Collecting Pathological Samples

Blood samples

When collecting blood samples, fill tubes containing anticoagulant to the fill line and mix gently immediately after collection. Vacutainers can be used for large animal blood sample collection, and a needle and syringe for small animals. The jugular vein is the preferred collection site using a wide bore needle to prevent haemolysis of the blood from using excessive suction during collection. The cephalic and saphenous veins are alternative venipuncture sites. Ensure the venipuncture site is clean and swabbed, wear gloves and ensure you have adequate restraint prior to collecting a blood sample.

Always label your sample tubes with permanent marker immediately with the animal's details and the date and time of collection. Serum samples can be processed by allowing the blood to clot in an upright position and removing the serum from the tube prior to sending the sample for analysis to avoid haemolysis (bursting of the red blood cells into the serum) during transit. A more ideal method of serum separation is to centrifuge the sample -balance the centrifuge by adding a tube filled with the same volume of fluid in the opposite well in the centrifuge for safety.

The following table is a guide to the sample type required for some specific laboratory tests - seek guidance from your processing laboratory for confirmation of sample requirements, which can vary depending on the equipment used to analyse the sample.

Test Required	Sample Required	Comments
Haematology	EDTA	Mix gently immediately after collection
Biochemistry	Serum	Centrifuge after permitting sufficient clotting time (min 2 hours)
Fibrinogen	Citrate	Mix gently immediately after collection
SAA	Serum	Needs to be processed quickly as the half life is very short
Electrolytes	Heparin	Mix gently immediately after collection
Glucose	Fluoride oxalate	Can use fresh whole blood if tested immediately
Insulin	Serum	Fasting sample required
Prothrombin clotting time	Citrate	A normal sample is required for comparison
Progesterone	Serum
Oestrone Sulphate	Serum
Testosterone	Serum	Request Testosterone before Inhibin for GCTs
IgG	Serum	Measurement techniques vary so check sample requirements
ACTH	EDTA plasma	Freeze plasma as soon after collection as poss (max 2 hours)
Selenium	Heparin/Serum	Send Heparin for deficiency testing, serum for toxicity testing.
Zinc	Serum	Avoid using tubes with a rubber bung
Drugs testing	Serum or Heparin	Send to HFL
Virus Titres e.g. EHV	Serum	Take 2 samples 14 days apart (freeze 1st)
Tapeworm ELISA	Serum
Fractional Excretion test	Serum and urine	Collect both samples simultaneously if possible and do not permit food or fluids until both are collected

Bacteriology Samples

Swabs

• Swab the area which is showing active signs of inflammation - avoid areas of pus as the neutrophilic destruction of bacteria which forms pus is less likely to produce a live culture.
• Use aseptic techniques and watch the swab tip until it has been safely replaced in the swab container to prevent accidental contamination prior to culture.
• Transport media containing charcoal is suitable for most samples with some notable exceptions such as swabs requiring Strangles PCR. Check with your submitting lad the first time you collect a swab for a specific diagnostic test to see if there are any special transport medium requirements. CEMO swabs must be placed in a charcoal medium for transportation.
• Label the swab immediately and indelibly with the patient details, site, and date.
• Fill in your external lab request form as completely as possible - the more information you include the better the chances of a diagnostic result. Most cultures are likely to give a mixed growth and if the pathologist can rule in or out organisms based on the information you give them then the pathological process becomes considerably more accurate.
• Refrigerate samples that are awaiting postage later in the day, as this will help to prevent an overgrowth of fast growing organisms at the expense of pathogenic organisms.

In House Culture

Accurate paperwork is essential when culturing in house samples so that changes noted on plates can be recorded over time. Always warm culture plates to room temperature prior to inoculation or cold shock can inhibit a delicate organism from growing on a plate. Different mediums can be a used to select for an organism of interest:

1. Blood Agar (BA) is the standard plate used for most samples - in most cases an additional plate is added to give more information about the identity of any organism that successfully grows.
2. COBA is selective for Streptococci species, which are ubiquitous in some species and frequently cause opportunistic infections. Tracheal washes, joint samples, and cervical swabs and potential Strep equi cases require this medium in addition to BA.
3. CLED with Andrade’s indicator is a useful medium when a swab is likely to grow E coli; the indicator will turn pink and the colonies will have a distinctive look and smell.
4. Sabaroud plates are selective for fungi and are useful for skin scrapes where dermatophytes are suspected - for many fungi special incubation requirements (e.g. temperature settings of 30°C for 48 hours) must be met so check the requirement based on the most likely organism to grow.
5. XLD and Brilliant green are used to encourage and identify Salmonella species; the colonies grow with specific characteristics on these two plates. Place your sample in Selenite broth prior to inoculating this media to reduce the number of contaminant organisms that will grow on the plate.
6. MRSA Chromogenic media will give a rapid (18 hour) diagnosis of MRSA infection – growth of the organism is associated with a distinctive colour change.
7. CEMO medium for Taylorella culture requires two plates of specialised agar which suppress the growth of contaminants; one plate contains added Streptomycin, the other is without Streptomycin as some strains of Taylorella have been found to be Streptomycin sensitive.

In addition to using these plates for your original inoculation, there are media available which can confirm the identity of an organism such as Pseudomonas aeruginosa that has grown on the standard media initially. Always test your growth media by maintaining known organisms and inoculating the media using normal conditions so that you can be sure that your methods are giving diagnostic results. Alternative methods of identifying an organism include biochemical tests such as oxidase or catalase reactions or latex agglutination tests.

Sensitivity testing

Sensitivity testing requires a standardised approach to plate inoculation and disc strengths used - the British Society for Antimicrobial Chemotherapy (BSAC) guidelines publish a methodology to ensure this. The plate requirement, dilution of the culture, strength of antibiotic disc used, interpretation of growth inhibition ring size and the culture conditions are all specified in these guidelines, which ensures in vivo antibiotic sensitivity. The remit is based on organism rather than species from which the sample was taken, so it is a valid approach to ensure accurate sensitivity tests in veterinary practice.

Sending Samples by Post

There are some absolute requirements for sending samples through the UK postal system. Blood samples cannot be posted outside the UK without an export license - use a large lab to send your sample as they will have the required licensing.

Packaging samples should follow the guidelines required by Royal Mail, namely:

• The maximum weight or volume permitted in any one package of diagnostic specimens is 50g/50ml. Samples that exceed this limit need to be packaged into several smaller packages rather than one large one.
• Use leak proof containers – seal the lid with a little parafilm or tape to ensure this. Label the sample accurately and in permanent ink including the sample type (e.g. blood, urine etc). Make sure samples preserved in Formalin are marked as such (in case of breakage during transport).
• Package the sample with sufficient tissue paper or cotton wool to absorb the entire contents of the sample should there be a leak whilst in transit. Always package bloods tubes/containers individually so they are kept separate from one another.
• Seal your wrapped sample in a separate outer packaging that is leak proof such as a sealable plastic bag.
• Place the sample and the lab request form into an envelope or jiffy bag that is clearly labeled in permanent ink ‘Diagnostic Specimen’.
• It is advisable (but not required) to add a returns address so that the practice can be contacted in the event of any problems.