Novel Cell Quantification Method Using a Single Surrogate Calibration Curve Across Various Biological Samples

Abstract

Quantitative polymerase chain reaction (qPCR) is generally used to quantify transplanted cell therapy products in biological samples. As the matrix effects on PCR amplification and variability in DNA recovery from biological samples are well-known limitations that hinder the assay’s performance, a calibration curve is conventionally established for each matrix. Droplet digital PCR (ddPCR) is based on the endpoint assay and advantageous in avoiding matrix effects. Moreover, the use of an external control gene may correct assay fluctuations to minimize the effects caused by inconsistent DNA recovery. In this study, we aimed to establish a novel and robust ddPCR method capable of quantifying human cells across various mouse biological samples using a single surrogate calibration curve in combination with an external control gene and DNA recovery normalization. Acceptable accuracy and precision were observed for quality control samples from different tissues, indicating the excellent quantitative and versatile potential of the developed method. Furthermore, the established method enabled the evaluation of human CD8+ T cell biodistribution in immunodeficient mice. Our findings provide new insights into the use of ddPCR-based quantification methods in biodistribution studies of cell therapy products. Graphical Abstract

Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues

“…However, the issue of matrix effects on cell quantification via qPCR must be considered for all tissue types of the body. The yield and quality of extracted genomic DNA can vary widely depending on the physical and biochemical nature of each tissue [ 16 , 31 ], and the tissue from which the DNA was extracted can have a significant effect on the efficiency, accuracy, and precision of the qPCR assay [ 34 ]. Therefore, current regulatory guidelines [ 24 , 25 ] and best practice recommendations [ 15 , 20 , 21 , 22 , 43 ] advise researchers to assess potential matrix effects by determining the recovery of the target DNA spiked into each tissue of interest during assay validation, whereby recovery rates in a wide range between 30% and 100% are considered to be expected [ 15 , 20 ].…”

“…Matrix effects have been studied predominantly in environmental microbiology, microbial food safety and forensic analyses, where the amounts of target nucleic acids are often extremely small and the matrices are particularly diverse and challenging [ 28 , 29 , 30 ]. In contrast, with the exception of forensically and microbiologically relevant body fluids and secretions, as well as food safety-relevant matrices such as muscle tissue and milk, the effects of mammalian matrices on qPCR results have been assessed and discussed only in a very limited manner and only for a limited number of tissue types [ 14 , 16 , 31 , 32 , 33 , 34 ].…”