Study of the factors affecting the immunophenotyping of CD34+ hematopoietic stem cells and their absolute count by applying ISHAGE protocol

Abstract

Background and objective: ISHAGE protocol is used for immunophenotyping CD34⁺ hematopoietic stem cells  to assess their number and viability. Several factors prevent its correct application. This study aimed to determine these factors and control them to obtain an accurate count  of CD34⁺ cells  which is the key of success of transplantation.Materials and Methods: Immunophenotyping of hematopoietic stem cells was performed using flow cytometry technicque by applying ISHAGE protocol with variables that effect the count of CD34⁺ cells. Factors were antibody anti-CD34 clone, its concentration, duration of incubation,  duration of sample storage,  Lysing solution and factors related to the device such as the flow rate, Forward Scatter threshold,  adjusting sectors and analysis gates. The study was applied on samples from 11 patients of the Children's Hospital prepared for autotransplantation using stem cells harvested from peripheral blood after G-CSF mobilisation. Samples were drawn from blood the day before harvesting, the day of  harvesting and from the apheresis  product. Results: The anti-CD34 8G12 clone was better than 581 and MY10 clone (P = 0.06, 0.03, respectively). The concentration of 10% of anti- CD34 antibody was optimal. The erythrocytes lysing solution with fixative had a significant effect on the cells count. These factors must be accompanied by a flow rate of 3000 cells/sec (P= 0.02, 0.04), a threshold of 100 with correctly set data analysis gates and device settings which  play a major role in obtaining a correct CD34⁺ cells count. The apheresis product contained 7 times more cells than the peripheral blood after stimulation (175 ± 29.98  versus 1250±300  cell /µl).

Conclusions: Factors influencing stem cells count are multiples. It is necessary to focus on these factors to improve the investment of ISHAGE protocol in obtaining the exact number of stem cells  because of its importance in the success of the transplantation.