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Improvement of tests before transfusion treatment

Enhance blood group antibody testing accuracy and efficiency. Learn about innovative techniques such as Chemiluminescence Test and Monocyte Monolayer Assay. Explore the significance of antibody titers through Continuous Flow Analyser. Discover the role of Recombinant Blood Group Proteins in identifying complex and rare antibodies. Improve antigen quantitation with synthetic A antigens and rBGPs. Delve into detecting specific blood types like MUT and Mur with advanced technology.

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Improvement of tests before transfusion treatment

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  1. Improvement of tests before transfusion treatment Ziyan Zhu Shanghai Blood Center 2018-09

  2. RBC 001 / ABO (319) 002 / MNS (47) 003 / P1(48) 004 / Rh (437) 005 / Lutheran (20) 006 / Kell (69) 007 / Lewis (74) 008 / Duffy (11) 009 / Kidd (26) 010 / Diego (91) 011 / Yt (4) 012 / Xg (2) 013 / Scianna (9) 014 / Dombrock (15) 015 / Colton (8) 016 / LW (3) 017 / CH/RG (7) 018 / Hh (108) 019 / Xk (35) 020 / Gerbich(10) HPA GP3A (16) GP2B (7) GP1A (7) GP1BA (2) GP1BB (1) CD109 (2) CD36(2) Mou(2) 021 / Cromer (15) 022 / Knops (31) 023 / Indian (4) 024 / Ok (5) 025 / RAPH (3) 026 / JMH (9) 027 / IGNT (13) 028 / GLOB (13) 029 / GIL (2) 030/RHAG(23) 031/FORS(2) 032/ Jr(23) 033/Lan(21) 034/Vel(2) 035/ CD59 036 / Aug HLA HLA-A (2368) HLA-B (3015) HLA-C (1846) HLA-E (13) HLA-F (22) HLA-G (48) HLA-H (12) HLA-J (9) HLA-K (6) HLA-L (5) HLA-P(5) HLA-V(3) HLA-DRA (7) HLA-DRB1 (1456) HLA-DRB2 -9(101) HLA-DQA1 (47) HLA-DQB1 (516) HLA-DPA1 (33) HLA-DPB1 (190) HLA-DMA (7) HLA-DMB (13) HLA-DOA (12) HLA-DOB (13) Plasma IGHG1 (4) IGHG2 (1) IGHG3 (13) Km (3) Gm(1) Am (2) HNA HNA-1 (3) HNA-2 (1) HNA-3 (1) NHA-4 (1) NHA-5 (1) Human blood group system that has been discovered Data s from： RBC：NCBI dbRBC.Updated 2013-08-11 HLA：European Molecular Biology Laboratory Updated 2013-07 , HPA ; European Molecular Biology Laboratory Updated 2013-07

  3. How to determine whether the antibodies have clinical significance. • Standard in vitro laboratory analysis • Antibody specificity • 37C Preheating experiment • Hemolysis experiment • In vivo analysis • 51Cr RBC survival in vivo • In vitro cell analysis • Chemiluminescence Test（CLT） • Antibody-dependent cytotoxicity test （ADCC） • Monocyte monolayer assay （MMA）

  4. Assays of Antibody Function Monocyte monolayer assay Rosette assay Recognition Phagocytosis Monocyte Lysis Antibody-dependent cell-mediated cytotoxicity assay Luminol Oxygenradicals light Chemiluminescence assay

  5. 巨噬细胞吞噬红细胞

  6. Monocyte Monolayer Assay （MMA）

  7. Isolation, washing of monocytes

  8. Mononuclear cells were plated on slides for 37C incubation (mononuclear adhesion)

  9. Add sensitized RBC, incubate at 37C

  10. Remove the supernatant and remove the small compartment

  11. Rinse the slide and remove the washer

  12. Wright's dye stain

  13. MMA positive result

  14. MMA negative result

  15. US Red Cross National Reference Laboratory MMA Results Statistics（ 1995-2011）

  16. Titer Vs Score Agglutination strength Score Titer 1/256 Score83

  17. Gold standard for anti-D and anti-c titer assay CAF • Continuous Flow Analyser (CFA) • Fully automated antibody quantitative analysis system, internationally recognized "gold standard“ • Quantitative determination of anti-D and anti-c • UK Guidelines for blood typing and antibody detection during pregnancy (2016), Antibody titers for RhD-negative pregnant women should be determined using CFA • When the anti-D>10~12IU/ml in maternal plasma is considered clinically significant

  18. Classification of the risk of anti-D in British pregnant women • Low risk: < 4 IU/mL: The fetus does not need continuous monitoring. • Moderate risk: 4 – 15 IU/mL: Expert assessment is required and continuous monitoring of the fetus is required. • High risk: >15 IU/mL: requires expert assessment and continuous monitoring of the fetus.

  19. 流式（FC）、效价积分（TS）、柱凝集（CAT）是否可替代连续流分析仪（CFA）流式（FC）、效价积分（TS）、柱凝集（CAT）是否可替代连续流分析仪（CFA）

  20. Antigen quantitation in IQC • Antigen quantitation • Diversity of A antigen expression between different Ax individuals • Synthetic A antigen (carbohydrate)Kodecyte • “plant” A antigen on O type RBCs • Can be used in quantitative detection of blood group antibodies

  21. Recombinant blood group proteins (rBGPs) • Institute of Transfusion Medicine and Immunohaematology, German Red Cross; Harvard Medical School, US • Beneficial tothe identification of complex antibodies and rare antibodies • Have got the CE certificate

  22. Current rBGPs

  23. Advantages of rBGPs • To detect the corresponding antibody by carrying a single blood group protein • To be used in the qualitative and quantitative detection of the blood group antibodies • To enhance the sensitivity of antibody identification • To be combined with solid Phase technique (ELISA, bead, microarray)

  24. Synthetic MUT+Mur antigen (Miltenberger) detection anti-MUT+ anti-Mur • Important blood type in Asian population • Molecular mechanism • GYPA-GYPB • Synthetic • FSL-MUT SQTNDKHKRDT • FSL-Mur TYPAHTANE • Can be used in tube test, CAT, ELISA, immunofluorescence detection

  25. Kodecyte predicts in vivo RBC in vivo survival rate mouse model

  26. Detection of anti-Dob contained in anti-Fya

  27. Feature of the reaction between blood group antigen and antibody • Interaction between proteins • Currently used precise detection methods • Surface Plasmon Resonance (SPR) • Biolayer Interferometry (BLI) • Microscale Thermophoresis (MST) • Back-scattering Interferometry(BSI)

  28. Surface Plasmon Resonance (SPR) • Have been used in the affinity detection of anti-A, anti-B, and anti-D • Have been more widely used in the detection of platelet antibody

  29. MST

  30. Thank SBC/WHOCC-BTS Email:zhuziyan@sbc.org.cn Tel/Fax:+85 021 62085329

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