Citoxlab Newsletter – March 2017

Dedicated experience in handling of obese minipigs weighing up to 100 kg at Citoxlab in Denmark

NL Bulle 5At Citoxlab Denmark, we have over 40 years’ experience in handling, housing and dosing of minipigs from birth (10.5 kg) to full sized adult, including obese animals weighing up to 100 kg. Each phase of the pig life (new born, newly weaned, young adult, fully-grown) and obese animals require special consideration and attention. However in a CRO the design and set up of the pens need to be flexible and adaptable to the changing needs of the animals, but also cost effective. Our facilities are specifically designed to accommodate the range of environments needed.

The farrowing units are designed to ensure the well-being and normal development of both sow and piglets, and include features such as temperature control and flexible pen set-up. These units allow for cross-fostering, evaluation of the animals health and dispensing of special feeding for both sows and piglets. In combination with an animal enrichment program, the stress level of both sow and piglets is greatly reduced.

Diet and enrichment programs are two critical aspects of the weaning process. The juvenile piglets are properly adapted to their new diet and go through enrichment programs with social housing that is essential to the continued health and well-being of the animals. Females are easily group-housed at all ages, however males are group-housed only after weaning and until the first signs of sexual maturity (approximately 4 months of age). Thereafter, the males are single-housed due to their dominant behaviour. At this stage, other enrichment practices are employed to ensure that the males are stress free.

At the upper end of the weight range, obese animals give many challenges with respect to housing, feeding, handling for body weight capture, blood sampling and handling in general. Screening is necessary for placement on studies and one important physical feature is to exclude animals with deviating leg positions, as lameness and other leg problems can be an issue once on study. Special enrichment programs are implemented with obese animals and include daily exercise outside of the pen. There is a large individual variability in willingness to move so exercise programs are individualized.

Citoxlab Denmark produces studies with scientifically sound data because they have been at the forefront of ensuring optimal housing and enrichment programs at all stages of the pig life, including obese animals.

Optimization of thyroid hormone measurements by LC-MS/MS and ELISA in mice and rats

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OECD Guidelines (OECD 421, 422, 443) now include a requirement for determination of circulating levels of thyroid hormones in order to identify potential endocrine disrupting properties of chemicals. These hormonal investigations are also required during the preclinical assessment of new drugs and can be critical in some mechanistic studies. This has led to a growing demand in recent years for the determination of T3, T4 and TSH in plasma samples from toxicology studies. To meet this demand, in particular in small animals such as mice and rats, our team of specialists in analytical techniques has developed and validated an LC-MS/MS method for T3, T4 and a Luminex immunoassay method for TSH measurement. Blood sample volumes required are modest since T3 and T4 are determined in the same LC-MS/MS analytical run and the Luminex volume requirement is as low as 25 ul/sample. Both assays are fully GLP compliant.

We have combined our analytical methods with a heparin-coated minivette capillary blood collection technique for microsampling in order to permit multiple serial blood samplings from individual animals. With this approach, we are capable of performing blood sampling from the same individual mice or rats on multiple occasions (up to 6 timepoints over 24 hours) both in short term and long term studies. Using this approach we have generated reference control data for the mouse which were recently presented at the Japanese SOT meeting (Nagoya: 29/6-01/07 2016)

Citoxlab strengthens its immunology laboratory biomarker capabilities with the introduction of SPARCL technology

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Spatial Proximity Analyte Reagent Capture Luminescence (SPARCLTM) is a technology used for homogeneous immunoassays, typically for the measurement of biomarkers in support of non-clinical and clinical programs. A SPARCL assay usually requires no wash steps, uses a single incubation, and takes less than one hour for assays to be completed (unlike enzyme-linked immunosorbent assays, ELISAs, that require 1 to 4 incubations, 1 to 3 wash steps, and take 2 to 4 hours to complete). The SPARCL technology is good for cost effective high throughput screening, whilst still maintaining assay sensitivity and a good dynamic range. Citoxlab North America has acquired a Molecular Devices SpectraMax® L microplate reader, and to demonstrate suitability of the SPARCL technology, has recently validated three biomarker assays: non-human primate and rat cardiac troponin I (cTnI), a cardiac injury biomarker, as well as non-human primate C-reactive protein (CRP) an inflammatory biomarker.

The validation involved using serum samples collected from animals which had undergone coronary ligature treatment to specifically induce increases in these cardiac biomarker levels. Interestingly, results showed that coronary ligature did indeed induce significant cTnI release (between 4 and 8 hours post-surgery), which was shortly followed by an inflammation response as seen by significant increases in CRP.

In conclusion, this assessment of the three cardiac biomarkers using the SPARCL technology demonstrated the advantages that this technology offers, including producing validatable quantitative methods with wide dynamic ranges that are relevant to the non-clinical matrices under investigation. This high throughput technology is a potentially a cost-effective solution for clients wishing to add additional biomarker endpoints to their non-clinical and clinical programs.

Citoxlab expands its capacity in molecular pathology by offering immunohistochemistry (IHC) and in situ hybridization new assays

NL Bulle 4Citoxlab has expanded its capacity in molecular pathology and offers complete customized IHC and in situ hybridization services, from the initial tissue sampling and method development to the expertly interpreted target expression-profiling studies. Contract research experiments are initiated in partnership with the Sponsor, and the objectives are defined based on an optimal scientific approach. The projects comprise of a preliminary step to set up and optimize the methods, followed by the main assay to immunolabel or hybridize the targeted tissues.

Our Molecular Pathology team is led by board certified DVM and MD pathologists who have extensive experience in tissue cross-reactivity studies and IHC labelling. Our list of markers is available for most laboratory animal species used in preclinical development and sample types such as FFPE and frozen OCT-embedded tissue blocks. This includes BrdU and Ki67 labelling for cell proliferation, hormone detection by IHC, and in situ detection of microRNA and messenger RNA, the latter, using the RNAscope technology®. These markers can be quantified by image analysis on full tissues or tissue microarray sections that have been digitized with an Aperio AT2 (Leica) high throughput whole slide scanning system. In addition, real-time digital slide sharing and discussion with the Sponsor’s team is carried out when needed, through the built-in web conferencing tool.

10-years biodistribution and shedding experience – extended capabilities for preclinical and clinical sample analysis

NL Bulle 3The development of viral based vaccines as well as gene and cell therapy products require detailed biodistribution studies. These studies aim at evaluating the persistence of viral vector DNA or RNA in tissues, and the risk of viral vector genetic material integration into the host genome. Shedding data can also be collected in the framework of preclinical studies to evaluate the risk of release of infectious particles in biological fluids of patients treated during clinical trials.

Several key factors are taken into consideration at Citoxlab to provide robust data and the highest assay sensitivity. We have already developed and validated numerous qPCR and qRT-PCR assays for a wide range of BSL1 and BSL 2 vectors such as plasmids, AAVs, adenoviruses, poxviruses, phages, and several RNA viruses. By controlling all critical steps, in particular specific necropsy procedures, robust PCR assay development and validation, and nucleic acids extraction processes, we are able to detect as few as 5 to 10 copies of vector DNA or RNA in many tissues and biological fluids, without significant contamination or PCR inhibition.

For the last ten years, we have been combining our experience in toxicology with our expertise in molecular biology to provide our clients with robust and fully integrated biodistribution and shedding studies. We recently extended our capabilities by acquiring an additional real-time PCR instrument in our facility in France, where we now also offer biodistribution and shedding services for clinical samples in compliance with GCLPs.

Citoxlab North America appoints Vittoria Badalone as VP of Business development and marketing

NL Bulle 6Citoxlab North America is pleased to announce the appointment of Vittoria (Vicky) Badalone as Vice President, Business Development and Marketing, effective October 17, 2016. In this role, Vicky will have overall responsibility for the North America’s marketing, sales and business development strategy.

After obtaining her BSc, Vicky started her career in 1990 as a toxicology technician at Bio-Research (which became CTBR and subsequently Charles River Laboratories). She gradually worked her way up the Toxicology ladder finally becoming a Senior Research Scientist, fulfilling the role of Study Director for 7 years. In 1998, still with CTBR, she had a change in career path, becoming the Manager, Software Validation and was mandated to implement new processes and procedures for compliance with FDA CFR Part 11. Another career change came about at CTBR in 2003, where she moved into a sales and marketing role, working her way up to Senior Account Manager.

After 18 years with Bio-Research/CTBR/CRL, Vicky left to undertake the role as Director of Business Development for IPS Therapeutics (1 year) and Insymbiosis (3 years). Prior to joining our Citoxlab North America team, Vicky was with ITR Laboratories for the last 5 years as the Director, Business Development, Marketing and Client Services.

As can be seen from her various roles, Vicky has an excellent understanding of our industry bringing with her a broad wealth of experience. In addition to heading-up Business Development and Marketing, Vicky will be a valuable member of our North American site’s Senior Management Committee (SMC).



The Citoxlab team would be delighted to meet you at the following scientific meetings:

> Society of Toxicology Annual Meeting and ToxExpo
March 12-16, Baltimore, MD Booth #2257
13 posters and a hosted session ”Neurotoxicology investigations in minipigs”

> Bio Europe Spring
March 20-22, Barcelona, Spain

> Global CRO Concil (GCC)
April 3rd, Los Angeles, CA
Presentation ”Bioanalytical Labs role in writing PK sample collection instructions”

> British Society of Toxicology (BTS)
April 3-5, Liverpool UK

> Workshop on Recent Issues in Bioanalysis (WRIB)
April 3-7, Los Angeles, CA Booth #39