THE HALO®-96 MeC and SEC STEM AND PROGENITOR CELL - QUALITY CONTROL (SPC-QC)
and
CAMEO™-96 STD
PLATFORMS
for measuring
Stem and Progenitor Cell Potency
in Transplantation and Cord Blood Bank Processing Laboratories
INTRODUCTION
The ability of a bone marrow, mobilized peripheral blood or umbilical cord blood units to produce short- and long-term engraftment and repopulation in a patient undergoing transplantation for a blood malignancy, is the most important function of a processing laboratory.
The "quality" of the stem cells transplanted must be ensured since the patients receiving the stem cell infusion have had their hematopoietic system partially or totally ablated by radiation and cytotoxic drugs and are at increased risk of dying if the transplanted cells do not engraft and repopulate their hematopoietic system. The first human autologous bone marrow transplantation (BMT) was performed by Kurnick et al in 1958. No assays to determine the "quality" of human transplanted cells with respect to their growth and engraftment potential were available until Pike and Robinson in 1971 applied the in vitro colony forming cell assay, first published in 1966, to human cells.
The cell processing laboratory is responsible for a quality product that is directly related to the success of the stem cell transplant. To this end, standards to maintain and enhance the quality and safety of the transplantation process through inspection and accreditation have been controlled by two groups in the United States, namely the American Association of Blood Banks (AABB) and Foundation for the Accreditation of Cellular Therapy (FACT) and in Europe by the Joint Accreditation Committee of ISCT-Europe and EBMT (JACIE). The U.S. Food and Drug Administration (FDA) has provided guidelines, especially since the implementation of gene therapy and ex vivo hematopoietic stem cell expansion protocols.
Methods to Determine Stem Cell "Quality"
There are 4 parameters that are normally used to assess the "quality"of a stem cell product prior to and after processing. These are nucleated cell count, viability and CD34+ cell number by flow cytometry and “progenitor cell assays” to monitor stem cell procedures and graft manipulations.
Prior to the introduction of flow cytometry, the colony-forming cell assay or “progenitor cell assay” as they have been called in the transplantation field were, and indeed still are, the only surrogate assays that have been used to detect growth potential of stem and progenitor cells during the cell processing procedure. Many transplant centers and umbilical cord blood storage facilities routinely perform the colony-forming cell assays for quality control purposes and clinical monitoring in a stem cell transplantation setting. However, their use has been called into question. In an article by Henon et al in 2001 the authors state, “Determination of the graft content in CFU-GM was the only one available until the end of the eighties. But, for technical reasons, and also because it does not actually evaluate the self-renewal potential of the cell products reinfused, it has now been commonly replaced by the determination of CD34+ cell amounts, which are known to contain the pluripotent hematopoietic stem cells.” Despite the availability of in vitro assays to detect stem cells with different degrees of “stemness” or primitiveness and therefore different degrees of self-renewal potential, the colony-forming assays suffer from many drawbacks. This has been discussed in the section on the Colony-Forming Cell Assays and The HALO® Platform versus the Colony Forming Assays.
Regulatory Issues
The potency of an assay is interpreted in the United States Code of Federal Regulations (21 CFR 600.3) to “mean the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result”. From the previous section, it follows that the CFC assay, as it was original designed and as it has remained, is certainly not an appropriate laboratory test, although it has been the only test available. The "controlled clinical data" is, in this particular case, the transplantation itself.
According to 21 CFR 610.10, “tests for potency shall consist of either an in vitro or in vivo test, or both, which have been specifically designed for each product so as to indicate its potency”. As stated at the beginning, the CFC was never specifically designed as a potency assay.
The latest edition of the FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing, and Administration states, in Section D6.13.1.3, that “For products undergoing manipulation that alters the final cell population, a relevant and validated assay, where available, should be employed for evaluation of the target cell population before and after the processing procedures". The CFCassay may be relevant, but it has never been validated as a potency assay, and in its present form, it is doubtful whether it ever will be validated.
Yet in a recent “Guidance for Industry” from the United States Food and Drug Administration (FDA) in which stem cell processing systems and storage are considered { }, the performance characteristics of the cord blood product prior to and after cryopreservation include nucleated cell count, viability, several phenotypic markers such as CD34 and so-called “total colony forming unit granulocyte macrophage (GM-CFC), total burst-forming unit-erythroid (BFU-E), if application, total colony for unit-granulocyte erythrocyte monocyte macrophage (CFU-GEMM), if applicable”. Although this guidance document actually defines the assays to determine performance characteristics, it actually appears to contradict both the Code of Federal Regulations (CFR) and the FACT-JACIE Standards. First it contradicts the latter for the requirement for a validated potency assay. Second it contradicts other FDA Guidances for Industry by the FDA since one of the issues such an FDA-released Guidance entitled, “Release Testing of Cell Therapy Products”, is not only that an assay should be a “rapid, sensitive and reliable test method”, but that the “product potency for living cell products may be compromised by extensive assay times”. One of the criticisms of the CFC assay, as it has been used in the stem cell processing laboratory is that it takes 14 days to complete the assay. This is approximately the same time as it takes a patient to engraft after transplantation. Therefore, the CFC assay is retrospective in nature and cannot be used forengraftment or reconstitution prediction purposes.
In addition, the term “validated potency assay” is key. Assay validation identifies sources of potential variability and addresses the quantification of these errors in the assay method. The assay validation procedure assigns acceptable values for the assay parameters (accuracy, precision, detection limits, quantitation limits, specificity, linearity, reproducibility and suitability) all of which will ultimately address the reliability and robustness of the assay procedure.
In a recent Forum on Bioassays, the Center for Biologics Evaluation and Research of the FDA, discussed potency assays for complex biological products. In the absence of a direct measurement for biological activity, one of the potency measurements used for in vitro cell and tissue culture is considered to be proliferation. Proliferation, as a surrogate measure of potency, is substantiated by a direct correlation with the results, i.e. biological activity, since without proliferation, the stem cell product will not be able to differentiate into the lympho-hematopoietic lineages needed for reconstitution. The use of a proliferation assay, rather than a CFC differentiation assay, provides all of the characteristics required to measure stem cell potency and can include proficiency testing under the auspices of independent agencies, a fact that has sadly been averted.
Differentiation and Proliferation Assays as a Measure of Stem Cell Potency
Differentiation is defined as the process whereby undifferentiated cells acquire the features of specialized cells.
Proliferation is defined as the expansion of cells by the continuous division of single cells into two identical daughter cells.
The relationship between proliferation and differentiation is:
Stem cells are, by definition, undifferentiated cells (see Stem Cells and the Blood-Forming System). The colony-forming cell assay is therefore NOT a proliferation assay, but rather a differentiation assay, since the end result of a colony-forming cell assay is the production of colonies containing functionally mature cells by which the colony can be identified under the microscope. Therefore, assessing the differentiation capability of cells as detected in the colony-forming cell assay, is secondary to their ability to initiate and sustain proliferation.
HALO®-96 SPC-QC - The First Proliferation Assay Specifically Designed to Measure Stem Cell Potency
The HALO®-96 MeC (Methylcellulose) Stem and Progenitor Cell - Quality Control (SPC-QC) Platform was the first specifically designed assay to measure stem cell potency. The rationale to develop the HALO®-96 SPC-QC Kit was four-fold.
Like the colony-forming cell assay, HALO®-96 MeC SPC-QC is a clonal assay. However, although detecting cells under clonal conditions has been the norm for many decades, it is not absolutely necessary for most applications. For this reason, HemoGenix® developed the HALO®-96 SEC (Suspension Expansion Culture) SPC-QC Platform, which as its name suggests, is performed not in methylcellulose, but in suspension culture. Because the cells are in close proximity and can form cell-to-cell interactions with each other under SEC conditions, the assay is completed more in only 5 days (as opposed to 7 days ) and is twice as sensitive as the HALO®-96 MeC SPC-QC.
CAMEO™-96 STD - The First Standardized Colony Forming Stem and progenitor Cell Assay that Measures both Proliferation and Differentiation
HemoGenix® realizes that the colony-forming cell assay has been used by investigators for more than 40 years and, for some, it is difficult to change not only an assay but a mentality. Because there is a direct correlation between the colony-forming cell assay, HALO®-96 MeC and HALO®-96 SEC, it was possible for HemoGenix® to develop the first stem and progenitor colony-forming cell potency assay that measures both proliferation and differentiation. More importantly, because a standardized, instrument-based, ATP bioluminesnce determination of proliferation is part of the procedure, the standardized ATP readout can also be used to calibrate manual enumeration of colonies in the same culture. This assay is now called CAMEO™-96 STD.
SUMMARY
HemoGenix® now produces three stem cell potency assay platforms:
Each platform can be used to measure single (CFC-GEMM) and duel (HPP-SP and CFC-GEMM) stem cell assays, 4-population hematopoietic assays and 7-population lympho-hematopoietic assays. In addition, there is also a primitive stem cell secondary re-plating assay.
