Influence of red blood cell lysing solution on comparative counting of CD34+ cells and its causes

Abstract:

Objective：To analyze the influence of the red blood cell（RBC） lysing solution on the comparative counting of CD34+ cells. Methods：A total of 101 peripheral blood stem cell collections were collected and stained by CD34-PE and CD45-FITC monoclonal antibodies. Then the RBCs were lysed by two commercially available lysing solutions，FACSTM lysing solution（FACS） and Optilyse C lysing solution（Optilyse C） and one self-prepared lysing solution. The washing procedure and ISHAGE gate strategy were used to de－tect the relative numbers of CD34+ cells，lymphoid cells，granulocytes，and monocytes，the percentage of CD45+ cells，and the various types of cell forward scatter（FSC） and side scatter（SSC） signals. Results：The percentage of CD34+ cells was the lowest in the FACS-treated samples，while the highest in self-prepared lysing solution（0.49±0.43 vs. 0.67±0.50 vs. 0.73±0.66，P=0.000）. The percent－age of CD45+ cells in the samples treated by FACS was much higher than that in the samples treated by Optilyse C and self-prepared lysing solution respectively（t=142.500，P=0.000）. The percentage of lymphocytes（F=35.340，P=0.000） had significant differences in samples treated by the three lysates solutions respectively，however，there was no statistically significant difference for the percentage of granulocytes（F=0.610，P=0.540） and monocytes（F=1.590，P=0.210）. After FACS treatment，the signal intensities of FSC and SSC of CD34+ cells and lymphocytes were weaker than those of the Optilyse C and self-prepared lysing solution treatments，while the SSC signals of granulocytes and monocytes were the strongest after FACS lysate treatment and were the weakest after Opti－clese C treatment. In addition，there was no correlation between the number of leukocytes after mobilization and the percentage of CD34+ cells（r=0.108，P=0.312）. Conclusion：The difference in the relative count of CD34+ cells caused by the RBC lysate may be related to the proportion of CD45+ cells，that is，the acquired effi－cient cell numbers. The effect of lysate on cell morphology is related to the significant difference in lymphocyte ratio. As different RBC lysates do have adverse effects on the relative counts of CD34-positive cells，the choice of RBC lysates reagents for the flow cytometry or counting should be careful.