Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or K₂EDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺, or CD56⁺CD16⁺ cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4⁺ cells were in general less affected by isolation and cryopreservation than diff CD8⁺ cells. Terminally diff effector CD4⁺ and CD8⁺ cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4⁺ and CD8⁺ cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4⁺CD25^hi expressing CD101 or CD129, CD4⁺CD25^hiCD127^-, and CD4⁺CD25^hiCD127^-FOXP3⁺, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4⁺CD25⁺CD127^- cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4⁺, CD8⁺, and CD25^hi lymphocytes are in general not influenced by isolation and long-term cryopreservation.

Anette Nilsson-Bowers

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