Preanalytical Variables

This article is the result of an initiative between the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECVAM). Its objectives are to provide the researcher in the safety evaluation laboratory with an up-to-date, easy-to-use set of data sheets to aid in the study design process whilst at the same time affording maximum welfare considerations to the experimental animals. Although this article is targeted at researchers in the European Pharmaceutical Industry, it is considered that the principles underpinning the data sets and refinement proposals are equally applicable to all those who use these techniques on animals in their research, whether in research institutes, universities or other sectors of industry. The implications of this article may lead to discussion with regulators, such as those responsible for pharmacopoeial testing. There are numerous publications dealing with the administration of test substances and the removal of blood samples, and many laboratories also have their own "in-house" guidelines that have been developed by custom and practice over many years. Within European Union Directive 86/609EEC1 we have an obligation to refine experiments to cause the minimum amount of stress. We hope that this article will provide background data useful to those responsible for protocol design and review. This guide is based on peer-reviewed publications whenever possible, but where this is not possible we have used "in-house" data and the experience of those on the working party (as well as helpful comments submitted by the industry) for a final opinion. The guide also addresses the continuing need to refine the techniques associated with the administration of substances and the withdrawal of blood, and suggests ways of doing so. Data-sharing between laboratories should be encouraged to avoid duplication of animal work, as well as sharing practical skills concerning animal welfare and scientific problems caused by "overdosing" in some way or another. The recommendations in this guide refer to the "normal" animal, and special consideration is needed, for instance, during pregnancy and lactation. Interpretation of studies may be confounded when large volumes are administered or excessive sampling employed, particularly if anaesthetics are used.

The purpose of this paper is to provide historical data pertaining to clinical chemistry and haematology parameters, obtained from control Sprague-Dawley rats, used in pre-clinical toxicity studies. Mean, standard deviation, minimum and maximum values for haematological and coagulative profiles, haemato-biochemistry and urine analysis data, and the differences per sex and study duration, 4 versus 13 weeks, are presented. The studies were conducted in agreement with the GLP (Good Laboratory Practice) regulations. Statistically significant differences, at the confidence level of 99%, for the red blood cell (RBC) parameters, the white blood cell (WBC) series parameters, plasmatic albumin/globulin (A/G), alanine amino-transferase (ALT), alkaline phosphatase (ALP), creatinine, globulin, glucose, sodium, total protein, tryglycerides, urea and urine volume were observed in males, when 4-week study values were compared with those obtained from 13-week studies. Female rats showed statistically significant variations, at the confidence level of 99% for RBC number and mean corpuscular haemoglobin (MCH), mean red blood cell volume (MCV), WBCs count and lymphocytes percentage, A/G, albumin, ALT, AST, ALP, creatinine, globulin, and sodium, when 4-week study values were compared to 13-week studies. Similar differences were observed comparing the female with male haematological and biochemical data for the two different times of the sample collection. These data could be useful as a reference for evaluation of background pathology in Sprague-Dawley rats, when used in studies performed to evaluate the toxicological profile of a new chemical entity (NCE) in agreement with requirements from international regulatory agencies.

Emotional, psychosocial, or anxiety-stimulated stress produces increased plasma concentrations of adrenal corticoids and other hormones though well-known neuroendocrine pathways. A direct consequence of these increased corticoid concentrations is injury to elements of the immunological apparatus, which may leve the subject vulnerable to the action of latent oncogenic viruses, newly transformed cancer cells, or other incipient pathological processes that are normally held in check by an intact immunological apparatus. This article describes studies that examine the adverse effects of increased plasma concentrations of adrenal corticoids on the thymus and thymus-dependent T cells, inasmuch as these elements constitute a major defense system against various neoplastic processes and other pathologies. The studies demonstrate that anxiety-stress can be quantitatively induced and the consequences measured through specific biochemical and cellular parameters, providing that authentic quiescent baselines of these conditions are obtained in the experimental animals by the use of low-stress protective housing and handling techniques.