Development of a bioanalytical method for circulating human T cells in animals using Arthrobacter luteus-based quantitative polymerase chain reaction and its application in preclinical biodistribution studies

Shimizu, Hisao; Kuze, Yoji; Higuchi, Tomoaki; Matsumoto, Shimpei; Yamamoto, Satoshi; Goto, Akihiko; Moriya, Yuu; Hirabayashi, Hideki

doi:10.1016/j.reth.2020.10.003

Cited by 3 publications

(6 citation statements)

References 20 publications

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“…Furthermore, we confirmed that the developed method was applicable to human cell quantification of in vivo samples (Fig. 4); the results were consistent with those of a previous report (14). In addition, 51Cr-labeled mouse T cells injected into mice were reported to show similar biodistribution (27).…”

Section: Discussionsupporting

confidence: 91%

“…In the present study, we set the acceptance criteria for accuracy and precision to be within 35% RE and < 35% CV, respectively, based on our previous report (14). These criteria were in line with the recommendations from the Global CRO Council in Bioanalysis (GCC) (29).…”

Section: Discussionmentioning

confidence: 96%

“…The qPCR-based method is highly sensitive and enables detection of CTPs without pre-labeling. qPCR targeting Arthrobacter luteus (Alu) elements (Alu-qPCR) is a highly sensitive cell quantification method as Alu elements account for > 10% of the human genome, with ~ 1.1 million copies (14)(15)(16). In our previous study, we demonstrated that the cell number in biological samples could be determined from the calibration curve prepared by spiking the standard cell lysates into the blank matrix (14).…”

Section: Introductionmentioning

confidence: 99%

“…qPCR targeting Arthrobacter luteus (Alu) elements (Alu-qPCR) is a highly sensitive cell quantification method as Alu elements account for > 10% of the human genome, with ~ 1.1 million copies (14)(15)(16). In our previous study, we demonstrated that the cell number in biological samples could be determined from the calibration curve prepared by spiking the standard cell lysates into the blank matrix (14). However, Alu-qPCR is subject to a matrix effect and/or variability in DNA recovery among different matrices.…”

Section: Introductionmentioning

confidence: 99%

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

Nakayama

Yamamoto

Hirabayashi

2023

AAPS J

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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

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Nakayama

Yamamoto

Hirabayashi

2023

AAPS J

Self Cite

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“…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 ].…”

Section: Introductionmentioning

confidence: 99%

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

Schröder

Niebergall-Roth

Norrick

et al. 2023

Cells

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Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

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DMPK perspective on quantitative model analysis for chimeric antigen receptor cell therapy: Advances and challenges

Goto,

Moriya,

Nakayama

et al. 2024

Drug Metabolism and Pharmacokinetics

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Development of a bioanalytical method for circulating human T cells in animals using Arthrobacter luteus-based quantitative polymerase chain reaction and its application in preclinical biodistribution studies

Cited by 3 publications

References 20 publications

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

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

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

DMPK perspective on quantitative model analysis for chimeric antigen receptor cell therapy: Advances and challenges

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