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Scheme Description

Scheme 4A2 is for laboratories that undertake DNA HLA based typing at the 2nd or 3rd field level (i.e. high resolution typing or next generation sequence typing).

Participants can choose at registration whether to be assessed at the 2nd field or 3rd field level.

Participants are requested to report Scheme 4A2 results using the correct nomenclature. The IMGT/HLA database update (https://www.ebi.ac.uk/ipd/imgt/hla/) from the April two years prior to start of the Scheme will be taken as the ‘reference’ allele baseline for the entire year and participants will be expected to report their findings in accordance with this report as a minimum (e.g. the IMGT/HLA release version 3.40 (2020-04) will be used throughout 2022-23).

Participants who register for assessment of results at the 3rd field resolution must be able to distinguish all nucleotide substitutions within the coding sequence for 3rd field assessment.  All ambiguities must be resolved.

 

Purpose

To assess participants’ ability to correctly determine HLA alleles to the 2nd or 3rd field level.

 

Samples

A total of ten blood samples will be sent each year as two distributions of five blood samples.

 

Reporting

Participants may register for any of the following: HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1 and DPB1, for 2nd or 3rd field assessment.

Participants must only use the reporting forms provided within the UK NEQAS for H&I Participant’s Portal and return results within 4 weeks.

 


 

HLA Typing to 2nd Field Resolution:

 

For typing to the 2nd field, HLA alleles should be assigned on the basis of differences in exons 2 and 3 for class I and exon 2 for class II, as a minimum requirement. Alleles with identical sequences over these exons which have not been excluded due to testing other areas of the gene should be reported.

Participants registered for 2nd field assessment should define all ambiguities that encompass a null allele wherever the polymorphism is located (see statement on EFI Standard below).

HLA alleles must be identified at the level of resolution which defines the first and second fields by at least resolving all ambiguities resulting from polymorphisms located within exons 2 and 3 for HLA class I loci, and exon 2 for HLA class II loci, as a minimum. Alleles with identical sequences over these exons which have not been excluded due to testing other areas of the gene should be reported in the same group e.g. allele x / allele y, where “/” means “or”, e.g. DRB1*01:01/01:02/01:04 means DRB1*01:01 or DRB1*01:02 or DRB1*01:04. Ambiguous typing combinations (heterozygous positions identified by sequencing, e.g. cis/trans ambiguities) that have not been excluded should also be reported.

Information on identical allele sequences and ambiguous allele combinations over exons 2 and 3 for HLA class I loci, and exon 2 for HLA class II loci are taken from the latest IMGT/HLA ambiguous allele combinations file release (https://www.ebi.ac.uk/ipd/imgt/hla/ambig.html).

Please report alleles fully, e.g. DRB4*01:02-01:03 and not as DRB4*01:02-03 which may be interpreted as DRB4*01:02-01:03 or as DRB4*01:02-03:01.

 

The European Federation for Immunogenetics (EFI) ’STANDARDS FOR HISTOCOMPATIBILITY & IMMUNOGENETICS TESTING version 7.0, standards state:

F1.1.4.1          HLA alleles must be identified at the level of resolution which defines the first and second fields according to WHO nomenclature by at least resolving all ambiguities:

F.1.1.4.1.1   resulting from polymorphisms located within exons 2 and 3 for HLA class I loci, and exon 2 for HLA class II loci.

F.1.1.4.1.2   that encompass a null allele, wherever the polymorphism is located, unless it can be demonstrated that an expressed antigen is present on the cells.

 

It is expected that EFI accredited laboratories that register for HLA typing to the 2nd field will have developed a strategy to comply with this Standard for the recognition of null alleles.

 

Assessment

Participating laboratories will be assessed on the loci they designate at registration.

The consensus full HLA genotype is determined by at least 75% of laboratories agreeing each allele. A “blank” forms part of the assessment if at least 75% of laboratories report a single allele at a locus. A reference result will be used for assessment for samples failing to reach the 75% consensus level.

Participants will only be assessed on those alleles that appear in the IMGT/HLA database update from the April two years prior to start of the Scheme (e.g. the IMGT/HLA release version 3.32.0 (2018-04) will be used throughout 2021-22).

For typing to the 2nd field, reports containing groups of alleles are considered acceptable if all alleles within the group have identical sequences over exons 2 and 3 for class I and exon 2 for class II. Alleles that differ within these exons are considered ‘unacceptable’.

Reports containing ambiguous allele typing combinations (e.g. cis/trans ambiguities) defined over exons 2 and 3 for class I and exon 2 for class II are considered acceptable.

For alleles reported above the 2nd field resolution, (e.g. 3rd or 4th field) the results up to the 2nd field will be used for assessment, unless registered for 3rd field assessment (see below).  Participant’s that report at 3rd or 4th field resolution and want to compare their results with other centres will need to do so manually using the results summary table.

 


 

HLA Typing to 3rd Field Resolution:

 

For typing to the 3rd field, HLA alleles should be assigned on the basis of any differences in the coding region as a minimum requirement, all ambiguities should be resolved before reporting. Alleles that differ in non-coding regions (i.e. 4th field results) may be reported for comparison with other laboratories, but the 4th field will not be assessed.

 

Assessment

Participating laboratories will be assessed on the loci they designate at registration.

The consensus full HLA genotype is determined by at least 75% of laboratories agreeing each allele. A “blank” forms part of the assessment if at least 75% of laboratories report a single allele at a locus. A reference result will be used for assessment for samples failing to reach the 75% consensus level.

Participants will only be assessed on those alleles that appear in the IMGT/HLA database update from the April two years prior to start of the Scheme (e.g. the IMGT/HLA release version 3.32.0 (2018-04) will be used throughout 2021-22).

For typing to the 3rd field, participants must report to 3rd field resolution with no ambiguities permitted. Participants must sequence all the relevant exons to produce an unambiguous 3rd field HLA type.  For example, DRB1*07:01:01/07:79 would be deemed ‘unacceptable’.  Likewise, DQB1*03:02:01/03:02:26 would also be deemed ‘unacceptable’ as ambiguities in the exon 4 have not be resolved in both cases.

For alleles reported above the 3nd field resolution, (e.g. 4th field) the results up to the 3nd field will be used for assessment.  Participant’s that report at 4th field resolution and want to compare their results with other centres will need to do so manually using the results summary table.


Assessment Procedure

Each full HLA genotype in agreement with the consensus/reference type – Acceptable

Each full HLA genotype not in agreement with the consensus/reference type – Unacceptable

Each sample/registered loci not reported – with valid reason – Not Assessed

Each sample/registered loci not reported / late submission of results – Unacceptable

 

Satisfactory Performance

Satisfactory performance is obtaining nine or more full HLA genotypes in agreement with the consensus/reference genotypes in a year.

 

Laboratories with unsatisfactory performance will receive written notification of their status and will be expected to reply to UK NEQAS for H&I detailing their corrective actions. For UK laboratories, unsatisfactory performance will be reported to UK NQAAP for Immunology. For UK laboratories, failure to reply or replies deemed unsatisfactory are likely to be further actioned by UK NQAAP for Immunology.

                                                                                                                  

Information/Analysis Provided to Participants

  • Anonymised summary table of all participant results, comments and methodology. Including sample assessment result (acceptable/unacceptable classification) for each participant.
  • End of year summary of participant performance.
  • Information detailing i) the sequences that are identical over exons 2 and 3 for class I and exon 2 for class II and ii) ambiguous typing combinations (heterozygous positions identified by sequencing) defined over these exons.