Evaluation of three media for the cryopreservation of peripheral blood mononuclear cells for ELISPOT
Abstract number: 1733_1504
Maier C., Bigler S., Bodmer T.
Background: ELISPOT is used in microbiology for the analysis of the cell-mediated immune response. Typically, this technique requires sample processing within hours in order to preserve the vitality of cells. This may limit its application in clinical settings. Therefore, cryopreservation of vital lymphocytes prior to testing would facilitate a wider use in clinical laboratories. The aim of this study was to evaluate an optimised protocol for cryopreservation of vital lymphocytes.
Methods: Blood was obtained from three healthy donors, and peripheral blood mononuclear cells (PBMC) were isolated using Vacutainer Cell Preparation Tubes (Becton Dickinson, Franklin Lakes, US). PBMC were split into four aliquots, one for direct processing and three for cryopreservation (liquid nitrogen, -160°C, 4 weeks) in medium A (FCS, 90%; DMSO, 10%), medium B (AIM-V, 50%; FCS, 35%; DMSO, 15%), and medium C (AIM-V, 90%; DMSO, 10%), respectively. Cell counts of the aliquots were assessed prior to and after four weeks of cryopreservation using a Cell Dyn 1200 System (Abbott, Laboratories, US); cell viability and background were tested using mitogen controls and negative controls of T-Spot.TB (Oxford Immunotec Ltd., Oxford, UK). The results were compared and the ideal medium formulation was identified. For the purpose of further validation, PBMC of three individuals with previous positive T-Spot.TB results were processed as described above, and tested including the TB-specific antigens of T-Spot TB after eight weeks of cryopreservation. Spot-forming cells were counted by the AID Elispot Reader System (AID GmbH, Strassberg, Germany).
Results: With respect of survival rate, cell viability, and technical requirements medium A was superior: survival rate was 61.7±12.7% and mitogen response as a surrogate for cell viability remained above the level of validity of T-SPOT.TB (≥20spots/well). Background signal was not influenced by FCS, as there was no increase in the extent of background signal (p = 0.19). All specimens, that had previously tested positive with the T-SPOT.TB, remained positive after cryopreservation (Panel A, p = 0.91; Panel B, p = 0.30).
Conclusions: We have identified a reliable protocol for the cryopreservation of PBMC to be tested in ELISPOT-assays. Successful cryopreservation of PBMC's facilitates a wider application of this technique in clinical laboratories, and will allow cell banking for prospective studies.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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