Sample Collection and Preparation

General

In order to derive maximum benefit from any laboratory diagnostic service, it is essential that the samples selected are the most appropriate and are adequately preserved.
Infectious agents are most likely to be present during the pre-clinical or early clinical stages of disease.
Sampling from the margins of lesions increases the chances of detecting viable microbes.
Swabs should be submitted in transport medium appropriate for the agent under test. Appropriate transport swabs for viruses, chlamydiae and some bacteria may be obtained from the Diagnostic Unit, Stormont (telephone 028-90525657) or the Omagh laboratory (telephone 028-82243337).

Enteric disease packages require faeces samples (minimum 10g) in a rigid container.
Plastic gloves are unsuitable as “containers” for faeces samples on Health and Safety grounds.
A sample of approximately 10g faeces is usually preferable to faecal swabs because the latter do not provide sufficient material for virological and parasitological examinations. However, faecal swabs placed in charcoal transport medium together with a 10g sample of faeces are useful for the diagnosis of swine dysentery.

Bacteriology

Samples for bacteriological testing should be collected using sterile instruments and containers. Contamination by environmental bacteria should be avoided and, where possible, the sample should be collected before antibiotics have been administered.
Swabs for bacterial culture should be submitted in bacterial transport medium. The Horserace Betting Levy Board Code of Practice on Contagious Equine Metritis indicates that swabs for examination for contagious equine metritis examination should be placed in Amies charcoal transport medium and submitted so as to arrive at the laboratory within 48 hours of swabbing.
Campylobacter transport swabs and enrichment media (for checking for Campylobacter fetus venerealis) are available on request from the diagnostic unit at the Stormont laboratory (telephone 028-90525649).

Milk samples should be collected aseptically because microbial contaminants such as streptococci and faecal coliforms can give misleading results.
Samples for microbiological examination should be packaged individually to prevent cross-contamination in transit.

Virology

Sera for paired serology for respiratory viruses should be taken 2-3 weeks apart. The first sera should be taken from new clinical cases or from in-contact animals with elevated temperatures but not yet showing clinical signs. Nasal swabs should be submitted in viral transport media available on request from the Stormont laboratory (telephone 028-90525649). Milk samples for bulk tank antibody testing should be placed in 20ml Universal containers with a "Lactab" added. Freezing reduces the viability of most viruses; refrigeration in transport medium is therefore preferable if a delay in submission is anticipated. Antigen Detection the fluorescent antibody test (FAT) can be used to detect viable and non-viable bacteria, viruses, chlamydiae or other agents in tissue specimens. Unfixed, (not frozen) samples or swabs are required for FAT testing. The chances of detecting viral antigens are maximised by sampling in the pre-clinical or early clinical stages.

Histology

Select a representative area of the lesion and try to include some adjacent normal tissue. Tissue slices should be less than 1cm thick in order to allow rapid penetration of fixative. A 10% solution of formalin (1 part 40% formaldehyde and 9 parts water) is an easily prepared general fixative although buffered formalin is preferable. The volume ratio of tissue to fixative should be at least 1:10. Do not freeze tissue samples intended for histological examination.

Reproductive Physiology

Semen samples should be submitted in sterile Vacutainers or equivalent. Milk samples for progesterone or oestrone sulphate testing should be placed in 20ml Universal containers with a "Lactab" added. Blood progesterone estimation requires a serum sample (although a clotted sample may suffice if submitted within 24 hours). PMSG estimation requires a clotted sample. Oestrone sulphate, testosterone and oestradiol assays can be carried out on clotted or heparinized samples.

Clinical Chemistry

Most biochemical tests can be carried out on a 10ml heparinized sample of blood. Serum from clotted samples can be used for many tests with the exception of the following:

Heparinized samples essential for GPX, lead and transketolase and preferred for copper
Blood glucose estimations require oxalate/fluoride samples
Oxalate/fluoride samples preferred for phosphorous

A clotted sample is essential for bile acids. Avoid rubber bung (eg Vacutainer) on samples for zinc estimation 100g of tissue are adequate for most tissue analyses. Samples which cannot be submitted within 24 hours should be stored in the fridge (not the freezer!).

Haematology

Use EDTA tubes especially when WBC differentials required (heparinized samples are acceptable if submitted within 24 hours). Do not use 10 ml EDTA tubes for small quantities of blood as the results will be inaccurate. Coagulation studies require blood collected with sodium citrate as anticoagulant.

How to Avoid Haemolysis

Haemolysis during and after blood collection can seriously affect the accuracy of biochemical and haematological tests. Small animal samples are especially susceptible and the causes listed below should be avoided.

Trauma

Excessive suction or pressure to draw or expel blood eg through a narrow-gauge needle.
Drawing blood into a large volume “Vacutainer” through a narrow needle - blood enters tube under excessive negative pressure and strikes the wall of the tube.
Negative pressure applied to blood already collected in the syringe while trying to relocate a “lost” vein.
Excessive “pumping” or squeezing of a limb to maintain blood flow.
Prolonged stasis of venous blood when vein is occluded.
Excessive shaking of the sample container.
Freezing the blood sample.
Excessive delay in despatching samples to laboratory
Excessive heat during storage eg in a car boot.
Contact with hypotonic solutions eg water in the syringe, needle or sample container.
Lipaemia increases the fragility of the RBCs (sample after fasting when possible).
Contact with chemicals including alcohol, ether, and acids.
Parasitology Ectoparasites: Send skin scrapings and samples of skin, hair or wool intact and dry.
Endoparasites: Send tissue or faecal samples without delay.

For the post-mortem investigation of diseases associated with nematode larvae in the intestinal wall, eg type-2 ostertagiosis in cattle or cyathostomosis in horses, submit unfixed representative portions of the affected wall along with a sample of contents. Worm larvae can be extracted from such tissue while still reasonably fresh. Do not forcibly detach parasites from tissues.

Details of samples required for laboratory testing are published periodically in our Vetline newsletter for veterinary practitioners.