Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units

Abstract

BackgroundRare hematopoietic stem cell populations are responsible for the transplantation engraftment process. Umbilical cord blood (UCB) is usually processed to the total nucleated cell (TNC), but not to the mononuclear cell (MNC) fraction. TNC counts are used to determine UCB unit storage, release for transplantation and correlation with time to engraftment. However, the TNC fraction contains varying concentrations of red blood cells, granulocytes, platelets and other cells that dilute and mask the stem cells from being detected. This does not allow the quality and potency of the stem cells to be reliably measured.Methods63 UCB segments and 10 UCB units plus segments were analyzed for the response of both primitive lympho-hematopoietic and primitive hematopoietic stem cells in both the TNC and MNC fractions. The samples were analyzed using a highly sensitive, standardized and validated adenosine triphosphate (ATP) bioluminescence stem cell proliferation assay verified against the colony-forming unit (CFU) assay. Dye exclusion and metabolic viability were also determined.ResultsRegardless of whether the cells were derived from a segment or unit, the TNC fraction always produced a significantly lower and more variable stem cell response than that derived from the MNC fraction. Routine dye exclusion cell viability did not correspond with metabolic viability and stem cell response. Paired UCB segments produced highly variable results, and the UCB segment did not produce similar results to the unit.DiscussionThe TNC fraction underestimates the ability and capacity of the stem cells in both the UCB segment and unit and therefore provides an erroneous interpretation of the of the results. Dye exclusion viability can result in false positive values, when in fact the stem cells may be dead or incapable of proliferation. The difference in response between the segment and unit calls into question the ability to use the segment as a representative sample of the UCB unit. It is apparent that present UCB processing and testing methods are inadequate to properly determine the quality and potency of the unit for release and use in a patient.

Development and validation of a rapid, aldehyde dehydrogenase bright–based cord blood potency assay

“…33 In addition, the use of the HALO adenosine triphosphate bioluminescence assay on segments and units indicates that potency assessment on the mononuclear cell fraction may be more robust and less variable than on the TNCC fraction. 34 Further refining of the CFU assay to decrease the length of the assay is also under investigation. 35,36 The creation of the Cord Blood Apgar scoring system, which utilizes precryopreservation and postthaw graft variables (TNCC, mononuclear cell, CFU, CD34…”

Improving Quality and Potency Testing for Umbilical Cord Blood: A New Perspective

“…More advance technology has been used recently to test the hypothesis that the quality and potency of primitive stem cell populations can be determined using the TNC fraction in both UCB segments and units. This “null hypothesis” was tested using a standardized and validated [37, 43, 44] ATP bioluminescence stem cell proliferation assay and verified against the CFU assay [45]. The results decisively rejected this hypothesis and instead demonstrated that the mononuclear cell (MNC) fraction, not the TNC fraction, should be used because the latter not only masked but significantly underestimated the presence, quality, and potency of UCB stem cells in both segments and units [45].…”

“…This “null hypothesis” was tested using a standardized and validated [37, 43, 44] ATP bioluminescence stem cell proliferation assay and verified against the CFU assay [45]. The results decisively rejected this hypothesis and instead demonstrated that the mononuclear cell (MNC) fraction, not the TNC fraction, should be used because the latter not only masked but significantly underestimated the presence, quality, and potency of UCB stem cells in both segments and units [45]. This is illustrated in Figure 2, which shows an example of the difference between the TNC and MNC fractions for both the unit and associated segment.…”

“…Another assumption negated by these studies was that dye exclusion viability provides a reliable method for demonstrating cell functionality. In fact, for both TNC and MNC fractions, dye exclusion viability did not correlate with metabolic viability and functionality [45]. As a result, cells may be viable using a dye exclusion method but may actually be dead metabolically.…”

“… Difference in stem cell proliferation ability (quality) and potential between the TNC and MNC fractions in a segment and unit from a single umbilical cord blood lot. Primitive hematopoietic stem cells (CFC‐GEMM) are represented by the dotted cell dose‐response linear regression lines, whereas solid lines represent the response of the more primitive lymphohematopoietic stem cells (HPP‐SP) [44, 45]. The quality of the stem cells is provided by the ATP concentration (in micromolars) at any specific cell concentration.…”