Blood Treatment Influences the Yield of Apoptotic Lymphocytes
Blood Treatment Influences the Yield of Apoptotic Lymphocytes
Purpose: No systematic investigation has been reported assessing the effect of cell isolation processes on postexercise apoptosis. Therefore, the effect of cell isolation procedures on apoptosis was evaluated in this study.
Methods: Untrained healthy individuals participated ( N = 13). Blood samples obtained at rest and immediately after an incremental exercise test to exhaustion were partitioned into three treatments: 1) whole blood smears made immediately after the sample was obtained (WB), 2) cells subjected to density-gradient isolation before smears were made (ISO), and 3) samples allowed to sit at room temperature (i.e., time-treated) before centrifugation and smearing (TT). Blood smears were stained using the May-Grünwald Giemsa procedure and lymphocytes were evaluated under a light microscope for characteristic features of apoptosis. Data were analyzed using a 2 × 3 ANOVA.
Results: A significant interaction effect existed ( P < 0.0001) such that at rest, no difference was detected in the amount of lymphocyte apoptosis among WB, ISO, or TT samples. However, after exhaustive exercise, the amount of apoptotic lymphocytes was significantly greater in WB compared with ISO and TT samples ( P < 0.0001).
Conclusion: Lymphocyte isolation results in a significant decrease in the percent of apoptotic lymphocytes after exhaustive exercise. This reduction is likely due to the time needed to isolate cells, rather than the isolation process itself. Because apoptosis is a time-sensitive process that occurs within minutes rather than hours, the length of time from initial sampling to the preparation of cells for assessment of apoptosis is critical and should be considered in future exercise studies.
Exercise is a type of physiological stress that has a marked effect on the cells of the immune system, and lymphocytes in particular. An increase in cell numbers is observed with the performance of high-intensity exercise, followed by a reduction such that the numbers become significantly lower than resting levels. It is probable that at least a portion of the postexercise decrease in lymphocytes is due to cell death or apoptosis.
Apoptosis is carried out by the activation of endogenous endonucleases and can be mediated through multiple pathways. Although apoptosis may be initiated by various events, the pathways converge to display the following common occurrences: cell shrinkage, the breakdown of DNA into characteristic lengths of 180 base pairs, orderly condensation of the nucleus, and membrane blebs, which are phagocytosed by surrounding cells. Compared with resting values, significantly elevated percentages of lymphocyte apoptosis have been reported immediately after high-intensity exercise in human subjects.
The method of blood treatment after sampling and assessment of apoptotic cells after exercise needs further scrutiny. To date, lymphocyte apoptosis after exercise has only been measured using isolated cells. No systematic investigation has been reported assessing the effect of the isolation process on apoptosis. Historically, the techniques used for lymphocyte isolation, that is, density gradients, have been used to return a high yield of viable cells (usually greater than 95%). It is possible that many apoptotic cells may actually be deleted during the isolation process. Furthermore, the isolation process itself requires at least 30 min of centrifuge time and 15 min for each additional wash thereafter. Because apoptosis is a process that is carried out relatively quickly once initiated, it is possible that cells could undergo cell death and be deleted before the isolation process is even completed. With these considerations, the validity of lymphocyte apoptosis measurements made after exercise is an issue that needs to be addressed. Therefore, the purpose of this study was to assess the influence of blood treatment on the yield of apoptotic lymphocytes after maximal exercise.
Purpose: No systematic investigation has been reported assessing the effect of cell isolation processes on postexercise apoptosis. Therefore, the effect of cell isolation procedures on apoptosis was evaluated in this study.
Methods: Untrained healthy individuals participated ( N = 13). Blood samples obtained at rest and immediately after an incremental exercise test to exhaustion were partitioned into three treatments: 1) whole blood smears made immediately after the sample was obtained (WB), 2) cells subjected to density-gradient isolation before smears were made (ISO), and 3) samples allowed to sit at room temperature (i.e., time-treated) before centrifugation and smearing (TT). Blood smears were stained using the May-Grünwald Giemsa procedure and lymphocytes were evaluated under a light microscope for characteristic features of apoptosis. Data were analyzed using a 2 × 3 ANOVA.
Results: A significant interaction effect existed ( P < 0.0001) such that at rest, no difference was detected in the amount of lymphocyte apoptosis among WB, ISO, or TT samples. However, after exhaustive exercise, the amount of apoptotic lymphocytes was significantly greater in WB compared with ISO and TT samples ( P < 0.0001).
Conclusion: Lymphocyte isolation results in a significant decrease in the percent of apoptotic lymphocytes after exhaustive exercise. This reduction is likely due to the time needed to isolate cells, rather than the isolation process itself. Because apoptosis is a time-sensitive process that occurs within minutes rather than hours, the length of time from initial sampling to the preparation of cells for assessment of apoptosis is critical and should be considered in future exercise studies.
Exercise is a type of physiological stress that has a marked effect on the cells of the immune system, and lymphocytes in particular. An increase in cell numbers is observed with the performance of high-intensity exercise, followed by a reduction such that the numbers become significantly lower than resting levels. It is probable that at least a portion of the postexercise decrease in lymphocytes is due to cell death or apoptosis.
Apoptosis is carried out by the activation of endogenous endonucleases and can be mediated through multiple pathways. Although apoptosis may be initiated by various events, the pathways converge to display the following common occurrences: cell shrinkage, the breakdown of DNA into characteristic lengths of 180 base pairs, orderly condensation of the nucleus, and membrane blebs, which are phagocytosed by surrounding cells. Compared with resting values, significantly elevated percentages of lymphocyte apoptosis have been reported immediately after high-intensity exercise in human subjects.
The method of blood treatment after sampling and assessment of apoptotic cells after exercise needs further scrutiny. To date, lymphocyte apoptosis after exercise has only been measured using isolated cells. No systematic investigation has been reported assessing the effect of the isolation process on apoptosis. Historically, the techniques used for lymphocyte isolation, that is, density gradients, have been used to return a high yield of viable cells (usually greater than 95%). It is possible that many apoptotic cells may actually be deleted during the isolation process. Furthermore, the isolation process itself requires at least 30 min of centrifuge time and 15 min for each additional wash thereafter. Because apoptosis is a process that is carried out relatively quickly once initiated, it is possible that cells could undergo cell death and be deleted before the isolation process is even completed. With these considerations, the validity of lymphocyte apoptosis measurements made after exercise is an issue that needs to be addressed. Therefore, the purpose of this study was to assess the influence of blood treatment on the yield of apoptotic lymphocytes after maximal exercise.
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