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Abstract
Background
The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining.
Methods
Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining.
Results
Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8±4.5%, 78.4±7.8%, and 65.5±8.1%; mononuclear cells, 94.4±1.7%, 90.8±4.2%, and 84.2±6.7%; and CD34-positive cells, 92.4±3.0%, 90.7±4.7%, and 89.3±7.0%. The viability using trypan blue was more than 90% until 48 hr after collection.
Conclusions
The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.
Figures and Tables
Fig. 1
Flow cytometric analysis results in determining the viabilities of total nucleated cells (TNC), mononuclear cells (MNC), and CD34+ cells. The 7-aminoactinomycin D (7-AAD) viabilities (%) of TNC (A), MNC (E), and CD34+ cells (D) are 100×viable TNC (F+G)/TNC (A), viable MNC (H+I)/MNC (E), and viable CD34+ cells (J+K)/CD34+ cells (D), respectively. The 7-AAD/annexin V viabilities (%) of TNC, MNC, and CD34+ cells are 100×viable TNC (G)/TNC (A), viable MNC (I)/MNC (E), and viable CD34+ cells (K)/CD34+ cells (D), respectively.
Fig. 2
Time-dependent alterations in the viability of total nucleated cells (TNCs) (●), mononuclear cells (MNCs) (▪), CD34+ cells (▴). Data shown in this figure were obtained using flow cytometric analysis with 7-aminoactinomycin D (7-AAD)/annexin V (A) and 7-AAD (B) and analyzed at T0, T0+24, and T0+48 hr. T0 represents data obtained immediately after receipt in the laboratory, which was within 6 hr after collection. The "+" sign indicates the additional time of incubation (hr) at room temperature after receipt in the laboratory. Error bars represent SD.
Table 1
Comparison of cell viabilities (%) measured using the trypan blue, 7-AAD, and 7-AAD/annexin V methods following elapsed time after collection
*T0 represents data obtained immediately after receipt in the laboratory, which was within 6 hr after collection. Statistical comparisons were made by one-way ANOVA techniques with post hoc analysis by the Tukey's method. †P<0.05 vs TNC at each time; ‡P<0.05 vs MNC at each time.
Abbreviations: 7-AAD, 7-aminoactinomycin D; TNC, total nucleated cells; MNC, mononuclear cell; NA, not applicable.
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