WHO/BS/2014.2246 Value Assignment of the proposed WHO 1st IS ADAMTS13, Plasma (12/252)

Overview

A collaborative study involving 32 laboratories from 14 countries has been undertaken to assign values for ADAMTS13 function and antigen to the proposed WHO 1st International Standard (IS) ADAMTS13 plasma (coded 12/252). Value assignment was based on assays relative to local pooled normal plasma preparations arbitrarily assigned 1.0 unit per ml. Most laboratories used either a Fluorescence Resonance Energy Transfer (FRET) assay (n=18) or an activity ELISA (n=9) to measure ADAMTS13 function and all laboratories used ELISA for antigen measurement. The candidate WHO IS was included in the study as coded duplicates (samples A & B). Comparison of the candidate WHO IS (samples A & B) with the local normal pools was associated with a high degree of validity in terms of parallelism of the dose-response relationships with only 9/117 function assays and 8/58 antigen assays excluded because of non-parallelism. Estimates of ADAMTS13 function for samples A & B were not significantly different and there was also no significant difference between the results by FRET and the activity ELISA. Combination of all results for function gave an overall mean of 0.91 units/ml for the candidate WHO IS with low inter-laboratory variability (GCV) of 12.4%. For estimates of ADAMTS13 antigen in the coded duplicates of the candidate WHO IS (samples A & B) there was also no significant difference and combination of all results gave an overall mean of 0.92 units/ml with inter-laboratory variability (GCV) of 16.3%. Two samples (C & D) from a patient with acquired ADAMTS13 deficiency due to an inhibitory autoantibody were also included in the study. The level of ADAMTS13 in sample C was below the limit of detection for assays of function in many cases (21/32) and calculated estimates were only possible in 11 laboratories. However, 31/32 results were consistent with a severe deficiency below 0.1 units/ml. Patient sample D contained a higher level of ADAMTS13 than sample C and only 8/32 data sets from the function assays were not amenable to quantification. The overall mean estimate for function in sample D was 0.15 units/ml with 23/24 laboratories agreeing levels below 0.3 units/ml. Ratios of function to antigen for samples C and D were greatly reduced at 0.11 and 0.24 respectively compared to normal plasma (0.99). This finding together with the large inter-laboratory variability of estimates for the patient samples is most probably related to the presence of circulating antibody-ADAMTS13 complexes. The availability of a common reference material (proposed WHO IS) could help to identify the methodological issues responsible for this variability. Assays of recombinant ADAMTS13 (sample E) indicated valid comparison of dose-response relationships with normal plasma (proposed WHO IS) but large interlaboratory of estimates for both function and antigen. This could indicate that the proposed WHO IS is not suitable for the assay of recombinant ADAMTS13.