What to Do When the Lab Is Closed: What a Blood Smear Can Tell You
World Small Animal Veterinary Association World Congress Proceedings, 2008
Sandra Forsyth, BVSc, DACVA
Institute of Veterinary, Animal and Biomedical Sciences, Massey University
Palmerston North, New Zealand

You can obtain a great deal of information about peripheral blood cells and potential differential diagnoses by examining a blood smear, so haematology is a useful skill to master.

Making the Smear

An important starting point is to make a good blood smear. There are several factors that determine the quality of a blood smear but one of the more important features is the standard of the slides that are used. The quality of the slide on which the blood is smeared is of less importance than the quality of the spreader slide. When selecting a spreader slide, run your finger along the edge and choose one that is extremely smooth to the touch. There is infrequently a difference between slides within a box, so it is more a factor of choosing a brand that provides smooth edged slides. Put a small dot of blood (not too small, or the smear will be too short, but only with practice will you learn what the ideal size of blood is) at one end of the slide, draw the spreader slide back into the dot at an angle of 30° to 45° and allow the blood to move side-ways under the spreader slide until it is about 1 mm from the edge. At this point push the spreader slide away from the dot. Two common errors are to put pressure on the spreader slide as it is advancing and to push the spreader slide too slowly. Ideally, the smear should be about ½ the length of the slide. A smooth edge on the spreader slide produces a smear with an even edge, whereas a rough edge produces an uneven feather-edge.

Making a good blood smear is important but equally is the need for adequate staining because it is very easy to miss WBCs and platelets when they are under-stained. Rapid staining systems (e.g., Diff-Quik) are commonly used in practice because of their ease of use. A consequence of frequent use without draining pots and replacing the solutions is dilution of the stains and poor quality pale staining, especially of the purple (blue) colour making nuclear features difficult to see.

Examining the Smear

Once the smear is stained and adequately dried, examination can begin. Scan the feather-edge of the blood smear at low power and look for platelet clumps which often accumulate there (more of this later) and for unusual cells (in both size or type). The ideal site to examine the cells in greater detail is just back from the feather-edge where there is a small space between the RBCs (the sweet zone). In thicker areas it is difficult to see RBC morphology and WBCs tend to be globoid rather than flat. On the other hand too near the feather-edge, RBCs lose their central pallor (dog) and WBCs are often damaged.

Red Blood Cell Changes

Shape and Size

Changes seen in the shape or size of RBCs or the presence of intracellular inclusions may be useful in determining the cause of a change in PCV. For example anaemia may be accompanied by spherocytosis, microcytosis, anisocytosis and polychromasia. The presence of some of these features may shorten the differential list (regenerative vs non-regenerative anaemia) or provide a possible answer as to the cause for the anaemia (spherocytosis).

Polychromasia (variation in colour of stained RBCs) and anisocytosis (variation in size of RBCs) are features of a regenerative anaemia. Polychromasia is due to early release of young cells from the bone marrow. These cells are usually blue (or blue-grey) in colour, due to staining of intracellular organelles such as ribosomes and mitochondria that are still present. The young cells are also larger and so increase the spread of cell size (anisocytosis). In a pre-regenerative or non-regenerative anaemia, polychromasia and anisocytosis will be absent.

A pre-regenerative anaemia occurs when a blood sample is assessed after acute haemorrhage or haemolysis and before the bone marrow has had time to respond--usually less than two to three days between RBC loss and time of sampling. A regenerative response would be expected a day or two later.

Variability in cell size (or diameter) can also be noted on a blood smear when small RBCs (microcytes) or globoid RBCs (spherocytes) are present.

Spherocytes result when there is an immune mediated removal of part of the RBC membrane that alters the shape of the cell from biconcave to globoid. The RBCs appear as small, dark cells with no central pallor. They are usually only noticed in dogs because the small feline RBCs lack central pallor normally. Spherocytosis can rarely be seen following multiple bee stings or snake evenomation.

Small RBCs (microcytes) must be very small before they can be appreciated on a blood smear. The most common cause for microcytosis is iron deficiency anaemia and pale cells (hypochromasia) may also be noted. Depending upon severity and duration of the iron deficiency varying severity of microcytosis, hypochromasia and regenerative response may be seen.

The presence of nucleated RBCs (metarubricytes) may be a normal part of a marked regenerative response but their presence in the absence of other signs of regeneration is abnormal and indicates possible bone marrow damage, hypersplenism, lead poisoning and uncommonly, hyperadrenocorticism.

Nucleated RBCs are sometimes a feature of splenic haemangiosarcoma as are red cell fragments (schizocytes). Schizocytes as well as keratocytes (helmet cells, apple stem cells) indicate RBC damage and result from shearing of the RBCs by intravascular trauma. They may be seen when cells pass through tortuous vessels (haemangiosarcoma) or intravascular fibrin meshes (DIC) or when the RBCs are especially fragile (iron deficient).

Burr cells (echinocytes) have multiple, short, evenly spaced surface projections. They are most commonly seen when a blood smear dries slowly but have been associated with lymphoma, rattle-snake envenomation and renal disease.

Other RBC morphological changes include moving of haemoglobin to one end of the cell and loss of central pallor (eccentrocyte) which is associated with oxidative damage (see also Heinz bodies); folding of the RBC membrane and target cell formation (leptocytes and codocytes) which are of little significance.

Stomatocytes are RBCs that have a slit like pale area in the centre rather than an area of central pallor. Small numbers may be seen in normal animals but large numbers are usually due to one of several hereditary disorders affecting some pure breed dogs.

Inclusions

Inclusions that may be seen in RBCs may or may not be significant. Howell-Jolley bodies are small dark spots that are commonly seen in cats, and in dogs with regenerative anaemia and reduced splenic function. They are nuclear remnants and are generally not of great significance.

Heinz bodies form when there has been oxidative injury to haemoglobin and they are commonly seen in small numbers in cat blood. They appear as small, pale staining, peripherally protruding from the surface of RBCs. With New Methylene Blue stain they stain positively and are much easier to see. In dogs, Heinz bodies commonly form after ingestion of large quantities of onions or garlic or large doses of Vitamin K but there are many other causes also. In cats Heinz bodies form readily after ingestion of some toxic compounds but they may also form in cats with diabetes mellitus, hyperthyroidism, lymphoma and other diseases. Haemolytic anaemia may result if many RBCs are affected.

RBC parasites are rare in dogs and usually only occur in animals that have been splenectomised. Cats, however, are found to have RBC parasites (Mycoplasma haemofelis) fairly often, sometimes incidentally, others associated with anaemia.

Rouleaux and Agglutination

Linear stacks of RBCs (rouleaux formation) are commonly seen in cats but rarely in dogs unless plasma protein concentration is elevated. Rouleaux must be differentiated from agglutination that causes cells to cluster in irregular clumps and is due to antibody interactions on the surface of the RBCs. Occasionally it may be difficult to differentiate the two and the addition of a drop of isotonic saline will disperse rouleaux but have no effect on agglutination.

White Blood Cell Changes

An estimate of WBC count can be made from the blood smear when it is not possible to get an automated count. Ten fields in the 'sweet zone' are examined and the number of WBCs seen counted and the average number of cells per field is calculated. This number is multiplied by 2.5 x 109/l.

Differential

A WBC differential count is carried out by categorising 100 cells in the smear as neutrophils, band cells and lymphocytes etc. The percentage of cells is multiplied by total WBC count to provide absolute cell numbers.

In cats and dogs neutrophils are the predominant cell type with smaller numbers of lymphocytes and monocytes. At low power, it can be seen if cell ratios are about right and if immature neutrophils (bands, or less mature cells), reactive monocytes or lymphocytes, and immature blast cells are present.

The presence of increased numbers of immature neutrophils (> 0.3 x 109/l) is called a left shift. Generally most of the cells will be band cells with fewer metamyelocytes and rare myelocytes etc. The extent of the left shift generally reflects severity of disease and the bone marrow's ability to respond. The response is favourable when there are more mature cells than immature. When there are more immature than mature cells and total neutrophil count is normal or close to normal then prognosis is generally poorer. Larger numbers of immature compared to mature cells and an increase in total neutrophil count may reflect recovery or deterioration of the patient and requires monitoring.

Cytoplasmic changes may be seen in mature and immature neutrophils when they are released from the bone marrow during periods of high demand (toxic change). These vary from Doehle bodies (pale grey bodies) denoting mild toxic change to basophilic stippling, basophilic, vacuolated cytoplasm and nuclear degeneration seen in more severe conditions. The presence of occasional Doehle bodies is not uncommon in normal cats.

Reactive changes may be seen in lymphocytes resulting from antigenic stimulation (infections, immune-mediated disease or neoplasia). The cytoplasm of these lymphocytes becomes deep blue in colour.

An extremely high leukocyte count (leukemoid reaction) with presence of immature cells may be seen with some inflammatory and paraneoplastic conditions and leukaemia.

Neoplastic Cells in Circulation

Grossly abnormal cells may be identified in circulation. Immature cells from the various cell lines and cells with nuclear-to-cytoplasmic asynchrony may be seen. Blasts are usually easy to identify and raise the suspicion of leukaemia but some conditions require skill to recognise the underlying problem and the samples that should be send to a commercial laboratory for verification.

Platelets

Examination of the feather-edge at low power should be carried out to determine whether platelet clumps are present as this will reduce subsequent counts. An estimation of platelet number can be made by counting the number of cells in 10 fields using the 100x objective, averaging the number and multiplying this by 15 x 109 (dog) or 20 x 109 (cat). The presence of large platelets suggests young platelets due to increased destruction or consumption. However, in the cat, platelets are often larger than RBCs. Thrombocytopenia may result from a BM defect or increased peripheral loss.

Summary

Examination of a blood smear can provide information that may be life-saving in a critical patient or merely useful in a stable patient. However, either way, blood cell recognition is useful skill to develop.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Sandra Forsyth, BVSc, DACVA
Institute of Veterinary, Animal and Biomedical Sciences
Massey University
Palmerston North, New Zealand


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