CFU-GM Assay Can Be Predictive of Umbilical Cord Blood Engraftment.

Abstract

Cord blood transplantation (CBT) has been considered a treatment modality to hematologic malignancies, marrow failure syndromes and genetic diseases. It has advantages such as immediate availability, lower risk of transmitting infections and higher tolerance to HLA disparities compared to bone marrow. The major drawbacks are slow hematopoietic recovery and a high incidence of graft failure or delayed engraftment, as a result of low stem progenitors (CD34+cells) yields. This study aimed at investigating if there was correlation between engraftment and number of total nucleated cells (TNC), number of CD34+ cells as well as number of granulocyte/monocyte colony forming units (CFU-GM) and erythroid burst forming units (BFU-E). Eighty four patients (age 0 to 55, M=7) with a variety of diseases (10 AML, 12 ALL, 31 Fanconi Anemia, 1 Hodgkin Lymphoma, 3 JMML, 3 Blackfan Diamond, 6 Wiskott Aldrich, 1 Krabbe Disease, 2 SAA, 4 CML, 3 Osteopetrosis, 3 SCID, 1 Diskeratosis Congenita, 1 Adrenoleukodystrophy, 1 Thalassemia Major, 1 Thrombocytopenia w/chromosome 7 monossomy) who underwent CBT were included in this study; 47.6% (40/84) of these patients had graft failure while 52.4% (44/84) showed successful engraftment. Regarding HLA-A, B and DRB1 matching, 20.2% (17/84) recipient/donor pairs were 6/6, 42.9% (36/84) were 5/6, 35.7% (30/84) were 4/6 and 1.2% (1/84) was 3/4. TNC and CD34+ cell counts in addition to hematopoeitic progenitor cells (CFU-GM and BFU-E) testing were performed in cord blood products after thawing and washing procedures. CD34+cells were analyzed by two color technique using FACS Calibur (San Jose, CA) and absolute counts were assessed by two-platform ISHAGE method. Colony assays for BFU-E and CFU-GM were performed by plating 3×104 mononuclear cells in methylcellulose medium with human recombinant cytokines (Methocult GF, Stem Cell Technologies); cultures were kept at 37°C in humidified incubator with 5% CO2 and after 14 days colonies were scored under an inverted microscope. Comparison of pre-thawing TNC [M=1.34×109(0.56–5.00)] and CD34+ [M=2.61×105/Kg (0.39–25.48)] cell counts, provided by cord blood banks, with post-thawing TNC [M=1.09×109(0.36–4.20)] and CD34+ [M=1.3×105/Kg(0.06–9.8)], performed by Wilcoxon signed rank test, showed a significant decrease in both cases (P<0.001). Descriptive statistics were tabulated separately for the number of TNC, number of CD34+ cells, numbers of CFU-GM and BFU-E colonies by whether patient engrafted or had graft failure, and comparison of the median cell counts between these two groups of patients was done by Wilcoxon Mann-Whitney rank sum test. TNC and CD34+ cell counts did not show significant difference between patients with graft failure [M=1.08×109(0.36–4.2) and M=1.28×105/Kg(0.09–9.8), respectively] and those who engrafted [M=1.12×109(0.46–2.49) and M=1.41×105/Kg(0.06–6.0), respectively]. Yet, the CFU-GM counts were significantly higher (P=0.005) for patients who engrafted [M=0.079×105(0.0017–0.75)] than for those who did not [M=0.04×105(0.0009–0.50)]. Although BFU-E counts did not differ with statistical significance between engrafted versus graft failure patients (P=0.059), higher counts could be observed in the group of patients who had successful engraftment. These data suggest that results of counts of CFU-GM colonies have a predictive value for engraftment in our center.

Factors influencing cord blood viability assessment before cryopreservation

“…By making these comparisons as a function of time and temperature before freezing we are able to examine how the differential susceptibility of WBC populations to necrotic/ apoptotic cell injury affects global measures of cell viability. We also reasoned that since engraftment and overall survival are related to TNC dose, 8,9 viable CD34+ cell dose, 20 and CFC content, 21,22 by comparing cell viability with recovery of TNCs, viable CD34+ cells, and CFCs we could determine which global viability measurements might be predictive of engraftment and overall survival following CB transplantation. It is appropriate to emphasize some of the analytical limitations of our study and other studies using CB with regard to viable CD34+ cell measurements by flow cytometry and CFC measurements by CFU assay.…”

Development of a novel assay to evaluate the functional potential of umbilical cord blood progenitors

“…A functional measure of progenitor activity is critical for selecting the highest quality UCB product for transplantation. Although there are some reports indicating that CFC assay results can be predictive of transplant outcome, 11‐13 the interlaboratory variability associated with the CFC assay when performed at multiple test sites prevents its general use for this purpose. Thus, a lack of a reliable potency assay to assess a unit of UCB leaves clinicians with the choice of selecting products for transplantation without knowing whether a high TNC also coincides with a high number of stem cells capable of engrafting.…”