Normal ranges and reference intervals

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5.1. Serum free light chain normal ranges

Normal range data for serum free light chains (sFLCs) have been published many times: those considered to be the most reliable are shown in Table 5.1. Serum results using polyclonal antibodies have varied considerably, indicating a degree of cross-reactivity with bound light chains in some of the assays. Perhaps unsurprisingly, assays using monoclonal antibodies have also produced varied results, the discrepancy between reports being more than tenfold ^[1][2][3][4]. It is unclear whether the variations were due to specificity, calibration or matrix differences, or a combination of these factors.

Table 5.1. Selected publications reporting normal sFLC concentrations (mg/L) and κ/λ ratios ^[11][3]. (^apolyclonal antibody against total light chains, ^cpolyclonal antibody against FLCs, ^dmonoclonal antibody against FLCs, ^elatex reagent, ^f 95% range).

Figure 5.1. (a) κ, (b)λ and (c) κ/λ ratios versus age (years) in 282 normal serum samples together with (d) cystatin C results in the same patients. sFLC/cystatin C ratios (e) and (f) show no change with age, confirming renal deterioration as the cause of increased sFLC levels in elderly individuals. (red = fresh sera, black = frozen sera). (Courtesy of J. A. Katzmann) .

The most detailed study of FLC concentrations in normal individuals was published by Katzmann et al. ^[12], and showed similar results to those previously reported by Bradwell et al. ^[11] The same assay procedures were used in both studies but a wider age range of individuals was studied by Katzmann et al. Serum samples were obtained from 127 healthy blood donors (21-62 years) and 155 older, normal individuals (51-90 years). A summary of the median values and reference ranges in the Katzmann study is shown in Tables 5.1 and 5.2.

The results of κ and λ measurements for all individuals in the Katzmann study are shown in Figure 5.1. There were no significant differences between results obtained using either fresh or frozen sera but there was a trend towards higher concentrations in elderly people (Figure 5.1 and Table 5.3). This occurred for both FLCs and also for the renal function marker, cystatin C, which was measured in the same samples. Calculations of cystatin C/sFLC ratios and κ/λ ratios on each sample normalised the elevated values (Figure 5.1, c,e,f). Therefore, the higher sFLC values seen in older people can be explained by small reductions in glomerular filtration rate ^[13].

An alternative method of presenting normal range data is shown in Figure 5.2. This shows the serum κ and λ results for each person plotted on a logarithmic scale. Such data presentation inherently includes the κ/λ ratios and is useful for visualising results from individual patients. It also allows easy comparison between different disease groups and is used extensively in later chapters for clinical comparisons.

One discrepancy between the results of Bradwell et al. and Katzmann et al. and those of many earlier researchers is that serum κ concentrations were found to be lower than serum λ, whereas traditionally κ levels were higher than λ (Table 5.1). Since there are nearly twice as many κ- as λ-producing lymphoid cells, this observation had seemed reasonable. Certainly, the total serum κ and λ levels reflect the normal ratio of κ to λ FLC synthesis (Table 5.2).

A plausible explanation for the inverted serum κ/λ ratio relates to the kinetics of FLC clearance. Since κ molecules are normally monomeric (25kDa), their renal clearance is faster than dimeric λ molecules (50kDa); consequently they accumulate less in serum. Calculation of the κ/λ clearance rate from serum and urine FLC concentrations ([κ urine]/[λ urine] ÷ [κ serum]/[λ serum]) produced a result of 3.0 ^[11]. In a study on the movement of dextran polymers across capillary membranes, it was shown that molecules of 20kDa were cleared 3.2 times faster than 37kDa molecules ^[14]. Admittedly, polysaccharides are different in charge, shape and flexibility from globular proteins of similar molecular weight, but differential glomerular filtration probably accounts for the inverse κ/λ ratios. This may not have been observed in some of the earlier FLC studies due to poor antisera specificity. Recent studies have shown more consistency in serum κ/λ ratios although Nakano et al. ^[3][4] remain uncertain about actual FLC concentrations (Table 5.1).

Table 5.2. Median values and ranges for free and total light chain concentrations and κ/λ ratios in the sera of 282 normal individuals.

The κ/λ ratio is the most important parameter when distinguishing monoclonal from polyclonal increases in sFLCs. Polyclonal increases result from increased synthesis or decreased renal clearance of normal FLCs (Chapter 20-21). These two processes increase both κ and λ concentrations equally, thereby maintaining a fairly constant κ/λ ratio. In patients with renal failure, the half-life of both FLCs is prolonged from a few hours to several days, so concentrations increase 20-fold or more. However, κ/λ ratios remain within fairly narrow limits. In contrast, in monoclonal gammopathies, only one of the FLC concentrations increases. Thus, κ/λ ratios distinguish monoclonal from polyclonal diseases.

5.2. Normal ranges in children

Figure 5.2. κ and λ FLC concentrations in 282 normal sera. A: axis at the normal κ/λ ratio of 0.6. B: axis at a κ/λ ratio of 1.00.

Figure 5.3.Changes in sFLC during childhood. Modified from Sölling ^[15] to allow comparison with adult concentrations. (Reproduced with permission from Scand J Clin Invest)

Table 5.3. Median values for sFLCs and κ/λ ratios in different age groups.

Only one study of sFLCs in children has been reported. This was conducted by Sölling, ^[15] in 1977 (Figure 5.3), who noted that sFLC concentrations fall after birth and then increase with B cell development at six months. It is likely, therefore, that diseases such as agammaglobulaemia could be detected in the newborn from low sFLCs.

5.3. Borderline results

All tests may produce borderline results, which should be considered in clinical context and alongside other laboratory test results. Borderline sFLC κ/λ ratios may be attributed to a variety of causes. Increases in FLC concentrations and borderline elevated sFLC ratios due to renal impairment in the absence of monoclonal gammopathy are well documented (see Chapter 20). For such patients, the use of a renal reference interval for the κ/λ sFLC ratio may reduce the number of false-positive results (see Section 5.4 below).

Borderline high sFLC ratios have also been reported in conditions associated with a polyclonal inflammatory response, such as cases of infection, inflammation and autoimmune disease ^[16][17][18]. In an audit reported by Marshall et al., borderline abnormal sFLC ratios (defined as κ/λ sFLC ratios between 1.67 and 3.2) were found in sera from 4.9% (47/955) of individuals tested ^[17]. In the majority of cases this was attributed to renal impairment or an inflammatory process. The authors commented that borderline low sFLC ratios were infrequently associated with renal impairment or inflammatory states, and that such borderline low ratios should prompt further investigation.

In addition to renal impairment and inflammatory conditions, borderline abnormal results may occur in a variety of monoclonal diseases encompassing the plasma cell dyscrasias, many lymphomas and leukaemias (Chapter 18), AL amyloidosis (Chapter 15) and FLC monoclonal gammopathy of undetermined significance (MGUS) (Chapter 19). It is now recognised that the probability of a malignant plasma cell disorder increases in relation to the degree of abnormality of the sFLC κ/λ ratio. Interpretation of sFLC ratios using likelihood ratios (LR) has been proposed to improve the clinical interpretation of sFLC results in the diagnosis of malignant plasma cell disorders ^[19]. Whilst a κ/λ sFLC ratio of 1.66–5.0 was found to be inconclusive (LR ±1), a sFLC ratio of 0.05–0.25 or >5.0-10.0 indicated the possible presence of a malignant plasma cell disorder (LR ±10) whilst an extreme ratio (<0.05 or >10) was suggestive of a malignant plasma cell disorder (LR ±50).

5.4. Renal reference interval

Figure 5.4 sFLCs in 142 patients presenting with dialysis-dependent, acute renal failure. 41 patients with κ (triangles) and λ (squares) MM are distinct from blood donors (red crosses: P<0.001) and from patients with acute renal failure from other causes (diamonds: P<0.001). Solid lines indicate κ/λ sFLC normal reference interval (0.26-1.65); broken lines indicate κ/λ sFLC renal reference interval (0.37-3.10).

sFLC levels are dependent upon the balance between production and clearance. In healthy individuals, although κ FLC are produced at a rate approximately twice that of λ FLC, the smaller κ FLC monomers are filtered more freely than the λ FLC dimers, such that the κ serum half life is shorter than λ. In cases of renal insufficiency, when glomerular filtration is reduced, clearance becomes more dependent upon the reticuloendothelial system, which shows no size preference. As a result, the serum half-life of κ FLC approaches that of λ FLC, and serum levels are more influenced by production rates, leading to minor increases in the sFLC ratio in the absence of monoclonal gammopathy ^[20].

In a study of 688 patients with chronic kidney disease (CKD) and no evidence of monoclonal gammopathy, median sFLC ratios were shown to increase progressively with increasing CKD stage from 0.6 to 1.1, with a 100% range of 0.37-3.1 (see Chapter 20) ^[20]. Therefore, to ensure good diagnostic specificity, a κ/λ reference interval of 0.37-3.1 was proposed for patients with renal impairment ^[21]. The impact of applying this modified renal reference interval was investigated in an audit of FLC results for 142 patients who presented with dialysis-dependent acute renal failure (see Section 13.4) ^[21]. Use of the standard reference interval for the κ/λ ratio (0.26-1.65) ^[12] identified all myeloma patients (41/41) plus 2 with MGUS and 5 "false-positives", providing an assay specificity for MM of 93%. When the renal reference range of 0.37–3.10 was used, the assay specificity increased to 99% (Figure 5.4) ^[21]. Mildly increased κ/λ sFLC ratios (up to 3.10) in patients with renal impairment have been confirmed in three additional studies, and further validate the utility of the renal reference interval ^[18][22][23].

Therefore, application of a modified renal reference interval for patients with renal impairment increases the specificity of κ/λ sFLC ratio for detecting monoclonal FLC production in patients with renal impairment.

5.5. Utility of serum free light chain assays for disease diagnosis

Using the reference intervals and diagnostic ranges shown above, Katzmann et al. ^[12], assessed the utility of sFLC measurements for identifying monoclonal FLC gammopathies (Table 5.4). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were estimated for the sFLC κ/λ ratios on the basis of both the central 95% interval and a diagnostic range that included 100% of normal results. Accuracy was calculated as the proportion of individuals classified correctly. PPV and NPV were calculated assuming a 15% prevalence of monoclonal proteins in the samples submitted for monoclonal protein studies. The patients in these studies are described in Chapter 6 for immunofixation electrophoresis (IFE), and in the respective chapters for MM, AL amyloidosis, light chain deposition disease (LCDD) and polyclonal hypergammaglobulinaemia. In the small group of samples selected, sFLC measurements had a higher sensitivity than IFE for detecting low concentrations of monoclonal sFLCs (Chapter 6).

The normal range data reported by Katzmann et al. ^[12] is the most detailed yet published and has been generally adopted (See Chapter 27 for implementation).

Table 5.4. Comparison of reference intervals and diagnostic ranges for sFLCs and κ/λ ratios. (Sera from the 282 reference individuals and 25 polyclonal hypergammaglobulinaemia patients as well as 66 sera from AL amyloidosis, LCDD and MM patients were used to calculate utility. (CL: confidence limits).

5.6. Urine free light chain normal ranges

In general, FLC values in urine (uFLC) have been similar in all studies even when they have included random, 24-hour or early morning samples. Furthermore, κ concentrations were higher than λ in all publications apart from one recent study (Table 5.5 ^[3]). This latter study also reported exceptionally high serum FLC concentrations (Table 5.1) suggesting there were problems with antibody specificity. It is logical that urine contains more κ than λ molecules because of its faster renal clearance.

Table 5.5. Selected publications reporting normal urine FLC concentrations in mg/L ^[11][3]. (^apolyclonal antibody against total light chains, ^burine FLCs in mg/24 hours, ^cpolyclonal antibody against FLCs, ^dmonoclonal antibody against FLCs, ^e latex reagent, ^f 95% range, ^gearly morning urine samples, ^hrandom urine).

Figure 5.5. Frequency distributions of FLC κ (A), λ (B) and κ/λ ratios (C) in 66 normal urines.

Figure 5.6. Comparison of FLC measurements in serum (from Figure 5.2) and early morning urine samples from healthy individuals.

The overall similarity of uFLC results suggests that reported concentrations are reasonably accurate. This is in part because normal urine contains trivial amounts of intact immunoglobulin molecules that can interfere with accurate FLC quantification. The higher uFLC levels shown in the study by Bradwell et al. ^[11] for both κ and λ were explained by the use of early morning urine samples, which are typically 2-3-fold more concentrated than 24-hour urine samples. The mean (±SD) free κ concentration was 5.4 ± 4.95mg/L (n = 66; range, 0.36-20.3mg/L), and the mean (±SD) free λ concentration was 3.17 ± 3.3mg/L (n = 66; range, 0.81-17.3mg/L) (Figures 5.5 and 5.6). The mean κ/λ ratio was 1:0.54 (95% confidence interval, 1:2.17-1:0.25). The mean normal uFLC excretion was 3.7 mg/g of creatinine for κ and 2.0mg/g of creatinine for λ. There was a positive but non-significant correlation of urine creatinine concentrations with κ (r = 0.22) and λ (r = 0.17) measurements.

As expected, the range of uFLC concentrations was much wider than for serum, and κ/λ ratios were more variable. Presumably, this reflects minor differences in renal handling, urine dilution and variations in mucosal secretion of FLCs. The wide range of normal uFLC concentrations is another argument in favour of serum measurements.

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