 2 had large amounts of serum and urine FLCs with good correlations between changes in concentrations. In patients 3 and 4, urine excretion was minimal and unchanging over many months while serum levels could be used to monitor the changing tumour burden. Despite similar sFLC concentrations, there were no uFLCs in these latter patients as there was no renal impairment and, therefore, no overflow proteinuria.

The concentrations of monoclonal sFLCs necessary to cause overflow proteinuria was studied by Nowrousian et al. ^[3] in a group of patients attending a myeloma clinic. In 131 samples from patients with elevated monoclonal serum κ concentrations, 82 had uFLCs by immunofixation electrophoresis (IFE) while 49 had FLC-negative urine (Figure 24.2). The median serum κ FLC concentration associated with monoclonal FLCs in urine was 113 mg/L (range 7-39,500) and for normal urine 40 mg/L (range 6-710). Monoclonal λ FLC-producing patients had median serum values of 278 mg/L (range 5-7,060) for FLC-positive urine and 44 mg/L (range 3-561) for FLC-negative urine. The wide range of renal thresholds observed presumably reflected different degrees of renal damage.

Thus, for κ-producing patients, median serum levels associated with abnormal uFLCs were 5-fold above normal (upper limit of normal range: 19.4 mg/L). For λ patients median sFLCs were 10-fold above normal (upper limit of normal range: 26.3 mg/L) when the urine contained monoclonal FLCs. The higher serum levels necessary for λ overflow proteinuria can be explained by the dimerisation of λ molecules. This reduces glomerular filtration compared with monomeric κ molecules (Chapter 3).

Thus, when FLC production is below the renal clearance threshold, serum tests are more reliable than urinalysis. This study by Nowrousian et al. ^[3] showed that the extra sensitivity of the serum tests translated directly into greater clinical sensitivity for evaluating disease stage (Figure 11.9). This is particularly relevant for identifying patients with residual disease when urine assessments indicate complete remission (Chapter 12), and has been incorporated into international guidelines (Chapter 25).

24.3. Problems collecting satisfactory urine samples

Figure 24.3 Serum and urine FLCs in a patient with LCMM. Disease relapse can be identified 3 months earlier using serum rather than urine samples. (Courtesy of G Wieringa).

Even if there is significant urine excretion of FLCs, accurate quantification requires a proper 24-hour urine collection. This may be particularly difficult because:

Hence, even if there is significant renal leakage of FLCs, urine measurements may not be as reliable as those in serum. Figure 24.3 compares serum and urine results in a patient with relapsing light chain multiple myeloma (LCMM). The concentrations of the FLCs in both fluids are considerably elevated indicating that the renal threshold is exceeded (compare with patients 3 and 4 in Figure 24.1). However, the urine measurements are highly variable and do not show a definitive rise until day 160. In contrast, the steady rise in sFLC concentrations from day 40 indicates relapse of the tumour 3-4 months earlier. Presumably, the 24-hour urine collections were inaccurate, but there may have been additional inaccuracies in the measurements of the monoclonal FLC by urine protein electrophoresis (UPE) (see below).

24.4. Problems measuring urine samples

Figure 24.4 Comparison of FLC immunoassays and IFE for detecting uFLCs. Median, 95% and ranges of FLCs are shown. (Courtesy of MR Nowrousian).

Figure 24.5 sFLCs in patients with low monoclonal immunoglobulin production rates. By electrophoretic tests, sFLCs are usually undetectable or unquantifiable in most of these patients. (See relevant chapters for details of the patient data)

uFLC measurements are normally based upon electrophoretic tests (Table 4.1 and Chapter 6). These may require samples to be concentrated up to 200-fold prior to analysis by either UPE and scanning densitometry or by IFE with visual interpretation.

Problems interpreting FLC bands in urine samples include (Chapter 6.6):

Consistent with the various technical issues that may affect urine results, Siegel et al. ^[4] reported that urine electrophoresis results can be highly susceptible to error. Analysis of 623 24h-urine collections revealed fluctuating urine electrophoresis results, and 19% of urine samples demonstrated spuriously increased monoclonal protein levels. No such fluctuations were observed for sFLC values.

As an alternative to urine electrophoretic tests, FLC immunoassays can be used on urine samples. Nowrousian et al. ^[3] compared the sensitivity of uFLC immunoassays with urine IFE (uIFE) in patients with multiple myeloma (MM). In 98 κ and 107 λ individuals that had abnormal serum κ/λ ratios, urine samples positive by IFE contained a median of 448 mg/L of κ (range: 5-70,800) and 313 mg/L of λ (range: 17-11,100) by FLC immunoassays (Figure 24.4), while urine samples negative by IFE contained a median of 23 mg/L of κ (range 0-251) and 9 mg/L of λ (range 1-196) by FLC immunoassays. Similar findings have been reported by others who concluded that uIFE was more reliable for detecting monoclonal diseases than uFLC analysis by immunoassay ^[5]. Alternatively, one could conclude that urinalysis by FLC immunoassays and IFE is complementary.

As the ranges of FLC concentrations and κ/λ ratios in normal sera are far narrower than in urine, they are clinically more reliable (Chapter 5.6). Furthermore, uFLC immunoassays do not solve any of the renal threshold, urine collection and urine measurement problems indicated above.

There are many other problems with urinalysis: urine is less easily handled than serum; samples may be unpleasant and need to be stored in large volumes if further analysis is required; and FLCs are more prone to precipitation in urine than in serum.

24.5. Clinical benefits of sFLC analysis

The improved sensitivity of serum over urine FLC measurements has had a major impact on the ease of diagnosis, monitoring and assessing risk of progression for many patients with the following diseases:

Figure 24.5 shows a set of sFLC concentrations in patients with low production rates at the time of clinical diagnosis. Samples from patients with NSMM are shown as white circles that, by definition, have no detectable monoclonal proteins by both serum and urine electrophoretic tests. Hence, other patients with monoclonal sFLCs at or below these concentrations but with other types of plasma cell dyscrasias are difficult to identify by conventional tests. The figure also includes samples from many patients with AL amyloidosis and IIMM who were in remission by IFE.

24.6. Elimination of urine studies when screening for monoclonal gammopathies

An extensive comparison of the relative diagnostic contributions of serum and urine studies for the detection of plasma cell proliferative disorders has recently been reported ^[6]. Samples from 1,877 untreated patients with various diseases (Table 24.1) received a full panel of screening tests (SPE, UPE, sIFE, uIFE and sFLCs). This facilitated the determination of the more sensitive combinations of screening tests and addressed the question of whether sFLC analyses could replace urine studies.

Table 24.1. Clinical diagnosis of the 1,877 patients studied by Katzmann et al.^[6]

Figure 24.6 Sensitivity of different testing panels for a range of lymphoproliferative disorders ^[6]. Urine IFE provides no additional sensitivity for MM and WM in comparison to SPE and FLCs.

The combined results from all five tests identified 1,851 (98.6%) samples as abnormal. Of those not detected 11 were AL amyloidosis (1.9% of total), 8 extramedullary plasmacytoma (80%), 3 plasmacytoma (10.3%), 3 LCDD (16.7%) and 1 POEMS syndrome (3%, see Chapter 18.8). The combination of serum and urine IFE in the absence of sFLC tests resulted in a further 6 cases of MM, 23 of AL amyloidosis and 1 of LCDD being missed. In contrast, the combination of sFLC and serum IFE in the absence of urine IFE failed to identify 6 cases of AL amyloidosis, 15 of MGUS, 1 of extramedullary MM and 1 of LCDD (in addition to those not detected by all five tests combined). It can be concluded that sFLC analysis can play an essential part in screening for monoclonal gammopathies by detecting a significant number of additional cases of malignant diseases, particularly MM and AL amyloidosis, which would not be otherwise detected by other tests, including urine IFE. Urine IFE allowed the detection of 6 AL amyloidosis patients and 1 LCDD patient who were normal by the serum tests; similar results have been reported by Palladini et al ^[7].

It may not always be practical for laboratories to perform serum and urine IFE on all samples and so the authors also considered the possibility of avoiding urine analysis by combination of SPE and sFLC analyses with follow up sIFE where indicated (Figure 24.6). This simplified algorithm showed the same sensitivity as sIFE plus sFLC for MM and Waldenström’s macroglobulinaemia (WM) but missed a further 44 MGUS patients, 7 POEMS, 5 AL amyloidosis, 1 plasmacytoma, 1 SMM and 1 LCDD. The 44 MGUS patients, by risk stratification (Chapter 19), were considered low risk for progression to malignant disease. The impact of savings from reduced follow-up of low-risk MGUS and reduced patient anxiety was considered a reasonable outcome of this screening algorithm. The overall findings of the study provided significant support for the IMWG guidelines ^[8], which recommended screening using a combination of serum electrophoresis plus sFLC tests, with additional uIFE only required in order to maximise sensitivity when AL amyloidosis is suspected.

The guidelines are supported by numerous smaller screening studies. One used a historical cohort of 428 unselected patient samples consisting of a variety of plasma cell proliferative diseases ^[9]. These were analysed with all 5 screening tests. Preselection criteria of the cohort included the requirement to have a monoclonal protein detected by uIFE. This facilitated the study aim of determining if serum tests could identify all samples found positive by uIFE.

Performance of the various tests and combinations are shown in Table 24.2 ^[9]. sIFE was the most sensitive serum test (93.5%) followed by sFLC κ/λ ratios (85.7%). Of the 61 uIFE-positive patients with normal sFLCs, 30 had intact monoclonal immunoglobulins in the urine but no urinary Bence Jones protein. The sFLC assays therefore missed 31 of the 61 urine FLC-positive monoclonal samples, producing a diagnostic sensitivity for sFLC detection of 93%. All but 2 of these patients were identified by sIFE. One missed sample was from a patient with a uFLC-only MGUS. The other was a monoclonal urine IgAĸ considered to be a contaminant (as it was not found in subsequent samples from the patient and was absent in the serum). Clinically, neither required medical attention.

Table 24.2. Diagnostic sensitivity of various tests for monoclonal proteins in patients with positive uIFE.

Twenty-eight patients in the study had negative sIFE results, 19 of whom had AL amyloidosis, 3 solitary plasmacytoma, 3 MGUS, 2 MM and 1 SMM. All these were identified both by sFLC analysis and by urine studies ^[9]. The authors concluded that by adding sFLC analysis to sIFE, urine screening tests were no longer necessary. Furthermore, since urine samples are frequently not included with initial diagnostic serum samples, sFLC testing has considerable diagnostic utility. For example, Robson et al.^[10] reported a urine compliance of <5% in a study carried out at New Cross Hospital, Wolverhampton (Table 24.3). The extremely poor provision of matched urine samples led the authors to comment that “the debate over the relative merits of the sFLC assay versus urine BJP analysis borders on the irrelevant”. This is in addition to its value as a prognostic marker in patients with MM, SMM, MGUS and AL amyloidosis etc.

Table 24.3. Published urine compliance data at five centres.

Fulton et al. ^[15] retrospectively analysed 219 sFLC requests that had matching SPE, sIFE, UPE and uIFE test results. They showed that the sFLC ratio was abnormal in 12% more samples than when using UPE plus uIFE. Furthermore, the combination of an SPE and sFLC analysis allowed the detection of 6% more abnormal samples than the combined use of SPE, sIFE, UPE and uIFE. The authors therefore proposed the incorporation of sFLC tests into a primary screening algorithm, thus reducing the use of labour intensive sIFE and uIFE.

A further screening investigation on 3,818 sera received for SPE analysis over a 1 year period has also been reported ^[16]. The authors used a series of criteria based on clinical presentation and other laboratory tests to reduce the need for sFLC analysis to 1,067 of the 3,818 samples. This testing strategy resulted in the detection of 95% of all new patients with MM, WM or AL amyloidosis. Overall, 4 patients with clinical disease were missed (1 with NSMM, 1 with plasma cell leukaemia and 2 with plasmacytomas), however all 4 were negative by urine electrophoresis.

A prospective screening study of 370 patients by Hill et al. ^[11] directly compared the clinical sensitivity of sFLC tests and UPE. Fifteen samples were positive for Bence Jones protein of which 11 were also sFLC positive. The 4 discordant samples (1%) were of no clinical consequence (Chapter 23.3), and no significant disease was missed.

A similar, prospective, serum versus urine study of 483 patients was recently published by Beetham et al. ^[13] Monoclonal proteins were detected in 105 (22%) patients, 34 of whom had Bence Jones proteins at greater than 5mg/L. Of these 34 samples, 8 had normal sFLC κ/λ ratios; however, 7 were found to be positive for intact monoclonal immunoglobulins by SPE/sIFE. Thus, of 105 patients with positive urines, only one was not identified by serum studies (0.2%) and this patient was considered to have a urine-only MGUS (<50mg/L) of no apparent clinical consequence. These results supported other published studies suggesting that SPE/sIFE and sFLC analysis can, in practice, replace urine studies.

However, while the serum tests gave the same practical results as urine tests, Beetham et al. ^[13] expressed some disquiet as to why monoclonal proteins were present in urine when sFLC κ/λ ratios were normal. There are a variety of mechanisms whereby this may occur, which are discussed below and elsewhere (see FcRn transport mechanism, Chapter 10). It might also have been useful if the urine of the discordant samples had also been assessed by quantitative FLC immunoassays, rather than relying on the electrophoretic techniques alone to provide correct results. Furthermore, no clinical details were provided for 5 samples in which abnormal sFLC κ/λ ratios were the only significant anomaly. The data of Katzmann et al. ^[17] suggest that AL amyloidosis or another subtle B-cell dyscrasia should have been considered.

24.7. Comparison of sFLCs and urinalysis for monitoring patients

Huge clinical benefits are derived from the improved sensitivity and specificity of sFLC analysis versus urine studies for monitoring patients with light chain diseases. The main studies are reported in detail in the appropriate clinical sections (listed in Section 24.5 above).

However, as an example, Avet-Loiseau et al. ^[18] in a large retrospective analysis of the IFM 2007-01 trial, compared the utility of urine electrophoresis and sFLC measurements in monitoring MM. In 50% of uIFE-positive IIMM patients, uIFE became negative after two cycles of therapy compared to 5% by sFLC κ/λ ratio. Similarly in LCMM, uIFE became negative in 50% of patients compared to 10% by sFLC. The study concluded that uIFE can lead to an overestimation of treatment responses in both IIMM and LCMM.

sFLC κ/λ ratios are occasionally normal when urine tests for Bence Jones protein are abnormal. Some of the discrepancies may be explained on technical grounds or as sampling errors (Chapter 6.6); for instance, when monitoring patients, 24-hour urine collections may be taken earlier than the corresponding serum samples. Since FLC concentrations can fall rapidly following treatment (Chapter 13), urine samples collected a few days before attending the clinic for sFLC analysis could produce quite different results. In a small proportion of AL amyloidosis patients, FLCs are normal by serum immunoassay but detectable by urine IFE. This is further discussed in Section 15.2.

sFLC assays are now recommended in international guidelines for the quantitatitve monitoring of patients with oligosecretory plasma cell disorders, including patients with AL amyloidosis, oligosecretory myeloma (serum monoclonal protein <10 g/L; urine monoclonal protein <200 mg/24 hours), and in nearly two-thirds of patients previously classified as NSMM (see Chapter 25).

24.8. Organisational, cost and other benefits of sFLC analysis

As well as improved clinical diagnosis, there are organisational, cost and other benefits of introducing sFLC assays. The laboratory issues were analysed at the Christie Hospital in Manchester, UK ^[19]. Superior analytical performance of the serum assays, faster reporting times and reduced laboratory costs were clearly identified (Table 24.5). Cost benefits in relation to clinical outcomes were not analysed in this study but they may accrue from earlier diagnosis and treatments that reduce morbidity.

Table 24.5. Analytical and cost/benefit study of sFLC and urine electrophoresis tests.

Hill et al. ^[11] compared the costs of urine electrophoretic tests with sFLC immunoassays in routine screening of patients for monoclonal gammopathies (Chapter 23). They considered that on a patient-by-patient basis the costs were increased by less than £5, but, in their study only 40% of serum samples were accompanied by urines so the overall costs increased considerably. However, better clinical governance was achieved and more clinical diagnoses were made.

Katzmann et al. ^[17] compared the costs of sFLC screening with urine testing. The 2006 Medicare reimbursement for sFLCs was $38 compared with $71 for urine assays (total protein, UPE and uIFE). With sFLC tests costing approximately half that of urine tests, considerable laboratory savings could be made.

There have been no direct assessments of clinical cost benefits. However, one relatively simple clinical situation that may be improved by measuring sFLCs is when determining the underlying pathology of patients presenting with acute renal failure (Chapter 13). If MM is suspected, the normal procedure is to perform SPE/IFE and UPE. A simpler and better approach would be to measure sFLCs. This would identify all patients with FLC as a cause of their renal impairment.

If a choice has to be made between serum or urine tests then serum is clearly preferable for the many reasons given above (Table 24.6) ^[20]. When both serum and urine tests are available, it is clinically reassuring to have two separate tests giving the same results. Clearly, samples do occasionally get incorrectly analysed, mislabelled or misplaced, so supporting evidence for making a diagnosis or changing treatment is always helpful. In the context of a stem cell transplant in MM patients, for example, the additional cost of performing both serum and urine tests is inconsequential. Furthermore, there are some patients with positive uFLCs and negative sFLCs although the clinical importance is unclear.

Table 24.6. Summary of clinical and analytical comparisons of sFLC and urine electrophoresis tests.

New national and international guidelines for patients with monoclonal gammopathies include recommendations on sFLC analysis in relationship to urine tests (Chapter 25).

Clinical case history No 11

References