Abstract
Objective:Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method.Materials and Methods:In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units.Results:Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant.Conclusion:In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.
Highlights
Recent scientific evidence demonstrates that different subtypes of CD34+ cells in the cord blood (CB) hematopoietic stem cell (HSC) niche have different engraftment potentials [1,2]
Our findings showed that in fresh samples total number of nucleated cells (TNCs), CD34+ cells, and aldehyde dehydrogenase (ALDH) correlated highly with counts of Colony-forming units of granulocytes/macrophages (CFU-GM), colony-forming unit (CFU)-erythroids/ granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively
In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, for post-thaw samples
Summary
Recent scientific evidence demonstrates that different subtypes of CD34+ cells in the cord blood (CB) hematopoietic stem cell (HSC) niche have different engraftment potentials [1,2]. Two different approaches can be used to assess the functionality and population-forming capacities of CB HSCs along with the gold standard method of the International Society of Hematotherapy and Graft Engineering (ISHAGE) [3]. Ex vivo colony-forming unit (CFU) assays are the most widely used tests for determining HSC functions, but they possess serious drawbacks such as difficulty in routine application, lack of standardization, labor-intensive nature, and long turnaround time [4]. One of the likely reasons for this is probably the fact that while being predictive of shortterm re-populating cells, CFU assays could not determine longterm populating cells effectively. Long-term populating cells have been shown to provide long-term immune reconstitution after CB transplantation (CBT); it is of crucial importance to assess their numbers. The measurement of aldehyde dehydrogenase (ALDH) activity can be much more accurate due to the intracellular presence of this enzyme [5]
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