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Scheme 1A HLA Phenotyping Scheme 1B HLA-B27 testing Deborah Sage

Scheme 1A HLA Phenotyping Scheme 1B HLA-B27 testing Deborah Sage. United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013. SCHEME 1A: HLA PHENOTYPING.

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Scheme 1A HLA Phenotyping Scheme 1B HLA-B27 testing Deborah Sage

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  1. Scheme 1A HLA Phenotyping Scheme 1B HLA-B27 testing Deborah Sage United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  2. SCHEME 1A: HLA PHENOTYPING Purpose: To assess ability to use serological and supplementary methods to correctly identify HLA specificities • 2 random donor samples sent x5 (5 cycles of 2 samples) • Register for HLA-A, B, C, DRB1, DQB1 typing or any combination • Assessment on loci tested using serological techniques ONLY • Stable testing profile, new overseas participants through year

  3. ASSESSMENT 2013 Consensus complete HLA phenotype determined by at least 75% of laboratories Assessment Procedure Each complete HLA type in agreement with consensus Acceptable Each complete HLA type not in agreement with consensus Unacceptable Satisfactory Performance 9 or more complete HLA types in agreement with consensus in a calendar year Labs with unsatisfactory performance will receive written notification of their status and be expected to detail their corrective actions For UK labs unsatisfactory performance is reported to UK NQAAP for Immunology

  4. Methods: Typing trays used

  5. Methods: Cell Preparation

  6. HLA Types in 2013

  7. Incorrect Assignments 2013

  8. Performance 2013 • 8 incorrect assignments (1 UK plus 7 non-UK labs) • 9 incorrect assignments in 2012 • 6/8 involved HLA B misassignment or failure to split B14 • 2/8 missed Cw14 • All laboratories achieved satisfactory performance • First year where there is no unsatisfactory performance

  9. Overall Accuracy Rates 2013 Antigen Error Assignments AI % 2012 HLA-A 0 512 100 99.5 HLA-B 6 512 98.8 99.1 HLA-C 2 148 98.6 99.3 HLA-DR 0 316 100 100 HLA-DQ 0 316 100 99.3 HLA-A, B, DR 6 1340 99.7 99.5 UK HLA-A 0 200 100 100 HLA-B 0 200 100 99.5 HLA-C 1 56 98.2 100 HLA-DR 0 180 100 100 HLA-DQ 0 180 100 99.4 HLA-A, B, DR 0 580 100 99.8

  10. Scheme 1A: Accuracy rates in UK 2000-2013

  11. SCHEME 1B: HLA-B27 TESTING Purpose: To assess ability to correctly determine HLA-B27/2808/B*27 status • 2 random donor samples sent x 5 • Report “HLA-B27” POSITIVE or “HLA-B27” NEGATIVE • 93-96 participants • 47/48 UK laboratories • B27 status determined by at least 75% agreement on presence or absence of HLA-B27

  12. ASSESSMENT Assessment Procedure Result in agreement with consensus HLA-B27 status Acceptable Result not in agreement with consensus HLA-B27 status Unacceptable Satisfactory Performance • Making 10 sample reports in agreement with consensus “HLA-B27” status in a calendar year • Labs with unsatisfactory performance will receive written notification of their status and be expected to detail their corrective actions • For UK labs unsatisfactory performance is reported to UK NQAAP for Immunology

  13. Techniques used

  14. Techniques used (all participants)

  15. Methods: Monoclonals

  16. Assignments out of Consensus *equivocal results not assessed

  17. Performance in 2013 • 4 samples HLA-B27 positive (1B05,08,09,10) • 4 unacceptable performers (1 UK lab and 3 non-UK labs) • 6 unacceptable performers in 2012 • 87 laboratories achieved satisfactory performance

  18. HLA-B27 testing Overall Accuracy rates 2000-2013

  19. Scheme 4A1 – DNA HLA Typing at 1st Field Resolution Dr Leigh Keen H&I - Filton United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  20. 4A1 – SCHEME SUMMARY Purpose To assess participants ability to correctly determine HLA alleles at the 1st field level. Samples Ten blood samples despatched each year in two batches of five. Reporting Participants can register for first field assessment of HLA-A, B, C, DRB1, DQB1 and DQA1. DRB3, 4 & 5 can be registered for 1st Field analysis or “presence of”. Results must be returned within 4 weeks of despatch.

  21. SCHEME 4A1 - ASSESSMENT Participating laboratories are only assessed on the loci that they are registered for. Consensus full genotype determined by at least 75% of laboratories agreeing each allele. Alleles failing to reach 75% consensus will not be assessed. Acceptable Result Each full HLA genotype in agreement with the consensus 1st field type. Unacceptable Result Each full HLA genotype not in agreement with the consensus 1st field type.

  22. 4A1 SCHEME - PERFORMANCE Satisfactory Performance Obtaining nine or more full HLA genotypes in agreement with concensus in a calendar year.

  23. 2013 – 4A1 Performance Summary (1) Poor Performers

  24. 2013 – 4A1 Performance Summary (2)

  25. SUMMARY Approx. 80% of laboratories submitting results scored 10/10. Laboratories submitting incorrect results used a mixture of SSP and SSOP approaches. The number of transcription errors was extremely low.

  26. Scheme 4A2: DNA Typing to 2nd Field Resolution Fotini Partheniou United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  27. Objective • To assess participant’s ability to correctly determine HLA alleles to the 2nd field level (formerly known as high resolution or 4-digit typing)

  28. Performance • 58 participating laboratories • 3 labs- no returns • 1 lab NT samples 01-05 & 06-10 • 2 labs NT samples 01-05 • 5 labs- Unsatisfactory performance

  29. Overall lab performance

  30. 2012 v 2013 • Overall there were: • more Class I Misassignments in 2013 than 2012 (7 v 4) • Less Class II Misassignments in 2013 than 2012 (14 v 16)

  31. Changes for 2014 • Labs can register for DPA1 assessment!

  32. Null Allele Questionnaire Results 2013 United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  33. Introduction The EFI Standard (D1.4) states: 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: resulting from polymorphisms located within exons 2 and 3 for HLA class I loci, and exon 2 for HLA class II loci. that encompass a null allele, wherever the polymorphism is located, unless it can be demonstrated that an expressed antigen is present on the cells. Questionnaire sent to participants of Scheme 4A2 to determine if they comply with this (64% return rate)

  34. Question 1: Do you comply with EFI Standard D1.4 on defining null alleles? • 90% of responses ‘Yes’

  35. Question 2: If Yes, how do you comply with the Standard on defining null alleles? • 12 (35 %) use Serology (+/- Molecular)

  36. Question 3: Should 4A2 participants be classed as unacceptable performers if they report a null allele? 18 Yes (47%) 16 No (42%) 4 No answer

  37. Summary • 90% of participants comply with EFI standard - for clinical samples • 35% of participants use serology to exclude null alleles • NEQAS for H&I are unable to supply enough fresh material to allow serological typing for scheme 4A2 • No clear feeling that participants should be unacceptable for reporting nulls • No changes to scoring for 2014 • Will continue to provide participants who report a null allele with a letter

  38. Scheme 4B-ABO grouping Jennifer Pepperall United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  39. Scheme 4B – ABO grouping To assess participants’ ability to correctly determine ABO blood groups using DNA-based methodology Uses scheme 4A1 samples 7 participants in 2013 All using PCR-SSP – 4 commercial kits

  40. Scheme 4B – ABO grouping • 1/7 has issue with 2 samples • Comment – With samples 01/2013 and 03/2013 we found a very doubtful result with our specific primers for the presence of blood group genotype O2. In this case we stated the doubtful result as negative, matching the blood group A1/Ax instead. As a phenotype we found blood group O in these samples. Routinely we would perform a absorption and dilution test to exclude the blood group Ax and ask for a new fresh sample to confirm our DNA results. • 1 of the 4 labs using commercial kit – however was a different kit from the other 3

  41. Scheme 5A – HFE Typing Jennifer Pepperall United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  42. Scheme 5A – HFE Typing To assess participants’ ability to correctly determine HFE mutations Must report on codon 63 and 282- may also report codon 65 2013 – 57 participants; 47% (n=27) reported codon 65 results The most common technique used was RT-PCR

  43. Scheme 5A – HFE Typing • No errors • Numbers slightly increased from last year with 1 lab reporting results for codon 168 • Just over 30% labs performing HFE testing participate in scheme 5B

  44. Scheme 5B - Interpretative: HFE genotype and hereditary haemochromatosis Alan Balfe United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2013

  45. PURPOSE of Scheme 5B To assess participants’ ability to make an accurate, clear and concise clinical report The report should be appropriate for the range of clinical staff involved in a patient’s care and treatment Report based on given HFE genotype plus other relevant clinical information supplied

  46. New Assessor in 2013 Team of three, since 2010: Gavin Willis (Norwich) Alan Balfe (Dublin) Dairena Gaffney (Glasgow) During 2013, Dairena retired Carol Hardy (Birmingham) appointed

  47. The Format of Scheme 5B Twice a year, 2 clinical scenarios HFE genotype provided, together with various pieces of clinical information; fictitious cases based on real or typical experience Participants are required to return their reports within 4 weeks Reports must be identical in format to that used for routine clinical reporting in participants’ laboratories

  48. Assessment and Scoring Procedure For each scenario, interpretative criteria expected to be covered by the report were identified and agreed by the expert assessors Criteria were classed under five general headings (describe the result, advise actions to be taken, state disease risk for patient, advise on risk to family members and on testing, and an open heading of common sense/other error)

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