CHARACTERISATION OF CD34+ HAEMATOPOIETIC PROGENITOR CELLS BY FLOW CYTOMETRY

Abstract

Cord blood transplantation is a potentially curative treatment for many conditions, including haematological malignancies, inborn errors of metabolism and immunodeficiencies. The CD34+ haematopoietic progenitor cell dose is well documented to be critical to a successful transplant outcome and insufficient haematopoietic progenitor cells can result in primary non-engraftment. In addition, increasing evidence is mounting that the presence of donor-specific HLA antibodies in the recipient can have a detrimental impact on donor cell engraftment. The majority of reports of HLA expression on haematopoietic progenitor cells date from 30 years ago. This project aimed to increase our understanding in this area, especially with regard to the expression of the HLA Class II antigens: HLA-DQ and -DP. Residual cells were isolated from 40 cord blood units after infusion to recipients at the Royal Manchester Children's Hospital and the Manchester Royal Infirmary. Haematopoietic progenitor cells were identified by flow cytometry using the ISHAGE sequential gating strategy and co-stained with HLA monoclonal antibodies. Results showed that almost all CD34+ haematopoietic progenitor cells express HLA-DR, however very few of these cells express HLA-DQ or -DP. In contrast, a higher percentage of CD45+ leucocytes expressed HLA-DQ and -DP than -DR. In addition, a higher percentage of CD34+ haematopoietic progenitor cells with homozygous HLA-DQB1*03:02 genotype had detectable levels of HLA-DQ compared to other genotypes, suggesting that not all alleles of the same HLA locus are expressed equally. Comparison of these samples' rs9277534 SNP genotypes did not support reports that this SNP controls expression level of HLA-DPB1, with all three allelic genotypes (A/A, A/G, G/G) showing similar expression of this antigen as opposed to the reported high expression for G/G genotype. Bland-Altman graphs indicated that proportional bias was present in CD34 enumeration as quantified by a single flow cytometry method for all samples tested compared to the values provided by each of the cord blood banks (p=0.0000). This demonstrated a lack of standardisation with potential clinical impact as these values are used for initial cord selection for transplantation. Data generated in this project potentially support a risk stratified approach to donor selection in the presence of donor-specific HLA antibodies according to the locus to which an antibody is directed. Further work to quantify HLA expression levels on CD34+ haematopoietic progenitor cells is necessary to establish the clinical significance of HLA-DQ and -DP antibodies in the context of cord blood transplantation.

Date of Award	20 Feb 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Valerie Kouskoff (Main Supervisor)