Normalization of cell associated antiretroviral drug concentrations with a novel RPP30 droplet digital PCR assay

Shetty Ravi Dyavar,Zhen Ye,Lee C Winchester,Anthony T Podany,Jonathan A Weinhold,Kimberly K Scarsi,Siddappa N Byrareddy,Courtney V Fletcher

doi:10.1038/s41598-018-21882-0

Abstract

Quantification of antiretroviral (ARV) drug concentrations in peripheral blood mononuclear cells (PBMCs) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMC) and rectum (RMC) is an important measure of bio-distribution. Normalization of drug concentrations is critical to represent tissue drug concentrations and to analyze both intra-individual and inter-individual variability in drug distribution. However, a molecular method to normalize intracellular drug concentrations in PBMCs and TIMCs methanol extracts is currently unavailable. In this study, a novel droplet digital PCR (ddPCR) assay was designed to amplify RPP30 gene sequence conserved in human and non-human primates (NHP). Genomic DNA (gDNA) isolated from 70 percent methanol embedded PBMCs and TIMCs was used as ddPCR template to quantitate precise RPP30 copies to derive cell counts. The novel molecular method quantitated RPP30 copies in human and rhesus macaque gDNA templates with greater accuracy and precision than qPCR. RPP30 ddPCR derived cell counts are strongly correlated with automated cytometer based cell counts in PBMC (R = 0.90, p = 0.001 and n = 20); LNMC (R = 0.85 p = 0.0001 and n = 22) and RMC (R = 0.92, p = 0.0001 and n = 20) and achieved comparable normalized drug concentrations. Therefore, the RPP30 ddPCR assay is an important normalization method in drug bio-distribution and pharmacokinetic studies in humans and NHPs.

Highlights

In the current era of antiretroviral therapy (ART), quantification of cell associated drug in peripheral blood mononuclear cells (PBMC) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMCs) and rectum (RMCs) is a measure of the drug’s cellular and tissue bioavailability and bio-distribution
The novel RPP30 assay quantitated RPP30 copies in as low as 100 pg of human and rhesus macaques (RMs) genomic DNA (gDNA) template in intra, inter-laboratory and inter-operational assays (Supplementary Figure S5A to S5F)
Prior to utilizing the methanol extracted PBMCs and LNMCs to extract gDNA templates for the RPP30 droplet digital polymerization chain reaction (PCR) (ddPCR) assay and other downstream processes, we examined cellular morphology, analyzed the quality of gDNA and quantitated the amount of drug released into the 70 percent methanol, the most widely used drug-extracting medium to analyze intracellular drug concentrations, with and without lysing the PBMCs

Summary

Introduction

In the current era of antiretroviral therapy (ART), quantification of cell associated drug in peripheral blood mononuclear cells (PBMC) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMCs) and rectum (RMCs) is a measure of the drug’s cellular and tissue bioavailability and bio-distribution. Unlike conventional cell counting methods, real-time polymerase chain reaction (RT-PCR) based molecular methods utilize genomic DNA (gDNA) extracted from cells, are highly sensitive and amplify low copy DNA sequences in the human genome, yielding higher specificity and a broader quantification range[3,4,5,6,7,8,9] These assays were non-quantitative[3,5,6,7,8] or semi-quantitative[4] and could not quantify absolute cell numbers. The assay was validated to determine the precise number of RPP30 copies in gDNA amounts as low as 100 pg to measure 50 mononuclear cells per sample with high accuracy and precision in both human PBMCs and TIMCs. The assay provides a strong correlation to cell numbers attained by automated and manual cytometers for cells isolated from PBMCs, LN and rectum and was useful to normalize antiretroviral drug concentrations in these tissues

Methods

Results

Conclusion