Korean J Hematol 1999; 34(3):
Published online September 30, 1999
© The Korean Society of Hematology
남정현, 강문수, 백병필, 최종원, 김진주, 배수환, 이홍복, 김철수, 임문환, 김순기
인하대학교 의과대학 임상병리학교실,
인하대학교 의과대학 내과학교실,
인하대학교 의과대학 산부인과학교실,
인하대학교 의과대학 소아과학교실
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 (LF) and compared the results with those of in-house method.
METHODS: CD34 analyses were performed in thirty units each ol 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.
Keywords Umbilical cord blood, Mobilized peripheral blood, Leukapheresis product, CD34, analysis, ISHAGE guideline
Korean J Hematol 1999; 34(3): 382-388
Published online September 30, 1999
Copyright © The Korean Society of Hematology.
남정현, 강문수, 백병필, 최종원, 김진주, 배수환, 이홍복, 김철수, 임문환, 김순기
인하대학교 의과대학 임상병리학교실,
인하대학교 의과대학 내과학교실,
인하대학교 의과대학 산부인과학교실,
인하대학교 의과대학 소아과학교실
Chung Hyun Nahm, Moon Soo Kang, Chul Soo Kim, Moon Hee Lee, Moon Whan Lim, Sun Ki Kim, Byung Pil Baek, Jong Won Choi, Jin Ju Kim, Soo Hwan Pai
Department of Clinical Pathology, Internal Medicine, Obsteterics and Gynecology, Pediatrics, College of Medicine Inha University, Inchon, Korea
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 (LF) and compared the results with those of in-house method.
METHODS: CD34 analyses were performed in thirty units each ol 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.
Keywords: Umbilical cord blood, Mobilized peripheral blood, Leukapheresis product, CD34, analysis, ISHAGE guideline
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