SOP - jmdp.or.jp

1 Ver. 1 1 (SOP) SOP .. 2 / .. 2 .. 3 CD34 .. 4 .. 7 .. 8 .. 14 .. 23 .. 26 .. 30 Ver. 1 2 / / Ver.

2 1 3 Sysmex, Siemens, Beckman Coulter (Trypan Blue) (Turk s solution) Acridine Orange/Ethidium Bromide (Hemacytometer) (Burker-Turk) (Improved Neubauer) CD45 Propidium Iodine 7 AAD Countess, Invitrogen 1.

3 Ver. 1 4 CD34 CD34 CD34 The International Society of Hematotherapy and Graft Engineering ISHAGE 1996 CD34 (FCM) Stem Cell Enumeration Committee 1999 Current Protocol in Cytometry CD34 (JCCLS H3-P ) ( ) CD34 CD45 CD45 CD34 CD45 CD34 CD45 CD45 CD34 trucount BD Flow-count(BC )

4 7-AAD 1. BEST STUDY#19-INTERLABORATORY EXCISE Final Protocol for Comments; May 2002 2. BD Technical Protocol CD34PE/CD45 FITC/7 AAD CD34 3. BD 4. CD34 -Procount 7 AAD Measurement of memeasurement of CD34 positive cells in the frozen cord blood: comparison of the procount and 7-AAD methods..2004; 50 : 605-612 5. CD34 6. CD34 (JCCLS H3-P ) ( ) Ver. 1 5 CD34 BD trucount (A) PharmLyse solution trucount CD34/CD45 20 L 7 AAD 10 L Sample 50 L Vortex 15 incubate PharmLyse solution mL Vortex 3 5 incubate Lot.

5 30 Lot . Lot . Sample Sample FCM CD34 Ver. 1 6 (B) FCM FCM CD34 CellQuest Run (Low ) FCM Threshold FSC/SSC Ver. 1 7 CD34 CD34 (CFU-GM colony forming unit-granulocyte/macrophage (CFU-GEoMM/CFU-Mix) BFU-e burst forming unit-erythrocyte CFU-Mix CFU-GM MethoCult GF H4434V (Methocult GF H4434V, Stemcell technoloies Inc., Vancouver, BC, Canada 2 1.))

6 Eaves C, et al: Atlas of Human Hematopoietic Colonies. StemCell Technologies Inc., 1995 2.. Ver. 1 8 . 1. 2. 1 DMSO AB 10% 2-4 107/ml 2) DMSO 3 108/ml GM CSF 3)

7 1983 Stiff 5%DMSO 6%HES -80 C 4 Makino Katayama, Kawano DMSO HES CFU-GM 5-7) CP-1 KK CP-1 Dimethyl sulfoxide DMSO Hydroxyl ethyl starch HES 4% CP-1 -80 C 12 CFU-GM 70% Ver. 1 9 1. Low Temp. Med. 1998; 24 : 171-174 2. Rowley SD: Hematopoietic stem cell cryopreservation. A review of current techniques. J. Hematother. 1992; 1 : 233-250 3. Rowley SD, et al: Effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation. Blood. 83: 2731-2736 4. Stiff PJ, et al: Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch.

8 Cryobiology. 1983; 20 : 17 5. Makino S, et al: A simplified method for cryopreservation of peripheral blood stem cells at -80 degrees C without rate-controlled freezing. Bone Marrow ; 8 : 239-244 6. Kawano Y, et al: Cryopreservation of mobilized blood stem cells at a higher cell concentration without the use of a programmed freezer. Ann. Hematol. 2004; 83 : 50-54 7. Katayama Y, et al: The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant. 1997; 19 : 283-287 Ver. 1 10 CP-1 (A) (B) ID CD34 CP-1 Lot 25% Lot CP-1 -80 C CP-1 25% Ver.

9 1 11 ID PBSC 1-1 PBSC CD34 1-2 2-1 CP-1 Lot 25% Lot 2-2 3-1 CP-1(100mL ) 4 C 3-2 CP-1 50mL 25% 32mL 100mL CP-1 181g) 4 C 4-1 4-2 5-1 5-2 50mL 5-3 100mL 2mL RPMI 102mL RPMI Lot 6-1 4 C CP-1 50mL 6-2 7-1 2 7-2 No 7-3 7-4 7-5 8-1 Ver.

10 1 12 9-1 9-2 9-3 -80 C 10-1 10-2 Ver. 1 13 PBSC ID ID 1-1 mL 2-1 mL CP-1 Lot 25% Lot 3-2 25% mL 4-2 1 2 5-2 mL 5-3 RPMI mL Lot RPMI Lot 7-2 1 2 7-3 1 mL 2 mL 7-5 mL x 10 9 x 10 9 /kg( ) CD34 x 106 x 106 /kg( ) 8-1 10-1 10-2 1 2 1 2 Ver. 1 14 . 1 ABO 2 3 Rh D Rh 4 ABO A O A B Rh D Rh Rh D Rh D Rh D D 1) Spectra SEPAX Ficoll 2) hydroxyl ethyl starch HES 3)