Quantitative detection of human umbilical cord blood lymphocytes in human-sheep chimerism by flow cytometry and fish

Abstract

In utero transplantation of hematopoietic stem cell (HCS) from human umbilical cord blood (UCB) into preimmune fetal sheep is a model for study of prenatal therapy for congenital hematologic and immunologic disorders. Varieties of methods have been utilized to detect donor cells engraftment after transplantation. This study proposed a design for the analysis of donor cell chimerism by Flow Cytometry (FCM) using mouse anti-human CD 45 monoclonal antibody specific to human lymphocytes compared with FISH analysis using DNA probe “CEP16”, specific to human chromosome 16. UCB was obtained from the pregnant women at King Chulalongkorn Memorial Hospital. The human and sheep lymphocytes were mixed in the test tube at different ratios of 1:100 1:500 1:1,000 1:5,000 and1:10,000. Comparing the results of both techniques, there was no significant difference at ratio between 1:100-1:1,000 (p˃0.05). But at lower concentration (1:5,000 and 1:10,000), there was significant difference of variation by FCM (p˂0.05). FISH analysis was shown to produce less variation than FCM. This in vitro approach was applied for detection of human donor cells in recipient lambs after in utero transplantation. Blood samples were drawn at 10 days and 1 month after birth. The results showed low percentages of donor cells in sheep peripheral blood ranging from 0.03%-0.63% (microchimerism). There were slightly decreases and no significant differences at the interval at 10 days and 1 month after birth by FCM (p˃0.05).