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HLA-B27 TYPING RESULTS OF SAMPLES 1B01&02/2017
UK NEQAS FOR H&I SCHEME 1B - HLA-B27 TESTING
DESPATCHED ON 14TH FEBRUARY 2017 Summary of Results Total tested (excl. Equivocal) Positive Negative NT/Equivocal % Positive % Negative Consensus
121
120
0 121 3 0.0% 100.0%
1 119 4 0.8% 99.2%
Donor HLA type
1B01/2017
HLA-A1, A24; B7, B8; Cw7, Cw-; DR15, DR-, DQ6, DQ-
Donor HLA type
1B02/2017
HLA-A2, A25; B7, B37; Cw6, Cw7; DR17, DR4, DQ2, DQ8
Negative
Negative
Lab No 1
Result 1B01/2017 Negative
Result 1B02/2017 Negative
Date received 15/02/2017
Date tested 27/02/2017
3 4 7 8 9 10 11 12 14 15 17 19 20 21 22 23 24 25 26 27 28 29
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 Negative Negative Negative Negative Negative Negative Negative
16/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 16/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 17/02/2017 17/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 14/02/2017 16/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017
16/02/2017 28/02/2017 15/02/2017 16/02/2017 21/02/2017 16/02/2017 21/02/2017 21/02/2017 15/02/2017 17/02/2017 27/02/2017 20/02/2017 07/02/2017 15/02/2017 16/02/2017 16/02/2017 20/02/2017 21/02/2017 16/02/2017 15/02/2017 21/02/2017 20/02/2017
Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological (e.g. flow cytometry)
31 32 34 35 37 39 40 41 42 45 46 47 48 54 55 57 58 62 66 67 68 72 76 77 78 83 84
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 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
15/02/2017 15/02/2017 15/02/2017
16/02/2017 15/02/2017 16/02/2017
15/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 22/02/2017 15/02/2017 15/02/2017 16/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 15/02/2017 16/02/2017 15/02/2017 16/01/2017 17/02/2017 15/02/2017 16/02/2017 15/02/2017 15/02/2017 15/02/2017
23/02/2017 17/02/2017 15/02/2017 17/02/2017 16/02/2017 27/02/2017 16/02/2017 15/02/2017 20/02/2017 07/02/2017 01/03/2017 16/02/2017 16/02/2017 15-23/02/2017 21/02/2017 15/02/2017 20/02/2017 28/02/2017 15/02/2017 16/02/2017 16/02/2017 16/02/2017 16/02/2017
Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry)
85 86 90 96 101 103 104
Negative Negative Negative Negative Negative Negative Negative
Negative Negative Negative Negative Negative Negative Equivocal
17/02/2017 16/02/2017 21/02/2017 16/02/2017 15/02/2017 15/02/2017 16/02/2017
21/02/2017 21/02/2017 21/02/2017 24/02/2017 16/02/2017 16/02/2017 16/02/2017
Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry)
106 107
Negative Negative
Negative Negative
16/02/2017 20/02/2017
27/02/2017 21/02/2017
Serological (e.g. flow cytometry) Molecular (e.g. PCR)
108 109 110 111 112 113 115 126 127 128 129 130 133
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative NT Negative Negative
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative NT Negative Negative
17/02/2017 15/02/2017 15/02/2017 16/02/2017 17/02/2017 15/02/2017 15/02/2017 20/02/2017 16/02/2017 16/02/2017
20/02/2017 26/02/2017 15/02/2017 16/02/2017 24-02-2017 16/02/2017 17/02/2017 20/02/2017 21/02/2017 21/02/2017
Molecular (e.g. PCR) Molecular (e.g. PCR) Serological, Molecular Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological & Molecular
Microarray Flow Cytometry, PCR-SSO / Luminex Commerical kit
One Lambda
Fluorescence
14/02/2017 16/02/2017
16/02/2017 17/02/2017
Serological & Molecular Serological & Molecular
Cytotoxicity, PCR-SSP Flow Cytometry, PCR-SSP
Olerup SSP Olerup
Gel Gel
137
Negative
Positive
15/02/2017
15/02/2017
Serological (e.g. flow cytometry)
Flow Cytometry
142 153 154 156
Negative Negative Negative Negative
Negative Negative Negative Negative
15/02/2017 15/02/2017 15/02/2017 14/02/2017
21/02/2017 20.02.2017 24/02/2017 20/02/2017
Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR)
RT-PCR
174 176 185 198
Negative Negative Negative Negative
Negative Negative Negative Equivocal
04/03/2017 20/02/2017 15/02/2017 15/02/2017
08/03/2017 20/02/2017 16/02/2017 16/02/2017
Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological & Molecular
209 219 223 224 225 232 234 236 245 252 255 256 257 269 273 276 278 279 288 292 295 300
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 Negative Negative Negative Negative Negative Negative Negative
15/02/2017 21/02/2017 15/02/2017 15/02/2017 20/02/2017 17/02/2017 20/02/2017 21/02/2017 15/02/2017 17/02/2017 15/02/2017 15/02/2017 14/02/2017
Molecular (e.g. PCR)
15/02/2017 14/02/2017 14/02/2017 14/02/2017 16/02/2017 16/02/2017 14/02/2017 22/02/2017
20/01/2017 24/02/2017 22/02/2017 16/02/2017 22/02/2017 22/02/2017 21/02/2017 22/02/2017 21/02/2017 22/02/2017 21/02/2017 15/02/2017 27/02/2017 22/02/2017 17/02/2017 22/02/2017 20/02/2017 15/02/2017 17/02/2017 16/02/2017 17/02/2017 24/02/2017
305 306 307 310 317 319
Negative Negative Negative Negative Negative Negative
Negative Negative Negative Negative Negative Negative
15/02/2017 15/02/2017 15/02/2017 16/02/2017 15/02/2017
08/03/2017 16/02/2017 21/02/2017 15/02/2017 27/02/2017 16/02/2017
Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR)
324 325 326 327
Equivocal Negative Negative Negative
Negative Negative Negative Negative
16/02/2017 16/02/2017 15/02/2017 20/02/2017
16/02/2017 21/02/2017 17/02/2017 27/02/2017
Serological (e.g. flow cytometry) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR)
Flow Cytometry
PCR-SSP
Commerical kit
BIONOBIS HLA B27 SSP réf: 101-531-48
Gel
329 334 339 340 349
Negative Negative Negative Negative Negative
Negative Negative Negative Negative Negative
16/02/2017 15/02/2017 15/02/2017 15/02/2017 16/02/2017
21/02/2017 15/02/2017 23/02/2017 16/02/2017 16/02/2017
Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry)
PCR-SSO
Commerical kit
AnDiaTec
Fluorescence
PCR-SSP
Own design
353 357 359 362 365 368
Negative Negative Negative Negative Negative NT
Negative Negative Negative Negative Negative NT
17/02/2017 16/02/2017 01/02/2017 15/02/2017 15/02/2017 15/02/2017
21/02/2017 16/02/2017 22/02/2017 16/02/2017 16/02/2017 ---
Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR)
Type of Method
Method
Molecular (e.g. PCR)
PCR-SSP
Primers and Probes PRIMERS MADE TO ORDER FROM ALTA BIOSCIENCE
PCR-SSO / Luminex
Commerical kit
Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological, Molecular Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR)
Luminex PCR-SSP PCR-SSP Luminex Flow Cytometry, PCR-SSP PCR-SSP Flow Cytometry, PCR-SSP Flow Cytometry Real-Time PCR PCR-SSP PCR-SSP PCR-SSO PCR-SSP Flow Cytometry PCR-SSP/SSO PCR-SSP/SSO
PCR-SSP Kit Manufacturer
One Lambda
Own design Own design Commerical kit Own design Own design without hybridization probes Own design Commerical kit
High Resolution Typing HLA-B*27 alleles 1B01/2017 1B02/2017
Comments
Fluorescence
Gel Gel Olerup
Olerup
Own design Commerical kit Commerical kit
Detection system Gel
Gel Gel Gel
1B02 is probably a B7
Gel Gel Gel
One Lambda Biofortuna
Gel Gel, BAG Healthcare reverse dot blot
Flow Cytometry
PCR-SSP PCR-SSP PCR-SSP
Own design Own design Own design
Gel Gel Gel
PCR-SSP Luminex PCR-SSP
Own design
Gel
PCR-SSP
Commerical kit
Invitrogen
Gel
PCR-SSP PCR-SSP/SBT RT-PCR
Commerical kit Commerical kit Own design
Innotrain Olerup
Gel Gel Fluorescence
Flow Cytometry PCR-SSP
Commerical kit
Tib MOLBIOL
Fluorescence
PCR-SSP
Commerical kit
Olerup
Gel
PCR-SSP
Commerical kit
Invitrogen
Gel
Own design
Gel
Molecular (SSP low resolution typing) also performed. PCR negative for both samples.
In case of a patient, the result "equivocal" should be confirmed. This confirmation is negative (PCR in an other laboratory). 1B 01/2017 was negative in exon 2 and exon 3, and 1B 02/2017 was negative in exon 2 and exon 3 PCR-SSP
Commerical kit
Olerup
Gel
Flow Cytometry, PCR-SSP Flow Cytometry
Commerical kit
Gel
PCR-SSP
Commerical kit
Gel
Unable to test samples before results deadline
Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Molecular (e.g. PCR) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Serological (e.g. flow cytometry) Molecular (e.g. PCR)
Commerical kit Commerical kit
Own design
Fluorescence
Real-Time PCR
PCR-SSP
In routine we perform FCM in any suspicious result we confirm using the SSP methodology Sample B01 and sample B02 were HLA-B27 and HLAB7 postitive. Sample B01 was B27 negative, sample B02 was B27 positive when they were tested with an antibody against an alternative epitope (MPC-11 clone). It is possible that HLA-B27 positivity of sample B01 is a result of cross reaction (ABCm3 clone).
Our real-time PCR is an in house method utilizing a commercial kit for control gene (Life Technologies), and a combination of published and in house primers and Taqman probes for HLA B27 detection
Commerical kit
Olerup
Gel Sample 1B 01/2017 was negative with both methods but HLA-B7 cross-reaction could be detected by flow cytometry. Sample 1B 02/2017 was repeatedly positive in flow cytometric measurements but using low resolution PCR we got negative result. Therefore we evaluated the sample equicocal with asking repeated sample from the patient.
PCR-SSP PCR-SSP PCR-SSP PCR-SSP PCR-SSP
Commerical kit Commerical kit Commerical kit Commerical kit Own design
Invitrogen BIOFORTUNA Olerup Olerup SSP
Gel Gel Gel Gel Fluorescence
PCR-SSP
Commerical kit
Olerup
Gel
PCR-SSP PCR-SSP PCR-SSP
Commerical kit Commerical kit Commerical kit
Olerup Euroimmun Euroarray Olerup
Gel Fluorescence Gel
PCR-SSP
Cytotoxicity Cytotoxicity Tested using: PCR SSP Inno-Train HLA Ready Gene B27
PCR-SSP
Commerical kit
Olerup
Gel
PCR-SSP
Commerical kit
Olerup
TapeStation PCR-SSP Commercial kit: Clonit HLA B27 Real time PCR
Fluorescence A positive diagnosis with our technique (Flow Cytometry) must be confirm be the reference technique (lymphocytotoxicity)
PCR-SSP Flow Cytometry RT-PCR
Commerical kit
Appolon Bioteck
Own design
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
Fluorescence Fluorescence
Method/Technique under evaluation/validation
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 1B01&02/2017 DESPATCHED ON 14TH FEBRUARY 2017 Lab No
Source of pos/neg control cells used (if applicable) 17
Control Cells: B7 Homozygous Cell, B27/B7 Positive cell, 20 B27/B7 Negative cell 21 Positive control: Crisp HLA-B27 positive control cells from Phoenix Flow Systems Negative control: ImmunoTrol 27 cells from Beckman Coulter 31 CRISP Cells Positive Controls -Crisp HLAB27 Supplier- Phoenix Flow Systems 68 110 N/A 111 128 133 In house 137 324 Internal control in each Duraclone B27 tube (Bc) (beads) 365
Monoclonal 1 Designation Specificity B27 FITC B27 B27-FITC, B27 and B7, Becton Dickinson
Manufacturer One Lambda
Designation CD3 PE B27-FITC, B27, One Lambda
Monoclonal 2 Specificity CD3
Manufacturer BD
Monoclonal 3 Specificity
Manufacturer
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
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-B27/B7 Anti HLA B27 FITC Anti HLA B27 FITC HLA-B27 (clone FD 705) PE
Designation
Flow Cytometry Pos/Neg Controls Detection system details Known patient positive for flow. In House Controls
Comments
Commercial Supplier
One Lambda BD Biosciences BC Immunotech Beckman Coulter Beckman Coulter
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 (clone HLAPE ABC-m3)
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
DuraClone B27, HLA-B27, Beckman Coulter
Commercial Supplier Commercial Supplier
BD Biosciences BC One Lambda Beckman Coulter Beckman Coulter
In house CD45 PerCP-Cy5.5, Lymphocytes Gating, Becton Dickinson Anti CD3 T cells BC
CD3 (clone UCHT1)
PE-Dyomics 649
Luminex
Beckman Coulter
NEQ-115 Issue 2 P. 2 of 2 Effective Date 10/10/16
