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UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING
HLA-B27 TYPING RESULTS OF SAMPLES 1B09&10/2016 DISPATCHED ON 27TH SEPTEMBER 2016 Summary of Results Total tested (excl. Positive Negative NT/Equivocal % Positive % Negative Consensus
115 72 43 10 62.6% 37.4%
119 3 116 6 2.5% 97.5%
Donor HLA type Donor HLA type
1B09/2016 1B10/2016
Not Assessed
Negative
1 3 4 7 8 9 10 11 12 14 15 16 17 19 20
Result 1B09/2016 Positive NT Positive Equivocal Negative Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive
Result 1B10/2016 Negative NT Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
Date received 28-Sep NT 27-Sep 28-Sep 27-Sep 28-Sep 28-Sep 28-Oct 28-Sep 28-Sep 28-Sep 28-Sep 06-Oct 28-Sep 29-Sep
Date tested 05-Oct NT 11-Oct 28-Sep 12-Oct 30-Sep 28-Sep 05-Oct 06-Oct 30-Sep 03-Oct 30-Sep 20-Oct 29-Sep 29-Sep
21 22 23 24 26 27 28 29 31 32 34 35 37 39 40 41 42 45 46
Negative Positive Positive Positive Negative Negative Positive Positive Negative Negative Positive Positive Positive Negative Positive Positive Positive Positive Negative
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
27-Sep 29-Sep 28-Sep 21-Sep 28-Sep 28-Sep 28-Sep 29-Sep 28-Sep 27-Sep 28-Sep 28-Sep 28-Sep 29-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep
28-Sep 06-Oct 10-Oct 05-Oct 28-Sep 28-Sep 04-Oct 04-Oct 28-Sep 13-Oct
47 48 54 55 57 58 62 66 67 68 72 76 78 83 84 85 86 90 93 96 101 103 104 106 107 108 109 110 111 112 113 115 126 127 128 129 130 131 133 137 142 153 154 156 158 174 176 185 198 199 209 219 223 225 232 234 236 245 253 255 256 257 269
Negative Positive Positive Negative Negative Positive Positive Positive Negative Negative NT NT Positive Negative Negative Negative Positive Negative Negative Positive Positive Negative Equivocal Negative Positive Positive Positive Positive Negative Positive Positive Positive Positive Positive Positive Negative Positive Negative Positive Negative Negative Negative Positive Positive Positive Positive Negative Positive Positive Positive Positive Negative Positive Positive Positive Positive Negative Positive Positive Positive Negative Positive Negative
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative NT NT Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Equivocal Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Positive Negative Equivocal
29-Sep 29-Sep 28-Sep 30-Sep 28-Sep 28-Sep 28-Sep 29-Sep 28-Sep 28-Sep 04-Oct 28-Sep 29-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Oct 29-Sep 28-Sep 28-Sep 29-Sep 29-Sep 04-Oct 29-Sep 28-Sep 28-Sep 29-Sep 30-Sep 28-Sep 28-Sep 03-Oct 28-Sep
07-Oct 29-Sep 28-Sep 30-Sep 05-Oct 29-Sep 13-Oct 30-Sep 04-Oct 30-Sep 29-Sep 11-Oct 06-Oct 03-Oct 11-Oct 29-Sep 30-Sep 13-Oct 28-Sep 10-Oct 10-Oct 04-Oct
04-Oct 28-Sep 28-Sep 28-Sep 28-Sep 29-Sep 28-Sep 28-Sep 30-Sep 04-Oct 03-Oct 03-Oct 28-Sep 28-Sep 03-Oct 29-Sep 06-Oct 28-Sep 04-Oct 03-Oct 03-Oct 05-Oct 28-Sep 03-Oct 29-Sep 28-Sep 27-Sep
07-Oct 29-Sep 30-Sep 30-Sep 29-Sep 04-Oct 29-Sep 30-Sep 17-Oct 11-Oct 08-Oct 03-Oct 04-Oct 30-Sep 06-Oct 03-Oct 07-Oct 04-Oct 07-Oct 07-Oct 04-Oct 06-Oct 04-Oct 07-Oct 04-Oct 28-Sep 27-Sep
273 276 278
Positive Positive Positive
Negative Negative Negative
29-Sep 28-Sep 03-Oct
04-Oct 10-Oct 13-Oct
SSP SSP/RT-PCR Microarray
279 285 288
Negative Negative Equivocal
Negative Negative Equivocal
28-Sep 28-Sep 28-Sep
30-Sep 29-Sep 29-Sep
Cytotoxicity Cytotoxicity Flow Cytometry
292 295 300 305 306 307 310 317 319 323 324 325 326 327 329 334 339 340 348 349 353 356
Negative Positive Negative Positive Positive Positive Negative Positive Equivocal Negative Negative Positive Negative Positive Positive Equivocal Negative Equivocal Negative Equivocal Positive Negative
Positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
30-Sep 28-Sep 20-Oct 27-Sep 28-Sep 28-Sep 29-Sep 10-Oct 28-Sep 30-Sep 29-Sep 28-Sep 29-Sep 05-Oct 28-Sep 28-Sep 03-Oct 28-Sep 03-Oct 28-Sep 28-Sep 04-Oct
30-Sep 29-Sep
Cytotoxicity Flow Cytometry SSP SSP SSP SSP Flow Cytometry SSP RT-PCR Cytotoxicity Flow Cytometry SSP Flow Cytometry SSP SSO Flow Cytometry SSP PCR-SSP Flow Cytometry Flow Cytometry SSP, SSO Flow Cytometry
Lab No
06-Oct 29-Sep 03-Oct 10-Oct 11-Oct 04-Oct 04-Oct 09-Oct 29-Sep 06-Oct 06-Oct 14-Oct 13-Oct 29-Sep 28-Sep 10-Oct 28-Sep 28-Sep
14-Oct 06-Oct 17-Oct 30-Sep 16-Oct 30-Sep 03-Oct 29-Sep 04-Oct 30-Sep 12-Oct 03-Oct 28-Sep 11-Oct 06-Oct 03-Oct 29-Sep 06-Oct 04-Oct
HLA-A1, A3; B27, B44; Cw2, Cw16; DR4, DR17; DQ2, DQ8 HLA-A24, A32; B7, B61; Cw2, Cw7; DR9, DR17; DQ2, DQ9
PCR-SSP
Method SSP NT SSP Flow Cytometry Flow Cytometry SSP Flow Cytometry Luminex SSP SSP PCR-SSP SSP, Luminex Flow Cytometry, SSP SSP, Luminex Flow Cytometry, SSP
Flow Cytometry RT-PCR SSP SSP SSP Flow Cytometry SSO SSP/SSO Flow Cytometry Flow Cytometry SSP SSP SSP SSP Flow Cytometry SSP Luminex SSP Flow Cytometry Flow Cytometry SSP SSP SSP Flow Cytometry SSP SSP, SBT RT-PCR - TAQMAN Flow Cytometry Flow Cytometry SSP Flow Cytometry SSP Flow Cytometry Flow Cytometry, SSP RT-PCR SSP Flow Cytometry Flow Cytometry SSP SSP Flow Cytometry Flow Cytometry Flow Cytometry SSP SSP SSP Flow Cytometry, SSP Flow Cytometry SSP SSP SSP RT-PCR Microarray 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 Serological SSO RT-PCR RT-PCR Flow Cytometry SSP SSP
Primers and Probes Alta Bioscience NT Own design
Detection system Gel NT Fluorescence
Own design
Gel
Own design Own design
Gel Gel
Olerup, One Lambda Olerup Own design
Gel, Fluorescence Gel Gel
High Resolution Typing HLA-B*27 alleles 1B09/2016 1B10/2016
Comments
No results returned Patient samples with such results would be referred for PCR analysis B*27:12 B*27:12
B*27:12 B*27:12
B*27:12/26
In-house Own design Olerup Own design
Gel Gel Gel
Sample showed unusual reactivity by flow cytometry. It was weakly positive with the use of Beckton Dickinson B27 antibody and negative with One Lambda B27 antibody. This prompted the test to be completed by PCR-SSP where the uncommon B*27:12 was detected (B*27:26 cannot be ruled out)
B*27:12 B*27:12
B*27:12 B*27:12
Biofortuna Phoenix Flow
Gel
Olerup Own design Own design Own design
Gel Gel Gel, Fluorescence Gel
Own design
Gel
Own design
Gel
Invitrogen Own design Hain Lifesciences
Gel Gel Fluorescence
Innotrain Olerup Own design
Gel
TIB MOLBIOL
Fluorescence
Commercial
Gel
Olerup Own design Invitrogen
Gel Fluorescence (Taqman probes) Gel
Olerup Olerup
Gel Gel
EuroArray Olerup LinkSeq HLA-B27 Commercial
Fluorescence Gel
Own design Olerup Olerup Own design
Gel Gel Gel Fluorescence
Olerup
Gel
1B09/2016 Weak/Neg reaction with some antisera - CDC. DNA testing - B*27:12
Olerup
Gel
We also confirm by SSP LR typing - Sample 1B09 the presence of ?B*27:12
B*27:12 B*27:12
B*27:12 B*27:12 Sample 1B09 peak has fallen directly on the decision line. Although the median is "Negative", this would require confirmation by genetic testing
B*27:12
Fluorescence
HLA-B27 testing currently being validated Unable to perform NEQAS or patient testing due to production facility failure
Determination by PCR: NEG
Gel
1B09 was negative by Flow cytometry
Olerup
Gel
Olerup
Gel Gel
Olerup
Gel
Linkafe Biosciences Commercial Clonit Invitrogen Commercial Olerup
Fluorescence Gel Fluorescence Gel Gel Gel
Sample 1B10 showed HLA-B7 crossreaction
Olerup EuroImmun, Euroarray
Gel
Olerup Linkage biosciences
Gel Melting Point
Samples seems to be HLA-B27 pos, but also some non HLA-B27:01 alleles are possible (B*07:197, 15:342, 37:02, 47:04, 47:05, 48:26)
Sample 1B09/2016: Exon2 - Neg, Exon3 - Pos Sample 1B10/2016: Exon2 - Neg, Exon3 - Neg
1B09: Equivocal (BD), Weak Pos (BC) 1B10: Neg (BD), Pos (BC) Technical issue?
Innotrain Commercial Sciences Biomark inc Olerup
Gel Gel Fluorescence Gel
Olerup
Gel
BAG Healthcare
Gel
Delivery delay due to customs
Discrepant results from different assays for sample 1B09
AnDiaTec
Gel Fluorescence
Own design Own design
Fluorescence Fluorescence
Sample 1B09 would be send to another laboratory We used two sets of primers, discordant results observed
Fluorescence Sample delivery delay
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. 1 of 2 Effective Date 10/10/16
UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING
HLA-B27 TYPING RESULTS OF SAMPLES 1B09&10/2016 DISPATCHED ON 27TH SEPTEMBER 2016 Lab No
Source of pos/neg control cells used (if applicable) 3 7 10 17 20 21 27
Enzo Life Sciences In-house In-house In-house Neg = Immunotrol cells (Beckman Coulter) Pos = Pheonix Flow CRISP HLA B27 pos cells
31 Positive control cells not used 32 40 46 Axxora 47 In-house 57 In-house 67 68 Crisp HLA-B27 76 83 HLA B27 CRISP positive control cells 84 Enzo Life Sciences 90 93 Beckman+One Lambda 103 Phoenix 104 106 110 111 In-house 128 131 Phoenix 133 BD 137 153 In-house, Commercial 198 256 288 295 In-house 310 In-house 324 326 In-house 334 348 349 356
Designation HLA-B27 FITC FITC HLA-B27 FITC B27 FITC Anti B27-FITC/CD3-PE HLA-B27 Duraclone B27
Monoclonal 1 Specificity Clone GS145.2 Anti-HLA-B27 HLA-B27 B27 B27+B7 HLA-B27 FITC CD3, HLA-B27, HLA-B7
BD BD One Lambda One Lambda BD One Lambda Beckman Coulter
MSIgG1 RDI/MsIgG2a FITC
Isotype control
PF/FITC HLA-B27 PE Anti HLA-B27 ABC m3 IgG2a IgG2a PE- anti-Human-HLA-B27 HLA-B27/B7 FD705 HLA-B27
B7/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
B7/B27 R-PE B27 HLA-B27 FITC/CD3 PE HLA-B27 FD705-9E1E10 Clone GS145.2 HLA-B27 HLA-B27
HLA-ABC m3/BB7.1 HLA-B27 FITC HLA-B27 testing on T-cells Anti-HLA-B27 Anti-HLA-B27 FITC Anti-HLA-B27 FITC Iotest HLA-B27 FITC/B7 PE HLA-B27 FITC Iotest HLA-B27 FITC, HLA-B7 PE HLA-B27 FITC CD3 PE/HLA-B27 FITC Anti-CD3 PE Duraclone B27
FITC HLA-B27 HLA-B27 clone FD705 and ABCm3 HLA-B27-FITC/B7-PE HLA-B27 FITC
HLA-B27/B7 IgG2b FD705 anti-HLA-B27 FITC + anti-CD3-PE HLA-B27 HLA-B27 Clone GS145.2 HLA-B27 HLA-B27 HLA-B27 HLA-B27 CD3, Clone UCHT1
CD3 PE PE/FITC CD3 PE CD3 PE Anti B27-FITC HLA-B27/HLA-B7
Monoclonal 2 Specificity Clone SK7 Anti CD3 CD3 CD3 B27 HLA-B27-FITC/HLA-B7-PE
Manufacturer BD BD BD BD One Lambda Beckman Coulter
Beckman Coulter
HLA-B27 FITC/B7 PE
B7/B27
Beckman Coulter
Beckman Coulter Beckman Coulter BD Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter
PE/FITC CD3-PE-DY649 Anti CD3 FD705 IgG2b IgG2b PE- Dyomics649- anti-Human CD3 HLA-B27 ABCm3 HLA-B7
IgG/IgG2 PE HLA-B27 HLA-B27 clone UCHT1 HLA-B27-FITC HLA-B27 PE
Beckman Coulter Beckman Coulter BD Beckman Coulter Beckman Coulter Beckman Coulter One Lambda Beckman Coulter Beckman Coulter
CD3 B27
PE-Dyomics649 B27
Beckman Coulter B7 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
HLA-B27 IgG2a-IgG1 anti-HLA-B27 FITC + anti-CD3-PE CD3 CD3 Clone SK7 HLA-B7 HLA-B7 HLA-B27
BD One Lambda Immunotech Beckman Coulter Isotype control BD BD Anti-HLA-B7 PE BD Beckman Coulter Beckman Coulter One Lambda Beckman Coulter
Manufacturer
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 Beckman Coulter BD Beckman Coulter Beckman Coulter Beckman Coulter Beckman Coulter BD BD Beckman Coulter
Designation
MPC-11 HLA-B27-FITC/B7-PE HLA-B27 control +B7 crossreaction Anti-CD3 Anti-CD3 PE Anti-CD3 PE Iotest HLA-B27 FITC/B7 PE HLA-B7 PE MO AB B27 FITC HLA-B7 PE Anti HLA-B27 FITC Duraclone B27
HLA-B27, Clone ABCm3
Designation
Monoclonal 3 Specificity
Manufacturer
BB7.1 IgG1 IgG1K unlabeled anti-Human B7
HLA-B7 CD3 Clone BB7.1
Beckman Coulter Beckman Coulter Beckman Coulter
BB7.1 CD3
HLA-B7 PC5
Beckman Coulter Beckman Coulter
Clone BB7.1
Beckman Coulter
Lymphocytes gating
BD
BD Beckman Coulter Duraclone B27
IgG2a-FITC and IgG1-PE Beckman Coulter HLA-B7
Beckman Coulter
HLA-B27, Clone FD705
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
