జర్నల్ ఆఫ్ స్టెమ్ సెల్ రీసెర్చ్ & థెరపీ

నైరూప్య

Quantitation of Megakaryocytic Progenitors in Apheresis Products by Flow Cytometry and Real Time PCR

Leinøe Eva, Nielsen Kaspar R, Baech John, Steffensen Rudi, Dybkaer Karen, Boegsted Martin and Johnsen Hans E

Background and aims: Quality assessment of autologous peripheral blood stem cell transplantation (PBSCT) may be improved by enumeration of CD34+/CD61+ megakaryocytic progenitors within the graft. Enumeration of subsets by flow cytometry (FC) has been difficult to standardize because of a low specificity which may arise from platelet or microsphere adherence. We aimed to analyse platelet adherence to haematopoietic stem cells and establish a quantitative real time polymerase chain reaction (RT-qPCR) assay for CD34 and CD61 gene transcripts. The analysis was used to study CD34 and CD61 as predictors for late platelet recovery following PBSCT in non- Hodgkin lymphoma (NHL).
 
Material and methods: FC analysis was performed at aphaeresis products harvested for autologous transplantation and confocal microscopy was applied on sorted cells. The clinical evaluation included analysis of the leukapheresis products of 21 consecutive NHL patients treated with high dose therapy and PBSCT. Early recovery was defined as an observed platelet count >20x10(9)/L before day 12 post transplant and late recovery as an observed platelet count <20x10(9)/L after day 12 post transplant. For RT-qPCR analysis CD34+ cells were sorted from thawed leukapheresis products and RNA extracted and reverse transcribed to cDNA for further analysis of CD34 and CD61 mRNA levels using TaqMan probes.
 
Results: CD34+/CD61+ cells identified by FC were shown to form a specific subset, with no signs of adherent mature platelets. CD34+/CD61+ cells expressed CD61 mRNA transcripts not found in complementary CD34+/ CD61- cells. No positive correlation between FC based enumeration and RT-qPCR analysis estimation of the megakaryocytic progenitor subsets was identified. Evaluation of the clinical impact by comparing samples from 21 patients with early and late platelet recovery revealed no predictive impact for CD61/BACT (β-actin), CD34/ BACT or CD61/CD34 mRNA, expression ratios amongst CD34+ sorted cells.
 
Conclusion and perspective: A specific subset of CD34+/CD61+ cells can be identified by FC and RTqPCR analysis; however enumeration of this subset did not correlate with platelet recovery after PBSCT. Future studies of the predictive value needs to be evaluated in a group of patients with engraftment failure in international collaboration within the European Blood and Marrow Transplantation Group (EBMT).