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Colony Forming Unit Assay

WHAT IS CFU ASSAY?

When a patient is diagnosed and treatment is established using cord blood stem cells, the bone marrow is destroyed by chemo +/- radiotherapy (myeloablation). In this process, the cellular lineages giving rise to Erythrocytes, Leukocytes and Thrombocytes are all destroyed. The cord blood stem cell unit is introduced for complete cellular hence functional haematopoietic reconstitution. As an outcome of cord blood stem cell transplant, the transfused cord blood unit is expected to regenerate all the three types of cells, which constitute the blood.

However, how can one be sure whether the stem cells will perform the function expected of them. Therefore, as a standard practice, every cord blood unit to be transfused is first assessed for its capability to repopulate the bone marrow adequately and with functionally active cells. This assessment, the ultimate proof of Proliferation and Multipotency, is the CFU Assay.

CFU Assay exhibits the capacity of a single stem cell, to not only differentiate into specialized cellular lineages but also indicates the proliferation capacity up to an acceptable number of each of the specialized cell types. Even though viability had been used as the yardstick of the engraftment capacity, just by judging whether a cell is alive or not, it gives us no indication of the functional capacity of the stem cells, which is defined only through Proliferative and Potency assays i.e. “CFU Assay”.

Therefore, it is not sufficient to just elect to store one’s cord blood, it is equally important to reassure oneself of the Proliferative capacity and Potency both before permanent storage as well as at after retrieval, since it would provide the transplant physician with the assurance that the stem cells will perform as intended and would increase the efficacy of the stem cells for the patient’s treatment.

Why is CFU Assay recommended?

Till date the cord blood units have been mostly selected for transplants based on the MINIMUM SELECTION CRITERIA set by FDA, AABB and FACT-NETCORD. The minimum selection criteria included TNC count, CD 34 and % viability of the cord blood unit and transplant success rates were assumed to be dependent on higher count of each parameter.

However, even the best of cord blood units selected based on the selection criteria, when transplanted, resulted in 20-24% failure in primary engraftment1. Detailed study showed that high viability did not mean that all cells were healthy and functional. % viability only showed how many cells were alive, but whether the cells were fully functional or not was not determined.

This gave rise to the question - how could one be sure whether the stem cells will perform the function expected of them?

That is why a fourth criterion - CFU Assay was recommended to be a part of the Minimum Testing Criteria for optimizing screening of Cord Blood Units for transplantation.

Why should you opt for CFU Assay at the time of storage?

Research carried out at Duke University2 showed evidence that Post processing and pre-freezing quality and potency was not equal to post thaw quality and potency and Cord Blood APGAR3 was developed which measured both pre-freezing and post-thaw graft characteristics and was thus predictive of engraftment after UCBT. According to this study, this difference was attributed to the various stress factors the stem cells go through especially the thermal stress during cryopreservation and thawing. This study suggested that Potency testing by CFU assay was not only recommended at the time of transplant but also before freezing. This would allow one to be assured of the quality of the stem cells i.e. potency and proliferative capacity and bring in a sense of security at the very incipient stage.

How is CFU Assay done?

A known number of Haematopoietic stem cells derived from umbilical cord blood, are introduced into an enrichment media, which under controlled conditions give rise to all the cells of the haematopoietic system, that too, in adequate numbers, expected from the initial cell sample after approx. 14 days of culture and a detailed report would be shared which would have the actual image of the colonies formed.
This test is an ex vivo representation of the expected outcome - engraftability, of the haematopoietic stem cells in vivo.




BFU-E: Burst forming unit-erythroid
CFU-GEMM: Colony-Forming Unit-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte

Reference:
1) Ivan N. Rich. Improving Quality and Potency Testing for Umbilical Cord Blood: A New Perspective. Stem Cells Translational Medicine 2015;4:967–973
2) Page et al. Total Colony-Forming Units Are a Strong, Independent Predictor of Neutrophil and Platelet Engraftment after Unrelated Umbilical Cord Blood Transplantation: A Single-Center Analysis of 435 Cord Blood Transplants. Biol Blood Marrow Transplant 17: 1362-1374 (2011)
3) Page et al. The Cord Blood Apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation. Transfusion. 2012 February ; 52(2): 272–283



With growing health care facility in India, it is very well expected that life expectancy of an individual will increase in coming years and can go up to Seventy-Five (75) years. Additional Storage Programme is a provision offered to parents to opt for extended storage of Umbilical Cord Blood Stem Cells for an additional period of Fifty-Four (54) years. This allows for secured storage for a longer duration in comparison to just 21yrs. The Additional Storage Period starts only after the completion of the primary Agreement i.e. on the Twenty-First (21st) birthday of the Baby. The Client has the option to either choose storage for 21yrs only or opt for additional 54yrs as well.

About HLA

Human leukocyte antigen (HLA) are proteins or markers found on most cells in our body. A patient’s immune system uses HLA markers to know which cells belong to their own body and which belong to the donor.

Why is HLA typing needed?

HLA typing is an important test on stem cells to identify a match for patients scheduled for cord blood stem cell transplants3. The best transplant outcome happens when a patient’s HLA and the HLA of the baby’s cryopreserved cord blood stem cells (donor) closely match.



There are 6 antigens normally tested for matching. Since the cord blood stem cells are immature, it poses lower risk of complications after engraftment, especially graft vs host disease (GVHD), a potentially serious complication which arises when the immune cells which are part of the donated stem cells, attack the patient’s body.

That is why, even a 4 out of 6 match is just as effective at curing patients as compared to a 6/6 match in bone marrow transplants.

Why is HLA typing recommended at the time of storage?

The entire process of HLA typing and matching is time-consuming and takes almost 6-9 weeks from decision for HLA typing of both stem cells and patient till transplant. HLA typing does not guarantee that the stored stem cells will be a match with the patient. Isn’t that a waste of time, when the patient is already critical waiting for treatment. Therefore, in order to avoid unnecessary delay, it is recommended to perform HLA typing on stem cells right at the time of storage for ready access whenever in requirement. If the HLA type is already available, all that the patient has to do is get his own HLA type done and match it against the available data. This takes just 2-3 weeks and saves crucial time for the patient in need and allows the family and physician to take steps accordingly. Getting HLA typing done at the time of storage also prevents wastage of precious stem cells since the test is done using cord blood sample leftover within the tubes instead of using one of the three cryopouch segments which would ideally be required for final testing at the time of release. One of the significant factors that influence the transplant outcomes is the TIME – how early the patient gets the matched stem cell unit for treatment.

Reference:
https://bethematch.org/transplant-basics/matching-patients-with-donors/how-donors-and-patients-are-matched/hla-basics/

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