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 FLC measurements. The preceding chapters have covered most of the issues, and in some detail, but they are scattered throughout the book rather than being focussed into a single coherent discussion. Since there is a residue of informed opinion that continues to favour urine over serum measurements, it is time to persuade them otherwise. An analogy with diabetes mellitus is helpful. 40 years ago, all patients were monitored using urine glucose tests. Now they are monitored using blood glucose because of its overwhelming clinical advantages. Because 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 free light chain excretion

Figure 24.1 Serum and urine FLCs in 4 patients with LCMM. 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 serum FLCs 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 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. In spite of similar sFLC concentrations, there was no urine FLCs in these latter patients because there was no renal impairment and, therefore, no overflow proteinuria.

The concentrations of monoclonal serum FLCs 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 urine FLCs by IFE while 49 had FLC negative urine (Figure 24.2). The median serum κ concentrations associated with monoclonal FLCs in urine was 113mg/L (range 7-39,500) and for normal urines 40mg/L (range 6-710). Monoclonal λ FLC producing patients had median serum values of 278mg/L (range 5-7,060) for FLC positive urines and 44mg/L (range 3-561) for FLC negative urines. 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 urine FLCs were 5-fold above normal (upper limit of normal range: 19.4mg/L). For λ patients median sFLCs were 10-fold above normal (upper limit of normal range: 26.3mg/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.

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 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 serum FLC 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 UPE (see below).

24.4. Problems measuring urine samples

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

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

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

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

As an alternative to urine electrophoretic tests, FLC immunoassays can be used on urine samples. Nowrousian et al. ^[3], compared the sensitivity of urine FLC immunoassays with urine IFE in patients with MM. 98 κ and 107 λ samples that had abnormal serum κ/λ ratios were studied. Urines 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). Urines negative by IFE contained a median of 23 mg/L of κ (range 0-251) and 9mg/L of λ (range 1-196) by FLC immunoassays. Similar findings have been reported by others who concluded that urine IFE was more reliable for detecting monoclonal diseases than urine FLC analysis ^[4]. Alternatively, one could conclude that urinalysis by FLC immunoassays and IFE are complementary.

Table 24.1. The sensitivity of serum electrophoretic tests combined with sFLC analysis means that urinalysis offers no additional benefits for disease diagnosis.

Because the ranges of FLC concentrations and κ/λ ratios in normal sera are far narrower than in urine, they are clinically more reliable (Chapter 5.3). Furthermore, urine FLC immunoassays do not solve any of the renal threshold, urine collection and urine measurement problems indicated above.

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

24.5. Clinical benefits of serum free light chain 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.

Table 24.1 shows the diagnostic sensitivity of serum tests (IFE plus FLCs) for various monoclonal gammopathies. There are no additional clinically significant diseases identified using urine tests. This indicates that sFLC analysis can replace urinalysis when the diagnosis of these diseases is being considered.

24.6. Elimination of urine studies when screening for monoclonal gammopathies

A direct comparison of the relationship between the diagnostic sensitivity of serum FLC analysis and urine studies, in a screening role, has been assessed by Katzmann et al. ^[5] They examined a large, historical cohort of unselected patients (428), with a variety of plasma cell proliferative diseases (Table 24.2), in whom 5 screening tests had been performed (UPE, uIFE, SPE, serum IFE and sFLCs). They then determined which patients had abnormal serum results and which patients had monoclonal proteins in the urine that would have been undetected in the absence of urine studies.

Table 24.2. Diagnoses for 428 patients with urine monoclonal proteins

The distribution of the monoclonal gammopathies was as expected apart from a relatively small proportion of patients with MGUS (Figure 7.0). By definition, all patients had positive uIFE for monoclonal proteins. Compared with serum studies, MGUS patients are relatively under represented because few have urine monoclonal FLCs (approximately 30%). Comparisons of the various tests are shown below (Table 24.3).

Serum IFE was the most sensitive serum test at 93.5% followed by sFLC κ/λ ratios at 85.7%. Of the 61 uIFE positive patients with normal sFLCs, 30 had intact monoclonal immunoglobulins in the urine but no urine FLCs. The sFLC assays, therefore, missed only 31 of the 61 urine FLC positive monoclonal samples, so that the diagnostic sensitivity for sFLC detection was 93%. All but 2 of these patients were identified by serum IFE. One missed sample was from a patient with a uFLC only MGUS. The other was a monoclonal urine IgAκ that was considered to be contamination as it was absent in the serum. Clinically, neither required medical attention.

28 patients in the study had negative serum IFE results of which 19 had AL amyloidosis, 3 solitary plasmacytoma, 3 MGUS, 2 MM and 1 ASMM. All these were identified both by sFLC analysis and by urine studies. The authors concluded that by adding sFLC analysis to serum IFE, urine screening tests were no longer necessary. Furthermore, since urine samples are frequently not included with initial diagnositic serum samples, serum FLC testing has considerable diagnostic utility. This is in addition to its value as a prognostic marker in patients with MM, ASMM, MGUS and AL amyloidosis etc.

Table 24.3. Diagnostic sensitivity of various tests for monoclonal proteins in patients with positive urine IFE.

A prospective screening study of 370 patients by Hill et al. ^[6], directly compared the clinical sensitivity of serum FLC tests and UPE. 15 samples were Bence Jones protein positive of which 11 were also sFLC positive. The 4 discordant samples (1%) were of no clinical consequence (Chapter 23.3), so no significant disease was missed.

A similar, prospective, serum versus urine study of 483 patients was recently published by Beetham et al. ^[7] Monoclonal proteins were detected in 105 (22%) patients of whom 34 had Bence Jones proteins at greater then 5mg/L. 8 of these 34 samples had normal sFLC κ/λ ratios. However, 7 were positive by SPE/sIFE for intact monoclonal immunoglobulins, so only one sample of 105 positive urines was not identified by serum studies (0.2%). This patient was considered to have a urine only MGUS of no apparent clinical consequence (<50mg/L). These results supported other published studies in 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. ^[7], expressed some disquiet as to why monoclonal proteins were present in urine when sFLCs κ/λ ratios were normal. There are a variety of mechanisms whereby this may occur, which are discussed below and elsewhere. Also, it might have been useful if the authors had assessed the urines of the discordant samples by quantitative FLC immunoassays rather than assume that the electrophoretic techniques gave the correct results. Furthermore, 5 samples had abnormal sFLC κ/λ ratios as the only significant anomaly, but no clinical details were provided. The data of Katzmann et al. ^[8], suggests that AL amyloidosis or another subtle B-cell dyscrasia should have been considered.

24.7. Comparison of sFLCs and urinalysis for monitoring patients

There are huge clinical benefits 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. However, as an example, Mazurkiewicz et al. ^[9], in a small UK study, considered that 11 of 16 patients might have had better treatment decisions based upon sFLCs rather than the urine studies that had been performed.

Table 24.4. Discordant results in 5 patients with AL amyloidosis who had abnormal urine FLCs by IFE and UPE but normal serum FLC κ/λ ratios (see Chapter 6).

Nevertheless, sFLC κ/λ ratios are occasionally normal when urine tests for FLCs are abnormal. Some of the discrepancies may be explained on technical grounds or on 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 serum FLC analysis could produce quite different results. The sensitivity of the urine measurements is also highly dependent upon technique. Obviously, highly sensitive uIFE gels identify more samples than UPE used in routine hospital practice.

The frequency of discordant results has been analysed in patients with AL amyloidosis ^[10]. 260 sera from patients with AL amyloidosis were studied alongside their corresponding urine samples. 5 samples had normal serum κ/λ FLC ratios but more than 200mg/day of urine FLCs by UPE (Table 24.4). The serum samples were taken during the period of the 24-hour urine collection to avoid errors of timing and all gels were carefully analysed retrospectively.

One of the serum FLC results in the 5 patients was borderline abnormal. Since the normal serum reference range for FLCs was based on a screening study that included all samples tested, it may be that when monitoring patients, narrower normal range criteria can be used (Chapter 5). Also, these patients usually have a degree of renal failure that needs to be considered in all patients with borderline results (Chapter 20). Slower clearance of monomeric κ molecules occurs when glomerular filtration is impaired so that serum κ/λ ratios increase slightly. In contrast, urine polyclonal FLCs increase, with a greater proportion of λ FLCs because of the impaired κ clearance. This may explain the predominance of λ discordant patient samples in Table 24.4.

Table 24.5. Analytical and cost/benefit study of serum and urine FLC tests.

Quantification of the urine using FLCs immunoassays showed lower concentrations than suggested by UPE and all 5 had normal κ/λ ratios (Table 24.4). It may be that UPE testing is inaccurate because polyclonal FLCs are included in the analysis. Moreover, low concentration urine monoclonal proteins in patients with normal serum FLCs and κ/λ ratios are of doubtful clinical importance. International guidelines indicate that monoclonal urine FLCs below 200mg/24 hours are of little clinical relevance (Chapter 25).

Table 24.6. Summary of clinical and analytical comparisons of serum and urine free light chain measurements.

These results suggest that it may be unwise to treat patients with chemotherapy based only upon minor urine monoclonal FLC bands detected by IFE when serum FLC tests are normal. In spite of these observations, there is support for the continuing use of 24-hour urinalysis for monitoring patients. This is based upon the clear evidence that urine tests can be positive when serum is negative, in some patients ^[11][12]. Further studies are required.

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 ^[13]. 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.

Hill et al., 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. ^[8], compared the costs of sFLC screening with urine testing. The 2006 Medicare reimbursement for sFLCs was $38 compared with urine assays at $71 (total protein, UPE and uIFE). With sFLC tests costing approximately half the urine tests per patient, 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 serum FLCs 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) ^[14]. When both serum and urine tests are available, it is clinically reassuring to have 2 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. And, there are some patients with positive urine FLCs and negative sFLCs although the clinical importance is unclear.

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

Clinical case No 11

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