image

39 - External quality assurance schemes for Freelite and Hevylite immunoassays

Chapter 39

Summary:

  • There are currently five external quality assurance schemes for sFLC analysis, and one scheme for Hevylite® analysis.
  • The UK NEQAS scheme has the highest number of participants, with over 380 laboratories registered.
  • When submitting sFLC results as part of an external quality assurance scheme it is important to treat the sample as routine (including antigen excess checks) and ensure that concentrations are reported using the correct units.

39.1. Introduction

It is important that all laboratories participate in external quality assurance (QA) schemes when performing monoclonal protein analysis. There are several national and international QA schemes for assessing serum free light chains (sFLC). Particular care should be taken when reporting sFLC results: in the USA mg/dL units are commonly used, while elsewhere, mg/L is the norm. Individual QA scheme results that are 10- or 100-fold different from the majority view can probably be attributed to errors in calculating or recording the sample concentration.

39.2. The Binding Site schemes

39.2.1. QA003

The Binding Site IMMPROVE™ serum paraprotein QA scheme (QA003) was the first scheme to include sFLC analysis. To date, there have been more than 70 distributions over 12 years, and over 300 laboratories worldwide currently participate in the scheme. Four serum samples are issued per year and results can be returned via the IMMPROVE website. Methodologies assessed include serum protein electrophoresis (SPE), serum immunofixation electrophoresis (sIFE) and sFLC assays (Chapter 4). Laboratories are able to return quantitative results for IgG, IgA, IgM, β2-microglobulin, κ sFLCs, λ sFLCs and the κ/λ sFLC ratio. A comprehensive report is returned to each laboratory in which sFLC results are categorised according to nephelometric/turbidimetric instrument type to allow user group comparisons to be made. The report also includes examples of electrophoresis gels and an interpretative comment regarding the sample and results.

Electrophoresis and sFLC results from a typical QA003 report (Distribution 129, August 2014) are shown in Figures 39.1, 39.2 and 39.3. Whilst 82% of laboratories reported that no monoclonal protein was visible by SPE or sIFE, the majority of participants reporting Freelite® results identified monoclonal κ FLCs of approximately 500 mg/L. A small sub-group of laboratories reported significantly lower κ sFLC concentrations. This sub-group primarily consisted of laboratories using another (non-Freelite) method for FLC measurement. This distribution illustrates that absolute FLC values reported by other FLC assays may be significantly different to those reported by Freelite (Chapter 8).

39.2.2. QA003.H

In May 2013 the Binding Site launched a pilot quality assurance scheme for Hevylite analysis (QA003.H). Four samples are distributed each year and over 40 laboratories currently participate in the scheme. The scheme requires samples to be initially screened by SPE and sIFE, followed by Hevylite assays with the HLC pair selected as indicated by the immunofixation results. Participants receive a comprehensive report that includes analysis of qualitative results for SPE/sIFE and quantitative results for HLC assays that correspond with the monoclonal intact immunoglobulin type. Examples from a typical report are shown in Figures 39.4 and 39.5.

39.3. United Kingdom National External Quality Assessment Service scheme

The United Kingdom National External Quality Assessment Service (UK NEQAS) for Immunology, Immunochemistry & Allergy (IIA) provides a Monoclonal Protein Identification scheme that is accredited by the United Kingdom Accreditation Service (UKAS). The scheme has incorporated sFLC analysis since 2005 and there are six distributions per year comprising unmatched serum and urine samples. Over 380 UK and international laboratories are registered for the scheme, and approximately 170 report quantitative sFLC results. Prior to June 2015 (up to and including distribution 152) quantitative sFLC were reported separately to other laboratory test results but have since been amalgamated into a single report. sFLC results are categorised by both instrument type and reagent manufacture.

Past distributions have highlighted the need for all participants to be aware of sFLC assay technical issues. For example, the Dade Behring BN™II Freelite κ sFLC results for UK NEQAS Distribution 095 were bimodal: peaks were observed at 143 mg/L and 1001 mg/L (Figure 39.6). It is likely that these two peaks corresponded to those participants who did not, and those who did, screen for sFLC antigen excess (following the dilution protocol in the product insert). The phenomenon of antigen excess is further discussed in Section 7.4.

Since 2012, Siemens N Latex FLC assays have been reported as a separate user group. This has allowed the absolute values reported by Freelite and N Latex FLC assays to be compared. For example in Distribution 136, N Latex FLC results were significantly lower than those reported by Freelite (Figure 39.7).

A recent review article by Carr-Smith et al. [868] compared sFLC results from the 2014 UK NEQAS distributions for Freelite on the Binding Site SPAPLUS® against N Latex FLC on the Siemens BN™II (Table 1). For all distributions, the average inter-laboratory variation (%CV) for κ FLC results was lower for Freelite compared with N Latex FLC assays. For λ FLCs, the mean %CV of Freelite results was superior (4/6 distributions) or equivalent (1/6 distributions) to N Latex FLC results for the majority of samples. The results also indicate that the absolute values reported by two assays do not compare well; this is discussed further in Section 8.5.3.

Instrument – Assay

(distribution)

κ FLC

λ FLC

n

Mean, mg/L

CV, %

n

Mean, mg/L

CV, %

SPAPLUS – BS (141)

35

5.59

10.3

35

17.39

8.6

BNII – SMS (141)

16

4.23

12.6

16

334.38

16.6

SPAPLUS – BS (142)

36

722.59

7

34

3.65

34.1

BNII – SMS (142)

13

152.25

44.5

13

0.36

12.7

SPAPLUS – BS (143)

39

9.67

9.7

39

13.21

7.5

BNII – SMS (143)

15

6.42

12.2

15

7.2

12.3

SPAPLUS – BS (144)

40

975.6

7.9

40

20.7

5.9

BNII – SMS (144)

19

171.2

68.8

19

22.21

10.5

SPAPLUS – BS (145)

44

82.4

16.2

41

2.9

10.5

BNII – SMS (145)

18

41

58.2

18

3.45

30.5

SPAPLUS – BS (146)

41

1.55

9.1

45

894

13.1

BNII – SMS (146)

23

1.21

19.8

24

545

13.1

Table 1. Summary of 2014 UK NEQAS Monoclonal Protein Identification scheme results for Freelite (BS) on the SPAPLUS and N Latex FLC (SMS) on the BNII [868].

39.4. College of American Pathologists scheme

The College of American Pathologists (CAP) has recently introduced a proficiency testing scheme for sFLCs. Three serum samples are distributed every 6 months, and currently over 250 participants are enrolled in the scheme. In contrast to The Binding Site and UK NEQAS schemes described above, the CAP sFLC scheme does not include proficiency testing for serum electrophoresis, which is tested separately.

39.5. Randox International Quality Assessment scheme

The Randox International Quality Assessment scheme (RIQAS) for Specific Proteins, incorporating FLCs, has been accredited by the UKAS since 2002 and is currently accredited to the ISO 17043 standard. Twelve liquid stable serum samples are distributed every 6 months with over 180 samples having been distributed to date. There are currently around 830 participants in the scheme with approximately 65 reporting results for FLCs.

39.6. German Institute for Standardisation scheme

The German Institute for Standardisation's Gesellschaft zur Förderung der Qualitätssicherung in Medizinischen Laboratorien scheme (abbreviated to INSTAND e.V.) provides a monoclonal protein scheme with over 760 participating laboratories reporting IFE results. Since 2007, sFLCs have been added and there are currently over 120 laboratories reporting sFLC results. Two plasma samples are distributed 4 times per year.

Questions

  1. How do absolute sFLC values reported by N Latex FLC assays and Freelite assays compare?

Answers

  1. EQA scheme results indicate that the assays do not compare well and are not interchangeable (Section 39.3 and Chapter 8).
VIEW ANSWERS

Figures