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Background:  Intracellular pharmacology requires an extraction step in order to liberate the analytes from cells. This step involves cells lysis which can be performed either at the clinical unit on fresh cells or at the analytical laboratory on frozen cells. The aim was to compare the 2 processes using natural deoxynucleotide triphosphates (dNTP) and triphosphate metabolites of nucleoside reverse transcriptase inhibitors (NRTI-TP), which compete on the reverse transcriptase.

Methods:  Peripheral blood mononuclear cells (PBMC) were isolated from blood of HIV-infected patients treated with lamivudine (3TC) and tenofovir (TDF) and of healthy subjects. The latter were either processed as blanks or incubated for 24 hours with d4T and 3TC (respectively, 5 and 4 µM). Cells were washed twice, evenly distributed and spin to 10⁷ cells. Samples were stored at –80°C either as pellet or as lysate (using 0.5 or 1 mL of tris/MeOH:  3 of 7). Extraction was performed by scrapping frozen cells whereas several methods (scrapping, vortex, or homogenization using a pipet) were tested on fresh ones. Blanks were eventually spiked before or after the extraction with NRTI-TP. NRTI-TP and endogenous nucleotides (3TC-TP, d4T-TP, TFV-DP, dA-TP and dT-TP) were quantified using a validated LC-MS/MS assay involving a TSQ Quantum Ultra (Thermo-Electron) with simultaneous monitoring.

Results:  Results are:  TP recovery is around 20% better using 1 mL extraction solvent vs 500 µL for TFV-DP, dA-TP, and dT-TP and 3TC-TP (each p < 0.05) but unchanged for d4T-TP. Variation between replicates was highly dependent of the lysis method; scrapping frozen cells and pipet homogenizing fresh ones leading to the best reproducibility (CV < 15%). All things being equal, cells lysed frozen gave higher recoveries for 3TC-TP, d4T-TP, and dT-TP (each p < 0.05) than cells lysed fresh. PBMC from patients were lysed either frozen in 0.5 mL or fresh in 1 mL; recovery of 3TC-TP and PMPApp were similar. NRTI-TP recovery from fresh cells was around 70%.

Conclusion:  These experiments suggest that nucleotide TP recovery is similar or better when the cells are lysed frozen rather than fresh. From our experience, the intracellular TP are stable when stored at –80°C as un-lysed cells. Moreover, lysing frozen cells in analytical laboratories allows an earlier addition of the internal standard allowing it to undergo every step of the assay. Hence, handling frozen cells exhibits relevant interests.

Keywords: nucleotides; recovery; PBMCs