The aim of this article is to introduce nurses to the most common cells seen on routine examination of a blood smear.

A routine haematology examination should consist of two parts: a quantitative measurement (determining the numbers of red blood cells and white bloods cells &tc.) and a qualitative examination (checking cellular morphology).

Quantitative parameters

The quantitative measurement is usually carried out by a wide range of commercial analysers and measures parameters, such as:

•   HCT (haematocrit) – the equivalent of the manual PCV

•   RBC (red blood cell count) – gives an estimation of the animal’s red cell mass

•   MCV (mean corpuscular volume) – gives the average size of the red blood cell and a normal level will indicate normal sized red blood cells

•   MCH (mean corpuscular haemoglobin) – indicates the mean quantity of haemoglobin inside a red blood cell

•   MCHC (mean corpuscular haemoglobin concentration) – shows the mean concentration of haemoglobin in the red blood cells.

Analysers will also give a white blood cell and platelet count. The platelet count should routinely be checked by a qualitative examination of a blood film as analysers can get it wrong owing to platelet clumps or the presence of giant platelets. A machine differential count is unable to show any morphological changes within the red and white blood cells.

Stains

There are several types of rapid ‘dunking’ stains available, with Diff-Quick being the most common. It is more than adequate for practice in-house use.

The following protocol can be used as a guide:

•   prepare a blood smear and let it air dry

•   use the first solution – a light blue fixative (methanol) and the slide is dipped five times for one second each time

•   use the second solution – a red stain (eosin) and again the slide is dipped five times for one second each time

•   the third – and last – solution is purple (methylene blue) and the slide is dipped seven times, again each dip lasting a second

•   the slide should then be rinsed with buffered or distilled water and left to air dry.

It is now ready for examination using an oil immersion lens.

Film examination

Smears should be examined in the thin area towards the feathered edge using the ‘battlement’ technique (Figure 1). The cells should be evenly distributed in a monolayer (not stacked on top on each other). Do not examine cells at the feathered edge as these may become distorted and clumped.

Figure 1: Smears should be examined using the 'battlement' technique

It is best to use a x50 or x100 oil immersion for a detailed look at the cells.

Red blood cells (erythrocytes)

Red blood cells are the most common cell seen on a blood smear. They are anucleate (do contain a nucleus) and usually stain pink. Canine erythrocytes have a central pallor, which is not normally seen in cats and other species. Anisocytosis and polychromasia are commonly reported on haematological examinations.

Anisocytosis refers to the variation in the size of the red blood cells and polychromasia is the level of regeneration demonstrated by the number of immature red cells seen. Polychromatophils stain blue/grey rather than pink and are larger than mature red cells, but make up less than one per cent of red blood cells seen in a normal animal.

White blood cells (leukocytes)

There are five types of leukocytes, which are divided into two groups: the granulocytes and agranulocytes. The granulocytes contain small granules in their cytoplasm and the agranulocytes do not.

White blood cell types Granulocytes

•   neutrophils

•   eosinophils

•   basophils Agranulocytes

•   lymphocytes

•   monocytes

The most common leukocytes seen in normal cats and dogs are neutrophils, followed by lymphocytes. Only small numbers of monocytes and eosinophils are seen in normal cats and dogs, and basophils are usually absent.

If you need to manually check a white cell count, use the x10 objective lens; count all the white blood cells in three high power fields (a high power field is the area you see on the microscope slide); work out the average and multiply the result by 150. Be aware, however, that this will only give you an idea as to whether the count is high, low or normal, rather than an accurate figure.

Neutrophils

Typically in mature neutrophils there are three to five segmented lobes or segments per cell (Figure 2). The cytoplasm stains faintly blue to pink depending on the type of stain used. Band neutrophils may be absent or present in very low numbers in peripheral blood and are identified by the U shaped nucleus with a constant width.

Figure 2: Two mature neutrophils

Neutrophils are the first line of defence against invading organisms. Their primary function is to kill bacteria, and this is done by phagocytosis (ingestion of the bacteria) followed by their destruction by enzymes within the neutrophil.

Lymphocytes

The second most common leukocyte in peripheral blood is the lymphocyte (Figure 3). Lymphocytes are generally round, slightly smaller than neutrophils, with a round to oval nucleus. There is also a small amount of light blue staining cytoplasm present.

Figure 3: A lymphocyte

There are two types of lymphocyte – B and T lymphocytes. B lymphocytes are responsible for producing antibodies and T lymphocytes help activate B cells and can also kill virus-infected cells.

Monocytes

Monocytes are absent – or present only in low numbers – in a normal blood film (Figure 4). These are large cells and the nucleus can be different shapes (oval with or without indentations is considered normal). The nucleus can also be kidney shaped. The cytoplasm stains blue/grey and may have multiple vacuoles.

Figure 4: A monocyte and two neutrophils

Monocytes circulate in the blood stream for a short time before they migrate into the tissues to become macrophages. Once they become macrophages their main function becomes the phagocytosis of dead cells and other foreign material such as fungi.

Eosinophils

Eosinophils are cells with poorly segmented purple/blue staining nuclei, and the cytoplasm contains round or rod-shaped reddish staining granules (Figure 5). Eosinophils may be absent or present in low numbers in a normal blood sample.

Figure 5: An eosinophil

The main function of eosinophils is to kill parasites, by secreting enzymes directly onto the surface of the parasite.

Basophils

Basophils have a very long, poorly segmented purple/blue staining nucleus with light purple staining cytoplasm (Figure 6). The cytoplasm can also contain purple staining granules. Basophils are rarely seen in normal blood films.

Figure 6: Basophils

Basophils numbers can increase when the body is responding to an allergic reaction.

Platelets

Platelets, or thrombocytes, are small round structures without a nucleus. Their main function is haemostasis. Following damage to a vessel, platelets are activated and form a plug that seals the leak. This process is known as primary haemostasis.

Machine-generated platelet counts are notoriously unreliable because of a number of factors. Firstly small clots called micro-clots can form in the EDTA tube, which are invisible to the naked eye but effect the overall count. Cats and some breeds of dogs produce ‘giant platelets’ which the machine will count as a red blood cell (Figure 7). If platelet clumps are present, the machine will give a low platelet count but the animal’s count will probably be normal.

Figure 7: A giant' platelet

To check a platelet count manually on a film, use the x100 oil immersion lens and count all the platelets in 10 high power fields (hpfs). Work out the average and multiply the result by 15. This will give a platelet count estimation in x109/l which is the same as an analyzer.

A manual differential count can give as much information about a case as a machine count. It will confirm the mechanical results as well as alert you to any morphological changes within the cells. A slightly elevated mechanical white blood cell count could be normal or could be caused by lymphoma; the patient is anaemic but is it regenerative? Is the body trying to combat the anaemia by releasing juvenile red blood cells from the bone marrow? A manual examination will tell the tale. 

Author

Matthew Garland

Cert Nat Sci (Open) VN

After qualifying as a VN in 2004, Matthew worked in small animal practice before moving to Torrance Diamond Diagnostic services in 2006. Now as laboratory manager of TDDS-Ringwood, he has developed a strong interest in haematology and biochemistry.

• VOL 25 • Nol • January 2010 • Veterinary Nursing Journal