Evaluation of 19 years of international external proficiency testing for high-resolution HLA typing

Abstract

The international high-resolution external proficiency testing (EPT) started in 2004 with high-resolution typing of human leucocyte antigen (HLA) class I (HLA-A,B,C) and HLA class II (HLA-DRB1, DRB345, DQB1, and DPB1) alleles, since possibilities for such an EPT within Europe were limited and all existing EPTs at that time made use of the comparison of HLA typing results without a reference. This EPT was set up as a collaboration between the HLA laboratory of Leiden, providing DNA samples to the participants, and the laboratory of Maastricht, performing the high-resolution typing as the reference result and evaluating the results of all participants according to the prevailing European Federation for Immunogenetics (EFI) standards. Once a year, 12 samples were sent to the participating laboratories, and evaluation and certificates were provided at the end of that same year. During the years, the EPT was extended to low-resolution HLA class I and II typing, high-resolution typing including DQA1 and DPA1, and allelic resolution typing for HLA class I, the latter one being unique in this field. Evaluation of the high-resolution typing results of the last 19 years showed a clear increase in the number of loci tested by the participating laboratories and a clear change of method from Sanger sequencing with additional other techniques (SSO/SSP) to the nowadays widely used next-generation sequencing method. By strictly using the EFI rules for high-resolution HLA typing, the participants were made aware of the ambiguities within exons 2 and 3 for class I and exon 2 for class II and the presence of null alleles even in a two-field HLA typing. There was an impressive learning curve, resulting in >98% correctly typed samples since 2017 and a 100% fulfillment of EFI rules for all laboratories for all loci submitted in the last 2 years. Overall, this EPT meets the need of an EPT for high-resolution typing for EFI accreditation.

Keywords: HLA high-resolution typing; NGS; accreditation; histocompatibility; immunogenetic tests; quality assessment; quality control; sequencing.

FIGURE 1

Percentage of laboratories submitting results for the HLA loci during the years.

FIGURE 2

Percentage of laboratories using the methods indicated for high-resolution HLA typing during the years. Footnote: SBT+ = SBT/SSP; SBT/SSO; SBT/SSP/SSO; SBT/RLB; SBT/SSP/RLB. NGS+ = NGS/SSP; NGS/SSO; NGS/qPCR. SBT/NGS+ = SBT/NGS/SSP; SBT/NGS/SSO; SBT/NGS/SSP/SSO.

FIGURE 3

Percentage of incorrectly typed samples split up into the different types of errors for HLA-A (A), HLA-B (B), HLA-C (C), HLA-DRB1 (D), HLA-DRB3/4/5 (E), HLA-DQB1 (F), HLA-DPB1 (G), and HLA-DQA1 (H). Error 1 is genotype ambiguity (i.e., ambiguities resulting from polymorphisms located within exons 2 and 3 for HLA class I loci and exon 2 for HLA class II loci), error 2 is null alleles not excluded^A, error 3 is incorrect allele type (i.e., allele different from consensus and missing allele or extra allele, the latter one also in case of a homozygous result), and error 4 is others (i.e., one-field typing till 2006, incorrect nomenclature from 2008)^B. Footnote: ^AError 2 not excluding the null alleles concerns the following null alleles: HLA-A: *01:01:01:02N, *03:01:01:02N, *26:01:01:03N, *31:01:02:03N. HLA-B: *15:01:01:02N. HLA-C: *03:03:01:50N, *03:03:01:52N, *07:02:01:17N, *15:02:01:08N. HLA-DRB4: *01:03:01:02N, *01:03:01:13N. HLA-DPB1: *04:01:01:24N. HLA-DQB1: *03:276N. ^BError 4 incorrect nomenclature (from 2008 on) concerns the following: DRB3/4/5: *03:01 instead of 3*03:01; 4*01:03N instead of 4*01:03:01:02N. DQB1: *06:03/39, *06:04/41 instead of *06:03/41, *06:04/39. DPB1: *04:01/105:01, *04:02/126:01 instead of *04:01/126:01, *04:02/105:01; *02:01/105:01, *04:02/416:01 instead of *02:01/416:01, *04:02/105:01.

FIGURE 4

Overview of the percentage of (A) laboratories that were 100% correct for all submitted loci, (B) laboratories that fulfilled the EFI criteria for all submitted loci, and (C) submitted loci that fulfilled the EFI criteria.