SOP - jmdp.or.jp

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

2 Ver. 1 3 Sysmex, Siemens, Beckman Coulter (Trypan Blue) (Turk s solution)

3 Acridine Orange/Ethidium Bromide (Hemacytometer) (Burker-Turk) (Improved Neubauer) CD45 Propidium Iodine 7 AAD Countess, Invitrogen 1.

4 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 )

5 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.

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 . 30 Lot . Lot . Sample Sample FCM CD34 Ver. 1 6 (B) FCM FCM CD34 CellQuest Run (Low ) FCM Threshold FSC/SSC Ver.

7 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.))

8 , Vancouver, BC, Canada 2 1. Eaves C, et al: Atlas of Human Hematopoietic Colonies. StemCell Technologies Inc., 1995 2.. Ver. 1 8 . 1. 2. 1 DMSO AB

9 10% 2-4 107/ml 2) DMSO 3 108/ml GM CSF 3) 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.

10 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. 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.