Quantitation of intracellular triphosphate metabolites of antiretroviral agents in peripheral blood mononuclear cells (PBMCs) and corresponding cell count determinations: review of current methods and challenges

Abstract

Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system and the target cells for human immunodeficiency virus, type 1 (HIV-1) infection. Nucleoside/nucleotide analogs for the treatment of HIV infection are prodrugs that require cellular activation to triphosphate (TP) metabolites for antiviral activity. A reliable method of PBMC isolation and subsequent cell counting, as well as an accurate bioanalytical determination of the TPs in PBMCs are important for understanding the intracellular pharmacokinetic (PK) of the TPs and its correlation with plasma PK, the drug effect, and dose determination. Areas covered: The authors review the challenges and solutions in PBMC sample collection, sample processing, cell lysis, cell counting methods, analyte extraction, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) quantitative analysis of the nucleoside reverse transcriptase inhibitor-triphosphate (NRTI-TP) metabolites, and analogs. Expert opinion: Analyzing large numbers of clinical PBMC samples for determination of NRTI-TPs and analogs in PBMCs requires not only a validated LC-MS/MS bioanalytical method but also reliable methods for PBMC isolation, counting, cell lysis, and analyte recovery, and an approach for assessing analyte stability. Furthermore, a simple, consistent, and validated cell counting method often involves DNA quantitation of the PBMCs samples collected from clinical studies.

Direct and indirect quantification of phosphate metabolites of nucleoside analogs in biological samples

“…During PBMC isolation, whole blood is layered over a density gradient made of Ficoll-paque. Based on their different densities, in the order from low to high; plasma, platelets, buffy coat/lymphocyte and monocyte band, density gradient fluid, gel barrier, and erythrocytes (red blood cells) and neutrophils layers are formed from top to bottom in the tube after centrifugation [18]. The buffy coat layer contains mostly PBMCs along with minor portions of granulocytes and erythrocytes.…”

“…Importantly, erythrocytes and granulocytes are capable of phosphorylating some NMs ex vivo after sample collection. To prevent erythrocyte contamination, an extra erythrocyte lysis step can be performed [18].…”

“…To express NMs concentration as amount per cell, the number of cells isolated should be quantified. Cell counting, however, can be a considerable source of variability, which may negatively affect the reliability of the quantitative analysis [18,22]. Conventional approaches for cell counting include hemocytometer, microscope, or flow cytometry, but other indirect approaches using protein [23] and DNA [24] determination have also proven to be useful [18].…”

“…Due to their hydrophilicity, chromatographic separation of NMs is challenging using traditional reverse phase chromatography. Therefore, alternative approaches are used for the chromatographic separation of NMs, which can be grouped into two categories, direct and indirect approaches [8,17,18]. Direct methods rely on the direct quantification of NMs under non-reverse phase LC conditions.…”

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“…Roswell Park Memorial Institute (RPMI) medium was prepared by mixing 40 mL of RPMI, 10 mL of fetal bovine serum (FBS) and 200 µL Antibiotic / antimycotic (Antibiotic antimycotic solution with Streptomycin 10 mg/20 mL, 10,000 U Penicillin, Amphotericin B and 0.9% normal saline). About 1 mL of RPMI medium was dispensed into falcon tubes, added 30 µL of Phytohemagglutinin (PHA), 100 µL of hPBMCs, and incubated with an atmosphere of 95% air and 5% CO 2 at 37 ºC for 4 h. Before incubation, counting the number of cells from 10 µL 15,16 .…”