Background:
Peripheral blood mononuclear cells (PMBCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 hours after the density gradient centrifugation on PBMC yield, purity and viability.
Methods:
PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 hours. PBMC yield, purity and viability were compared.
Results:
The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was only affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24h delay, and viability did not further decrease after 48h (64%/44%[p<0.0001]/40%[p<0.0001]).
Conclusions:
In conclusion, our data suggests that post-centrifugation delays of up to 48h might have only a minor effect on cell yield and purity. However, at the same time, a relevant decrease in cell viability was observed, which could be partially compensated by the use of CPT if the isolation was resumed latest the day after blood withdrawal.
A QUICK AND EASY METHOD FOR THE ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM WHOLE BLOOD BY DENSITY GRADIENT CENTRIFUGATION USING HISEP LSM MEDIUM.
