Dear Sir. Concerning your issue about the anticoagulant for PBMC extraction. Several parameters involved in blood collection, processing and shipping may influence immunological function of the resulting cells, including anticoagulant type, time from venipuncture to PBMC isolation/cryopreservation, method of PBMC isolation and procedure for sample shipping. We examined these parameters in single and multiple site studies, and found the length of time from venipuncture to cryopreservation is the most important parameter affecting performance of T cells in immunological assays. Comparing blood processed at 24 hours after venipuncture with that processed within eight hours, we observed on average a modest reduction in PBMC viability (~8% decrease), a greater loss in cell recovery (~32%), and between 36-56% loss in IFN-γ T cell frequencies by ELISpot assay. We also describe three cold shipping methods that maintain immunological function in appropriately cryopreserved PBMC. These data indicate that cryopreservation of PBMC should occur within eight hours of venipuncture for optimal performance. This narrow window for specimen processing has important implications in selecting and monitoring clinical sites with laboratory capacity to perform these procedures in future clinical trials. Samples may be collected in Heparin, sodium citrate or EDTA tubes. Use care when using heparin as an anticoagulant since an excess of heparin will give falsely high values. Use no more than 10 IU of heparin per mL of blood collected.Human Plasma can be frozen at -80°C and tested later.
I think the following below links and the attached file may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904432/pdf/nihms-22817.pdf
https://pdfs.semanticscholar.org/a2ad/6f393d9c5d667b49fe0e58b80e2d664bac3b.pdf
https://www.miltenyibiotec.com/_Resources/Persistent/6d7e0fb8a3eab613958d142051e3c7050eb0c8a9/SP_MC_PB_density_gradient.pdf
Thanks
I guess it depends on how early you plan to process the blood (within 8 hours or within 24 hours) and what you plan to do with the PBMCs after (look at functionality or not).
There is literature out there that shows that PBMC viability and functionality are maintained for up to 24 hours post blood collection if using heparin as an anticoagulant. If using EDTA, this was reduced to within 8 hours. This becomes more critical if you plan to freeze the PBMCs and look at functional responses one day.
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