Scheme 1B - 2017-07-08

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UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING HLA-B27 TYPING RESULTS OF SAMPLES 1B07&08/2017 DESPATCHED ON 15th August 2017 Summary of Results Total tested (excl. Positive Negative NT/Equivocal % Positive % Negative

121 120 1 6 99.2% 0.8%

Consensus Lab No 1 3 4 7 8 9 10 11 12 14 15 17 19 20 21

Donor HLA type Donor HLA type

117 117 0 10 100.0% 0.0%

1B07/2017 1B08/2017

HLA-A2, A2; B27, B57; Cw1, Cw6; DR103, DR15, DQ5, DQ6 HLA-A1, A2; B8, B27; Cw1, Cw7; DR103, DR17, DQ2, DQ7

Positive

Positive

Result 1B07/2017 Positive

Result 1B08/2017 Positive

Date received 16-Aug

Date tested 23-Aug

Molecular

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

Positive Positive Positive Equivocal Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

16-Aug 16-Aug 17-Aug 16-Aug 16-Aug 16-Aug 15-Aug 17-Aug 16-Aug 16-Aug 16-Aug 18-Aug 16-Aug 16-Aug 16-Aug

16-Aug 29-Aug 18-Aug 17-Aug 18-Aug 16-Aug 23-Aug 23-Aug 21-Aug 18-Aug 21-Aug 19-Aug 18-Aug 16-Aug 17-Aug

Serological Molecular Serological Serological Molecular Serological Molcecular Molcecular Molcecular Molcecular Serogical & Mol Molecular Serological Serological Molecular

without hybridization probes

Fluorescence

Positive Positive Positive Positive NT Positive Positive

Positive Positive Positive Positive NT Positive Positive

15-Aug 16-Aug 16-Aug 16-Aug 17-Aug 16-Aug 16-Aug

24-Aug 24-Aug 22-Aug 17-Aug

Molecular Molecular Molecular Molecular

Own design Commerical kit

Gel Gel

21-Aug 17-Aug

Molecular Molcecular

Commerical kit Commerical kit

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

16-Aug 16-Aug 16-Aug 16-Aug 17-Aug 17-Aug 16-Aug 16-Aug 16-Aug 17-Aug 16-Aug

17-Aug 16-Aug 21-Aug 16-Aug 23-Aug 21-Aug 23-Aug 22-Aug 18-Aug 18-Aug 17-Aug

Serological Serological Molcecular Molecular Molecular Molecular Serological Molcecular Molcecular Molecular Serological

Positive Positive Positive Positive Positive Positive Positive Positive Positive NT Positive Equivocal

Equivocal Positive Positive Positive Positive Positive Positive Positive Equivocal NT Positive Equivocal

17-Aug 17-Aug 16-Aug 17-Aug 15-Aug 16-Aug 21-Aug 16-Aug 16-Aug 16-Aug 18-Aug 16-Aug

17-Aug 22-Aug 18-Aug 22-Aug 16-Aug 23-Aug 21-Aug 25-Aug 16-Aug 21-Aug 23-Aug 17-Aug

Serological Molecular Molecular Molecular Serological Molecular Molecular Molecular Serological Serological Molecular Serological

Equivocal

Equivocal

16-Aug

18-Aug

Serological

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive NT Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Positive

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive NT Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Equivocal

16-Aug 17-Aug 16-Aug

18-Aug 17-Aug 16-Aug 18-Aug 18-Aug 31-Aug 22-Aug 18-Aug 16-Aug 18-Aug 18-Aug 23-Aug 22-Aug 22-Aug 16-Aug 18-Jul 22-Aug 22-Aug 22-Aug 28-Aug 18-Aug 24-Aug 25-Aug 17-Aug 18-Aug 15-Aug

Molecular Serological Serological Molecular Molecular Serological Molecular Molecular Serological Serological Molcecular Molecular Molcecular Molecular Serological & Mol Serological Molecular Molecular Molecular Molecular Molecular Serological & Mol Molecular Serological & Mol Serological Serological

Positive Positive Equivocal Positive Positive Positive Positive

Positive Positive Equivocal Positive Positive Positive Positive

Method Primers and Probes PRIMERS MADE TO ORDER FROM ALTA

PCR-SSP Kit Manufacturer

Detection system Gel

High Resolution Typing HLA-B*27 alleles 1B07/2017 1B08/2017

Results for 2nd sample were within indeterminant zone (21.4) Commerical kit

One Lambda

Own design Own design Commerical kit Own design Own design

Fluorescence

Gel Gel Olerup

Gel Gel Gel Method: Genotyping of HLA0B27 by Real-Time PCR without Hybridization Probes, Controls: previous NEQAS scheme samples.

22 23 24 25 26 27 28 29 31 32 34 35 37 39 40 41 42 45

Olerup

Own design

Gel Not tested due to incorrect storage One Lambda Biofortuna

Gel Gel, BAG Healthcare reverse dot blot

B*27:03/27:05

B*27:03/27:05

Late submission due to staff shortages Own design Own design Own design

Gel Gel Gel

Own design

Gel

Own design

Gel 1B08 - Although reported as positive, as results fall close to decision line sample would be referred for confirmation by genetic screening

46 47 48 54 55 57 58 62 66 67 68 72 76

Commerical kit

Invitrogen

Gel

Commerical kit Commerical kit Own design

Innotrain Olerup

Gel Gel Fluorescence

Commerical kit

Tib MOLBIOL

Fluorescence

NT due to discrepanc with current antibodies in use Both assays gave equivocal results, normal practice is to refer there for confirmation by PCR Both samples performed identically giving insufficient definition between a positive or negative result. In this situation, we would refer the sample to NBTS for molecular confirmation.

77 78 83 84 85 86 90 96 101 103 104 106 107 108 109 110 111 112 113 115 126 127 128 129 130 133 137 142 153 154 156 174 176 185 198 209 219 223 224 225 232 234 236 245 252 255 256 257 269 273 276 278 279 288 292 295 300 305 306 307 317 319 324 325 326 327 329 334 339 340 348 349 353

16-Aug 18-Aug 17-Aug 16-Aug 16-Aug 18-Aug 17-Aug 22-Aug 18-Aug 16-Aug 16-Aug 17-Aug 17-Aug 17-Aug 16-Aug 22-Aug 16-Aug 18-Aug 23-Aug 16-Aug 17-Aug 15-Aug

Commerical kit

Olerup

Gel

Commerical kit

Invitrogen

Gel

Commerical kit

Olerup

Positive in exons 2 and 3

17-Aug 24-Aug 20-Aug 26-Aug 21-Aug 17-Aug 17-Aug 17-Aug 25-Aug 21-Aug 24-Aug 25-Aug 23-Aug 29-Aug 23-Aug 21-Aug

Serological Molecular Molecular Molecular Serological Molecular Serological Molecular

16-Aug 16-Aug 17-Aug 16-Aug 17-Aug 16-Aug

28-Aug 18-Aug 23-Aug 23-Aug 17-Aug 21-Aug 28-Aug 19-Aug 17-Aug 18-Aug 17-Aug 23-Aug 29-Aug 24-Aug 24-Aug 22-Aug 27-Aug 17-Aug 18-Aug

Molecular Molecular Molecular Molecular Molecular Serological Molecular Molecular Molecular Serological Molecular Molecular Molecular Molecular Molecular Serological Serological Serological Serological Molecular Molecular Molecular Molecular Molecular Molecular Serological Molecular

21-Aug 16-Aug 17-Aug 16-Aug 16-Aug 18-Aug 16-Aug 17-Aug 17-Aug

22-Aug 28-Aug 17-Aug 21-Aug 17-Aug 18-Aug 18-Aug 18-Aug 17-Aug

Molecular Molecular Serological Molecular Molecular Serological Serological Molecular Molecular

Gel

Commerical kit

Gel qPCR assay used with IH designed primers

Commerical kit

One Lambda

Fluorescence

Commerical kit Commerical kit

Olerup SSP Olerup

Gel Gel

Commerical kit

Olerup

Gel

Commerical kit

Olerup

Gel

Commerical kit Commerical kit Commerical kit Commerical kit Own design

Invitrogen BIOFORTUNA Olerup Olerup SSP

Gel Gel Gel Gel Fluorescence

Commerical kit

Olerup

Gel

Commerical kit Commerical kit Commerical kit

Olerup Gel Euroimmun Euroarray Fluorescence Olerup Gel

16-Aug 15-Aug 16-Aug 18-Aug 17-Aug 16-Aug

21-Aug 23-Aug 29-Aug 21-Aug 18-Aug 22-Aug 22-Aug

Molecular Molecular Serological Molecular Serological Serological Molecular

Fluorescence

Positive in exons 2 and 3

Commerical kit

Olerup

Gel

Commerical kit

Olerup

TapeStation

Commerical kit Commerical kit

BIONOBIS HLA B27 AnDiaTec

Gel Fluorescence

Own design

Positive in exons 2 and 3

Fluorescence

The difference between the sample result and cut off is not sufficient to exclude a false positive due to cross reactivity. Results needs confirming by molecular biology.

357 359 362 365 368 370 372 373

Positive in exons 2 and 3

Gel

Commerical kit

Own design 16-Aug 17-Aug 17-Aug 23-Aug 21-Aug 16-Aug 16-Aug 16-Aug 23-Aug 16-Aug 17-Aug 22-Aug 18-Aug 22-Aug 22-Aug 17-Aug 21-Aug 16-Aug 16-Aug 16-Aug 22-Aug 16-Aug 15-Aug 22-Aug 16-Aug 17-Aug 18-Aug 16-Aug 21-Aug

Comments

Commerical kit Own design

Appolon Bioteck

Fluorescence

Late submission due to internet problem

Fluorescence

IH HLA-B27

UK NEQAS for H&I is operated by Velindre NHS Trust, a UKAS proficiency testing provider No 8351. © Confidential report; no data may be published without permission from UK NEQAS for H&I

NEQ-115 Issue 2 P. 1 of 2 Effective Date 10/10/16

UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING

HLA-B27 TYPING RESULTS OF SAMPLES 1B07&08/2017 DESPATCHED ON 15th August 2017 Lab No

Source of pos/neg control cells used (if applicable)

10 IH 17 20 Control Cells: B7 Homozygous Cell, B27/B7 Positive cell, B27/B7 Negative cell 21 27 Positive control: Crisp HLA-B27 positive control cells from Phoenix Flow Systems Negative control: ImmunoTrol cells from Beckman Coulter 31 CRISP Cells 68 Positive Controls -Crisp HLA-B27 Supplier- Phoenix Flow Systems 76 HLA-B27 pos CRISP cells supplied by Enzo Life Sciences. 110 N/A 111 128 133 In house 137 324 334 365 Internal control in each Duraclone B27 tube (Bc) (beads)

Monoclonal 1 Designation Specificity HLA-B27 145.2 HLA-B27 B27 FITC B27 B27-FITC, B27 and B7, Becton Dickinson

Manufacturer BD One Lambda

Designation CD3 SK-7 CD3 PE B27-FITC, B27, One Lambda

Monoclonal 2 Specificity CD3 CD3

Manufacturer BD BD

Monoclonal 3 Designation Specificity HLA-B27 FD705 9E1010 HLA-B27 N/A

HLA-B27-FITC One Lambda Beckman Coulter Duraclone

HLA-B27-FITC / HLA-B7-PE Beckman Coulter

IgG2a-FITC, IgG1-PE, Isotype Control, Beckman Coulter HLA B27-FITC/HLA B7-PE, Beckman Coulter

IOTest HLA-B27-FITC HLA-B7-PE, HLA-B27 and HLA-B7, Beckman Coulter HLA B27-FITC, One Lambda

DuraClone B27, HLA-B27, Beckman Coulter

FD705

ABCm3

BB7.1

HLA-B27

Anti HLA B27 FITC FD705-9E1E10 Anti HLA B27 FITC Clone GS145.2 IOTEST HLA-B27-FITC, HLA-B27, Beckman coulter Anti CD27 CD27 HLA-ABCm3/BB71 HLA-B-27HLA-B7 Anti HLA B27 FITC Anti HLA B27 FITC HLA-B27 - FITC HLA-B27 HLA-B27 (clone FD 705) PE

Beckman Coulter One Lambda BD Biosciences BC Immunotech Beckman Coulter Beckman Coulter Beckman Coulter

Manufacturer One Lambda

HLA-B27

CD3 PE Clone SK7 IOTEST HLA-B7-PE, HLA-B7, Beckman coulter Anti CD7 CD7 MPC-11 HLA-B27 HLA-B7-PE HLA-B7-PE HLA-B27 - PE HLA-B27 HLA-B27 (clone HLA-ABC-m3) PE

Beckman Coulter

HLA-B7

Beckman Coulter

BD Biosciences BC One Lambda Beckman Coulter Beckman Coulter

CD45 PerCP-Cy5.5, Lymphocytes Gating, Becton Dickinson Anti CD3 T cells BC

CD3 (clone UCHT1)

PE-Dyomics 649

Beckman Coulter

NEQ-115 Issue 2 UK NEQAS for H&I is operated by Velindre NHS Trust, a UKAS proficiency testing provider No 8351. © Confidential report; no data may be published without permission from UK NEQAS for H&I

P. 2 of 2 Effective Date 10/10/16