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2014 Participant Meeting

Participate in the UK NEQAS for H&I 2014 Annual Meeting for Histocompatibility and Immunogenetics. Assess your ability in HLA phenotyping and HLA-B27 testing. Understand the assessment procedures, acceptable performance criteria, methods used, and past performance data to improve accuracy rates.

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2014 Participant Meeting

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  1. 2014 Participant Meeting United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  2. UK NEQAS for H&I Scheme Director: Chris Darke Scheme Manager: Deborah Pritchard Deputy Scheme Manager: Melanie Bartley UK NEQAS Officers: Geraint Clarke & Luke Gardner

  3. 2014 Steering Committee • Deb Sage (Chairman) • Jeanette Ayers • Alan Balfe (Lead Expert Advisor Scheme 5B) • Patrick Flynn • Carol Hardy (Expert Advisor Scheme 5B) • Mark Hathaway • Leigh Keen • Helen Lee (BSHI Rep to UK NQAAP) • Edwin Massey (Clinical Representative) • Fotini Partheniou • Jennifer Pepperall • Ruhena Sergeant • Gavin Wills (Expert Advisor Scheme 5B)

  4. Scheme Assessment • All Schemes (except 5B – Interpretative HFE) are assessed on a consensus basis • All use 75% consensus level i.e. 75% of reports must agree on a result for it to be assessed • Scheme 3 also uses a 95% consensus for antibody absence

  5. Scheme 1A HLA Phenotyping United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  6. 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; increase in overseas participants this year

  7. ASSESSMENT 2014 • 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

  8. Methods: Typing trays used

  9. Methods: Cell Preparation

  10. HLA Types in 2014

  11. 2014 Incorrect Assignments

  12. 2014 Incorrect Assignments

  13. 1A Performance 2014 • 34 incorrect specificities reported by 28 labs (3 UK & Ireland) • 18 reports of broad, not split specificity (e.g. DQ1 not DQ5) • 6 reports of wrong specificity (e.g. DR1 not DR103) • 4 reports of incorrect split specificity (e.g. B57 not B58) • 3 reports using incorrect nomenclature (e.g. DQ03:02) • 3 reports missed specificities (e.g. reported blank) • 8 with Unacceptable Performance - 7/8 of these were new to this scheme for 2014

  14. Scheme 1A: Accuracy rates in UK 2000-2014

  15. Scheme 1B HLA-B27 Testing United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  16. 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 • 103-107 participants • 49-51 UK laboratories • B27 status determined by at least 75% agreement on presence or absence of HLA-B27

  17. 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

  18. Techniques used

  19. Methods: Monoclonals

  20. 2014 Incorrect Assignments

  21. Performance 2014 • 5 HLA-B27 positive samples distributed • 4 Unacceptable Performance (2 UK & Ireland)

  22. HLA-B27 testing Overall Accuracy rates 2000-2014

  23. Scheme 2A Cytotoxic Crossmatching United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  24. Scheme Summary Purpose To assess participants’ ability to correctly determine cell/serum cytotoxic crossmatch status Participants in 2014 76 for PBL/T cells 60 for B cells Changes in 2014 split O/S into lymphocytes and whole blood

  25. Methodology - PBL/T cells Incubation times Pre C’ 20 to 60 Post C’ 30 to 120 14 different combinations 30+60; 60+60; 45+90 Visualisation of dead cells PI, EB/AO (UK), Flouroquench (O/S), Stain Fix Beaded cells used by 50% 58 no results returned Technical (3), viability (41), no reason supplied (10), staff shortages (4)

  26. Methodology – B cells Incubation times Pre C’ 30 to 60 Post C’ 30 to 120 14 different combinations 30 + 60; 60+60; 45+90 Visualisation of dead cells PI, EB/AO (UK), Flouroquench (O/S), Stain Fix 61 no results returned Technical (5), viability (43), staff shortages (6) no reason supplied (7)

  27. Performance 2014 PBL/T Cells UK&I and RoW receive different blood samples to ensure enough cells for testing

  28. Performance 2014 B Cells UK&I and RoW receive different blood samples to ensure enough cells for testing

  29. 2014 Results NA = Not assessed, FP=False Positive

  30. Scheme 2A Performance

  31. Scheme 2A with DTT • Results with DTT were not assessed in 2014

  32. Changes for 2015 • Results with DTT will be assessed. • DTT results will be considered independently from results without DTT • RoW participants will be split - 3 different blood samples distributed for each sample e.g. 2A01/15 UK&I, 2A01/15 RoW whole blood, 2A01/15 RoW lymphocytes) • PBL/T-cell & B-cell assessment will be linked – both must be in line with the consensus results for acceptable performance

  33. Scheme 2B Crossmatching by Flow Cytometry United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  34. The purpose: To assess participant’s ability to correctly determine cell/serum flow cytometry crossmatch status Satisfactory performance: Achieve 85% of reports on all sera in agreement with the consensus findings in a calendar year

  35. UK – T Cell ResultsN = 23  Consensus 2B01 2B02 2B03 2B04 2B05 2B06 2B07 2B08 2B09 2B10 Not consensus 40 cell / sera combinations – 31 assessed Not tested 1/9 samples not assessed also not assessed B cell FCXM 

  36. UK – B Cell ResultsN = 20 Consensus Not consensus 2B01 2B02 2B03 2B04 2B05 2B06 2B07 2B08 2B09 2B10 Not tested 40 cell / sera combinations – 34 assessed

  37. Overseas – T Cell ResultsN = 47 / 48 depending on cycle        Consensus 2B01 2B02 2B03 2B04 2B05 2B06 2B07 2B08 2B09 2B10 Not consensus Not tested 7/12 samples not assessed also not assessed B cell FCXM 40 cell / sera combinations – 28 assessed 

  38. Overseas – B Cell ResultsN = 44 / 45 depending on cycle Consensus 2B01 2B02 2B03 2B04 2B05 2B06 2B07 2B08 2B09 2B10 Not consensus Not tested 40 cell / sera combinations – 25 assessed

  39. Satisfactory Performance Satisfactory Unsatisfactory

  40. Performance 2014 T Cells UK&I and RoW receive different blood samples to ensure enough cells for testing

  41. Performance 2014 B Cells UK&I and RoW receive different blood samples to ensure enough cells for testing

  42. Unsatisfactory Unsatisfactory 2014 Performance

  43. 2014 Results NA = Not assessed

  44. Scheme 2B Performance

  45. Changes for 2015 • As with Scheme 2A, T-cell & B-cell assessment will be linked – both must be in line with the consensus results for acceptable performance

  46. Scheme 3 HLA Antibody Specificity Analysis United Kingdom National External Quality Assessment Service Histocompatibility and Immunogenetics Annual Participant Meeting 2014

  47. Scheme 3 • Specificities reported by ≥ 75% labs are assessed as ‘present’ • Specificities reported by < 5% of labs are assessed as ‘absent’

  48. Scheme 3 Methods 78 Participants (25 UK&I) 57 (21) Luminex only 18 (4) Luminex & CDC 1 (0) ELISA & Luminex 1 (0) CDC & ELISA & FC & Luminex 1 (0) CDC only • Luminex Kits • 53 (68.8%) use One Lambda kits only • 12 (15.6%) Use Lifecodes Only • 9 (11.7%) Use One Lambda & Lifecodes • 4 (5.2%) information not provided

  49. Bead - Serum Volume Variation

  50. Other Test Variables Sera Treatment • 17 labs (8 UK&I) used EDTA • 2 labs (1 UK&I) used heat inactivation MFI cut off value • LABScreen cut off value (n=58) ranged from 250-2500 MFI • 52% 1000MFI

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