Screening for the Megakaryocytopoietic Potential of Cord Blood Units.
Authors: Eskola Mikko , Rekunen Sari , Mottonen Sari , Aroviita Pekka , Kekomaki Riitta 
Published: October 2019

OBJECTIVE: Delayed platelet engraftment is a common problem after hematopoietic stem cell transplantation. However, although aiming at high quality, the megakaryocytopoietic potential of cord blood (CB) units is not routinely studied in CB banks. In this study, a simple method for the evaluation of the megakaryocytic growth of unfrozen CB units was designed. The results were compared with cell characteristics of the units, including mean platelet volume (MPV) and platelet concentration in unprocessed CB as possible markers of thrombopoietic potential.

MATERIALS & METHODS: Buffy coat (BC) samples from 24 randomly selected CB units were cultured on two double-chamber slides per unit in a semisolid assay designed to support megakaryocytic cells (MegaCult-C, StemCell Technologies Inc., Vancouver, Canada). After 13 days, the cells were fixed and stained using a monoclonal CD41 antibody and the alkaline-phosphatase anti-alkaline-phosphatase technology. Based on the appearance of megakaryocytic colonies, the CB units were scored on a scale 0–3 by three independent investigators. The scores were compared with CB bank data of the same units. The nonparametric Kendall coefficient of concordance (W) and simple linear regression were used for statistics.

RESULTS: The study sample was confirmed representative of all CB units processed during the same time period. All CB units received a mean score of at least 0.7, indicating megakaryocytic growth. The scores given by the three investigators appeared similar and a high concordance was observed (W= 0.91; p < 0.0001), although the distribution of scores varied slightly between the investigators. Using simple linear regression, a clear yet not statistically significant correlation was discovered between the megakaryocyte (MK) score and the volume proportion of platelets in unprocessed CB (“plateletcrit”), defined as MPV × platelet concentration.A correlation (although not statistically significant) was also observed between the MK score and both MPV and platelet concentration.

MK score and markers of thrombopoiesis (n=24) Mean (Range) R p* *Two sided Plateletcrit, % 0.23 (0.13–0.35) −0.21 0.32 MPV, fl 8.9 (8.0–9.7) 0.27 0.20 Platelet concentration, 109/l 258 (136–374) −0.25 0.23

In addition, a negative correlation between MPV and platelet concentration was discovered (R= −0.35; p= 0.091). No significant correlation was observed between the MK score and total colony-forming unit (CFU) number, total CD34+ cells or total nucleated cells (TNC) in BC.

MK score and CB unit cell contents (n=24) Mean (Range) R p* *Two sided Total CFU, 106 2.4 (0.8–4.5) 0.004 0.98 Total CD34+ cells, 106 5.4 (1.4–11.5) 0.04 0.84 TNC, 107 124 (69.4–212) 0.13 0.54

CONCLUSIONS: As the megakaryocytopoietic potential of a CB unit does not necessarily correlate with data used to select units for banking and transplantation, screening for this potential may be of clinical significance. The screening method used in this study was simple and required no extra sample from the CB unit; however, more studies are needed to identify the best procedure for a routine setting. The associations observed between the MK score, MPV and platelet concentration may reflect the autoregulation of thrombopoiesis as well as the reliability of the screening procedure. The development of screening methods for the megakaryocytopoietic potential of a CB unit may be an important step in improving platelet engraftment after transplantation.