P33

Exploring the heterogeneity of the hematopoietic stem cell pool: A simultaneous staining protocol for the Side population, Aldehyde dehydrogenase and CD34 in Cord Blood

Sofia Ulrika Frändberg1,*, Susann Li2, Cecilia Boreström2, Lars Palmqvist2

1Stem cell laboratory, CLINICAL IMMUNOLOGY AND TRANSFUSION MEDICINE SAHLGRENSKA UNIVERSITY HOSPITAL GÖTEBORG, 2Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine Sahlgrenska Academy, Göteborg, Sweden

Introduction: Choosing cord blood unit (CBU) for transplantation is based on tissue typing and the stem cell potential of the unit. CBUs are assessed at collection with regards to total nucleated cells, CD34+count and cultivation protocols such as the Colony-Forming-Unit assay. Efforts are made towards finding better ways of defining stemness of CBUs

Objectives: Side population phenotype (SP) and activity of the Aldehyde Dehydrogenase enzyme (ALDH) are functional markers of stemness that are assayed using flow cytometry. We have developed a protocol for the simultaneous determination of CD34+, SP and ALDH+ in relation to immature leucocytes.

Methods: 30 CBUs. 200µl cord blood buffy coat was sampled from each CBU. Nucleated cells were stained with Hoechst 33342 for 90 minutes at 37°C. After a few minutes on ice, cells were stained with ALDH reagent for 30 minutes at 37°C . Subsequently, the cells were put on ice and stained with 7-AAD and antibodies against CD45 and CD34 for 30 minutes. Samples analyzed on a FACSAriaII with near UV-laser. Calculations: IBM SPSS v. 21.

Results: The CD45dimSSClow population (immature leucocytes) was approx. 10% of the number of 7-AAD negative events. The CD34+ and ALDH+ populations amounted to a few percent of the CD45dimSSClow population and were approximately of the same size. The SP population was smaller.There was no overlap between the SP and ALDH+ in any of the investigated units. The SP cells were CD34 negative, whereas the ALDH+ was dominated by CD34+ events.

Conclusion: We show that simultaneous staining for CD45, CD34, SP and ALDH+ cells is feasible using small amounts of cord blood buffy coat in a time frame possible to implement in routine laboratory work. In our study, the sizes of the ALDH+ and CD34+ populations in relation to the number of CD45dimSSClow events were approximately the same whereas the SP was smaller. There were also differences in immunophenotype between the SP and ALDH+ populations inferring that they represent cells with diverse biology.

Keywords: Blood, Method