Complete, Bioluminescence Assay to Optimize and Standardize Methods
and Procedures to Ensure Hematopoietic Stem Cell Health for Cellular Therapy
Stem Cell Quality
- Stem cell "quality" is defined as the proliferation ability of stem cells at a specific point in time and at a particular cell dose.
- Stem cell "quality" cannot be measured using minimal testing criteria, (e.g total nucleated cell (TNC) count, viability, viable CD34 or a CFU assay), because these do not quantitatively measure the proliferation ability of stem cells.
- To quantify the proliferation ability or status ("quality") of stem cells in a stem cell product. Please note that this is not the same as stem cell potency.
- To compare stem cell "quality" before and after a specific proecdure (e.g. red blood cell depletion of cord blood).
- To determine stem cell "quality" pre- and post cryopreservation.
- To determine the effect on stem cell "quality" of equipment and bags used for stem cells.
- To determine stem cell processing stability.
- Gene targeting.
- Culture and expansion of stem cells in vitro and in bioreactors.
- To promote "Best Practice Criteria Testing".
- Quantitatively measures viability, proliferation ability and cellular functionality that cannot be enumerated using total nucleated counts (TNC), viability or flow cytometric detection of membrane expression markers.
- Determines the "quality" of mature, hematopoietic stem cells (SC-GEMM) alone or SC-GEMM and more primitive lympho-hematopoietic stem cells (SC-HPP).
- Instrument-based, incorporating an external standard and controls for assay calibration and standardization.
- Validated according to FDA bioanalytical method validation guidelines and can be validated in-house.
- Compare results over time for intra- and inter-laboratory comparison studies.
- Assay can be completed in just 5 days or can be extended to 7 days with a 3-fold increase in sensitivity.
- Simple, time efficient and more accurate than any traditional CFU assay, even using automated colony counting.
- CFU substitute proliferation assay allowed by the FDA, AABB and NetCord-FACT. Results can be uploaded to the NMDP cord blood database.
- Incorporates proprietary Suspension Expansion Culture™ (SEC™) Growth Medium and ATP bioluminescence technology, the most advanced and cost-effective lympho-hematopoietic in vitro assay system available for the stem cell processing laboratory.
An example of an ATP standard curve and control results are shown in the diagram below and actual values for the kit being used are given in the Technical Manual. If the values obtained are within the ranges given in the Technical Manual, the user can process the samples knowing that the results will be trustworthy.
- In vitro, hematopoietic multipotential stem cell (SC-GEMM 1)
- In vitro, lympho-hematopoietic primitive stem cell (SC-HPP)
|Assay Type||Cell Type||Catalog Number||Number of
|HALO® SPC-QC||SC-GEMM 1||K2-1QC-1||1||16|
|HALO® SPC-QC||SC-GEMM 1||K2-1QC-2||2||32|
|HALO® SPC-QC||SC-GEMM 1||K2-1QC-4||4||64|
|HALO® SPC-QC||SC-GEMM 1 + SC-HPP 2||K2-2QC-2||2||16|
|HALO® SPC-QC||SC-GEMM 1 + SC-HPP 2||K2-2QC-4||4||32|
- Mobilized peripheral blood
- Umbilical cord blood
- Bone marrow
- Purified cells from the above tissues
The recommended cell purity is a mononuclear cell (MNC) fraction or higher purity. A total nucleated cell (TNC) fraction is not recommended as this contains high concentrations of cell impurities, such as red blood cells, neutrophils, platelets and other cells that dilute, mask and severely underestimate and even inhibit the detection of rare primitive stem cells.
Examples of Measuring Stem Cell "Quality" Using HALO® QC
Normally, the SC-GEMM 1 (methylcellulose, CFC-GEMM 1 equivalent) stem cell population would suffice for routine assessment. However, a stem cell product may contain different proportions of stem cell populations. A product with few, if any, primitive stem cells will only result in short-term engraftment and reconstitution. A product with few multipotential stem cells may be misinterpreted as containing few, if any, stem cells, even if sufficient primitive stem cells were present, but not detected. These and other possibilities could occur during the processing procedure if the latter had not been properly evaluated or may occur during the cryopreservation or cell thawing process, if these had not been sufficiently evaluated. The results shown in the upper graph demonstrate that umbilical cord blood mononuclear cells exhibit the lowest proliferation ability compared to bone marrow and mobilized peripheral blood (mPB) CD34+ cells for both the primitive and more mature stem cell populations. Please note that mPB cells are shown at 1,000 CD34+ cells/well, while bone marrow and cord blood are shown at 5,000 MNC/well.
The lower graph illustrates a number of important aspects of the HALO® Platform and HALO® QC, in particular. HALO® QC can be setup so that results can be obtained on day 5, 6 or 7. If greater assay sensitivity is required, the cultures can be processed to measure intracellular ATP on day 6 or 7, instead of day 5. For stem cells, there is a 3 fold increase in proliferation ability within 2 days when measured on days 5 and 7. This is because the cells are proliferating exponentially. However, after day 5, differentiation begins to increase. Preferred Cell Systems™ recommends completing the assay on day 5, but if scheduling is a problem, it can be extended to day 7. Please note, however, that once day 5, 6 or 7 has been elected as the day of processing, ALL future assays should be processed on the same day, otherwise it will not be possible to properly compare results between assays.
The exponential increase in proliferation seen between days 5 and 7, and which continues to increase until about day 10, can only be detected using a proliferation assay such as HALO®. The CFU/CFC assay will not provide this information, since once a colony is formed, it will only get larger. The size of the colony is an indication of the primitiveness of the cells forming that colony.This information is very difficult to quantify and is lost to the user. The number of colonies counted between days 7 and 10 remains the same.
HALO® QC and HALO® PCA
Please note that although both these assays incorporate SEC™ and bioluminescence technology, they do not measure the same cell populations and therefore one cannot be used in place of the other.
Luminescence plate reader or multimode plate reader with "glow" luminescence measuring capability.
- HALO® QC Master Mix for SC-HPP and/or SC-GEMM and/or background control
- ATP standard
- ATP high and low controls
- ATP Enumeration Reagent
- Sterile, 96-well plates
- Non-sterile, 96-well plates
- Sterile, adhesive foil covers
Download the Luminometer Setup and RLU to ATP Conversion
Download the Certificates of Analysis (CoAs). Some CoAs will be included with the assay kit.
Download SDS Sheet for HALO® QC Master Mix.
Download SDS Sheet for ATP Enumeration Reagent
Download SDS Sheet for ATP Standard and Controls