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1 BD FACS TM Review Vol.7 BD FACSAria TM BD FACSAria FACS HLA-FACS Key words: HLA FACS 1988 Eliane Glukmann Fanconi % 30% mixed chimerism HLA human leukocyte antigen HLAFACS fluorescence activated cell sortor HLA-FACS I. fluorescence in situ hybridization FISH X/Y-FISH 2 short tandem repeat STR PCR STR-PCR 3 FACS FACS 1 HLA 2 HLA HLA FACS3 FACS
2 1. HLA graft versus host disease GVHD major histocompatibility complx; MHC HLA-A BDR 6 A B DR HLA1 2 GVHD HLA 2 795% MHCI HLA-A B 1 HLA HLA HLA-A2 HLA-B5 HLA HLA 2. HLA HLA 3. FACS FACS StanfordLeonald A. Herzenberg FACS-I Stanford FACS Facility FACS Pattern Sorter APC-Cy7 PE-Cy7 FACS Becton Dickinson BD FACS 1995 BD FACSVantage SE 2003 BD FACSAria BD 1 BD FACSAria 9 10 HLA HLA HLA HLA-AB HLA-DRDNA 1964 UCLA Paul I. Terasaki lymphocyte cytotoxicity test; LCT HLA HLA HLA HLA HLA LCTHLA-A B HLA HLA cross-reactive group One Lambda Terasaki HLA HLA HLA 5, 6 HLA HLA HLA 7 2
3 II acute myelogenous leukemia AML M mllineage ml AML/ETO FISH 2 myelodysplastic syndrome MDS RAEB mllineage STR-PCR HLA-FACS HLA One LambdaPacific Orange-CD14 Invitrogen BD HLA-A11 biotin 1 Stain 1 Stain 3 SA-PE Stain 2 SA-APC 2 HLA- A11 biotinhla-a26 1) HLAFITC PE lineage 1 Stain 1 Stain 2 2) HLA 1 Stain 3 FITC PE PE-Texas Red PerCP-Cy5.5 PE-Cy7 APC APC-Cy7 Pacific Blue Pacific Orange Detector Blue-E Blue-D Blue-C Blue-B Blue-A Red-B Red-A Violet-B Violet-A Stain 1 HLA-A24 HLA-A11 PI CD8 CD3 CD56 CD19 CD4 CD14 Stain 2 HLA-A24 CD11b PI HLA-A11 Stain 3 HLA-A24 HLA-A11 PI HLA-DR CD33 CD34 CD56 CD45 CD HLA-A11 biotin1 Stain 1 Stain 3 SA-PE Stain 2 SA-APC 2 HLA-A11 biotinhla-a26 Alexa Red Laser APC(Red-C), Alexa700(Red-B), APC-Cy7(Red-A)Alexa700PMT 685LP, 712/21BP 3
4 3. FACS 1) 5 10 ml 2) 50 ml Ficoll 15 ml 15mL Ficoll 4 mlpbs(-) 1.5 2,000 rpm 20OFF 3) Ficoll 50 ml 4) PBS(-) 1 PBS(-) 1 5) 100 µl 5% PBS(-) 6) FACS ) PBS(-) µl PBS(-) 1 µg/ml propidium iodide PIBD FACSAriaPI PE-TR FCS FlowJo Treestar III. 1. 1lineage T 4 1 TCD4 + T 2 BNK % % 3 5 NK ( 3) ml Stain 1 Stain 2 1PI BD FACSAria TBNK 1 TT
5 ml Stain 1 Stain 2 1 FSC-SSC PI CD4 + TCD8 + TBNK ml Stain
6 2. CD34-CD45 49 CD34 - CD45 + CD34 + CD45 dim 4A 4B 4AG2 G8 2 G2 G8 HLA HLA 4A CD34-CD45 G1 G8 G1CD34 Bright G6 CD34-CD45 4B 4A Stain 3 1 4A 49PIPI-CD34 CD45 G1 G8 8 PI- HLA G2 CD34 + CD45 dim G8 CD34 - CD45 + 4B G1 G8 CD14 CD34-CD45 4B 140 4B 6
7 3. 2 GVHD A CsA CD8 + T IFN B HLA-FACS STR-PCR STR-PCR 3 7 HLA-FACS 5 STR-PCR HLA-FACS CD34 + CD45 dim CD34 + CD45 dim 0.3% 1% FISH AML1/ETO 5% HLA-FACS 320 CD34 + CD45 dim 6 CD56 HLA-DR CD567 AML1/ETO 0% 7 100% 7 CD34 + CD45 dim FACSSTR-PCR CsACD8 + T IFN-CsA CsA CsA CD8 + TIFN- CsA PMA4CD8 IFN
8 ml Stain 3 1 AML-M2CD34 + CD45 dim CD34-CD45CD34 + CD45 dim 7 CD34 + CD45 dim ml Stain 3 1 AML-M2 CD34 + CD45 dim HLA-DR CD56 AML1/ETO FISH 8
9 IV. HLA-FACS 1 2 HLA-FACS BD FACSAria Windows BD FACSAria BD FACSAria Takahashi S, Ooi J, Tomonari A, et al. Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen. Blood. 2007;109: Turkiewicz D, Gorczynska E, Toporski J, et al. Monitoring of hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation (allosct) by dual-color FISH analysis of X and Y chromosomes. Leuk Res. 2003;27: Thiede C, Florek M, Bornhauser M, et al. Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection. Bone Marrow Transplant. 1999;23: Sijpkens YW, Doxiadis, II, De Fijter JW, et al. Sharing cross-reactive groups of MHC class I improves long-term graft survival. Kidney Int. 1999;56: Pei R, Chen T, Orpilla J, Lee JH. A simultaneous negative and positive selection method that can detect chimerism at a frequency of 1 per 10,000 by flow cytometry. Tissue Antigens. 1997;50: Metes D, Logar A, Rudert WA, et al. Four-color flow cytometric analysis of peripheral blood donor cell chimerism. Hum Immunol. 2003;64: Piazza A, Poggi E, Ozzella G, et al. Public epitope specificity of HLA class I antibodies induced by a failed kidney transplant: alloantibody characterization by flow cytometric techniques. Transplantation. 2006;81: Mascher B, Schlenke P, Seyfarth M. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J Immunol Methods. 1999;223:
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