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Background - Cellular Therapies
 
  • Turning Failures

    into Success 

  • Stem cell "Quality" Testing

  • Stem Cell Potency Testing

Background to Cellular Therapeutics

 

Hematopoietic Stem Cell Transplantation
and Cord Blood Banking:Turning Failures into Success

Download the article "Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units" published in the Journal of Translational Medicine (2015) 13:94. Click here to download or go to PubMed.

Read the article entitled "Current Testing Fails to Assay Cord Blood Quality & Potency" in the Parent's Guide to Cord Blood Foundation, May 2015.

 

Introduction

Transplantation of hematopoietic cells dates back more than 60 years. However, it was not until the 1970's that transplantation of human bone marrow stem cells became a routine procedure. In the 1980s, and with the introduction of commercially available human, recombinant granulocyte colony-stimulating factor (G-CSF) to mobilized stem cells out of the bone marrow and into the circulation, it became possible to use mobilized peripheral blood as a stem cell source for transplantation. In 1989, umbilical cord blood ceased to be a waste product and became the third source of hematopoietic stem cells used for transplantation into patients with blood maligancies.

Consider This:

More than 35,000 cord blood transplants have now been performed worldwide for more than 100 different applications.

But:

Of these 35,000 cord blood transplants from more than 730,000 umbilical cord blood units collected and worldwide, not a single unit has been evaluated to ensure that the stem cells are of "high-quality" and potency.

And the failures keep mounting:

  • Up to 1 in 4 patients succumb to graft failure due to low or lack of stem cell potency.
  • Failure to consider cord blood stem cells as the only crucial entities upon which cord blood stem cell transplantation is based.
  • Failure by the cord blood community to accept that assumptions do not make for a successful cord blood transplant, or any stem cell transplant for that matter.
  • Failure to understand the limitations of current cord blood processing and testing "minimum guidelines".
  • Failure by the cord blood community to consider anything else except total nucleated cell (TNC) count for cord blood storage and use.
  • Failure to use scientific measurements to determine cord blood quality and potency.
  • Failure to properly understand the meaning, use and measurement of potency.
  • Failure of U.S. public cord blood banks to comply with the Stem Cell Therapeutic and Research Act of 2005 and its reauthorization in 2010 to ensure that the stem cells in all cord blood units collected (approx. 206,000) are of "high-quality".
  • Failure of the Advisory Council on Blood Stem Cell Transplantation (ACBSCT), authorized by the Stem Cell Therapeutic and Research Act to define what is meant by "high-quality umbilical cord blood units".
  • Failure by the National Marrow Donor Program (NMDP), which is sub-contracted by the U.S. Government under the Stem Cell Therapeutic and Research Act of 2005 and 2010, to change its "inventory requirements for new cord blood units" and allow cord blood banks to persue and use their own assays to ensure "best practices" for cord blood unit quality and potency.
  • Failure by the NMDP Cord Blood Advisory Group to promote, and instead activiely impede, the "rapid adoption of new technologies".
  • Failure by the U.S. Food and Drug Administration (FDA) to update their "current thinking on the topic" to improve "non-binding recommendations" of "purity and potency" that are now known not to work.
  • Failure by NetCord-FACT to promote "best practices".
  • Failure by the the cord blood community to acknowledge the problems with cord blood testing and remain silent.
  • Failure by regulatory agencies, standards organization and the cord blood community in general to make the patient their #1 priority.

Complacency leads to failures that are paid for by the patient
START TURNING FAILURE INTO SUCCESS
It's in everyone's interest

 

Regulatory Considerations

In 2005, the United States Congress passed the Stem Cell Therapeutic and Research Act. The proper title is: "To provide for the collection and maintenance of human cord blood stem cells for the treatment of patients and research and to amend the Public Health Service Act to authorize the C. W. Bill Young Cell Transplantation Program". The Act specifically requires public cord blood banks to ensure the collection and maintenance of "high quality cord blood units". This means that the stem cells in the cord blood unit must be of "high-quality". The Statute was re-authroized in 2010. However, not a single umbilical cord blood unit collected and uned for transplantation has ever been tested to ensure that the stem cells in the unit are of "high-quality" and potency; in fact, stem cells are not test at all.

In 2009, the FDA designated umbilical cord blood a "drug". This is because cord blood transplantation results in systemic effects to the patient. It is also the only hematopoietic stem cell product that is stored for long periods of time. This required cord blood banks that supplied cord blood units for transplantation to to apply for a license from the FDA . As of April 2015, only 5 cord blood banks in the U.S. have been approved.

The FDA Guidance for Industry with the title "Biologics License Application for Minimally Manipulated, Unrelated Allogeneic Placenta/Umbilical Cord Blood Intended for Hematopoietic and Immunologic Reconstitution in Patients with Disporders Affecting the Hematopoietic System" was published in March 2014. Both this and a previous Guidance published in 2009 give "non-binding recommendations" for cord blood tests of "purity and potency" as:

  • Total nucleated cells (TNC) of Hematopoietic Progenitor Cells (HPC), Cord Blood (pre-cryopreservation),
  • Viable nucleated cells of HPC, Cord Blood (pre-cryopreservation), and
  • Viable CD34+ cells (flow cytometry) of HPC, Cord Blood (pre-cryopreservation).

These are considered "Minimal Testing Criteria", rather than "Best Practice Criteria Testing"  by AABB, NetCord-FACT and the National Marrow Donor Program (NMDP) Current Inventory Requirements for New Cord Blood Units.

Since only "Minimal Testing Criteria" are presently used, none of the recommendations and standards actually address the characterization and properties of cord blood stem cells to ensure "high-quality umbilical cord blood units" as mandated by the Stem Cell Therapeutic and Research Act. In addition, since potency has to be measured prior to use of the product and none of the recommended tests for cord blood purity and potency comply with U.S. Code of Federal Regulations for potency assays (see Stem Cell Potency Testing), the FDA BLA guidelines for cord blood directly contradict other U.S. Statutes.

The failures listed above are a direct consequence of these actions by regulatory and standards organizations.

 

Promoting "Best Practice Criteria Testing" 

Cellular therapy and regenerative medicine are rapidly evolving fields. Characterization of a therapeutic product is key to understanding and predicting the intended response and effect. “Minimum testing criteria” rarely allows the necessary characterization that defines the “quality” and potency of the product prior to use in the patient. HemoGenix® advocates and promotes the use of “Best Practice Criteria Testing” for cellular therapy and regenerative medicine products for use in patients. Here are a few recommendations that will help you implement “Best Practices” in your cell processing laboratory.

  1. The quality of results directly correlates with the quality of the material and the assay(s) used for testing.
  2. Consider the origin of the sample to be analyzed and whether it actually represents the total product. For example, umbilical cord blood segments will differ widely from each other and from the unit they are supposed to represent.
  3. Where possible, always use a standardized and validated assay. Assay validation is the key to trustworthy results.
  4. Consider the purity of the cell sample to be characterized. Never use a total nucleated cell (TNC) fraction for a stem cell product. The TNC fraction contains cell impurities that will dilute and mask the rare, primary stem cells from being measured. This will seriously underestimate the “quality” and potency of the product and will lead to a false interpretation of the results. Take cord blood processing to the mononuclear cell (MNC) level.
  5. Consider how viability is measured. Dye exclusion viability (trypan blue, propidium iodide, acridine orange, 7-AAD) can result in high, false positive results. In other words, the cell might exhibit a high viability, but their metabolic viability may indicate low sustainability or that most of the cells are dead.
  6. What needs to be measured to obtain an accurate and reliable analysis of the product prior to use in the patient? If the cells in a product have to proliferate in order to exercise their effect, a functional assay that measures cell proliferation is required. Other assays will not be relevant.
  7. Potency is a quantitative measure of biological activity of the “active” components that result in the intended response and/or effect. Tests such as cell count, viability and membrane expression markers are neither specific nor do they measure biological activity and are therefore not potency assays. In addition, the measure of potency is the potency ratio, which can only be determined when the sample is compared to a reference standard of known potency.

Stem Cell "Quality" for Engraftment and Reconstitution

 

Download the article "Dtecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units" published in the Journal of Translational Medicine (2015) 13:94. Click here to download or go to PubMed. This article used HALO®-96 SPC-QC.

 

What is Stem Cell "Quality"?

Stem cell "quality" is the proliferation ability of the stem cells measured at a specific cell dose and point in time. Since one of the properties of stem cells is to proliferate, stem cell "quality" can only be measured using a stem cell proliferation assay. Stem cell "quality" is therefore a specific biological function that characterizes the stem cell cells. 

Regulatory Considerations

The Stem Cell Therapeutic and Research Act of 2005 requires that all public cord blood banks ensure that the cord blood units collected and maintained are "high-quality cord blood units". Present tests, notably:

  • Total nucleated cells (TNC),
  • Viability, and
  • Viable CD34+ cells, together with the
  • CFU assay

are considered "Minimal Testing Criteria" and do not determine if a cord blood unit is actually of "high-quality". The assays described below promote "Best Practice Criteria Testing" and have been designed to measure stem cell "quality". 

Assays for Stem Cell "Quality" 

HemoGenix® has developed 2 distinct assays to measure stem cell "quality".

 

 

Using STEMpredict to predict cord blood quality, viability and functionality

 

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STEMpredict™

STEMpredict™ is part of the HALO® family of assays for hematopoietic stem cell therapy. STEMpredict™ is the most rapid stem cell assay available. It is completed in just 3 days. As its name implies, STEMpredict™ predicts the ability of hematopoietic stem cells to demonstrate proliferation. It uses ATP bioluminescence technology to compare the stimulation and growth of stem cells to a background control (see the graph). STEMpredict™ is used to help determine whether a cord blood unit can be permanently banked and if a donor or patient has been properly mobilized for peripheral blood stem cell collection. 


HALO®-96 SPC-QC

HALO®-96 SPC-QC is a 5 - 7 day assay used to measure stem cell proliferation ability ("quality") before and after a processing procedure in order to standardize and optimize the method. HALO®-96 SPC-QC assay kits are available to measure either primitive hematopoietic stem cells (CFC-GEMM) alone or in combination with primitive lympho-hematopoietic stem cells (HPP-SP). Like STEMpredict™, it incorporates ATP bioluminescence technology required to detect and measure rare, stem cell populations.

For the mesenchymal stem cell system, the equivalent assay to HALO®-96 SPC-QC is MSCGlo™-96 HuQC

Assays for Engraftment and Reconstitution

After infusion of a hematopoietic stem cell product into a patient (the transplantation procedure), the stem cells will try to "home" or find their way to the bone marrow. There, they will lodge and start to proliferate. This is called the engraftment process. Shortly after early engraftment, some of the stem cells will enter the differentiation lineages that are responsible for producing red blood cells, neutrophils and platelets. This process is called reconstitution. A patient is said to have engrafted if the number of neutrophils is equal to or greater than 500,000/µL for 3 days or more and the number of platelets is equal to or greater than  20,000 - 50,000/µL for 3 days or more. The time at which these numbers are reached is call the Time to Engraftment. 

The cells from which the neutrophils are derived are the granulocyte-macrophage progenitor cells. Similarly, platelets are derived from megakaryocyte progenitor cells and red blood cells are derived from erythropoietic progenitor cells. The assay used to determine these progenitor cells is called the colony-forming unit (CFU) or colony-forming cell (CFC) assay. Since the assay takes 14 days until results are obtained and the time to engraftment for bone marrow or mobilized peripheral blood is similar to or less than this time period, the CFU/CFC assay is not a routinely used by many transplantation centers for these tissues. Umbilical cord blood, however, is always used from a cryopreserved source. It takes much longer for thawed cord blood cells to start reconstituting a patient (usually 20 days or longer) and as a result, the CFU/CFC assay has been maintained as a requirement by the cord blood community.

Usually, the CFU/CFC assay is performed prior to cord blood unit cryopreservation and does not provide a good indicator for thawed cells. Indeed, the assay is not only difficult to perform, but exhibits notoriously high variations (coefficients of variation, CVs) that make inter-laboratory comparisons extremely difficult. In their Current Inventory Requirements for New Cord Blood Units, the NMDP only requires a sample tested using the CFU/CFC assay to be reported as "Growth" or "No Growth" with the actual colony count. That the threshold for cord blood unit release cannot be defined has led to the conclusion that the assay should not be used or a different assay should be used.  

 

HALO®-96 PMT

HALO®-96 PMT is an alternative the CFU/CFC assay. It is another member of the cellular therapy assay family designed to predict time to engraftment and determine "global" reconstitution. Early prediction of engraftment can provide much needed time for the transplantation physician to re-treat the patient if engraftment is not predicted engraftment or is delayed. Therefore, the greater the predictive value of the assay, the lower the risk to the patient.

 

Patient monitoring after a stem cell transplant using HALO-96 PMT 7-Population "Global" assay

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In addition, HALO®-96 PMT for 4-, 5- or 7-populations can be used to monitor hematopoietic or lympho-hematopoietic reconstitution. The graph shows results of a 7-population assay for both bone marrow and cord blood detected after 7 days in culture. If results are obtained after only 5 days, the response of cord blood will be much lower than that of bone marrow. This indicates that cryopreserved cord blood cells require about 2 days longer to reach the same level of proliferation ability than bone marrow cells.

Stem Cell Potency Testing

  

Promoting "Best Practice Criteria Testing" for Hematopoietic Stem Cell Therapy products

 

 

 

Published articles on measuring hematopoietic stem cell potency

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Regulatory Considerations

The potency of a drug predicts its dose. Traditional drug potency testing has been a routine procedure for many years. Potency testing of cellular therapeutic and regenerative medicine products is a new and often misunderstood and confusing experiance. Since regulatory agencies, i.e. FDA and the European Medicines Agency (EMA), are new to this field, they rely on industry to provide them with details. This is particularly the case for stem cell biologics and hematopoietic stem cell therapy in particular.

The EMA published its guidelines on potency testing in May 2008 entitled "Guideline on the Potency Testing of Cell Based Immunotherapy Medicinal Products for the Treatment of Cancer", while the FDA published its "Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products" in January 2011. The tissue most affected by these guidelines at the present time is umbilical cord blood since this was designated as a "drug" by the FDA in 2009. As such, the potency of cord blood stem cell products that are minimally manipulated and used for allogeneic transplantation should be assessed when a unit has been identified and prior to use in the patient. This is because "All potency assays used for release testing of licensed drug products must comply with applicable biologics and cGMP regulations including":

 

Potency Assay Requirements Code of Federal Regulations
Indicates biological activity specific to the product 600.3;610.3;210.3(b)(16)(ii)
Results allow for realease of the product 610.1; 211.165(a)
Provides quantitative data 211.194
Meets pre-defined acceptance/rejection criteria 211.165(b)(16)(ii); 211.160
Includes reference material, standards and controls 210.3(b)(16)(ii); 211.160
Demonstrates validation 211.165(e); 211.194(a)(2)
Measures identiy & activity of the active ingredients 211.164(a)

 

In contrast to these regulations, non-binding recommendations by the FDA for purity and potency of cord blood represent "Minimum Testing Criteria" and include: 

  • Total nucleated cells (TNC)
  • Viable nucleated cells
  • Viable CD34+ cells
  • All tested on the pre-cryopreservation product.

These same recommendations are mirrored in the NetCord-FACT 5th Edition Standards, but in addition include the statement, "CFU or other validated potency assay". This assumes that the colony-forming unit or CFU is a potency assay and is validated, neither of which are correct.

Since the potency and general characterization of umbilical cord blood stem cells has never been addressed by either the FDA or standards organizations (NetCord-FACT, AABB, NMDP), cord blood testing has not been in compliance with the Stem Cell Therapeutic and Research Act of 2005.

 

How does "Minimum Testing Criteria" differ from "Best Practice Criteria Testing"?

The 'minimum testing criteria" refers not only to the most basic type of testing that can be performed, but also to tests and assays that are not standardized or validated and, as a result, produce highly variable data that cannot be used to produce statistical significance. "Best practice criteria testing" are often more complex assays that allow the method to be standardized and validated so that consistency, reliability and reproducibility can be obtained. Implementing "best practice criteria testing" includes, but is not limited to, the following considerations: 

  • Intsrument-based signal detection readout
  • Quantitative data
  • Uses standardized analysis of biological assay procedures described in the U.S. Pharmocopeia
  • Inclusion of internal and/or external standards that allow a standard curve to be performed
  • Inclusion of controls
  • Determination of acceptance/rejection values
  • Determination of value ranges for specific assay parameters
  • Potential to validate the assay (accuracy, sensitivity, selectivity, reliability, reproducibility, robustness)
  • Ability to accurately and reliably measure the "active" target components
  • Ability to compare data over time. 

The key to "best practice criteria testing" is standardized measurement. If an assay is not standardized, it cannot be validated and the results cannot be trusted.

 

What is Stem Cell Potency?

Stem cell potency is the quantitative and validated measurement of the biological activity of the "active" stem cell components that are responsible for the intended response or effect.

From the regulatory requirements for a potency assay, it is clear that "minimal criteria testing" cannot and does not provide the compliance needed to measure potency of a hematopoietic stem cell therapeutic product. To measure stem cell potency, the assay has to conform to "best practice criteria".

 

 

When should stem cell potency be measured?

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When should cord blood stem cell potency be measured?

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When Should Potency be Measured?

The timing of conducting a potency assay is key to providing the transplant center and physician with the most important information. When an umbilical cord blood unit is collected, it must be cryopreserved within 48 hours of collection. When the unit reaches the processing laboratory a sample is taken on which numerous tests and assays are performed, including HLA typing, sterility, infectious diseases as well TNC, viability, CD34 and CFU, i.e. "minimal testing criteria". It can take up to three weeks to obtain all the results from a single, by which time the unit has been frozen. "Minimal testing criteria" are usually performed on the fresh cells and the decision to upload the umbilical cord blood unit to the NMDP cord blood inventory is based on parameters such as cell number, volume, weight etc., rather than the properties of the stem cells present in the product. Prior to freezing, the cord blood unit is red cell reduced. However, the properties of the cells are quite different between the fresh and frozen state. In fact, stem cell proliferation ability will actually decrease 3-4 fold between the fresh and frozen state. It therefore follows that the "minimum testing criteria" recommended by the FDA as purity and potency characteristics on a pre-cryopreserved sample cannot be used to measure cord blood stem cell potency. Indeed, due to these testing method not complying with the Code of Federal Regulations for potency assays as well as other considerations, the "minimum testing criteria" should never be considered as potency assays.

For umbilical cord blood, stem cell potency must be performed on a sample after the unit has been cryopreserved (see diagram). This allows the results from the potency assay to be uploaded to the cord blood inventory database so that the transplant physician can asses whether the units with exhibit "high-quality" and high potency to predict engraftment. Since cord blood units may remain frozen for more than 15 years, a second potency assay should be performed when the unit has been identified as compatible with a patient and prior to leaving the cord blood bank for the transplant center. This allows results to be compared with the original potency assay to determine if stem cell "quality" and potency have changed and provides the transplant physician with the assurance that the unit can be used. 

 

Potency Assays that Conform to "Best Practice Criteria Testing"

HemoGenix® has developed two potency, quality, release assays that are compliant with both potency assay regulations and "best practice criteria testing". These are HALO®-Potency for hematopoietic stem cell products, MSCGlo™-Potency for mesenchymal stem cell products and STEMGlo™-Potency for ES, iPS and primary stem cell populations. Both incorporate ATP bioluminescence technology and like all HemoGenix® ATP bioluminescence assays, they incorporate standards and controls that promote "best practice criteria testing". In addition, they also allow an internal proficiency test to be performed evry time prior to measuring a sample. HemoGenix® has a Master File for HALO®-Potency with the FDA.

To perform a potency assay for a hematopoietic stem cell product (cord blood, mobilized peripheral blood, bone marrow or purified populations from these tissues), it is first necessary to reduce or remove unwanted cells (red blood cells, granulocytes, platelets) since these dilute and mask the rare stem cell populations that need to be determined.

HALO®-Potency, MSCGlo™-Potency and STEMGlo™-Potency are all performed by measuring the slope of the cell dose response of the stem cells being measured. The slope is a measure of stem cell proliferation potential. The steeper the cell dose response, the greater the stem cell proliferation potential, the more primitive the stem cells and the greater their potency. As seen in the diagram below, stem cell self-renewal, primitiveness and potency all correlate with stem cell proliferation potential. The greater the potency, the greater the probability of engraftment. Thus, a stem cell potency assay predicts the ability of the stem cell to engraft in the patient. This is called stem cell engraftment potential.

 

 

The hierarchy and organization of the blood-forming system and the properties and tests used to measure different cells

 

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The stem cell transplantation process and why stem cell potency must be measured before transplantation

 

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Stem Cell Potency / Engraftment Potential is not the same as Time to Engraftment

Time to engraftment is measured by the number of neutrophils, platelets and red blood cells in the circulation. These functional, mature cells are derived from lineage-specific progenitor cells, which, in turn, are derived from the hematopoietic stem cells. The number of these progenitors (GM-CFC, Mk-CFC and BFU-E) is indicative of time to engraftment. Since the presence of these progenitor cell populations and their functionally mature progeny are part of the reconstitution program as a result of engraftment, these populations provide no indication of stem cell engraftment potential and therefore cannot be used to measure the potency of the stem cell populations responsible for engraftment. The difference beteen engraftment potential and time to engraftment is shown in the lower diagram.