Triplicate samples of 1 x 106 cells are stained with CD45-FITC/CD34-PE (Becton Dickinson, San Jose, CA) for each different analysis. A single isotype control is concurrently stained with CD45- FITC/IgG1- PE not present on human leukocytes. Cells are incubated with antibody at an approximate concentration of 0.5ug per 1 x 106 cells at 4°C for 15 minutes. After incubation, mature erythrocytes are lysed by incubating for 10 minutes with a commercial lysing reagent (10% FACS Lysing Solution (Becton Dickinson, San Jose, CA) in deionized water). Washing out the lysing solution using Dulbecco's Phosphate Buffered Saline (PBS- 1X, pH 7.4, Irvine Scientific, Santa Ana, CA) with 5% goat serum (Sigma, St Louis, MO) prepares the cells for analysis by flow cytometry.

Using the EPICS Elite ESP Flow Cytometer and Expo for Elite software package (Beckman Coulter, Miami, FL), a minimum of 75,000 CD45 cells are acquired and analyzed using the four parameter flow methodology adopted by the International Society of Hematotherapy and Graft Engineering (ISHAGE) ⁽⁹⁾. The percent CD34 is determined by dividing the number of events in the defined region for CD34 positive cells by the total number of CD45 positive cells aquired above a noise discriminator (threshold) set on forward light scatter. Cells from the isotype control sample that fall within the gated region for CD34 are subtracted out as a non- specifically stained cells.

The % CD34 cells determined by the above procedure are used together with the total nucleated cells cryopreserved and the patient's weight to calculate the total CD34 cells /kg that were cropreserved. A test vial of the PBSC is thawed after cryopreservation, and the % viable cells recovered determined by trypan-blue dye exclusion. The CD34+ cells/kg cryopreserved x % viable cells on test thaw/100 = Viable CD34+ cells/kg.