Scheme 1B - 2016-03-04

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UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING HLA-B27 TYPING RESULTS OF SAMPLES 1B03&04/2016 DESPATCHED ON 08 March 2016 Summary of Results Total tested (excl. Positive Negative NT/Equivocal % Positive % Negative

117 1 116 3 0.9% 99.1%

116 114 2 4 98.3% 1.7%

Donor HLA type Donor HLA type

1B03/2016 1B04/2016

Negative

Positive

Lab No 1 3 4

Result 1B03/2016 Negative Negative Negative

Result 1B04/2016 Positive Positive Positive

Date received 10-Mar 09-Mar 09-Mar

Date tested 17-Mar 11-Mar 23-Mar

7 8 9 10 11 12 14 15 16 17 19 20

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

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

09-Mar 09-Mar 09-Mar 10-Mar 07-Mar 08-Mar 09-Mar 09-Mar 11-Mar 10-Mar 09-Mar 09-Mar

09-Mar 09-Mar 10-Mar 10-Mar 14-Mar 14-Mar 14-Mar 10-Mar 15-Mar 22-Mar 14-Mar 09-Mar

Luminex SSP SSP Luminex SSP Flow Cytometry, SSP SSP Flow Cytometry

21 22 23 24 26 27

Negative Negative Negative Negative Negative Negative

Positive Positive Positive Positive Positive Equivocal

10-Mar 11-Mar 09-Mar 09-Mar 09-Mar 09-Mar

10-Mar 15-Mar 15-Mar 17-Mar 09-Mar 10-Mar

Flow Cytometry RT-PCR SSP SSP SSP Flow Cytometry

28 29 31 32 34 35 37 39 40 41

Negative Negative Negative NT Negative Negative Negative Negative Negative Negative

Positive Positive Positive NT Positive Positive Positive Positive Positive Positive

09-Mar 10-Mar 09-Mar

14-Mar 14-Mar 09-Mar

09-Mar 10-Mar 09-Mar 11-Mar 09-Mar 09-Mar

11-16-Mar 10-Mar 14-Mar 10-Mar 22-Mar

SSO SSP/SSO Serological Flow Cytometry SSP SSP SSP SSP Flow Cytometry SSP

42 45 46 47 48 54 55 57 58 62 66 67 68 71 72 78 83 84 85 86 90 93 96 101 103 104 106 107 108 109 110 111 112 113 115 126 127 128 129 130 131 133 137

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

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

09-Mar 10-Mar 10-Mar 09-Mar 11-Mar 09-Mar 03-Mar 09-Mar 09-Mar 10-Mar 10-Mar 09-Mar 09-Mar 09-Mar 16-Mar 09-Mar 09-Mar 09-Mar 09-Mar 09-Mar 16-Mar 09-Mar 10-Mar 09-Mar 09-Mar 10-Mar

14-Mar 11-Mar 14-Mar 09-Mar 21-Mar 17-Mar 15-Mar 09-Mar 11-Mar 10-Mar 23-Mar 09-Mar 15-Mar 10-Mar 29-Mar 11-Mar 09-Mar 09-Mar 10-Mar 11-Mar 16-Mar 09-Mar 17-Mar 18-Mar 11-Mar 10-Mar

14-Mar 10-Mar 09-Mar 09-Mar 09-Mar 11-Mar 09-Mar

21-Mar 11-Mar 14-Mar 10-Mar 10-Mar 14-Mar 14-Mar

14-Mar 09-Mar 09-Mar 21-Mar 10-Mar 09-Mar 10-Mar 09-Mar

21-Mar 14-Mar 10-Mar 31-Mar 10-Mar 11-Mar 11-Mar 09-Mar

142 153 154 156 158 174 176 185

Negative Negative Negative Negative Negative Negative Negative Negative

Positive Positive Positive Positive Positive Positive Positive Positive

10-Mar 09-Mar 09-Mar 11-Mar 11-Mar 12-Mar 14-Mar

14-Mar 10-Mar 11-Mar 16-Mar 22-Mar 15-Mar 14-Mar

198 199 209 219 223 225 232 234 245 253 255 256 257 269 273 276 278 279 285 288 292 295 300 305 306 307 310 317 319 323 324 325 326 327 329 334 339 340

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative 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 NT

09-Mar 15-Mar 09-Mar 15-Mar 09-Mar 14-Mar 10-Mar 14-Mar 10-Mar 14-Mar 10-Mar 09-Mar 16-Mar 17-Mar 09-Mar 09-Mar 14-Mar 09-Mar 08-Mar 10-Mar 11-Mar 09-Mar 11-Mar 09-Mar 09-Mar 10-Mar 09-Mar 14-Mar 09-Mar 10-Mar 10-Mar 09-Mar 09-Mar 22-Mar 09-Mar 09-Mar 11-Mar 09-Mar

Consensus

10-Mar 22-Mar 17-Mar 22-Mar 14-Mar 17-Mar 14-Mar 16-Mar 15-Mar 18-Mar 21-Mar 10-Mar 16-Mar 22-Mar 10-Mar 11-Mar 29-Mar 10-Mar 10-Mar 11-Mar 11-Mar 09-Mar 16-Mar 22-Mar 10-Mar 12-Mar 09-Mar 16-Mar 10-Mar 10-Mar 10-Mar 17-Mar 10-Mar 23-Mar 16-Mar 09-Mar 15-Mar 09-Mar

HLA-A2, A3; B7, B18; Cw7, Cw-; DR4, DR16, DQ5, DQ8 HLA-A2, A-; B27, B62; Cw2, Cw10; DR1, DR12, DQ5, DQ7

Method

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

SSP Flow Cytometry SSP Flow Cytometry Flow Cytometry SSP

Luminex SSP Flow Cytometry Flow Cytometry SSP SSP SSP Flow Cytometry SSP SSP, SBT RT-PCR - TAQMAN Flow Cytometry Flow Cytometry Flow Cytometry SSP SSP Flow Cytometry Flow Cytometry, SSP RT-PCR SSP Flow Cytometry Flow Cytometry SSP SSP Flow Cytometry Flow Cytometry Flow Cytometry SSP SSP SSO Flow Cytometry, SSP Flow Cytometry SSP SSP SSP SSP EuroArray Flow Cytometry, Luminex SSO Cytotoxicity, SSP Flow Cytometry Flow Cytometry, SSP Flow Cytometry

RT-PCR Flow Cytometry SSP RT-PCR SSP SSP/SSO, Microarray Cytotoxicity SSP

Flow Cytometry RT-PCR SSP SSP SSP SSP SSP Molecular SSO RT-PCR RT-PCR Flow Cytometry SSP SSP SSP Microarray Cytotoxicity Cytotoxicity Flow Cytometry Cytotoxicity Flow Cytometry SSP SSP SSP SSP Flow Cytometry SSP RT-PCR Cytotoxicity Flow Cytometry SSP Flow Cytometry SSP SSO Flow Cytometry SSP Molecular

Enzo Life Sciences Control included in assay In-house

Primers and Probes Alta Bioscience

PCR-SSP Detection system

High Resolution Typing HLA-B*27 alleles 1B03/2016 1B04/2016

Comments

Gel

Own design

Fluorescence, SYBR GREEN and Melting Curve

Own design

Gel

Own design Own design

Gel Gel

Olerup Invitrogen In-house

Gel Gel Gel

In-house, Commercial In-house In-house

B27:03/05/07/09/11/13/14/17/19/25/27/28/32/3437/39/41/43

In-house (previous NEQAS scheme samples Own design Olerup Own design

Gel Gel Gel

B27:01/02/05/08+

Biofortuna

Gel

Olerup Own design Own design Own design

Gel Gel Gel, Fluorescence Gel

Own design

Gel

B27:01-04/05:02-05:31/06-21/23-90/92-136/137

Own design

Gel

B27:03 B27:05/47/116/117

Invitrogen Own design Hain Lifesciences

Gel Gel Fluorescence

Innotrain Olerup Own design

Fluorescence

TIB MOLBIOL Commercial

Fluorescence Gel

Olerup Own design Invitrogen

Gel Fluorescence (Taqman probes) Gel

Olerup Olerup

Gel Gel

EuroImmun Olerup

Fluorescence Gel

Neg = Immunotrol cells (Beckman Coulter) Pos = Pheonix Flow CRISP HLA B27 pos cells In-house

B27:03/05

Commercial No results returned

In-house Axxora In-house

In-house Secore

Gel B27:05/08/13/37/38/67/70

Crisp HLA-B27

HLA B27 CRISP positive control cells Enzo Life Sciences In-house In-house Beckman+One Lambda Olerup Phoenix In-house, Commercial

Commercial

Gel

Own design Olerup Olerup Own design

Gel Gel Gel Fluorescence

Olerup

Gel

Olerup

Gel

In-house

In-house

Phoenix BD

No results returned

B27:01-05/13/15/25/28 1B03/2016 positive for B27 and B7 with ABCm3 and BB71 clone), but negative for B27 with clone MPC-11)

In-house, Commercial Olerup

Gel

Olerup

Gel Gel

Olerup

Gel

Linkafe Biosciences Commercial Clonit Invitrogen Olerup Olerup

Fluorescence Gel Fluorescence Gel Gel Gel

Olerup EuroImmun Olerup LinkSeq HLA B27

Gel

Innotrain Commercial Sciences Biomark inc Olerup

Gel Gel Fluorescence Gel

Olerup

Gel

BAG Healthcare

Gel

AnDiaTec

Gel Fluorescence

Own design

Fluorescence

Invitrogen In-house

In-house UK NEQAS 1B

Commercial

B27:01-05:08/05:10-05:15/05:17-05:22/05:2405:30/08:10:13:15:17:25:28:36-40:42:44-45:4769/71-74/79-80/82-84/86-90:03/93-100/103105/108-118/120-124/126/128/131-135 1B03 - B7 crossreaction

Gel

In-house

In-house In-house

In-house Bionobis

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

Method under validation

NEQ-115 Issue 1 P. 1 of 2 Effective Date 10/03/16

UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING HLA-B27 TYPING RESULTS OF SAMPLES 1B03&04/2016 DESPATCHED ON 08 March 2016 Lab No 3 7 17 20 21 27 31 32 40 46 47 57 67 68 71 83 84 90 93 103 104 106 110 111 128 131 133 137 153 198 256 288 295 310 324 326 334

Monoclonal 1 Designation HLA-B27 FITC FITC B27 FITC Anti B27-FITC/CD3-PE HLA-B27 Duraclone B27 MSIgG1 RDI/MsIgG2a FITC PF/FITC HLA-B27 PE Anti HLA-B27 ABC m3 IgG2a IgG2a ABC m3 HLA-B27/B7 Duraclone B27 HLA-B27 B7/B27 HLA-B27 B27 HLA-B27-FITC/HLA-B7-PE Anti-HLA-B27 HLA-B27 Anti-HLA-B27 FITC Anti-HLA-B27 FITC Iotest HLA-B27 FITC HLA-B27-FITC/HLA-B7-PE HLA-ABC m3/BB7.1 HLA-B27 FITC HLA-B27 testing on T-cells Anti-HLA-B27 Anti-HLA-B27 Anti-HLA-B27 FITC Iotest HLA-B27 FITC/B7 PE HLA-B27 FITC Iotest HLA-B27 FITC, HLA-B7 PE HLA-B27 FITC

Monoclonal 1 Specificity Clone GS145.2 Anti-HLA-B27 B27 B27+B7 HLA-B27 FITC CD3, HLA-B27, HLA-B7 Isotype control B7/B27 FITC HLA-B27 HLA-B27 HLA-B27 HLA-B27-FITC/B7-PE FITC B7/B27 R-PE B27 HLA-B27 FITC/CD3 PE HLA-B27 FD705-9E1E10 Clone GS145.2 HLA-B27 HLA-B27 HLA-B27/B7 IgG2b FD705 anti-HLA-B27 FITC + anti-CD3-PE HLA-B27 FITC Clone GS145.2 HLA-B27 HLA-B27 HLA-B27

Monoclonal 1 Manufacturer BD BD One Lambda BD One Lambda Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter BD Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter One Lambda Beckman Coulter BD One Lambda One Lambda BD Beckman Coulter Beckman Coulter BD Immunotech One Lambda BD BD BD and BC BD Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter

Monoclonal 2 Designation CD3 PE PE/FITC CD3 PE Anti B27-FITC HLA-B27/HLA-B7

Monoclonal 2 Specificity Clone SK7 Anti CD3 CD3 B27 HLA-B27-FITC/HLA-B7-PE

Monoclonal 2 Manufacturer BD BD BD One Lambda Beckman Coulter

HLA-B27 FITC/B7 PE PE/FITC CD3-PE-DY649 Anti CD3 FD705 IgG2b IgG2b FD705 HLA-B27

B7/B27 IgG/IgG2

Beckman Coulter Beckman Coulter Beckman Coulter BD Beckman Coulter Beckman Coulter Beckman Coulter One Lambda

Monoclonal 3 Designation

Monoclonal 3 Specificity

Monoclonal 3 Manufacturer

Details of Pos/Neg conrol cells used Crisp B7 homozygous control Phoenix flow crisp

PE HLA-B27 HLA-B27 HLA-B27 HLA-B27-FITC

BB7.1 IgG1 IgG1K BB7.1

HLA-B7 CD3 HLA-B27

Beckman Coulter Beckman Coulter Beckman Coulter

HLA-B7

PE

Beckman Coulter

CD3

PC5

Beckman Coulter

CD3 B27

PE-Dyomics649 B27

Beckman Coulter Beckman Coulter

B7

Clone BB7.1

Beckman Coulter

HLA-B27

HLA-B7/B27

Immunotech

CD3 PE Iotest HLA-B7 PE

Clone SK7 HLA-B7

BD Beckman Coulter

CD45 PerCp-Cy5.5

Lymphocytes gating

BD

HLA-B27 IgG2a-IgG1 anti-HLA-B27 FITC + anti-CD3-PE CD3 PE Clone SK7 HLA-B7 HLA-B7 HLA-B27

BD One Lambda Immunotech Beckman Coulter BD BD BD Beckman Coulter Beckman Coulter One Lambda Beckman Coulter

MPC-11 HLA-B27-FITC/B7-PE HLA-B27 control +B7 crossreaction Anti-CD3 Anti-CD3 Anti-CD3 PE Iotest HLA-B27 FITC/B7 PE HLA-B7 PE MO AB B27 FITC HLA-B7 PE

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

Isotype control

IgG2a-FITC and IgG1-PEBeckman Coulter

Anti-HLA-B7

PE

Crisp Known positive samples

Crisp

BC 1 Pos, 1 Neg

NEQ-115 Issue 1 P. 2 of 2 Effective Date 10/03/16