CD34 Analysis according to ISHAGE Guideline / 대한혈액학회지

ABSTRACT

BACKGROUND: Flow cytometric analysis for CD34 has been widely used for hematopoietic stem cell enumeration. The procedure is simple and rapid for clinical use but the lack of standardization resulted in great intralaboratory variations. In 1995, a guideline for CD34 analysis was established by International Society of Hematotherapy and Gene Engineering (ISHAGE) for reliable testing. We performed CD34 analysis using the ISHAGE guideline in umbilical cord blood (UCB), mobilized peripheral blood (MPB) and leukapheresis product (LP) and compared the results with those of in-house method. METHODS: CD34 analyses were performed in thirty units each of UCB, MPB and LP according to the ISHAGE guideline and in-house method and the results were analyzed by the t-test. Both methods used CD45FITC/CD34PE and its isotype controls. In ISHAGE guideline, among CD34+/ CD45+ cells, only those with low forward scattering, low to intermediate side scattering and low to intermediate CD45 fluorescent intensity were identified as stem cells, and the percentage of those cells among CD45+ cells was calculated. In in-house method, cells expressing both CD34 and CD45 antigens were selected by isotype control and the percentage of CD34+/CD45+ cells among CD45+ cells were calculated. RESULTS: Significant differences were observed in the percentages of CD34+ cells in UCB, MPB and LP between ISHAGE guideline (0.25%, 0.42%, 0.80%) and in-house method (0.40%, 0.55%, 1.20%) (P<0.001). So were the CD34+ cell counts mean values of CD34+ cells in microliter of UCB, MPB and LP were 20, 40, 1,392 by ISHAGE guideline, and 35, 62, 2,079 by in-house method (P<0.001). CONCLUSION: ISHAGE guideline for CD34 enumaration was considered as a simple, rapid and reliable method for clinical setting and to have economic benefits because no additionalmonoclonal antibodies were required.