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20151107 workshop HPA, HLA, HEA genotype
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- 2. • Introduction toHEA • Introduction to HPA • Introduction to HLA • Application of HEA genotype • Application of HPA genotype • Application of HLA genotype • FluoGene / marked automation for molecular HLA-, HPA-, RBC- typing Outline
- 3. • 1900：Landsteiner－ Plasmafrom some individuals would agglutinate the red cells from others. ＞＞ABO • 1927：Landsteiner & Levine－ Only those antibodies directly agglutinate red cells could be studied. ＞＞MN & P • 1945：Coombs、Mourant & Race：Developed antiglobulin test, thus non- or weak- agglutining antibodies could be detected and the science of blood group serology blossomed. ＞＞ Other 20 major system RBC blood group brief (Lancet 1945,2:15 & Br J Exp Pathol 1945, 26:255-266)
- 5. 國際輸血學會(ISBT)的紅血球血型抗原命名委員會為了方便電腦化作業,將 20世紀發現的血型,自從1990年(242抗原)加以整理整合及命名在1995年將 血型分為三類: 截至今日35個血型系統至少超過有270個抗原 ; 6個血型集合有12個抗原 (200系列)、700系列有17個抗原、901系列有7個抗原。 35個血型系統(為單一基因或很接近的基因控制下產生的抗原,這些抗原在 serology或biochemistry上關係密切). 6個血型匯合(Collection在genetic,serology或biochemistry上相關,但沒有 完全符合System的條件. 血型系列(series) a.低頻率700系列(頻率<1%) b.高頻率901系列(頻率>99%) ISBT nomenclature http://www.isbtweb.org/working-parties/red- cell-immunogenetics-and-blood-group- terminology/
- 6. • RBC antigens( ABO antigens, Rh, Lewis, Duffy ….) • Human leukocyte antigen (HLA) • HLA class I • Human platelet antigens (HPA) • 28system 34 antigens • Glycoprotein IV (CD36, Naka ) • 亞洲及台灣約1.5% negative , 可分為type I, type II Human platelet antigens
- 7. HPA British Journal ofHaematology Volume 161, Issue 1, pages 3–14, 1474 1 11 80,000 mol/plt 25,000 mol/plt9600 mol/plt 1,000 mol/plt
- 8. MHC locus On chromosome6 HLA class III. are soluble molecules as complement, TNF, HSP Many other proteins involved in antigen presentation HLA genes
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- 10. Application of HEAgenotype • Transfused recently • No anti-sera eg.anti-HUT, anti-Mur, anti-Luth3, Dib, Wrb…… • High prevalence Ag or null form confirm eg.JK3, D--, Dnull, Knull …… • ABO HDN prediction • Blood group subgroup eg. ABO subgroup, D variant
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- 12. Antibodies to highprevalence antigen All manual Polybrene screening lcells: positive <3 mouth transfusion, DO NOT do MP AC Classic AHG IAT +AC MP and /or classic AHG IAT +AC, if < 3 mouth no transfusion AC negative AC positive All reactive same strength ≦ 1+ 2X dilution~256 Titer ≧32 High titer low avidity HTLA antibodies All same reactive strength Titer < 32 IAT positive IAT negative Auto immune antibodies Drug-induced antibodies, almost drug dependent AC > IAT Auto immune antibodies Drug-induced antibodies, some drug independent. If forward type O type, check anti-H H(-), paraBombay or Bombay H(+) All cell reactive, but not same reactive strength AC < IAT Only one or few panel cells not reactive Ethnic relative high prevalence antigen Phenotype D, s, Lea, Leb, Fya. Multiple antibodies Dosage effective single Ab to high prevalence Ag See : multiple antibodies identification procedure anti-Dib(Medipro SRC) anti-Wrb(Some panel), anti-k, anti-Kpb Check phenotype Dia, Wra, K, Kpa All same mix field reactive strength Anti-Lub, (DTT destroyed) Anti-Sda( G-P urine inhibition) Differentiated by common panel cell Drug-induced antibody investigation Thermo aptitude study Donath-Landsteiner test Cold agglutinin titration Warm auto immune hemolytic anemia study AC positive ≧2+ Eluent and serum Classic AHG screening cells positive Anti-Sda: mixed reaction, neutralization by GP urine Anti-Rg/Ch: plasma inhibition Check phenotype RhD, RhCE, Kidd, Duffy , MNSs, Kell, Rh: anti-Hr0 Kidd: anti-Jk3 Duffy: anti-Fy3 MNSs: anti-U Kell: anti-Ku, anti- Kx ( weak Kell) Ficin( papain), 0.2M DTT treated cells cord i cells, IgG1, IgG3 hemolytic potent evaluation IgA/IgG subclass Enzyme +, DTT+ Anti-Jra Anti-Vel(cordi w) Anti-p Anti-Tja Enzyme +, DTT 0 Anti-Kpb Anti-AnWj (cordi 0) Anti-Jsb (EGA 0) Anti-Lub Anti-Lu3~Lu21 (exp.Lu8) Enzyme 0, DTT 0 Anti-JMH (cordi w) Anti-Inb Anti-Yta Enzyme 0,DTT + Anti-Ch/Rg +: reactive, 0: non-reactive, w: weak reaction enzyme : papain/ ficin HTLA Ab is not an antibody or antigen Enzyme 0, DTT 0: anti-JMH Enzyme 0, DTT +: anti-Ch/Rg Enzyme +, DTT +: anti-Csa Enzyme 0, DTT w: Knop Ab (anti-Kn, McCa, Sla, Yka) Yka can be destroyed by Neuraminidase If doubt of drug-induced antibody; eluate, fresh serum and drugs were prepared for study
- 13. RBC-FluoGene kits ARTICLE REACTIONEN/TESTTESTS/KIT RBC-FluoGene ABO basic 17 40 / 10 RBC-FluoGene vERYfy RHD: Exone 1, 5, 10, psi; RHCE: C, C W , c, E, e; KEL1(K), KEL2(k), JK1(Jk a ), JK2(Jk b ), FY1(Fy a ), FY2(Fy b ), FYnull(Fy a -, Fy b -), FYX(Fy bweak ), MNS1(M), MNS2(N), MNS3(S), MNS4(s), DO1(Do a ), DO2(Do b ) 32 30 / 10 RBC-FluoGene CDE RHD: Exone 1-7, 9, 10, psi, DNB, D cat VII, DHMi, DAU; RHCE: C, C w , c, E, e 24 40 / 10 RBC-FluoGene D weak/variant weak D type 1, 1.1, 2, 3, 4.0/ 4.1, 4.2 (DAR), 5, 11 (M295I), 14, 15, 17, K409K, IVS3+1G>A 16 48 / 40 / 10 RBC-FluoGene KKD KEL1(K), KEL2(k), JK1(Jk a ), JK2(Jk b ), FY1(Fy a ), FY2(Fy b ), FYnull(Fy a -, Fy b -), FYX(Fy bweak ) 13 10 RBC-FluoGene MNS MNS1(M), MNS2(N), MNS3(S), MNS4(s) 6 10 RBC-FluoGene Rare DI1(Di a ), DI2(Di b ), DI3(Wr a ), DI4(Wr b ), YT1(Yt a ), YT2(Yt b ), LU1(Lu a ), LU2(Lu b ), KEL3(Kp a ), KEL4(Kp b ), KEL6(Js a ), KEL7(Js b ), CO1(Co a ), CO2(Co b ), KN1(Kn a ), KN2(Kn b ) 32 30 / 10 RBC-FluoGene Vel-Screen Für Vel + und Vel - Screening. 2 48
- 14. Application of HPAgenotype • Platelet transfusion refractory • Neonate Allo Immune Thrombocytopenia Purpura NAITP Neonatal alloimmune thrombocytopenia (NAIT) Platelet transfusion refractoriness (PTR) Post-transfusion thrombocytopenic purpura (PTP) Passive alloimmune thrombocytopenia (PAT) Transplantation-associated alloimmune thrombocytopenia (TAATP) Dr. N.H. Tsuno presented in 24th regional congress of ISBT
- 15. Platelet transfusion strategy DtschArztebl Int. 2014 Nov; 111(48): 809–815.
- 16. Application of HLAgenotype • Pharmacogenetics of disease • HLA-B*58:01, HLA-B*57:01, HLA-B*15:02, HLA-A*30:01 • Defense or protective/sensitive gene • Leukemia HLA-DRB1*12:01 increase, HLA-DRB1*15:01 decrease • GWAS HBV HLA-DP, -DQ • Disease association • HLA-B27, HLA-A*06:01 • Transplantation • Platelet refractory • NAITP( few case) • TaGVHD/TRALI investigation.
- 17. • 為什麼血小板的輸注及檢驗可以不像輸紅血球一般做pretransfusion test? • 為什麼紅血球交叉配血(最低)不相容需通知醫師，但血小板交叉不合不 需要通知醫師? •血小板輸注無效不會影響病人安全，頂多再多輸一點血小板也可以改善 病人出血狀況? • 輸血小板都是非常急迫的，不能等待血小板的交叉結果及血小板的抗原 genotype? • 血庫的人力不足，根本無法接受platelet antibody screening , cross match, 及HPA/HLA genotype的檢驗工作? • 醫檢師存在的價值在於提供有價值的檢驗資料. Platelet transfusion refractory (PTR)
- 18. Comparison of HPAand HLA antibody in Taiwan Lin et al. 2003 Chang et al. 2009 Chu et al. 2013 People with Positive Antibody 57 23 51 Positive HLA Antibody Only 38 (66.7%) 8 (34.8%) 7 (13.7%) Positive HLA & HPA Antibody 11 (19.3%) 6 (26.1%) 24 (47.1%) Positive HPA Antibody Only 8 (14.0%) 9 (39.1%) 20 (39.2%)
- 19. Allo immune ofPTR 2008 (40 case study) 2014 (23 cases study) HLA 12 33.3% 2 8.6% (11.7%) HLA+ platelet specific 22 55% 11 47.8% (64.7%) Platelet specific 5 12.5% 4 17.4% (23.5%) unidentified 1 2.5% 6 Total 40 23
- 20. FluoGene FluoGene New technology platformof DNA testing for HEA, HLA, HPA 20 瀚 揚 有 限 公 司 0 2 - 8 7 5 1 - 1 3 4 5 0 7 - 5 5 5 - 7 9 2 7
- 21. 21 • SSP利用不同的PCR primer針對各 HLA分型的特異位置進行放大，藉由 特定PCR產物的有無，判斷基因型別 •與SSO或SBT法相比，SSP的 • 優點：不需要特別昂貴的儀器 技術門檻低。 適合少量檢體或緊急需求。 • 缺點：需耗用較大量的DNA檢體 自動化程度低 大量的人工操作 難以同時進行大量檢體。 HLA-SSP (SEQUENCE-SPECIFIC PRIMERS) FluoGene
- 22. 22 FluoGene FLUOGENE TECHNIQUE 以 TaqManprobe 偵測PCR反應 當試劑的primer、 probe與檢體DNA相 符，PCR反應中probe被水解而釋出可被 激發螢光 qPCR為了定量，需用定量PCR儀在反應 中讀取每個cycle的螢光強度 FluoGene為定性檢驗，可使用一般的 PCR儀器，在反應完成後讀取最終的螢 光強度
- 23. 23 • FluoGene：endpoint qPCR •反應後測量終端螢光強度，取代電泳步驟 • 大幅縮短實驗時間 (無須配膠、電泳、照相) • 減少汙染 (不須開啟PCR產物、無EtBr染色) • 軟體自動分型 FLUOGENE FluoGene
- 24. 24 • 試劑盤 • 96孔白盤，附有條碼與顏色識別標記 •定量PCR用光學封膜 • 三色TaqMan螢光組，乾燥於盤底 • Channel 1：HGH control • Channel 2：HLA(or RBC、HPA)識別 • Channel 3：第二組的識別探針 • 可見染劑：NTC淡紅色，specific淡藍色 • 試劑 FluoMix • 含polymerase 兩倍濃度預混PCR試劑 • 每檢體一管，不須分裝 FLUOGENE 產品特點 FluoGene
- 25. 25 FluoGene Pre-read FluoMix + DNA +ddH2O PCR Post-read Automation analysis
- 26. 26 開啟FluoVista與軟體、取出試劑回到室溫 每支檢體各使用一支MasterMix ABC 780 μL, DRDQ 270 μL 在NC(黑色標記)加入7.5μL Master Mix 在NC(黑色標記)加入7.5μL nuclease-free water 加入DNA及nuclease-free water ABC ： 780 ng DNA 補水至 780 μL (總體積1560 μL) DRDQ： 270 ng DNA補水至 270 μL (總體積540 μL) 混合後，除NC外 每個well 加入15μL。以光學透明膜封妥 預備反應
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- 29. 29 PCR 反應 Initial 95℃,2 min 40 x Cycles 95℃, 15 sec 60℃, 60 sec Cool down 20℃, 3 min 20℃, 15 min ~80 min 98℃, 2 min 98℃, 20 sec 60℃, 60 sec 20℃, 3 min 20℃, 15 min For Bio-Rad C1000 S1000
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- 31. 31 FluoGene (post-read – pre-read)> cut-off = positive reaction “+” (post-read – pre-read) < cut-off = negative reaction “-” 分別條列 CWD 與 rare 對應的血清學分型標示 顯示可能的高解析結果 自動分析結果
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- 33. 33 HLA-ABC TEST SYSTEM =3 primer-/probe mixes 1 = contamination control 96 reactions per plate; 10 tests per kit HLA-A HLA-B HLA-C 32 56 31 1 2 3 4 5 6 7 8 9 10 11 12 A 8 16 24 8 16 24 32 40 48 8 16 24 B 7 15 23 7 15 23 31 39 47 7 15 23 C 6 14 22 6 14 22 30 38 46 6 14 22 D 5 13 21 5 13 21 29 37 45 5 13 21 E 4 12 20 4 12 20 28 36 44 4 12 20 F 3 11 19 3 11 19 27 35 43 3 11 19 G 2 10 18 2 10 18 26 34 42 2 10 18 H 1 9 17 1 9 17 25 33 41 1 9 17 Lot: F973065 2017-05 0123 衛部醫器輸字 第027088號 HLA-FluoGene
- 34. 34 32 reactions;1tests per plate; 10 tests per kit 3 tests per plate; 30 tests per kit 1 2 3 4 5 6 7 8 9 10 11 12 A 8 16 24 32 8 16 24 32 8 16 24 32 B 7 15 23 31 7 15 23 31 7 15 23 31 C 6 14 22 30 6 14 22 30 6 14 22 30 D 5 13 21 29 5 13 21 29 5 13 21 29 E 4 12 20 28 4 12 20 28 4 12 20 28 F 3 11 19 27 3 11 19 27 3 11 19 27 G 2 10 18 26 2 10 18 26 2 10 18 26 H 1 9 17 25 1 9 17 25 1 9 17 25 HLA-DRB1/DRB345/DQ 22 5 9 HLA-DR/DQ TEST SYSTEM = 3 primer-/probe mixes 1 = contamination control 0123 衛部醫器輸字 第027204號 Lot: F977045 2017-03 HLA-FluoGene
- 35. 35 FluoGene FLUOGENE 產 品特 點 • 檢體用量少 – 780ng DNA for ABC – 270ng DNA for DRDQ SSP試劑DNA標準量 ABCDRDQ (容許範圍 ±50%) Inno-Train 1,050 ng Brand T : 6,060 ng Brand I : 8,350 ng Brand O: 15,000 ng • 低汙染 – 每個檢體個別使用已備 妥之單管試劑 – 反應後不須開啟，環境 無PCR產物汙染 – 不須使用有害的EtBr • 多用途 – HLA、RBC、HPA typing
- 36. 36 FluoGene • 省時、省人力 – 不須製膠、電泳、染色、 照相、判讀、輸入等步驟 沒有額外的費用!! –程式自動分析 FLUOGENE 產 品 特 點 • 使用單位 – 人力較少的單位 – 新建置HLA實驗室 – 緊急檢驗 – 血庫
- 37. 37 許可證字號 有效日期 中文品名英文品名 申請商 製造廠 1 衛部醫器輸字 第027088號 109/03/05 一諾人類白血球抗原 ABC分型套組 INNO HLA-FluoGene ABC 瀚揚有限公司 INNO-TRAIN DIAGNOSTIK GMBH 2 衛部醫器輸字 第027203號 109/04/09 一諾人類白血球抗原 ABDR分型套組 INNO-TRAIN HLA-FluoGene ABDR 瀚揚有限公司 INNO-TRAIN DIAGNOSTIK GMBH 3 衛部醫器輸字 第027204號 109/04/09 一諾人類白血球抗原 DRDQ分型套組 INNO-TRAIN HLA-FluoGene DRDQ 瀚揚有限公司 INNO-TRAIN DIAGNOSTIK GMBH 4 衛部醫器輸字 第027205號 109/04/13 一諾人類白血球抗原 B27試劑 INNO-TRAIN HLA-FluoGene B27 瀚揚有限公司 INNO-TRAIN DIAGNOSTIK GMBH 5 衛部醫器輸壹 字第015045號 109/03/26 "一諾" 螢光讀取分析儀 (未滅菌) "Inno-train" FluoVista Analyzer (Non-sterile) 瀚揚有限公司 INNO-TRAIN DIAGNOSTIK GMBH 西藥、醫療器材、含藥化粧品許可證 HLA-FluoGene
- 38. 38 HPA-FLUOGENE HPA-FluoGene 8 HPAreactions + 1 NC / typing HPA-1 a/b, -2 a/b, -3 a/b, -4 a/b, -5 a/b, -6 a/b, -9a/b, -15 a/b 單一反應檢驗對偶基因的SNP 一對引子雙重探針偵測三種結果 aa, ab, bb a …GCCCTGCCTC C GGGCTCACCT… b …GCCCTGCCTC T GGGCTCACCT… F-primer R-primera-probe F-primer R-primerb-probe
- 39. 39 System Glycoprotein CDSNP Antigen Original names Frequency in Taiwanese HPA-1 GPIIIa CD61 176T>C HPA-1a Zw a , Pl A1 99.55 HPA-1b Zw b , Pl A2 0.45 HPA-2 GPIbalpha CD42b 482C>T HPA-2a Ko b 96.49 HPA-2b Ko a, Sib a 3.51 HPA-3 GPIIb CD41 2621T>G HPA-3a Bak a , Lek a 55.81 HPA-3b Bak b 44.19 HPA-4 GPIIIa CD61 506G>A HPA-4a Yuk b , Pen a 99.75 HPA-4b Yuk a , Pen b 0.25 HPA-5 GPIa CD49b 1600G>A HPA-5a Br b , Zav b 98.50 HPA-5b Br a , Zav a , Hc a 1.50 HPA-6w GPIIIa CD61 1544G>A HPA-6a -- 97.75 HPA-6b Ca a , Tu a 2.25 HPA-9w GPIIb CD41 2602G>A HPA-9a -- HPA-9b Max a HPA-15 CD109 CD109 2108C>A HPA-15a Gov b 53.71 HPA-15b Gov a 46.29 HPA SYSTEM HPA-FluoGene
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- 41. 41 RBC-FluoGene RBC-GENOTYPING • 紅血球抗原基因分型 • 以血清學方式可以快速鑑定RBC抗原 •但是對於 最近有輸血的病患 反應較弱的抗體 或罕見型別就較難以鑑定 • 歐美已推廣使用基因分型 • 產前診斷(Rh-) • 鐮狀紅血球病患配血 • 稀有血型鑑定
- 42. 42 ARTICLE Rxn/TEST RBC-FluoGene ABObasic 01,02, B, A, A2 17 RBC-FluoGene vERYfy RHD: Exone 3/ 5, 10, psi; RHCE: C, C W , c, E, e; KEL1(K), KEL2(k), JK1(Jk a ), JK2(Jk b ), FY1(Fy a ), FY2(Fy b ), FYnull(Fy a -, Fy b -), FYX(Fy bweak ), MNS1(M), MNS2(N), MNS3(S), MNS4(s), U+var(P2), U+var(NY), DO1(Do a ), DO2(Do b ) 32 RBC-FluoGene CDE RHCE: C, C w , c, E, e RHD: Exone 1-7, 9, 10, psi, DNB, D cat VII, DHMi, DAU 24 RBC-FluoGene D weak/variant weak D type 1, 1.1, 2, 3, 4.0/ 4.1, 4.2 (DAR), 5, 11 (M295I), 14, 15, 17, K409K, IVS3+1G>A 16 RBC-FluoGene KKD KEL1(K), KEL2(k), JK1(Jk a ), JK2(Jk b ), FY1(Fy a ), FY2(Fy b ), FYnull(Fy a -, Fy b -), FYX(Fy bweak ) 13 RBC-FluoGene MNS MNS1(M), MNS2(N), MNS3(S), MNS4(s) 6 RBC-FluoGene Rare DI1(Di a ), DI2(Di b ), DI3(Wr a ), DI4(Wr b ), YT1(Yt a ), YT2(Yt b ), LU1(Lu a ), LU2(Lu b ), KEL3(Kp a ), KEL4(Kp b ), KEL6(Js a ), KEL7(Js b ), CO1(Co a ), CO2(Co b ), KN1(Kn a ), KN2(Kn b ) 32 RBC-FluoGene Vel-Screen Vel + and Vel - Screening 2 RBC-FLUOGENE RBC-FluoGene
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- 44. 44 RBC-FLUOGENE VERYFY Rhesus rare MNSKKD RHD psi RHD exons 1, 5 & 10 C, c, CW, E, e MMS1 (M) MNS2 (N) MNS3 (S) MNS4 (s) Dombrock a/b KEL1, KEL2 JK*A, JK*B FY*A, FY*B FY*X, FY*null serological unclear Rhesus typings samples from poly transfused patients patients with allo-antibodies RBC-FluoGene
- 45. 45 RBC-FLUOGENE CDE For unclearserological rhesus typings RHD Allel: overall RHD result RHD: Exon 1, 2, 3, 4, 5, 6, 7, 9, 10 and RHD Ψ RH-CATPA: “Category and Partial” RHCE: C, c, E, e and Cw RBC-FluoGene CDE
- 46. 46 HLA HPA RBC KIR SSP FluoGene SBT Software,Instrument HLA HLA HPA RBC 德國 法蘭克福 1998, FIRST IVD RBC GENOTYPING KIT
- 47. 47 Thank You HPA FluoGeneReadyGene SuBiTo RBC HLAKIR ReadyPlate