Practical Laboratory Techniques

Figure 7.2 Veterinary nurse carrying out buccal mucosal bleeding time (BMBT) in house.


Figure 7.3 In house viral testing kit for feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV).


Packed Cell Volume and Total Protein

PCV (or haematocrit) and TP are important tests in the emergency and critical patient. They are quick and simple to perform and give relevant information about the status of the patient. PCV and TP should always be interpreted together to interpret the patient’s status accurately. Whole blood should be placed into either anticoagulant coated capillary tubes, or drawn from a filled EDTA tube. Once the sample has been spun the three layers of cells will be evident, the PCV should be measured first then, using a refractometer, the TP can be measured. This is performed by splitting the capillary tube above the buffy coat (the layer of white blood cells and platelets), the plasma protein layer can then be ‘blown’ onto the surface of the refractometer using a 1-ml syringe. The TP is measured by looking at the scale labelled g/dl (usually on the left-hand side, with the specific gravity scale being on the right-hand side) (see Figure 7.4). The line where the shaded area meets the coloured area is the TP reading. As with SG measurement, the refractometer should be calibrated regularly using distilled water which has an SG of zero.

Figure 7.4 Refractometer reading of total protein level.


Saline Agglutination

Auto-agglutination of red blood cells indicates anti-red blood cell antibodies are present and is a commonly used test to indicate immune-mediated haemolytic anaemia. The test is performed by mixing one drop of blood with one drop of isotonic saline on a microscope slide. The slide should be gently rocked from side to side in order to mix the saline and blood, and then observed for signs of macroagglutination (obvious to the naked eye) (see Figure 7.5). Once this has been performed, the blood–saline can be covered with a coverslip and the slide examined under the microscope. Rouleaux can be differentiated from agglutination by examination under a microscope, and the addition of saline should disperse any rouleaux.

Figure 7.5 Positive saline auto-agglutination test.


Prepare and Examine Blood Films

Most practices have in-house analysers available. However, these should always be used in conjunction with blood film examination, and this is a particularly useful technique for the emergency patient. In particular, WBC evaluation is not always reliable, nor is the platelet count always correct, especially in cats which have relatively small red blood cells and quite large platelet – most analysers differentiate these cell types by their size. Furthermore, platelet clumping also falsely lowers the platelet count. Breed also needs to be taken into consideration when assessing platelet counts, e.g. Cavalier King Charles Spaniel may have abnormally low number of platelets (thrombocytopenia) and oversized platelets (macrothrombocytopenia).

Blood film examination will detect these errors and provides much information that is not available from the analyser. For white blood cells, a left shift, toxic neutrophils, reactive lymphocytes and neoplastic cells can all be identified on a manual blood smear. When assessing anaemic patients, examination of red blood cell morphology is vital, both to distinguish between regenerative and non-regenerative anaemia and sometimes to identify the cause of the anaemia (e.g. spherocytes found in immune-mediated haemolytic anaemia). Finally, by counting the platelets seen on the blood film, the analyser can be verified or corrected.


Romanowsky’s stains include Wright–Giemsa and rapid ‘dipping’ kits (Diff-Quik kits). The latter are more than adequate for in-house use (see Figure 7.6). These kits have a three-stage staining procedure, which incorporates a fixative pot (usually five dips), orange–pink dye in the second pot (usually three dips) and a blue–purple dye in the third pot (usually six dips). The stain is stored in glass jars which should be cleaned regularly to avoid the build-up of stain precipitate. Periodically they should be scrubbed out using methanol to remove all stain. Stain-containing precipitate can be filtered or discarded and replaced with fresh stain. With time, the red–orange dye can be inadvertently carried over into the blue–purple dye resulting in weak staining. The only solution is to replace the blue–purple dye.

Figure 7.6 Rapid dipping kit in an in-practice lab.


Film Examination

To ensure all cell lines are examined properly, a set procedure of blood film examination should be routinely followed. Initially, the smear is checked for large platelet clumps by examining the feathered edge at low power (20×). The smear is then examined at higher power (40× or 100×) in the thin area in from the feathered edge, where the cells are evenly distributed in a monolayer. The red blood cells, white blood cells and platelets should then be examined in turn.

Examination of Red Blood Cells

Erythrocytes, commonly known as red blood cells, are the oxygen carrying cells within the blood. When these cells are low in number, the patient is anaemic; this can be determined by measuring PCV or haematocrit. Examination of red blood cells in blood smears should include an assessment of colour, size and shape, and a search for inclusions. Canine red blood cells (see Figure 7.7) have a pale area in the centre of the cells (central pallor) which is not obvious in feline red blood cells (see Figure 7.8), which are smaller. If the animal is anaemic, blood film examination helps determine if the anaemia is regenerative.

Figure 7.7 Normal red blood cells (canine).


Figure 7.8 Normal red blood cells (feline).


Features of Regeneration

  • Polychromasiathese are young red blood cells which stain a purple–lilac colour due to the presence of ribosomes in the cytoplasm. Occasional polychromatophils (one every 2–4 1000× field) are seen in films from healthy animals. Several per field would indicate a robust regenerative response.
  • Anisocytosis with large (young) red blood cells.
  • Howell–Jolly bodiesthese are small but prominent dark inclusions which are remnants of nuclear material.
  • Nucleated red blood cellsdo not confuse these with lymphocytes. Nucleated red blood cells have smaller nuclei and the cytoplasm is a similar colour to polychromatic red blood cells (see Figure 7.9).

Figure 7.9 Nucleated red blood cells.


Evaluation for a Possible Cause of Anaemia

The blood film can help identify several causes of anaemia, including immune-mediated haemolytic anaemia, babesiosis, feline infectious anaemia, oxidant injury, microangiopathic haemolytic anae­mia and iron deficiency.

Immune-Mediated Haemolytic Anaemia

This disease usually leads to circulating spherocytes (see Figure 7.10). These appear smaller than normal red blood cells with a darker and/or denser cytoplasm lacking central pallor. Care should be taken to look in the examination area monolayer and avoid looking at the tail of the smear where cells are flattened and lose their normal central pallor, giving a false impression of spherocytes. Spherocytes are difficult to see in cats′ blood because normal feline red blood cells have minimal or no central pallor. NB Small numbers of spherocytes may be seen with other causes of anaemia.

Figure 7.10 Spherocytes.



Babesia canis (see Figure 7.11) appear as large pear-shaped organisms, usually in pairs. Babesia gibsoni organisms are much smaller, circular bodies. These parasites are more often seen in capillary blood (e.g. from an ear prick) and most frequently found along the edges of films. However, these organisms are not always visualised and serology or polymerase chain reaction (PCR) are more sensitive methods for diagnosis.

Figure 7.11 Babesia canis.


Feline Infectious Anaemia (Haemotropic Mycoplasma Infection)

Provided that samples are obtained during an episode of parasitaemia, organisms may be identified using the Romanowsky stains. The organisms stain blue–grey to pale purple with Romanowsky stains and appear as small cocci singly or in chains. Again, PCR is a more sensitive diagnostic technique. Figure 7.12 demonstrates the presence of Mycoplasma felis on a blood smear.

Figure 7.12 Mycoplasma felis.


Oxidant Injury

Ingestion of oxidants, such as onion or zinc, can lead to the formation of Heinz bodies or eccentrocytes with resultant anaemia. Heinz bodies are seen as non-staining round bodies, usually protruding from the surface of the cell. Eccentro­cytes (see Figure 7.13) have a pale area on one side of the cell which is devoid of haemoglobin, but with a cell membrane visible around this pale area.

Jul 30, 2017 | Posted by in GENERAL | Comments Off on Practical Laboratory Techniques

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