BD FACS TM Review Vol.7 BD FACSAria TM BD FACSAria FACS HLA-FACS Key words: HLA FACS 1988 Eliane Glukmann Fanconi 1 5 10% 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
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 1960 1971 StanfordLeonald A. Herzenberg FACS-I1 2 1990 Stanford FACS Facility FACS 19958 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 4 1975 One Lambda Terasaki HLA HLA HLA 5, 6 HLA HLA HLA 7 2 www.bd.com/jp/
II. 1. 2 1 acute myelogenous leukemia AML M2 1 2 3 4 8 10 mllineage 159 281 3202 ml AML/ETO FISH 2 myelodysplastic syndrome MDS RAEB 11 5 10 mllineage STR-PCR HLA-FACS 49140 2. 1 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 CD14.1 9 HLA-A11 biotin1 Stain 1 Stain 3 SA-PE Stain 2 SA-APC 2 HLA-A11 biotinhla-a26 Alexa700 10 Red Laser APC(Red-C), Alexa700(Red-B), APC-Cy7(Red-A)Alexa700PMT 685LP, 712/21BP www.bd.com/jp 3
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) FACS2 10 6 4 20 7) PBS(-) 1 200 µl PBS(-) 1 µg/ml propidium iodide PIBD FACSAriaPI PE-TR FCS FlowJo Treestar III. 1. 1lineage 1 2 3 T 4 1 TCD4 + T 2 BNK 1 2 2 1.5% 3 6 10% 3 5 NK ( 3) 1 1 1 2 3 4 2 10 ml Stain 1 Stain 2 1PI BD FACSAria TBNK 1 TT 2 2 4 www.bd.com/jp
2 1 2 10 ml Stain 1 Stain 2 1 FSC-SSC PI CD4 + TCD8 + TBNK 3 2315 1 5 ml Stain 1 1 1 2 3 6 10 3 www.bd.com/jp 5
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 2 49140 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 www.bd.com/jp
3. 2 GVHD A CsA 2842 56 5 2CD8 + T IFN- 5 140 49 4B HLA-FACS STR-PCR STR-PCR 3 7 HLA-FACS 5 STR-PCR HLA-FACS 4. 5 1 159 CD34 + CD45 dim 0.226281 5.71 6 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 5 2 1 12FACSSTR-PCR CsACD8 + T IFN-CsA CsA 10 810 CsA CD8 + TIFN- CsA PMA4CD8 IFN- 8 www.bd.com/jp 7
6 5 1 1592813202 ml Stain 3 1 AML-M2CD34 + CD45 dim CD34-CD45CD34 + CD45 dim 7 CD34 + CD45 dim 1 320 2 ml Stain 3 1 AML-M2 CD34 + CD45 dim HLA-DR CD56 AML1/ETO FISH 8 www.bd.com/jp
IV. HLA-FACS 1 2 HLA-FACS BD FACSAria Windows BD FACSAria BD FACSAria 1 2 1. 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;109:1322-1330. 2. 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;27:993-998. 3. 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;23:1055-1060. 4. 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;56:1920-1927. 5. 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;50:197-201. 6. Metes D, Logar A, Rudert WA, et al. Four-color flow cytometric analysis of peripheral blood donor cell chimerism. Hum Immunol. 2003;64:787-795. 7. 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;81:1298-1305. 8. Mascher B, Schlenke P, Seyfarth M. Expression and kinetics of cytokines determined by intracellular staining using flow cytometry. J Immunol Methods. 1999;223:115-121 www.bd.com/jp 9
1. BD FACSAria 10 FITC Alexa Fluor 488 PE PerCP-Cy5.5 PE-Cy7 PE-Texas Red APC Alexa Fluor 700 APC-Cy7 Pacific Blue Alexa Fluor 405 Pacific Orange a) FITC PEAPC HLA-FACS HLAFITC PE APC b) PE-Cy7 Alexa Fluor 700 APC-Cy7 Pacific Blue Alexa Fluor 405 Pacific Orange APC- Cy7-CD19 Pacific Blue-CD4 c) PE-TR FITC PE PerCP-Cy5.5 PE-Cy7 Pacific Orange 610/20PI d) PerCP-Cy5.5 APC-Cy7 PE-Cy7 X Y Log Bi-exponential Version 4.1DiVa PC PC e) Pacific Blue Alexa Fluor 405 Pacific Orange f) Pacific Orange 488nm 2. a) 5 6 BD TM CompBeads BD TM CompBead 100 CD4 PE-Cy7CD8 PE-Cy7 b) 10 www.bd.com/jp
FITC or Alexa488: PE PE-TR Pacific Orange PE: FITC PerCP-Cy5.5 PE-Cy7 PE-TR PerCP-Cy5.5: PE-Cy7 APC Alexa700 APC-Cy7 PE-Cy7: PE PerCP-Cy5.5 PE-TR APC-Cy7 PI: FITC PE PerCP-Cy5.5 PE-Cy7 APC PLOFILE APC: PerCP-Cy5.5 Alexa700 APC-Cy7 Alexa700: APC APC-Cy7 APC-Cy7: PE-Cy7 APC Alexa700 Pacific Blue or Alexa Fluor 405: Pacific Orange Pacific Orange: FITC PE PerCP-Cy5.5 PE-TR FACS BD FACSAria BD FACSDiVa FITC PE HLA MHCI 2 FACS 1998 2004 FACS FITC-PE 2 www.bd.com/jp 11
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