2 time volume of PBS/EDTA buffer (including 0.5% of HSA: Human Serum Albumin) was added into 50-120ml of PBSPC obtained in 1 to 2 collections and the supernatant was removed after centrifuge. The volume was adjusted to 90-100ml and then 7.5ml of CliniMACS CD34 reagent was added. It was put into a rotator (25rpm) at 19-25℃ and cultured for 30 minutes, then washed with PBS/EDTA buffer twice. The final volume was made to be 100 to 300ml.
The above PBSPC leukapheresis products were connected with CliniMACS. "CD34 selection 1" programme was selected and the purification of CD34+ cells would start automatically. 42 to 45ml of final product was retained. The final purified CD34+ products were infused to 5 patients immediately after separation, while the other 22 purified products were stored in liquid nitrogen at -196oC after sustained programmed cryopreservation.
1.4 Blood cell counting and flow cytometry
The final purified product were diluted with the ratio of 1:20 for original solution to PBSPC 1:20. The cells were counted with cytometer ( CELL-DYN 1500, Abbott) and 50ul of the diluted solution was added into different tubes and then different McAbs labeled by fluorescent-marks such as CD45-Fitc/CD34-PE、CD3-Pc5/CD4-Fitc/CD8-PE、CD3-Fitc/CD19-PE、CD3-Fitc/CD56-PE etc. (IMMUNOTECH)and propidium iodide(PI，SIGMA)were added into the tube to be cultured for 30 minutes. Non-detergent hemolysin(IMMUNOTECH)was added to lyse erythrocytes. 20 minutes late, the percentage of CD3+、CD8+、CD4+、CD19+、CD56+ were detected by counting 20,000 cells with EPICS ELITE flow cytometer(BECKMAN-COULTER). The percentage of CD34+ cells were counted by adjusted with CD45+ cells and analyzed with ELITE 4.5 software. Died cells were excluded with PI(1.25ug/tube)and the number of died cells were counted by the strong emission of FL3 channel.
1.5 Methods for transplantation
The patients were protected in laminar air flow room and then treated by conditioning regimens (Table 2 shows the details). Haploidentical PBSPC were infused to recipients promptly after CD34+ purified in 3 cases. The cryopreserved purified CD34+ cells from other two haploidentical PBSPCs and from all of autologous donors were quickly thawed at 42℃ and then infused. Antibiotics and gamma globulin were used to prevent inflammation for the patients who suffered from neutropenia. If peripheral blood platelets were less than 20×109/L, suspension of platelets were infused to prevent bleeding. 5ug/Kg/d of G-CSF was given in 3 to 5 days after infusion until the number of
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