Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Marco-Casanova, Paola; Lukashchuk, Natalia; Lombardi, Benedetta; Munugalavadla, Veerendra; Frigault, Melanie M.; Barrett, J. Carl; Pierce, Andrew J.

doi:10.3791/60776

Cited by 3 publications

(5 citation statements)

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“…The proof-of-principle demonstration of this new DDR-2 assay for quantifying the DNA damage response in non-stimulated PBMCs isolated from individuals shows the potential for use of the assay in providing pharmacodynamic markers for kinase inhibitors in clinical trials [45]. To be successful in this setting, there are some challenges that must be overcome.…”

Section: Discussionmentioning

confidence: 99%

“…Another challenge is overcoming preanalytical variables during sample collection. Notably, some phosphosignaling is susceptible to ischemic effects in tissue collection [56], potentially introducing preanalytical variations [45]. Translation of quantitative phosphorylation assays would require strong validation studies to ensure stability of the phosphopeptides.…”

Section: Discussionmentioning

confidence: 99%

“…As discussed above, analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials for POM and PD studies, to help guide dose and scheduling of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers [45]. We sought to evaluate the utility of the DDR-2 assay in pharmacodynamic profiling in both proliferating and nonproliferating primary human PBMCs in the presence/absence of DDR kinase inhibitors.…”

Section: Pharmacodynamic Profiling Of Kinase Inhibition In Primary Human Cellsmentioning

confidence: 99%

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Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Whiteaker

Wang

Zhao

et al. 2021

Cancers

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The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Pharmacodynamic Profiling Of Kinase Inhibition In Primary Human Cellsmentioning

confidence: 99%

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Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Whiteaker

Wang

Zhao

et al. 2021

Cancers

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“…Different numbers of MCF-7 cells (10, 50, 100, and 200 cells) were spiked into a solution containing peripheral blood mononuclear cells (PBMCs) or spiked into the whole blood treated by red blood cell lysis buffer. For PBMCs preparation, the procedure was based on the published reference . For whole blood preparation, 1 mL of red blood cell lysis buffer was added to the blood sample for 5 min and incubated on ice to remove red blood cells.…”

Section: Methodsmentioning

confidence: 99%

“…For PBMCs preparation, the procedure was based on the published reference. 39 For whole blood preparation, 1 mL of red blood cell lysis buffer was added to the blood sample for 5 min and incubated on ice to remove red blood cells. After incubation, the solution was centrifuged at 300g for 3 min.…”

Section: ■ Introductionmentioning

confidence: 99%

Facile Preparation of Three-Dimensional Wafer with Interconnected Porous Structure for High-Performance Capture and Nondestructive Release of Circulating Tumor Cells

Liu

Wang

et al. 2022

Anal. Chem.

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Efficient isolation and downstream bioinformation analysis of circulating tumor cells (CTCs) in whole blood contribute to the early diagnosis of cancer and investigation of cancer metastasis. However, the separation and release of CTCs remain a great challenge due to the extreme rarity of CTCs and severe interference from other cells in complex clinical samples. Herein, we developed a low-cost and easy-to-fabricate aptamer-functionalized wafer with a three-dimensional (3D) interconnected porous structure by grafting polydopamine (PDA), poly(ethylene glycol

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A missense mutation in human INSC causes peripheral neuropathy

Yeh,

Chao,

Hong

et al. 2024

EMBO Mol Med

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PAR3/INSC/LGN form an evolutionarily conserved complex required for asymmetric cell division in the developing brain, but its post-developmental function and disease relevance in the peripheral nervous system (PNS) remains unknown. We mapped a new locus for axonal Charcot–Marie-Tooth disease (CMT2) and identified a missense mutation c.209 T > G (p.Met70Arg) in the INSC gene. Modeling the INSCM70R variant in Drosophila, we showed that it caused proprioceptive defects in adult flies, leading to gait defects resembling those in CMT2 patients. Cellularly, PAR3/INSC/LGN dysfunction caused tubulin aggregation and necrotic neurodegeneration, with microtubule-stabilizing agents rescuing both morphological and functional defects of the INSCM70R mutation in the PNS. Our findings underscore the critical role of the PAR3/INSC/LGN machinery in the adult PNS and highlight a potential therapeutic target for INSC-associated CMT2.

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Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Cited by 3 publications

References 0 publications

Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

Facile Preparation of Three-Dimensional Wafer with Interconnected Porous Structure for High-Performance Capture and Nondestructive Release of Circulating Tumor Cells

A missense mutation in human INSC causes peripheral neuropathy

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