Preanalytical and analytical variation in animal clinical pathology: how to handle your samples.

Dai Davies
Head of Clinical Pathology, ZENECA Pharmaceuticals, Macclesfield, Cheshire, England

Clinical Pathology, consisting of the evaluation of haematology and blood/urine chemistry, is a very important part of drug safety evaluation. Generally, good controlled experiments with homogeneous groups of animals and a randomised block design provides a way of increasing precision and thus maximising the chance of detecting a true treatment effect. Having optimised study design, then it is important to ensure that quality samples, each handled in an identical fashion, are analysed in the laboratory using precise, well controlled assays. While the physical environment can be well controlled and every effort can be made to standardise the experimental procedures, every test system is, however, subject to experimental variance. These experimental variances can be considered under four main topics, of which the first three will be considered in detail:
1. Biological Variation.
Several preanalytical variables will be considered including species, gender, age, chronobiochemistry (or bio-rhythms), nutrition and stress. All these variables are directly related to the animals and an awareness of these changes is important in the accurate interpretation of experimental results. Variability associated with the day-night cycle is well described for hormones and other bodily functions (e.g. urine production) but changes from day to day can also be recognised. Some parameters, such as haemoglobin concentration and serum electrolyte values, some minimal variation while circulating serum enzyme values can vary significantly for consecutive samples from the same animal. Seasonal breeding animals show much longer cycles of change associated with reproduction while some neuropeptides can change from minute to minute.
2. Pre-analytical Variation.
Additional variability can be introduced by study personnel not taking adequate care to adopt consistent procedures associated with sample collection, handling, separation and storage. All of the following can significantly influence the final test results and examples of each will be presented during the lecture: time of sampling; sample volume; site of sampling; agents used for anaesthesia and euthanasia; assay interference; frequency of sampling and the use of appropriate anticoagulant.
3. Analytical Variation.
In a modern laboratory with current instrumentation, the goal of achieving an analytical imprecision of less than one-half of the average within animal biological variation, is often comfortably achieved. Use of appropriate internal and external quality control samples helps the laboratory to standardise its approaches to improve both precision and accuracy. Special consideration is required, however, to ensure that the analytical method employed is appropriate for the species being evaluated and that compounds under evaluation do not interfere with the assay.
4. Pharmacological Variability
No two individuals respond in an identical manner to an administered drug. Similarly, when a group of animals are tested for a new drug entity it is not uncommon to see variability in physiological and pharmacological responses to the test compound.
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