Implementation and interpretation of free light chain assays

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26.1. Why measure free light chains in serum?

Figure 26.1 sFLCs in several clinical conditions and changes seen during chemotherapy. Results at presentation (A), after high-dose melphalan showing treatment response and bone marrow suppression (B), in complete remission (C) and in relapse with renal impairment from FLC toxicity (D). For explanations of E-F: G-H see text. X----Y: mid point of normal κ/λ ratios.

It is logical to measure serum FLCs in the situations listed below. Justification can be found in the relevant chapters. Most of the indications are well established while others are still under evaluation. A selection of uses include:-

FLCs are preferably measured in serum rather than urine. Urine samples have a wider normal range, are more difficult to collect and process and are less sensitive when FLC production is low (Chapter 24). Both FLCs should be measured and κ/λ ratios calculated. Results should be reported on log/log graphs that include normal range data and results from a variety of clinical conditions (Figure 26.1).

26.2. Getting started

Figure 26.2 Serum κ and λ concentrations in a selection of clinical conditions. Patients are categorised according to FLC concentrations and κ/λ ratios. See Table 26.1 for interpretation. The axes are truncated for clarity compared with Figure 26.1.

There are many implementation issues to be considered: clinical, technical, educational, political, etc. Laboratories are familiar with the introduction of new tests so it is not appropriate for these issues to be discussed here. Implementation of FLC tests normally come under the category of “service development”. Because FLCs are widely measured in urine they can usually be introduced without seeking new test approval.

In analytical terms, FLC molecules are stable in serum, kits are available for many routine laboratory instruments and clinical interpretation of the results is well established. Practical issues directly related to FLC analysis such as the most appropriate instrumentation and QA schemes are discussed below and in Chapter 27 and 28.

Serum or plasma samples can be used and FLCs are stable for many weeks when stored at 4^oC. Longer-term storage should be at -20^oC with preservatives (Chapter 4). There is minimal variation in FLC concentrations in samples taken from patients at different times of the day.

FLC kits are available for use on many instruments. Results are generally more precise on the large clinical chemistry analysers. Details can be found in Chapter 27. External quality control schemes are available and should be used (Chapter 28).

Reporting of results for diagnosis should be on κ/λ log plots that include normal range data and results from a variety of clinical situations (Figure 26.1). Serial monitoring should include both FLCs and κ/λ ratios. It may be useful to include a renal function marker such as serum creatinine or cystatin C.

26.3. Use and interpretation of serum free light chain results

Serum FLC analysis should be used alongside SPE and sIFE tests and then >99% of patients with monoclonal gammopathies are identified. Results are considered abnormal when they are outside the following normal ranges (Figure 26.2):-

Patients’ results separate into different categories depending upon several factors: whether the clone is κ or λ, the presence of renal failure or polyclonal hypergammaglobulinaemia and the degree of bone marrow impairment from the growing tumour or from drug therapy (Figure 26.2). An accompanying table for this figure provides a simplistic guide to interpretation of results (Table 26.1).

Table 26.1. Classification of monoclonal gammopathies according to serum FLC concentrations (also see Figure 26.2). BM: bone marrow. MG: monoclonal gammopathy. pIg: raised polyclonal immunoglobulin.

renal impairment (common).

over-production of polyclonal FLCs from inflammatory conditions (common).

biclonal gammopathies of different FLC types (rare).

Many clinical studies have shown that urine FLC tests offer no additional benefits over serum FLC tests in assessing patients with MM, AL amyloidosis and MGUS. Laboratory comparison of the sensitivity of serum FLC tests with urine tests indicates greater sensitivity for serum tests. Occasional patients have normal serum FLC results but minor monoclonal FLCs in the urine. However, their clinical relevance is doubtful (Chapter 24).

Patients with monoclonal gammopathies can be monitored serially using the tumour FLC and κ/λ ratios. When sFLC concentrations are very high, the level of the individual monoclonal light chain can be used for monitoring changes in disease. This is particularly important when the non-tumour FLC concentrations are very low and measurement precision is poor. As levels trend to normal or there is renal impairment, κ/λ ratios become better monitoring tools. As an alternative, the numerical value of the concentration of the tumour (involved) FLC, minus the non-tumour (uninvolved) FLC appears satisfactory. This technique of subtracting the polyclonal FLC component is included in the new response criteria for monitoring MM (Chapter 25, Table 6). Also, it is important to ensure that sFLC concentrations are given in consistent units. All results outside the USA are in mg/L whereas within the USA results may be either in mg/L or mg/dL (Figure 28.7).

Results from sFLC measurements can be presented in different ways. Figures 26.1 and 26.3 show changes in serum FLC concentrations in a patient with MM from the time of presentation to disease relapse. In Figure 26.1, at presentation (A), κ concentrations were highly elevated at 3,500mg/L and the non-tumour FLC was mildly elevated because of FLC deposition in the kidneys. While under treatment (B), both sFLC concentrations fell because of bone marrow suppression but there was selective tumour cell killing reflected in a reduction of the κ/λ ratio. Successful treatment is shown by return of the FLC concentrations and κ/λ ratio to normal (C). At subsequent relapse (D), the tumour FLC and the κ/λ ratio increased, as might be expected, but the alternate FLC also increased as a result of renal impairment from chemotherapy and FLC deposition in the nephrons.

The mid point of normal κ/λ ratios is marked as X---Y. Changes away from this axis indicate decreasing (E) or increasing λ tumour FLCs (F). Changes parallel to the κ/λ ratio axis and both FLC concentrations increasing (G) indicate renal impairment. Similarly, decreasing concentrations indicate renal function recovery, with the addition of bone marrow suppression if below the normal range (H).

Another way to present the results is against time. Data should include κ, λ and κ/λ ratios (Figure 26.3), perhaps with the addition of other markers such as intact immunoglobulins. Examples of patients being monitored using serum FLCs can be found in the relevant chapters. A computer programme based upon Microsoft Access and Excel spread-sheets has been described.

26.4. Limitations of serum free light chain analysis

Figure 26.3 Data from the patient shown in Figure 26.1. Results at clinical presentation (A), after high-dose melphalan showing treatment response and bone marrow suppression (B), in complete remission (C) and in relapse with renal impairment from FLC damage (D).

Figure 26.4 Serum FLC concentrations in 5 patients with biclonal gammopathies. (Courtesy of I Ramasamy)

While serum FLC immunoassays have many advantages over electrophoretic tests, there are limitations. When results give doubt, monoclonal FLCs should be assessed by SPE and IFE. Difficulties that may occur are described below. Details can be found in the relevant chapters of the book, particularly Chapter 4.

1. sFLC assays do not measure intact monoclonal immunoglobulins.
This may seem a rather obvious statement, yet there are several publications in which the authors criticise the sFLC assays for missing patients with monoclonal gammopathies ^[1][2][3]. FLC antisera do not, by definition, react with intact monoclonal immunoglobulins and will fail to identify these patients if monoclonal FLCs are absent.

2. Clonality assessment
Using FLC immunoassays, monoclonal FLCs are determined by abnormal κ/λ ratios. This is clearly different from the assessment of bands on electrophoretic gels. Arguably, κ/λ ratios are preferable because numerical limits are easily established while visual impressions of bands on gels are more difficult to assess and quantitate. However, some find this difficult to accept because of its novelty.

In some situations analysis of κ/λ ratios is clearly preferable. For example, patients with NSMM have highly abnormal κ/λ ratios yet electrophoretic tests are normal. In AL amyloidosis (Chapter 15) and LCDD (Chapter 17) , serum FLCs are the actual molecules causing the disease so quantification is preferable.

Rare patients have monoclonal FLC bands when tested by urine IFE that are not abnormal by serum FLC immunoassays (Chapter 24). These monoclonal bands are of little clinical relevance and can probably be safely ignored.

3. Inaccuracy of sFLC measurements
Each monoclonal protein is structurally unique, so results depend upon how well the antibodies recognise molecular variants and polymeric configurations. Some of these variants give rise to inaccuracies in measurement. Historically, this line of argument has been used to discredit measurements of IgG, IgA and IgM by nephelometry. However, experience has shown that this has not invalidated their utility and widespread acceptance in a clinical setting. The same can now be said for sFLC immunoassays. Nevertheless, in some patients huge discrepancies are seen between monoclonal FLCs measured by immunoassays and other techniques, particularly in urine samples. In the latter fluid, FLC immunoassays may give higher concentration values than other methods of FLC measurement (Chapter 4 and 6).

4. Standardisation
While current standards may well be accurate, there are no international standards or traceable international materials and none will be available in the short term. Although every effort might be made by the manufacturer to ensure good quality, the assays may drift with time and in an unpredictable manner. Local standards and reference materials should, therefore, be used with the assays, when possible. Initially, a set of local serum samples should be compared with published reference ranges and this is usually satisfactory. If not, the manufacturer should be contacted to determine if the cause is due to local inexperience or an instrument related problem. When results are still unsatisfactory, a local reference range can be established ^[4].

5. Non-linearity of monoclonal free light chain proteins
This refers to variations in quantification that occur when a sample is diluted and the result is different from the starting value. A number of factors may be involved. Details can be found in Chapter 4 and include the following:-

• Monoclonal FLCs of unusual shape, partially missed by the antisera (see below).
• Antibody bias to one form of the FLC proteins when polymerisation or fragments are present.
• Antisera cross-reactivity with intact immunoglobulins.
• Non-specific assay interference (lipids, haemoglobin etc).
• Use of unsuitable materials for assay calibrators.

6. Different batches of antisera
Consideration should be given to apparent changes in serum FLC concentrations that might occur when changing to a different batch of antisera. Batches are made to react in a characterised manner with a variety of standards and control sera. These are manufactured to defined limits that are achieved during kit production. Within these limits there is some small but quantifiable variation.

In addition, each monoclonal FLC is unique and will react in its own particular manner in the assays. While great effort is made during manufacture to maintain lot-to-lot consistency, antisera cannot be made to recognise every individual monoclonal FLC equally. Some structurally abnormal molecules may not, therefore, be reproducibly measured. In such circumstances, the previous sample should be re-run alongside the new sample, using the new batch of antiserum, and the results compared ^[5][6][7].

7. Presence of polyclonal free light chains
Polyclonal FLCs in all sera lead to overestimations of monoclonal FLCs. This is particularly apparent in patients with renal failure in whom polyclonal, non-tumour FLCs are greatly elevated (Chapter 20). However, κ/λ ratios usually remain abnormal if monoclonal FLCs are present. Borderline results may need to be corrected against serum creatinine (or a better marker of impaired glomerular filtration such as cystatin C) in patients with renal failure.

8. Biclonal gammopathies of different light chain types
Approximately 1-2% of patients with MM have bi-clonal gammopathies. When the LC types differ (~50%), so that the patient has both types of FLC, κ/λ ratios can be normal. Since it is likely that both FLC concentrations would be elevated, and in different amounts, the clinician would usually be alerted to an abnormality (Figure 26.4) ^[8]. The issue can be resolved by testing the sample using IFE and identifying two monoclonal bands of different FLC types. Renal function should also be determined to assess the degree of polyclonal elevation of sFLCs from reduced glomerular clearance.

References