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

Abstract

This article critically reviews current methods to test and characterize umbilical cord blood (UCB) for hematopoietic stem cell transplantation. These tests include total nucleated cell (TNC) count, viability, viable CD34-positive content, and the colony-forming unit assay. It is assumed that the data obtained are sufficient to perform a UCB stem cell transplant without actually determining the quality and potency of the stem cells responsible for engraftment. This assumption has led not only to a high graft failure rate attributed to low or lack of potency, but also to noncompliance with present statutes that require UCB stem cells to be of high quality and, indeed, potency for a transplant to be successful. New evidence now calls into question the quality of the data, based on the UCB processed TNC fraction because using this impure fraction masks and significantly underestimates the functionality of the stem cells in both the segment and the unit. It is proposed that UCB units should be processed to the mononuclear cell fraction and that new cost-effective technology that measures the quality and potency of UCB stem cells be implemented to achieve better practices in UCB testing. These changes would provide the transplant physician with the assurance that the stem cells will perform as intended and would reduce risk and increase safety and efficacy for the patient.

Significance: Current stem cell transplantation of umbilical cord blood cells requires testing that includes four basic parameters that do not determine whether the stem cells are of high quality, as required by the Stem Cell Therapeutic and Research Act of 2005. No cord blood units collected or transplanted so far have been tested for stem cell quality or potency. New scientific evidence calls into question cord blood processing and testing practices required by regulatory agencies and standards organizations. A new perspective is described that includes stem cell quality and potency testing that could reduce graft failure rates.

Figure 1.

Umbilical cord blood testing paradigm. This diagram shows current minimum criteria testing on the left of the cord blood processing and use flowchart, whereas best practice criteria testing and new perspectives in UCB testing are shown on the right. Better practices involve processing UCB units to the MNC fraction and cryopreservation in this state. The TNC fraction is not a suitable starting material to implement best practices (Fig. 2). It also involves testing stem cell metabolic functionality and viability prior to cryopreservation to store the stem cell units predicted to have the highest quality. After cryopreservation, the first stem cell potency assay would be performed and the results uploaded to the cord blood inventory. When a UCB unit has been identified based on compatibility with the patient, a second potency assay would be performed to confirm the stability of the stem cells in the unit and to allow for its release. Both potency assays and perhaps one performed by the transplant center would provide predictive information regarding the UCB stem cell engraftment response. Assays to predict time to engraftment could still be performed but could not be used to determine potency. Abbreviations: CBB, cord blood bank; CFU, colony-forming unit; GM, granulocyte-macrophage; MNC, mononuclear cell; RBC, red blood cell; TNC, total nucleated cell; UCB, umbilical cord blood.

Figure 2.

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. Proliferation potential is determined by the slope of the cell dose-response linear regression. The steeper the slope, the greater the proliferation potential, the more primitive the stem cell population, and the greater its potency and engraftment potential [–40]. The results demonstrate not only that the TNC fraction masks and underestimates the functional capacity of the stem cells but also that the segment is not a good representative of the unit when based on the MNC fraction. Abbreviations: CFC-GEMM, colony-forming cell-granulocyte, erythroid, macrophage, megakaryocyte; HPP-SP, high proliferative potential-stem and progenitor cell; MNC, mononuclear cell; TNC, total nucleated cell.

Figure 3.

The potency calculated for stem cell populations determined from umbilical cord blood (UCB) segment MNC fractions. Potency can be measured only by using a validated assay and the inclusion of a reference standard (RS) of the same material against which the sample can be compared. This comparison provides the measure of potency, the potency ratio, for each stem cell population (CFC-GEMM and HPP-SP). The potency ratio is calculated by the slope of the dose-response linear regression for each stem cell population from the segment sample divided by that for the RS. The UCB RSs were aliquots of cryopreserved MNCs from a single batch (lot) of processed umbilical cord blood. The potency of the RS is always 1. The potency ratios of both stem cell populations should be greater than 1 to provide assurance that the stem cells demonstrate sufficient engraftment potential when transplanted into the patient [–40]. Abbreviations: CFC-GEMM, colony-forming cell-granulocyte, erythroid, macrophage, megakaryocyte; HPP-SP, high proliferative potential-stem and progenitor cell.