Validation of a quantitative PCR (qPCR) assay for the detection and quantification of replication competent lentivirus for patient monitoring in lentiviral vector-based cell therapy studies

AAPS PharmSci360 2024 -- Lentiviral vectors are a common delivery system for various cell therapies, such as CAR/TCR-T cell therapies. There is a potential risk for replication competent lentivirus (RCL) in patients receiving lentiviral vector-based cell therapies. There is a critical need for regular RCL monitoring in patients. PCR-based assays for RCL detection are recommended by the FDA. Our objective was to validate a qPCR assay based on quantification of vesicular stomatitis virus glycoprotein (VSV-G) envelope protein gene sequence for RCL.

Alternative approaches to animal testing for ecotoxicity assessments: Validating the OECD319 regulations

SETAC North America 2024 -- Fish bioconcentration studies are carried out to determine whether substances have the potential to bioaccumulate or to bio-magnify throughout different trophic levels. As the requirements to conduct these studies on a broad variety of industrial chemicals, pharmaceuticals and crop protection products increase across the globe, so does the numbers of fish needed to fulfil these investigations. Prior to the revision to the OECD Test Guideline (OECD 305: 2012), the preferred method of testing required three doses of chemical (including a control) with up to 100 fish per dose. Latterly, there is now the option to test on one concentration and the control only, providing there is scientific justification. This has the potential to decrease the numbers of fish used in these tests by one third (100 fish per study). Here we present our approach to assess the selection of positive controls to be used in these assays using pyrene, the reference chemical stated in the guidelines, together with testosterone and diclofenac as both undergo Phase I hydroxylation reactions in the presence of S9 and hepatocytes, similar to pyrene.

Per- and polyfluoroalkyl substances (PFAS): Validation of methodology for the determination of residues in fruit, fruit processed commodities and fish tissues

SETAC North America 2024 -- Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic organofluorine compounds that have become a concern due to their persistence in the environment following extensive use over the last few decades. Due to their high degree of chemical stability as well as their widespread use, PFAS compounds are increasingly being detected in the environment and appear to persist for extended periods of time. Furthermore, exposure to these chemicals in everyday objects such as packaging and cookware, as well as within certain areas of industry, has led to the apparent bioaccumulation in certain areas of the population. Because of the concerns over health and environmental issues associated with PFAS chemicals, there has been a need to be able to monitor the occurrence of these materials in food commodities. The work described in this poster was a project to develop and validate an LC-MS/MS method for the analysis of a “typical” suite of PFAS analytes in fruit (strawberries and grapes), fruit processed commodities (jam and wine) as well as in fish tissues (edible muscle fillet). A simple and robust method was required, that could be easily applied to routine analysis at low concentrations, using commonly available equipment and approaches.

Enabling gene therapy access: Labcorp's first cell-based companion diagnostic (CDx)

The emergence of novel gene therapies has necessitated the development of cell-based CDx assays, posing multiple technical and operational challenges. Labcorp worked with a sponsor to develop the nAbCyte™ CDx assay to determine patient eligibility for a novel gene therapy program BEQVEZ™ (fidanacogene elaparvovec). nAbCyte is an FDA-approved in vitro CDx device that detects neutralizing antibodies to the AAVRh74var capsid in serum specimens from patients previously diagnosed with hemophilia B. This case study illustrates how Labcorp worked with the client and regulatory agencies to overcome development hurdles with a customized approach combining scientific expertise, collaboration, strategic engagement and governance.

Navigating the complexities of the Biocidal Products Regulation

Biocidal products are used to control unwanted organisms (e.g., pests, such as rats, or micro-organisms, like bacteria) that are harmful to the health of humans, animals or the environment. They play an important role in everyday life and are regulated in the European Union (EU) and European Economic Area (EEA) by the European Chemicals Agency (ECHA). A biocidal product must be authorized before it can be placed on the market, although exemptions apply for active substances included within ECHA’s Review Programme.

Biocide product authorization process

From initial planning to post-submission support, Labcorp can help you navigate every stage of authorization for your biocide.

Multiparameter characterisation of human alveolar macrophage-epithelial co-culture (ImmuLUNG™) following exposure to whole smoke

CORESTA Congress 2024 -- ImmuLUNG™ cultures were exposed to whole smoke (WS) generated from 1R6F Kentucky Reference using a Vitrocell® VC10® smoking robot at varying airflows. The effects of WS on cytotoxicity, oxidative stress, epithelial barrier permeability, cytokine secretion, gene expression and individual cell phenotype via high content imaging analysis were assessed. The ImmuLUNG™ epithelial component exhibited high TEER values. Concentration-dependent viability and oxidative stress responses were observed, as indicated by 8-isoprostane/LDH release. Gene expression analysis revealed relevant macrophage RNA markers and upregulation of genes controlling macrophage production, differentiation, and function. Proteomic analysis showed increased expression of inflammatory signaling proteins and protein expression changes associated with oxidative stress control. High-content image analysis of ImmuLUNG™ macrophages revealed morphological changes, indicating assessment of macrophage endpoints are compatible with WS exposure. 

Utilizing the in vitro micronucleus assay microflow endpoint on V79 cells after treatment in 24-well plates

CORESTA Congress 2024 -- Within the standard genotoxicity test battery, the in vitro micronucleus (IVMN) test is an option for detecting chromosomal damage. As standard this is performed through slide preparations and manual scoring. Improving timelines and increasing cell numbers analysed is achieved through flow cytometry and staining methods. This method was utilised with V79 cells cultured in 24-well plates. These were treated with the OECD 487 test chemicals including nicotine and Total Particulate Matter (TPM) (ISO 20778:2018) generated from 3R4F Kentucky Reference, eluted in DMSO. Endpoints were measured using the Litron™ MicroFlow kit. Cytotoxicity was measured by relative population doubling (RPD), supported by the use of Cell Sorting Set-up Beads (Invitrogen™) during flow cytometry assessment. The Litron™ MicroFlow kit was used to stain and lyse the cells, then micronucleus (MN) induction was assessed on a flow cytometer. Results showed dose related increases in MN induction for all expected positive OECD test chemicals. 3R4F TPM at 3+21 -S9 showed a slight increase, prior to cytotoxicity. 3+21 +S9 showed dose-related induction. 24+0 -S9 showed increases to MN induction. This validation study confirmed the correct functioning of a high throughput IVMN assay by flow cytometry, with treatment in 24-well plates

Validation of the Vitrocell® 48 2.0+ 24-well mammalian module for assessing cigarettes and eHTPs: Evaluation of dilution airflow, dose resolution and 3D tissue suitability

CORESTA Congress 2024 -- The use of 3D in vitro airway tissue models that closely mimic the physiological architecture and microenvironment of the human respiratory system is critically important when studying aerosol exposure. Presence of a pseudostratified mucociliary epithelium and mucus secreting goblet cells help to provide a more accurate representation of the deposition, dissolution and clearance patterns of inhaled aerosols during human exposure. The objective of this study was to validate the Vitrocell® 48 2.0+ 24-well module by quantifying delivered dose across a range of dilutions from both reference cigarettes and electronically heated tobacco products (eHTPs). The study findings revealed low variation within dilution airflow, overall good dose resolution and supports the use of this module in relevant toxicological investigations and risk assessments.

A new way to fight cervical cancer: How artificial Intelligence is transforming pap test screening

Screening is critical for effective prevention and treatment of many cancers, and cervical cancer is no different. Each year in the U.S., an estimated 26.4 million Pap tests are ordered, and an estimated 11,500 new cases of cervical cancer are diagnosed.