The Only Family of
STEM CELL
QUALITY CONTROL,
POTENCY and
PATIENT MONITORING ASSAYS
Specifically Designed for
Stem Cell Transplantation Processing Laboratories
Download the Quality Control Brochure here.
IMPORTANT NOTICE
In a surprising turn of events, Stem Cell Technologies has acknowledged the basic concept of the HALO® Platform:
At the International Society of Cellular Therapy meeting in San Diego, May 3-6, 2008, Stem Cell Technologies claimed to have developed a "fast and simple" colony-forming cell (CFC) assay as a potency assay forhuman umbilical cord blood.
In fact there is nothing "new" about the assay. Essentially the same results were published in 2003 by the Founder and CEO of HemoGenix® (Current Opinion in Drug Discovery & Development 6:100-109). The assay is certainly not easier because the same method is being used and manual enumeration of colonies under a microscope is still necessary. And even though the assay may be as fast as HALO®-96 MeC and completed in 7 days, it still produces the same subjective and meaningless results as with the 14 day assay. For reasons described below, no CFC assay, regardless of whether it is performed at 7 days or 14 days can be used as a potency assay.
INTRODUCTION
Stem cell transplantation has been part of the clinical community since the mid 1970s. Starting with bone marrow transplantation followed by the use of mobilized peripheral blood when recombinant G-CSF was introduced in 1987 and then with umbilical cord blood in 1989, procedures and regimen for treating patients with life-threatening hematological disorders have improved dramatically. New regulatory guidelines require ever-stricter improvements in most aspects of the stem cell transplantation process. Instrumentation has significantly improved both in sensitivity and in sophistication. Yet, the basic tests and assays required to determine the “quality” of the product prior to transplantation have remained unchanged for decades.
The exception has been HLA typing. However, this highly advanced technology is coupled with four parameters that, at best, provide limited correlative information to reduce risk and improve outcome for the patient. The total nucleated cell (TNC) count is usually used to determine the number of cells that need to be transplanted. But TNC contain cells that do not proliferate and mask the mononuclear cell fraction which contain the stem cells. Viability is performed by dye exclusion that detects membrane integrity, but provides no information on cellular and mitochondrial integrity indicative of cell functionality. Although other membrane markers for stem cells are now being used, CD34 membrane expression, is still thought of as a “stem cell marker”. However, lineage-specific progenitor cells, which have no stem cell characteristics, also express this antigen. Despite this, TNC, viability and CD34 enumeration can be standardized and validated. In contrast, the colony-forming cell (CFC) assay, which has been employed in the field since 1971, is so subjective, it has never been possible to validate as a quality control assay, let alone a potency assay required by regulatory agencies.
HemoGenix® has been the leader in hemotoxicity testing since it was founded in 2000. HemoGenix®® has also been proactive and innovative in developing a family of quality control, cell potency and patient monitoring assays specific for the processing laboratory that are not only aimed at regulatory compliance by being standardized and validated, but are also rapid and easy to use.
What is Standardization and Validation?
Those who propagate the colony-forming cell (CFC) assay for clinical use maintain that the assay is standardized. They point to “standardized” and “consistent” reagents as evidence. Yet standardization is more than ensuring that reagent batches are similar and that they work properly. Assay standardization is defined as the process of checking or adjusting, by comparison with a known standard, the accuracy of the test. Until the development of CAMEO™-96 STD, there was no known external standard to which the CFC assay could be compared.
Validation is defined as establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes. Part of the assay validation procedure involves quantitatively demonstrating accuracy, selectivity, specificity, reliability, reproducibility and robustness. The CFC assay fails miserably for accuracy, reliability, reproducibility and robustness.
ALL HALO® QUALITY CONTROL, STEM CELL POTENCY and PATIENT MONITORING ASSAYS FROM HemoGenix® ARE STANDARDIZED and VALIDATED and DESIGNED TO BE COMPLIANT WITH REGULATORY AGENCY GUIDELINES.
HALO®-96 SPC-QC (Stem and Progenitor Cell – Quality Control) was specifically developed to routinely assess the proliferation potential of bone marrow, mobilized peripheral blood or umbilical cord blood in the busy cell processing laboratory. HALO®®-96 SPC-QC is only available to measure either the mature hematopoietic stem cell population, CFC-GEMM, or both the CFC-GEMM and the more primitive lympho-hematopoietic stem cell population, HPP-SP, since these are the cell populations upon which stem cell transplantation relies. The CFC-GEMM cell population will provide an excellent indication for potential short-term engraftment, while measuring both mature and primitive stem cell populations will provide information on both short- and long-term engraftment potential without having to use the more laborious and costly LTC-IC assay.
HALO®-96 PQR (Potency, Quality Release) was specifically designed as a stem cell potency and release assay for umbilical cord blood units prior to transplantation.
REGULATORY REQUIREMENTS
In the United States, potency is defined in the Code of Federal Regulations (21 CFR 600.3) as “the specific ability or capacity of a 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”. Potency is defined by the European Medicines Agency (EMEA) as a “quantitative measure of biological activity”. A test for potency has also been defined in the U.S. Code of Federal Regulations (21 CFR 610.10), but the EMEA’s definition is probably easier to understand as an in vivo or in vitro test that is “appropriately validated” and “based on a defined biological effect as close as possible to the mechanism(s) of action / clinical response”.
Potency measurements have been used for decades to determine the activity and therefore the administration dose of a drug, vaccine, growth factor etc. Potency measurements are now being required for cellular therapy products because:
• It ensures consistency during production/manufacture of biological products.
• It shows product stability.
• It predicts product performance and assurance.
• It allows evaluation and/or correlation with the clinical response.
• It avoids product failure due to toxicity or improper potency.
What is Needed to Measure Potency?
There are three basic requirements for measuring potency. These are:
1. An “appropriate validated” assay.
2. A reference standard (RS) of the same material.
3. A dose response relationship.
As shown in the diagram below, 3 growth factor samples are compared to the same growth factor reference standard to produce statistically parallel dose responses for the linear portion of curves. The horizontal displacement to the right indicates a lower potency ratio than the RS, while a displacement to the left indicates a higher potency ratio and therefore greater activity than the RS.
Can any of the Presently Used Tests be Used as a Potency Assay?
NO. Tests for TNC, viability, CD34 and other membrane or intracellular markers detected by flow cytomtry cannot be used as potency assays because they do not conform to the requirements.
Can the Colony-Forming Cell (CFC) Assay be used as a Potency Assay?
NO. It is not standardized and it cannot be validated.
HALO®-96 PMT (Patient Monitoring after Transplantation) helps you ask the question:
ARE THE TRANSPLANTED CELLS ACTUALLY REPOPULATING THE PATIENT?
Whereas HALO®-96 SPC-QC and HALO®-96 PQR were developed to assess the “quality” and potency of the stem cell product prior to transplantation and may even predict engraftment potential, HALO®-96 PMT is used to monitor reconstitution of the patient after transplantation.
Although an assay may be predictive based on results, the disease, conditioning regimen and many other factors can greatly influence engraftment and reconstitution of the patient. During the early phase after transplantation, hematopoietic reconstitution is of prime importance. At later times, lymphopoietic reconstitution is important in order to try and predict lymphoid involvement. Reconstitution may also be “unbalanced” in that cells of one or more lineages may be produced either preferentially or at levels that cannot sustain the patient.
HALO®-96 PMT can help monitor the lympho-hematopoietic status of the patient after transplantation using peripheral blood or a bone marrow sample of less than 500μl.
Like HALO®-96 SPC-QC, HALO®-96 PMT is also available to culture cells either under clonal conditions in methyl cellulose, or using Suspension Expansion Culture (SEC) technology. Unlike HALO®-96 SPC-QC however, HALO®-96 PMT is designed to detect and measure either 4 or 7 cell populations simultaneously. The cell populations detected are:
• 4-Population Assay. CFC-GEMM, BFU-E, GM-CFC and Mk-CFC.
• 7-Population Assay. HPP-SP, CFC-GEMM, BFU-E, GM-CFC, Mk-CFC, T-CFC and B-CFC with a background control.
The 4-population assay can monitor hematopoietic reconstitution, while the 7-population assay provides a “global” picture of both the hematopoietic and lymphopoietic repopulation.
HOW TO STANDARDIZE THE COLONY-FORMING CELL (CFC) ASSAY - Use CAMEO™-96
Having reagents that show batch consistency does not mean that the assay is standardized. Furthermore, training personnel to count similar types and numbers of colonies produced in the CFC assay also does not mean that the assay is standardized. In order to standardize the CFC assay, it has to be calibrated against an external standard. Since no external standard exists to which the CFC assay can be calibrated, it can not be standardized.
This problem has been solved by HemoGenix®, which has developed CAMEO™-96 for stem cell processing laboratories. The assumption that allows CAMEO™-96 to be used to standardize the CFC assay is that target cells are cultured using the same reagents under exactly the same conditions. CAMEO™-96 combines a 14 day CFC differentiation assay for human bone marrow, mobilized peripheral blood or umbilical cord blood cells, cultured in methylcellulose in a 96-well plate, with an 14 day ATP bioluminescence proliferation assay performed on the same cells in the same plate. After the 14 day incubation period has elapsed, the total number colonies in each well are manually counted using an inverted microscope. After counting, the same cultures are then processed to measure the intracellular ATP concentration using the reagents included in the kit. Prior to processing, an ATP standard curve and high and low controls are performed. When the total colony count is plotted against the iATP concentrations, a direct correlation between the 2 parameters is obtained. Because of the correlation produced, total colony counts can be expressed directly as standardized iATP concentrations.