Cord Blood (CB) CD34 + and CD3 + Cells with Decreasing Levels of Their Mitochondrial Potential Are Not Detectable with 7-AAD, Have Impaired Function and Ultrastructural Morphology Characteristic of Early Apoptosis.

Background: Banked CB is an effective source of hematopoietic stem cells for allogeneic bone marrow reconstitution. Critical indices of engraftment potential and quality of a CB graft are the nucleated cell dose, CD34 + cell content and total CFU. Determination of CD34 + cell viability by flow cytometry depends on 7-actinomicin D (7-AAD) exclusion, but its diffusion into cells depends on permeability of the cell membrane. Earlier stages of the death pathway (apoptosis) escape detection by 7-AAD. Aims of our study: to establish a sensitive method for detection of early apoptosis in CB cells and to evaluate their ultrastructural morphology, to determine the extent to which early apoptosis may disclose reduced functionality of CD34 + and CD3 + cells and therefore, whether early apoptosis should be investigated routinely in CB grafts before transplantation and to investigate the presence and degree of apoptosis in CD34 + and CD3 + cells upon CB storage at RT and after cryopreservation/thawing, as well as the effect of post-thaw DMSO removal. Results: After CB storage over 48 hrs, evaluation by multiparameter flow cytometry with the mitochondrial potential (MP) marker DILC, Annexin V, 7-AAD and surface cell markers, identified 3 populations with decreasing MP and negative 7-AAD: 1. DILC with high MP (MP High )/Annexin(−); 2. DILC with intermediate MP (MP Interm )/Annexin(−) and 3. MP Interm /Annexin(+). Groups were sorted for CD3 + and CD34 + cells, ultrastructural morphology was evaluated by electron microscopy (EM) and their function by culture with Phytohemaglutinin/IL-2 (PHA) for CD3 + and by CFU assay for CD34 + cells. EM revealed lymphocyte shrinkage, nuclei with condensed chromatin and polarized clusters of organelles, findings suggestive of early stages of apoptosis. In addition, CD3 + cells with MP High had higher stimulation than those with MP Interm and impaired more when cells became Annexin(+) (ratio MP High /MP Interm =54; p + cells with MP High showed higher CFU than those with MP Interm /Annexin(−) and those with Annexin(+), (CFU ratio MP High /MP Interm = 17.7 and 35.6 respectively, p + and CD34 + cells upon CB storage over 48hrs and after cryopreservation/thawing showed decreased CD3 + cell viability (p + cells (range 37–79%). In contrast, there were no significant variations in viability or function of CD34 + cells after cryopreservation/thawing. Thawed cells with DMSO dilution down to 2% concentration or DMSO removal (Dextran/Albumin wash) effected no significant change in viability or function. Summary: CB cells with decreased mitochondrial potential have impaired function and morphological characteristics of early apoptosis; A combination of 7-AAD with a marker of mitochondrial potential (DILC) allows for the detection of early apoptotic cells which are not detected by 7-AAD; CB-lymphocytes have immature appearance by ultrastructural morphology compared to those of adult peripheral blood; Viability and function of CB-CD34 + cells were not significantly reduced by cryopreservation/thawing but those of CD3 cells were. Lower viability and decreased function of CB-CD3 cells may affect the immunological interactions in double or multiple CB transplants as well as clonal expansion post-transplant.