Major Histocompatibility Complex 2013; 20 (: 13 16 QCWS # 1. ワークショップの経過 24 1 QCWS HP QCWS 24 2 57 DNA-QC 53 QC 38 1 20 QCWS DNA-QC QC 4 QCWS 4 4 4 16 QCWS 5 21 57 DNA-QC 53 QC 37 5 6 7 HP 8 25 HP 2. QCWS のテーマおよび試料選択について DNA-QC DNA 2 DNA DNA HLA Ambiguity 5 QCWS HLA HLA 4 DNA QC 3 4 HLA HLA-C IgM HLA # QCWS 2) 3) 4) 5) 6) 7) 8) 5) 9) 10) 1 12) 13) 2) 3) 4) 5) 6) 7) 8) 9) 10) 1 12) 13) 13
MHC 2013; 20 ( 2 QC QC DNA-QC 3. QCWS 集会参加および参加証明書発行 QCWS QCWS QCWS 77 4. 解析方法 DNA-QC Luminex SSO SSO SSP SBT QC FlowPRA Lab Screen WAK Flow ICFA 4 HLA-QC 1 Luminex SSO SSO SSP SBT HLA 表 1 第 16 回 QCWS 参加施設 14
MHC 2013; 20 ( 2 FlowPRA Lab Screen HLA WAK Flow ICFA 3 DNA 5. QCWS サンプルの総合結果 DNA DNA HLA-A, B, C, DRB1, DQB1 1 DNA Ambiguity IMGT/HLA Database Sequence Alignments based on Release 3.7.0 Jan- 2012 HLA 2010 1.1 2 QC 3 2 8 1 3 2 4 SH2402 IgM ** 3 表 2 DNA 15
MHC 2013; 20 ( 表 3 16
Major Histocompatibility Complex 2013; 20 (: 17 18 DNA Liminex 1. 概況 Luminex 53 31 58.4% LABType 10 32.3% LABType HD 7 22.6% WAKFlow 17 54.8% 6 19.4% LIFECODES 1 3.2% HLA-A, B HLA-C 26 86.7% DRB1 30 96.8% DRB3/4/5 3 9.7% DQA1 7 25.8% DQB1 15 48.3% DPA1 4 12.9% DPB1 8 25.8% 2. 解析方法 3 HLA-A, B, C, DRB1 %CV Pmin/Nmax P/N 16 QC LIFECODES 1 3. 結果と考察 結果の表記 Luminex ambiguity ambiguity 2) 反応データ HLA-C 2 %CV 56% 4 H2401 H2404 H2402 2 159 3 2543, 2270, 2304 P/N HLA-DRB1 P/N 3 7 3 3) アサインミス 7 HLA-A 2 HLA-B 2 HLA-C 1 HLA-DRB1 2 3 No. 3, 4, 7 17
MHC 2013; 20 ( No. 1, 2 1 No. 5 7 2 DRB1*08:02 DRB1*04:10, DRB1*16:02/17/19 Luminex LABType NormalValue 4. まとめ 18
Major Histocompatibility Complex 2013; 20 (: 19 検査法別解析 DNA タイピング SSO 法 (INNO-LiPA) 1. はじめに INNO-LiPA SSO RELI QCWS 4 RELI RELI 2. 検査状況 4 1 24D18 H2401 H2402 INNO-LiPA SSP HLA-C DRB3/4/5 DQB1 1 24D08 3. 解析結果 3.1 クラス I INNO-LiPA ambiguity 4 ambiguity H2401 H2402 H2403 4 24D04 24D18 H2401 1 1 ambiguity H2404 A 3 B 3 2 1 2 3.2 クラス II DR 1 Decoder DRB3/4/5 DQ DRB1 1 H2401 H2404 1 4. まとめ 2 1 1 19
Major Histocompatibility Complex 2013; 20 (: 20 DNA SSP 1. 概要 SSP 27 50% 7 SSP 18 9 2 1 SSP Major Histocompatibility Complex 2013; 20 (: 13 34 2. 参加部門 SSP SSP 3. 試薬 low resolution 4 high resolution 3 Micro SSP OneLambda low resolution Micro SSP JPN 19 4. 解析結果 QCWS Consensus Allele miss assign false positive false negative 判定ミス (miss assign) false positive false negative 2) 相対的反応データの不備 false positive false negative 5 miss assign 5. まとめ SSP low resolution 2 Ambiguity 1 low resolution SSP 20
Major Histocompatibility Complex 2013; 20 (: 21 22 検査法別解析 DNA タイピング SBT 法 1. はじめに (HP 掲載結果 : 図 2) SBT 6 HLA-A, B, C, DRB1 HLA-DQB1, DPB1 3 AlleleSEQR HLA typing kits CELERA 6 1 SeCore Sequencing Kits invitrogen Assign AlleleSEQR HLA typing kits CELERA Assign QCWS IMGT/ HLA3.7.0-12/04/2012 Assign3.6+ 1 3, 4 2. 解析結果と考察 Assign 1 2.1 Assign 解析ソフトにおけるリファレンスの更新ミス ( 図 6) SBT Conexio Genomics Assign IMGT-HLA 2.2 表記ミス ( 図 7) 1 H2404 DRB1 allele1 DR14 allele2 DR8 DR13 HLA II.I 2 DRB1*08:02/13:47/+ DRB1*14:02/06/52/+ AlleleSEQR HLAtyping kits CELERA SeCore Sequencing Kits invitrogen HLA-DRB1 2Forward 2Reverse Codon86 3 8 Codon86 DRB1 86 GTG Assign 9 Codon86 GTG QCWS H2404 Codon86 HLA- DRB1*08:02 -DRB1*14:06 2.3 キットによる違い QCWS AlleleSEQR HLAtyping kits CEL- ERA SeCore Sequencing Kits invitrogen 2 21
MHC 2013; 20 ( 2 HLA-A B C DRB1 DQB1 DPB1 10 ClassI HLA-A B C 2 3 4 ClassII DRB1 2 DQB1 2 3 DPB1 AlleleSEQR HLA-DPB1 typing kits 2 SeCore DPB1 Locus Sequencing Kits 2 3 4 AlleleSEQR typing kits SeCore DPB1 Locus Sequencing Kits H2402 HLA-DPB1 SeCore Sequencing Kits 11 H2402 HLA-DPB1 IMGT-HLA HLA-DPB1*135:01 HLA-DPB1*02:01:02 HLA-DPB1*135:01 Assign Pending IMGT HLA SBT H2402 DRB1 SeCore DRB1 Locus Sequencing Kits Assign 2 Assign 3. まとめ SBT QC 22
Major Histocompatibility Complex 2013; 20 (: 23 24 DNA 1. 概要 16 QCWS 53 A B C DRB1 DRB3/4/5 DQB1 DPB1 DQA1 DPA1 A B 53 100% DRB1 52 98.1% C 41 77.4% HLA 2010 1.1 1.1 改訂箇所 ( II ambiguity 2. 2 4 2 / 2 HLA-A2/210 H2401 A*02:01/02/03/+ A2/203 A*02:03 A203 A*24:02/03/04/+ A24/2403 A*24:02 A24 3) 改訂箇所 (3) IV HLA HLA-C HLA WHO HLA HLA 1 HLA HLA-C HLA-C*12 C*18 HLA 1 HLA HLA / 3 4 /+ 2) 改訂箇所 (2) IV HLA HLA DNA HLA HLA HLA / HLA-DRB1*04:03/05/06/+ HLA-DR4 HLA-A*02:06/10/21/+ 表 1 DNA ambiguity 15 A*24 L 15 N 15 : 5 A*2602 A*24:02/02L/03/+ A*24:02/09N/10/+ 5 24:02/03/04/+ * 5 24:02/03/04/+ 5 DRB1 15 undefined 5 Blank 5 A* 10 DRB1*14:54/01/02/+ 23
MHC 2013; 20 ( 10 2. 結果 表 2 HLA / 10 10 B15 B62/B15 B62 B15 1 10 Cw12 blank undefined 5 DRB 10 * DRB1 Cw14* DNA HLA A B 3. 考察 DNA HLA HLA 2 HLA 2010 24
Major Histocompatibility Complex 2013; 20 (: 25 26 FlowPRA 1. 概要 FlowPRA Negative Control Positive Control FCS FCS FlowJo Tree Star, Inc. USA FlowPRA 2. FlowPRA 法の検査状況 FlowPRA Screening IgG test HLAClass I 19 HLAClass II 18 9 16 5 FlowPRA Screening IgM test HLAClass I 2 2 2 2 HLAClass II 2 2 2 2 FlowPRA Single Antigen IgG test HLA- Class I 3 HLAClass II 3 2 3 1 11 8 3. FlowPRA Screening IgG test 解析結果 4 Sample HLAClass I IgG SH2401 SH2403 SH2404 SH2402 SH2401 100% SH2402 84.2% 2 1 SH2403 100% SH2404 84.2% 2 1 HLAClass II IgG SH2401 SH2404 SH2402 SH2403 4 Sample 100% % HLAClass I & II FCS Negative Control Anti-Human IgG-FITC FCS Control Beads 1 SH2401 SH2403 Gate HLA- Class I Beads SH2402 SH2404 Sample 4. FlowPRA Screening IgM test 解析結果 4 Sample HLAClass I IgM SH2401 50% SH2402 100% SH2403 100% SH2404 50% HLAClass II IgM 4 Sample 100% HLAClass I IgM SH2401 SH2404 Nega- 25
MHC 2013; 20 ( tive Control IgM Negative Control serum IgG Negative Control serum Lot. IgM 5. FlowPRA Single Antigen IgG test 解析結果 FCS Cut-off 6. まとめ FlowPRA Screening % FlowPRA Single Antigen test Cut-off 26
Major Histocompatibility Complex 2013; 20 (: 27 LABScreen HLA 1. はじめに LABScreen 21 14 13 13 1 1 Mixed Multi PRA Single Antigen SA 9 SA 11 Mixed 6 PRA 4 Multi 1 1 HLA Fusion 2. 結果の解析および考察 2.1 判定の不一致 Class I SH2402 Class II SH2402 SH2403 SH2402 Class I 14 6 1 SH2402 Class II 14 3 2 SH2403 Class II 16 2 cutoff Baseline Normalized Value BNV 2.2 cut off の設定について HLA Fusion cut off BNV500 1,000 cutoff Threshold cutoff cutoff cutoff BNV900 BNV600 2.3 実測値のバラツキ Positive Control Beads BNV 10,000 beads QCWS 2.4 その他の判定不一致 QCWS 2 3. まとめ LABScreen HLA cutoff cutoff SA SA PRA HLA 27
Major Histocompatibility Complex 2013; 20 (: 28 29 WAK Flow ICFA 1. はじめに WAKFlow HLA WAKFlow MR I 10 II 6 ICFA 5 Table 1 I M0A 1 L0B 7 3 II L0A 3 L0B 2 K0B 1 Table 2 16 QC HLA WAKFlow ICFA 2. WAKFlow-MR クラス I 2.1 ビーズ反応性の比較 BB PB M0A Fig. 1 BB, PB PB S04 S17 S05 Fig. 2 2.2 サンプルごとの反応性について HLA Table 3 6 Fig. 3 6 Index Index Fig. 7 11 WAKFlow-MR C SH2403, 2404 C Fig. 10, 11 2.3 施設間での結果の不一致例について Fig. 12 16 Fig. 12 Fig. 13 15 Fig. 16 3. WAKFlow-MR クラス II HLA 1 I Table 28
MHC 2013; 20 ( 13 Fig. 17, 18 4. ICFA 法 ICFA I 5 II 1 I 4 25 II 4 ICFA Table 14 ICFA Index Fig. 22 24 SH2401 ICFA LABScreen Single Antigen LS-SA BNV 5,000 Fig. 22 SH2403 LS-SA Cw9, 10, 1, 14 ICFA Cw1, Cw14 Fig. 23 SH2404 LS-SA Cw5, Cw8 ICFA Cw5 SH2403 C Fig. 24 5. まとめ II 1 WAKFlow-MR IgM WAKFlow-MR I C ICFA ICFA LS-SA BNV 5,000 WAKFlow-MR I LS-SA BNV 15,000 C C WAKFlow-MR HLA 29
Major Histocompatibility Complex 2013; 20 (: 30 検査法別解析その他検査法およびクロスマッチ 1. その他検査法 HLA FlowPRA LABScreen LCT LIFT LCT AHG-LCT 2 LIFT1 MPHA4 LIFT HLA 2. クロスマッチ 9 13 6 11 SH2401 LCT4 LIFT5 ICFA9 SH2401 B35 LABScreen Single BNV 1,500 ICFA Index 2.0 A24 LABScreen Single BNV 6,000 LIFT QC A24 HLA α1 α2 SH2401 HLA H2401 H2403 QC 11 15 DNA-QC QC QC HLA 30
Major Histocompatibility Complex 2013; 20 (: 31 32 第 16 回 HLAQC ワークショップレポート 部門別解析 DNA-QC および結果評価 1. はじめに DNA-QC QCWS HLA HLA DNA 1 DNA-QC 53 56 37 24 19 7 22 1 2. 使用タイピング法について SSO Luminex SSP 54.1% 2 SSO INNO-LiPA 100% 3 HLA 4 HLA-A B DRB1 100% HLA-C SSO INNO- LiPA 4 50% HLA-DRB3/4/5 HLA-DQB1 HLA SSO Luminex SBT SSP SSO INNO-LiPA 3. 結果評価 3.1 概要 DNA-QC HLA-QC QCWS 3 3.2 判定結果の評価 HLA-QC HLA HLA 60 0 QCWS 56.5 2 54.8 SSO/SSP 14th QCWS 57.3 15th QCWS 53.4 16th QCWS 56.5 15th QCWS SSO/SSP 3.3 結果表記の評価 3 2 31
MHC 2013; 20 ( DNA HLA HLA-C HLA HLA-C*12 C*18 HLA 40 QCWS 38.7 3 QCWS 38.5 3.4 試験 検査状況の評価 HLA A B C 5 SSO INNO-LiPA B 4. 総合評価 4 95.3 92.8 100 A 60 100 B 0 60 C A 26 B 27 C 0 C 0 4 14th QCWS 45 QCWS 5 14 16th QCWS QCWS 32
Major Histocompatibility Complex 2013; 20 (: 33 34 部門別解析および結果評価 ( 抗体部門 ) 1. 概要 22 23 15 15th 1 37 37 34 FlowPRA 18 LABScreen 14 WAKFlow 9 LABScreen single antigen 8 5 2. 部門別解析 15th 4 I II 97% 83.0% 100% SH2402 Consensus IgM 100 Class I 86.7% 86.4% 65.2% Class II 73.3% 63.6% 60.9% 15th 3. 結果評価 3.1 抗体 QC 結果評価に対する考え方 15th 0.67 2/3 SH2402 I HP 3.2 評価内容 1 A 80 32 86.5% B 40 80 5 13.5% SH2404 Cw8+Cw5 LABScreen single antigen 2 27 A 21 77.8% B 3 11.1% C 40 3 11.1% A B 38.9 33
MHC 2013; 20 ( 36.8 33.0 3 3 Class I 42 Class II 23 Class I 42 7 27 25.9% 7 1 1 4 42% 78.6% Class II 1 65.2% Class I 4 1 4 Consensus Result Consensus Result Score 1 Score 8 Consensus Result Score 8 Score 1 2 27 21 77.7% LABScreen FlowPRA single antigen MFI 1,000 Score 8 HP 4. 結語 16QCWS 15QCWS C 3 IgG/M Score 8 Single antigen QCWS Score 8 1 0 8 4 Score QCWS QCWS 34