Blood Sample Collection

Introduction

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.

Sampling

Anticoagulated blood is required for cell counts. If there are any visible clots, even small ones, the sample is discarded, as the WBC and platelet counts will be inaccurate. EDTA is the best anticoagulant to preserve cell detail but this does not provide such good cell morphology as a well prepared fresh smear. Heparin can be used but produces poor staining of cells. EDTA tubes should be completely filled to the level indicated. An excess of EDTA causes red cell shrinkage and decreased PCV. Overfilling may lead to clotting. Blood should be analysed within 2-3 hours or kept refrigerated. After 6-24 hours storage, RBCs swell leading to increased PCV and MCV with reduced mean corpuscular haemoglobin concentration (MCHC). Other red cell indices are not affected. Artefactual changes may result from excess suction on the syringe, over zealous agitation of the EDTA tube and high environmental temperatures, which can all cause haemolysis.

Sample ageing also increasesthe likelihood of haemolysis. Samples submitted to the laboratory should arrive within 2 days. Delays often resultin cell deterioration and lysis, therefore submission of a fresh smear is advisable.

References: Nation Wide Laboratories

Sample Type Required table

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

Preparation of Blood Smears

Blood smears should be prepared immediately to avoid artefact due to exposure to anticoagulant. Smears prepared from an EDTA sample can be used for a routine differential count but the cell morphology is not ideal. Well prepared fresh smears are particularly important for examining atypical cell morphology.

If parasitaemia is a concern, smears should be made from capillary blood (ear prick) since the concentration of parasites is higher in peripheral blood.

Nation Wide Labs

Well prepared blood smears should have an area where the blood cells are in a monolayer. Place a small drop of blood on the slide towards one end. Draw the spreader slide back to contact the drop and then move it forwards before the blood has spread along the width of the spreader slide. This will prevent the smear extending to the extremities of the slide, when cells can be lost overthe edges. The smear should terminate before the end of the slide (Fig 1).

The area to be examined is the monolayer where 50% or less of cells are in contact.

The differential count is performed, and cell morphology is assessed in the monolayer (Fig 2).

References: Nation Wide Labs

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Nation Wide Labs

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Feathered Edge

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Monolayer