Quality assurance for serum free light chain analysis

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28.1. Introduction

It is essential that all laboratories participate in external quality assurance schemes when performing monoclonal protein analysis. Guidelines are described in many publications and are summarised in Chapter 25. There are several national quality assurance (QA) schemes for assessing monoclonal immunoglobulins. Typically, distributed samples comprise serum or urine and contain combinations of intact monoclonal immunoglobulins and monoclonal FLCs. Results are generally satisfactory for serum samples but less good for urine, particularly when FLC concentrations are low. The explanations for poor urine results are:- variations in electrophoretic techniques, the requirement to concentrate urine samples and difficulties with interpretation of the gels (Chapter 25) .

Many laboratories measuring monoclonal proteins could improve their results by the addition of FLC immunoassays. If the QA schemes encouraged the use of serum rather than urine tests, this might further improve clinical diagnostic accuracy. Wide availability of QA schemes for sFLC analysis should be encouraged.

28.2. The Binding Site QA scheme (QA003)

Figure 28.1 SPE and sIFE in a patient with IIMM. SPE comprised normal sera (lanes 1-4) a patient sample (IgGκ) applied 3 times (lanes 5-7) and a κ FLC positive urine (lanes 8-10). The κ sFLC concentration was 420 mg/L

Figure 28.2 (A) SPE, (B) IFE and (C) sFLC concentrations in a patient with NSMM. SPE comprised a normal sera (lanes1-4) and the patient sample (lanes 5-8). sIFE was normal.

This was the first sFLC QA programme and was initiated to fulfill customer demands in the absence of any national schemes at that time. To date, there have been over 40 distributions in 7 years. There are currently 140 participants for the sFLC component and 70 for the urine tests. Figure 28.1 shows the results of analysis of one of these samples, from a patient with IIMM, that had a κ sFLC concentration of 420 mg/L. The monoclonal band was undetectable by SPE and IFE.

Another of the distributed samples was from a patient with NSMM and contained no detectable monoclonal protein by all serum and urine electrophoretic tests (Figure 28.2). This included uIFE on highly concentrated samples. sFLC immunoassays showed 250 mg/L of κ FLC and 11 mg/L of λ FLC. This was identified correctly only by those laboratories using sFLC immunoassays. It should be noted that weak, false-positive bands for FLCs may sometimes be seen by IFE. So-called “free” light chain antisera used in IFE usually have some cross-reactivity with bound light chains in order to improve IFE sensitivity (see information leaflet in IFE kits).

28.3. College of American Pathologists (CAP) QA scheme

The College of American Pathologists (CAP) produces a serum paraprotein QA scheme with over 700 participants. 2 samples are distributed twice a year. Reporting methods include SPE, IFE and monoclonal protein quantification and “Binding Site Freelite” participants. Unfortunately, the scheme does not report the FLC results.

One of the earlier distributed samples was particularly difficult to characterise for monoclonal immunoglobulins, yet contained a monoclonal IgA λ of 6g/L (Figure 28.3). This was missed by 65% (593/916) of laboratories using SPE and 6% of laboratories using IFE but only 43% (398/916) of laboratories used the latter method. However, sFLC measurements showed an elevated λ of 39mg/L (normal range 5.7-26.3mg/L) and an abnormal κ/λ ratio of 0.24 (normal range 0.26-1.65) indicating monoclonality (Figure 28.3). This abnormal FLC result would have alerted many laboratories to the presence of a monoclonal plasma cell disease.

28.4. La Agencé Francaise de Securité Sanitaire des Products de Santé QA Scheme.

Figure 28.3 College of American Pathologists QA Scheme. SPE, IFE and sFLC concentrations in a patient with IgAλ MM in relation to other patients with IgAMM (Figure 11.2). SPE comprised 3 normal sera (lanes 1-3) and the patient sample applied 4 times (lanes 4-7).

Figure 28.4 La Agencé Francaise de Securité Sanitaire des Products de Santé QA Scheme. sFLC concentrations in a patient with IgDλ MM (black circle) and results from other patients with IgD MM (blue squares) (see Figure 11.2).

France has a national quality control scheme for monoclonal immunoglobulins La Agencé Francaise de Securité Sanitaire des Products de Santé (143- 147 Boulevard Anatole, 93285 St Denis cedex, France). One distributed serum sample was from a patient with IgD multiple myeloma. Almost 95% of laboratories identified the IgDλ monoclonal immunoglobulin correctly, but only 17% of 1,118 laboratories reported the associated λ FLC. However, sFLC immunoassays showed a λ FLC concentration of 254mg/L and a κ/λ ratio of 0.0096 - the latter result being 30-fold outside the normal range (Figure 28.4).

28.5. UK NEQAS Monoclonal Protein Identification Scheme.

The UK National External Quality Assessment Service for Immunology and Immunochemistry (PO Box 401, Sheffield, S5 7XY, UK) provides a scheme for monoclonal proteins and FLCs. Approximately 80 laboratories currently participate in the FLC component and the usual quality assessment parameters are analysed. There are 6 distributions per year containing unmatched serum and urine samples. Antigen excess has been identified by many laboratories as an issue. Any sample with an elevated κ/λ ratio should be diluted to check for antigen excess, even when the instrument is not indicating a problem (see FLC kit package insert).

28.6. Randox Laboratories Ltd. UK

This is a recent, international FLC scheme and the first clinical external quality control scheme to gain UKAS accreditation. 12 serum samples are distributed per 6-monthly cycle. There have been 22 samples distributed, to date, and there are currently 17 participants. All reported FLC results have been within the normal range.

28.7. Instand e.V. Laboratories (Germany)

This company provides a monoclonal protein scheme with over 180 participating laboratories reporting IFE. Since 2007, sFLCs have been added and there are currently over 70 users. 2 plasma samples are distributed 4 times per year.

28.8. Practical aspects of The Binding Site QA scheme QA003

Figure 28.5 Binding Site QA003 Scheme: SPE and UPE tests on samples containing low concentrations of λ FLCs.

Figure 28.6 Binding Site QA003 Scheme: Frequency distribution of reported λ sFLC concentrations from QA003 97. The reporting laboratory’s result is indicated by an arrow.

Figure 28.7 Combined results of The Binding Site Paraprotein Quality Assurance Scheme (QA003) distribution 97.

There are 6 sets of samples issued per year and each contains unmatched serum and urine samples. The results that are returned to the laboratories include photocopies of the electrophoretic gels, comparisons with other laboratories' results and an “expert opinion” regarding the sample and results. Typical reports are shown in Figures 28.5 and 28.6.

The left hand side of Figure 28.5 shows an SPE gel with normal sera for reference on the 4 left-hand lanes, then 3 lanes loaded with the unknown serum sample and on the right, 3 lanes loaded with the urine sample. In these samples the serum is negative but the urine positive by protein electrophoresis, while a λ band is visible by urine IFE. Figure 28.6 shows the λ results reported from all laboratories with the mean, standard deviation and the result from the reporting laboratory. It should be noted that an aberrant sample result from one laboratory was 1000 fold different from the majority view. Results 10-fold or 100-fold different may be due to mathematical errors in calculating the sample concentration. In the USA, mg/dL is commonly used, while elsewhere mg/L is the rule. Care should be taken when calculating the results.

Laboratory and clinical reports accompanying results from The Binding Site QA003 Scheme (Figures 28.5-28.7).

Serum sample: IgG 7.9g/L, IgA 1.6g/L, IgM 0.77g/L, κFLC 13.7mg/L (NR 3.3-19.4), λ FLC 70mg/L (NR 5.7-26.3), κ/λ ratio 0.096 (NR 0.26-1.65) and β2 microglobulin 1.8mg/L. The sample appeared normal by SPE but a λ FLC band was detected by IFE.

Urine sample: κ FLC 4.2mg/L (NR 0.36-20.3mg/L), λ FLC 62mg/L (NR 0.81- 17.3mg/L), κ/λ ratio 0.07. UPE showed a minor band on concentrated urine and IFE confirmed an abnormal λ FLC band.

Comment: The serum sample contained elevated λ FLCs of 70mg/L with no evidence of immunosuppression. The FLC band was not visible by SPE since the concentration was below the detection limit. By IFE, a λ monoclonal band was detected with no coincident heavy chains for IgG, A, M, D or E. sFLC assays showed an elevated λ concentration and a reduced κ/λ ratio. Tests on the urine sample showed similar results by electrophoretic tests and uFLC immunoassays. The latter can often be used to decypher urine electrophoretic test results that are difficult to interpret.