Recommendations For Serum Free Light Chains (SFLC) Measurement In Routine Laboratories

Transcription

1 ROYAL PRINCE ALFRED HOSPITAL A tradition of excellence since 1882 Recommendations For Serum Free Light Chains (SFLC) Measurement In Routine Laboratories Louise Wienholt Laboratory Manager Royal Prince Alfred Hospital, Sydney.

2 Why do we need guidelines? Despite the wide utility of serum free light chains (SFLC) results from the 2012/13 RCPA QAP Immunochemistry Paraprotein program indicated large variability between laboratories, and for different manufacturers assays and platforms. In light of this, the RCPA convened a working party to develop guidance recommendations for SFLC measurement by routine laboratories.

3 Variability

4 Working party Information was gathered from WP-SFLC members working in routine laboratories and clinics, the manufacturers producing SFLC kits (Freelite, The Binding Site and N Latex, Siemens Healthcare), who provided input through their recommendations for dilutions, and the literature Jill Tate, Sue Jovanovich (Chair), Peter Mollee,Weldon Chiu, Louise Wienholt, David Gillis, Lindsay Reibelt & Odette Youdell

5 What did we want to address? 1) What are acceptable imprecision goals? 2) What reference intervals should be used? 3) What sample dilutions should be used to detect antigen excess and nonlinearity? 4) What is the optimal reporting of results?

6 Before you begin (or change) The question I m asked the most is what I think of the Siemens assay. You re going to have to repeat a lot of previous samples and probably have to run in duplicate for 6 months. You may have to refer clinical trail samples When introducing the serum FLC assay the minimal verification study should include: 1) A 5-day imprecision study, using patient specimens of pooled serum, to verify a manufacturer s claims and requires that runs are performed twice a day, for five days, with a minimum time separating each run (CLSI EP15-A2; 5) 2) Verification of the manufacturer s reference intervals according to CLSI C28- A3, paragraph 11.2, by examining 20 reference individuals from a laboratory s own subject population.

7 1. What are acceptable imprecision goals? Biological variability (BV) of SFLC can help in defining allowable analytical limits: Intra-individual variation (CVi): κ FLC, 8.1%; λ FLC, 7.0%; κ/λ FLC ratio, 8.2% Between-individual variation (CVg): κ FLC, 21%; λ FLC, 30%; κ/λ FLC ratio, 36.6%

8 Performance goals for SFLC measurement For diagnosis, the desirable Total Error (99%) is: κ FLC concentration <12.3% λ FLC concentration <13.5% κ/λ FLC ratio <16.1% For monitoring, the minimum analytical imprecision (CVa) is 0.75* CVi: κ FLC concentration 6.1% λ FLC concentration 5.2% κ/λ FLC ratio 6.2%

9 Performance goals for SFLC measurement

10 What does this show us? There is still room for improvement in current FLC assays to attain minimum performance goals (derived from biological variations) of between 5.2% and 6.2% for FLC concentrations and ratio for monitoring purposes.

11 Recommendations 1. Laboratories should use a serum-based control either within the normal range or close to SFLC upper reference limit values to monitor assay imprecision and any reagent lot-to-lot variation. 2. Manufacturers SFLC quality controls matched to specific kit lots should be within 20% CV of the quoted values.

12 2. What reference intervals should be used? In practice few laboratories can determine their own FLC reference intervals It is relatively easier to validate previously established reference limits for the population that laboratories service by examining 20 reference individuals from a laboratory s own subject population (CLSI document C28-A3).

13 Manufacturers ranges

14 Recommendations 1. The WP recommends using the manufacturers κ and λ FLC reference intervals and κ/λ ratio diagnostic ranges and for laboratories to validate manufacturers values according to the CLSI document C28- A3. 2. In end stage renal failure a different κ/λ ratio range needs to be applied when using the Freelite assay but not when using the N Latex FLC assay.

15 What sample dilutions should be used? Unfortunately there is no easy answer to this question!! One default dilution for FLC measurement will not suit all samples. Measuring range for FLC is from ~1 to 100,000 mg/l and above. Antigen excess, nonlinearity, interferences, matrix effect, and aggregation are interference effects described for serum FLC measured by Freelite and N Latex immunoassays.

16 Manufacturer s recommended sample dilution procedures for serum FLC measurement using Binding Site Freelite TM

17 What sample dilutions should be used? Laboratories should adopt a consistent approach to sample dilution procedures for FLC measurement and follow either the manufacturers recommendations (or use a validated in-house dilution procedure) to determine the linear ranges where FLC reactivity for most monoclonal FLC parallels the polyclonal calibrator response

18 Antigen excess Although antigen excess is relatively uncommon ( %) it can be easily missed if the analyser does not do automated antigen excess testing (and sometimes even if it does) Higher manual dilutions still need to be performed when antigen excess is suspected ie. a high clinical suspicion of a monoclonal light chain disease or a paraprotein is detected in serum or urine on IFE.

19 Example of excess/non linearity

20 When to check? The Binding Site FLC kit insert recommends further testing if: An initial sample gives either a FLC concentration or a κ/λ ratio outside of the quoted reference range; Sample is from a patient who has previously demonstrated antigen excess; or Sample gives a FLC concentration or ratio that does not agree with other clinical or laboratory findings.

21 Binding Site FLC dilutions

22 Recommendations Laboratories follow the manufacturers dilution procedures for FLC measurement. Investigate further if required.: Freelite FLC assays: If the second result is greater than [4x initial result] it is recommended to report the final result unless the second result is less than [4x initial result] in which case the initial result should be reported. N Latex FLC assays: For problematic samples further sample dilutions may be helpful for interpreting results. Dilute samples serially until two or more dilutions give FLC values within ± 20% deviation from the mean of the values.

23 Recommendations Use the same FLC assay and the same platform when monitoring disease response. If there is a change of assay or platform, rebaseline FLC on one or two samples when monitoring disease progress. Use the same dilution (if possible) for subsequent samples The WP recommends that the assay type (Freelite or N Latex) be mentioned in the report.

24 4) What is the optimal reporting of results? Reporting of serum FLC to one or more decimal places would convey an unrealistic impression of their precision. Reporting in whole numbers from 0 to 100 mg/l is recommended. Values >100 mg/l can be reported to two 2 significant figures after rounding. However, individual κ or λ FLC raw values from the analyser should be kept at 1 decimal place before κ/λ ratio is calculated.

25 Interpretive commenting Haematologists, General Practitioners, renal and cardiac specialists are ordering FLC and protein electrophoresis for PCDs. Comments need to be tailored for specific groups and some laboratories will have a more educative approach to commenting where the comments do not only pertain to SFLC testing but also to protein electrophoresis, IFE and immunoglobulin levels

26 General recommendations Consistency in reporting is essential This is not an easy assay and results need to be examined for discrepancies between collections as well as with other assays Both assays have merits and flaws It s only the beginning for SFLC.