Laboratory FAQ
Laboratory questions about serum testing for free light chains
Here you will find answers to questions about serum testing for free light chains.
- Which machines can run these assays?
- Can I replace intact immunoglobulin measurements with serum FLC measurement?
- How long does it take to run the assay?
- Is the serum FLC assay as sensitive as IFE for detecting minimal residual disease?
- Are serum FLC levels prognostic?
- Why are there so many abnormal FLC results in patients with no malignancy?
- I have many equivocal results with IFE on urine samples - can serum FLC assays help?
- Should sFLC tests be used as a screen for monoclonal gammopathies instead of SPE?
- Will any monoclonal proteins detected by SPE and/or IFE be missed by sFLC tests?
- If the sFLC concentration is many thousands of mg/L why is there no band on SPE?
- Many sera tested by SPE have bands that are barely visible and I worry that I might be missing myeloma patients. At present I ask for IFE on these samples. Can serum Freelite testing help?
- Serum albumin levels are reduced in patients with nephrotic syndrome and gross proteinuria. Are serum Freelite levels also reduced in patients with proteinuria?
- How can clonality be judged using FLC assays?
- How do we report borderline results?
- What is the frequency of false positive and false negative results, and how are they dealt with?
- Why is the free κ/λ ratio different from the total κ/λ ratio in normal subjects?
- Why should I change from using a total light chain measurement to a more expensive free light chain assay?
- How does the sensitivity of serum Freelite tests compare with capillary zone electrophoresis (CZE)?
- Since the Freelite reagents use polyclonal antibodies, how is batch to batch variation minimised?
- If I am going to use sFLC tests how do they fit into my laboratory protocols?
Which machines can run these assays?
Freelite assays can be run on:- Dade Behring BNII and ProSpec, Beckman IMMAGE, Roche/Hitachi 911, 912, 917 and Modular P, Olympus AU400, 640, 2700, 5400 (not AU600), Radim Delta, Bayer Advia.
Can I replace intact immunoglobulin measurements with serum FLC measurement?
In many of the retrospective studies others and we have conducted, the intact immunoglobulin measurements gave no information in addition to that given by serum FLC measurements and in many cases gave less information. However, at initial screening, use of SPE to detect intact immunoglobulin is essential as 5% of patients have normal FLC at presentation. It is probable that, for the purpose of monitoring this group of patients, intact immunoglobulin measurements are necessary. When monitoring a patient for relapse, both SPE and FLC assays are needed to ensure detection of either paraprotein at relapse i.e. intact + FLC relapsing as just intact, or just FLC.
How long does it take to run the assay?
The assays take 5-18 minutes depending on the analyser being used.
Is the serum FLC assay as sensitive as IFE for detecting minimal residual disease?
Serum FLC assays are more sensitive than IFE for detecting minimal residual disease. A large subset of patients who are in complete remission (CR) by IFE have been shown to be abnormal by serum FLC assay. It has been shown that an abnormal κ/λ ratio during IFE CR is indicative of a higher risk of early relapse.
Are serum FLC levels prognostic?
In some instances yes. Elevated serum FLC levels have been shown to be an independent risk factor for progression to full multiple myeloma in monoclonal gammopathy of undetermined significance (MGUS).* A >50% reduction in serum FLC levels in response to therapy has been shown to predict longer remission in AL amyloidosis patients. Abnormal FLC ratios during IFE CR in post-transplant patients has been shown to be predictive of earlier relapse in two separate studies. Further work is being carried out to fully evaluate the importance of the last observations.
* In the USA diagnostic use of this product is restricted to those stated in the product insert
Why are there so many abnormal FLC results in patients with no malignancy?
Serum FLC levels are affected by rate of FLC production and rate of FLC removal (rate of glomerular filtration). Both of these factors can be affected in conditions other than malignancy. For example the rate of glomerular filtration can be reduced by a non-malignant condition such as non-AL amyloidosis, this will lead to elevated levels of serum FLC but normal FLC ratio. Also, production of polyclonal immunoglobulin is elevated in various autoimmune conditions and some bacterial infections, both have associated elevated levels of polyclonal FLC production which leads to elevated FLC levels with normal FLC ratios. An abnormal FLC ratio is highly diagnostic for B cell malignancy.
I have many equivocal results with IFE on urine samples - can serum FLC assays help?
Yes, because low levels of urinary monoclonal FLC (and therefore equivocal results) are usually associated with highly elevated serum FLC levels and abnormal FLC ratio. An equivocal IFE with a normal FLC result can reasonably be interpreted as "no monoclonal protein detected". An equivocal result with an abnormal FLC result should be used as trigger for further studies.
Should sFLC tests be used as a screen for monoclonal gammopathies instead of SPE?
No. SPE detects intact immunoglobulin monoclonal proteins and some FLC monoclonal proteins. In contrast, sFLC assays detect FLC monoclonal gammopathies, either alone, or in association with intact monoclonal immunoglobulins. Approximately 95% of patients with intact monoclonal immunoglobulins have abnormal sFLCs, but not all, particularly those patients with low concentration MGUS.
Will any monoclonal proteins detected by SPE and/or IFE be missed by sFLC tests?
Yes. Monoclonal proteins can be intact immunoglobulins or FLCs. Since sFLC assays are >100 times more sensitive than electrophoretic tests it is most unlikely that FLCs will be detected in serum by SPE or IFE yet be normal by FLC assays. However, intact immunoglobulin monoclonal immunoglobulins by tradition (especially MGUS) are detected by IFE and SPE, but may have normal FLC concentrations. Studies indicate that all LCMM and ~95% of AL amyloidosis patients are correctly identified by sFLC assays.
If the sFLC concentration is many thousands of mg/L why is there no band on SPE?
The sensitivity of SPE for monoclonal bands depends upon the width of the band and its position in the gel in relation to other plasma proteins. Narrow monoclonal bands in the gamma region in association with hypogammaglobulinaemia will be visible at 200-400mg/L. The same band in a beta position, perhaps superimposed on transferrin, will be invisible. The monoclonal protein may need to be over 2,000mg/L to be visible in this area of the gel. In addition, monoclonal FLCs may be polymerised to different extents and then they migrate on electrophoresis gels as diffuse bands. This is frequently found in NSMM and is well-documented in LCMM. In these patients, even 5,000mg/L of monoclonal protein may be difficult to detect above the background of the other plasma proteins.
Many sera tested by SPE have bands that are barely visible and I worry that I might be missing myeloma patients. At present I ask for IFE on these samples. Can serum Freelite testing help?
Yes, the sFLC assays will detect all patients with LCMM and most patients with NSMM and AL amyloidosis. IFE will not detect many of these patients. Since sFLC immunoassays are 100-fold more sensitive than serum IFE, sFLC abnormalities, visible by IFE, will be exceptionally rare if the sFLC tests are normal. Urine IFE or uFLC measurements may be helpful in these rare cases.
Serum albumin levels are reduced in patients with nephrotic syndrome and gross proteinuria. Are serum Freelite levels also reduced in patients with proteinuria?
No. Renal damage never increases the glomerular filtration rate of small molecules such as FLCs or creatinine since they normally pass relatively unhindered through the glomerular pores. Molecules as large as albumin are not normally filtered by the kidney but they are cleared in nephrotic syndrome as the glomerular pores become damaged. The extra protein leakage overwhelms the proximal tubular reabsorption mechanisms allowing many different proteins to appear in the urine. The protein leakage damages the tubules in the process which become sclerotic. Renal clearance of all small proteins is then reduced. This leads to an increase in sFLC levels (and creatinine). In the early stages of the process, FLCs are increased in the urine because of increased competition with albumin for reabsorption by the proximal tubules.
Renal impairment leads to increases in both κ and λ FLCs in the serum. Therefore, when both are elevated the likely cause is a reduction in glomerular filtration. There is a correlation between changes in the concentrations of serum creatinine, cystatin C and FLCs during changes in renal function.
How can clonality be judged using FLC assays?
Using electrophoresis methods, clonality is judged by the appearance of a narrow protein band. Using FLC assays, clonality is judged by the numerical ratio of free κ to free λ concentrations. In a similar manner, B-cell clonality in leukaemia is assessed by cellular κ/λ ratios using flow cytometry. Arguably, numerical FLC ratios are more accurate than visual assessments of stained bands on electrophoresis gels. Furthermore, in NSMM, clonality may not be apparent by any electrophoretic procedure but is usually identified by serum κ/λ ratios. In the situation of biclonal gammopathies, with increased synthesis of both free κ and λ molecules, free κ/λ ratios may be normal but the concentrations of both FLCs will be raised.
How do we report borderline results?
All tests have borderline results. For FLCs, clinical results should be judged against normal and disease state sera from the laboratory, and from national and international reference ranges. The normal range recommended for the free κ/λ ratios is greater than that used for most tests in order to provide a large safety margin for normal individuals.
Since FLC results are quantitative, less experience is required compared with protein electrophoresis. This leads to less subjective interpretation of results.
What is the frequency of false positive and false negative results, and how are they dealt with?
All tests produce false positive and false negative results and these need to be assessed for clinical significance. Reference ranges have been developed in collaboration with The Mayo Clinic and include individuals up to 90 years of age. Some of these individuals have minor degrees of renal impairment. This increases the concentrations of the FLCs, and the κ/λ ratios, and is apparent on a κ/λ log plot. The
difference between the normal and abnormalsamples is then selected using standard deviations from the mean. If all of the 282 normal samples in the Mayo Clinic study are used, this represents four standard deviations from the mean and is greater than normally chosen cut off levels. Therefore, test samples outside this range will most likely indicate patients with monoclonal gammopathies.Negative sFLC results occur in a few patients with NSMM, AL amyloidosis and LCDD. Also, rare patients have monoclonal proteins in the urine detected only by IFE. The molecular form of these FLC molecules is unknown but they may be abnormal in shape or size and this may prevent their detection by the FLC antibodies.
Why is the free κ/λ ratio different from the total κ/λ ratio in normal subjects?
Approximately twice as many κ molecules are produced as λ. Since free λ is mostly in dimeric form it has a half-life (determined by glomerular filtration) that is approximately three times that of monomeric κ FLCs. This causes free λ molecules to accumulate in the serum more than free κ molecules and alters the free κ/λ ratio from 1.8 to 0.6. When the light chains are bound to immunoglobulins they are metabolised as
the whole immunoglobulin, which is independent of light chain type, so total κ/λ ratio is 1.8:1.Why should I change from using a total light chain measurement to a more expensive free light chain assay?
Normal serum FLC concentrations are 10-30mg/L, which is much lower than total light chain concentrations of 1,000-3,000 mg/L. Patients who have levels of FLC above 30mg/L and below the normal range of total light chains can be diagnosed and monitored with Freelite assays but not total light chain assays. Since this applies to most patients with AL amyloidosis, nonsecretory myeloma and many light chain myelomas, Freelite assays have considerable clinical application in these diseases.
How does the sensitivity of serum Freelite tests compare with capillary zone electrophoresis (CZE)?
CZE of serum is more sensitive than SPE but less sensitive than IFE for detecting monoclonal proteins. In a recent study, it was shown that sFLC assays detected all monoclonal FLCs from patients with LCMM that were missed by CZE but detected by IFE. If CZE is used for initial detection of monoclonal proteins, sFLC assays will detect additional patients.
Since the Freelite reagents use polyclonal antibodies, how is batch to batch variation minimised?
The FLC antisera are produced by immunisation with many different monoclonal FLC proteins. These are not representative of all monoclonal FLCs but the antibody target is the constant region of the molecule that has little structural variation. However, tumour produced monoclonal FLCs may be truncated, have amino acid substitutions or additions and may be abnormally polymerised. Therefore, occasional patient's
monoclonal FLCs may not be detected reliably by the immunoassays or may be detected differently with different antiserum batches. It is, therefore, ideal laboratory practice to assay current and previous samples alongside each other. This is no different from the situation when measuring IgG with different antiserum batches.To minimise batch-to-batch variation, antisera pools are large, are prepared from multiple immunisations and are carefully controlled to maintain consistency. Many monoclonal proteins are tested when new batches are prepared but there is a limit to the number of different monoclonal proteins that can be used.
Monoclonal antibodies have been assessed in some studies to measure FLCs but they have proved to be unreliable. Polyclonal antisera are superior since they detect more monoclonal FLC molecules and they detect them more reliably.
If I am going to use sFLC tests how do they fit into my laboratory protocols?
The preferred option is to measure FLCs alongside SPE at the time of the presentation blood sample. SPE will identify IIMM patients while FLC assays will identify LCMMs, most NSMMs and other FLC diseases such as AL amyloidosis. Low concentration, intact immunoglobulin MGUS sera (less than 2-5g/L) will not be detected using these two procedures. A strategy of performing SPE and IFE as a screen for FLC monoclonal proteins and not FLC immunoassays will result in some patients with LCMM, NSMM and AL amyloidosis being missed. FLC assays, performed on a patient's presentation sample, are also important for providing a baseline for subsequent disease monitoring. For easy interpretation, results should be reported using a logarithmic κ/λ plot, alongside existing clinical data.
When monitoring patients with FLC diseases, results should be reported alongside other analyses. IFE may add little to the combined use of SPEand FLC tests apart from identifying some low level intact immunoglobulin MGUS samples.
