Blood smear examination is a vital part of practice medicine that is often overlooked due to over-reliance on automated analysers. The accuracy of an analyser identifying cell types, even newer generation laser-based systems, is often not good. They also do not provide clinically useful information that a good blood smear does. A complete haematology or full blood count for an animal requires a blood smear examination, which may be best performed by a specialised veterinary haematologist if the situation demands it. However, whether in-practice or externally, it can only be performed on a consistently good smear.

Sampling

The blood is typically placed into EDTA. The tube should be filled, without the needle attached, to the correct level. The blood should be gently mixed by inversion and rolling not shaking. At least one fresh blood smear is then made.

Blood Smear Preparation

1.  Slides: Label one clean slide with the relevant details. A 'manufactured' spreader slide is created by breaking the corner off a normal slide.

2.  The EDTA sample is gently resuspended / mixed and then a small drop placed at one end of the labelled slide using a pipette/capillary tube.

3.  The spreader slide is held at about 30 degrees between your thumb and second finger, your index finger gently on top, and then slid back to meet the drop.

4.  The blood is allowed to spread. When fully along the edge, the spreader slide is advanced smoothly and briskly. An even 'square' feathered edge should naturally be produced about 2/3 to 3/4 of the way along the labelled slide (Figure 1).

5.  Allow the smear to air-dry fully before staining.

Figure 1. Blood smear

Producing a good quality smear requires practice and consistency, but is essential for reliable and accurate examination. Artefact from a poor smear affects numbers and cell morphology. Examples with possible causes are shown In Figures 2–6.  

Figure 2. Too little blood / spreader too steep / advancement too fast.

Figure 3. Too much blood / spreader too shallow / advancement too slow.

Figure 4. Too much blood / spreader too steep / advancement too fast.

Figure 5. Greasy slide / spreader dirty or damaged.

Figure 6. Uneven pressure / advancement not smooth.

If the animal has altered red cell numbers then the technique, especially the angle, will need modifying to produce a good quality film. The spreader slide should be wiped clean after each use and intermittently with water, and be replaced on a regular basis.

Staining

In-house staining is typically rapid based. Generally follow the manufacturer's guidelines. However, check the colour scheme as otherwise critical staining, such as polychromasia, is often missed. These stains do not show some useful cell features. As a guide, dunk in the orange dye until an even orange sheen, and then similarly in the blue dye. It is best to rinse with dedicated buffered or distilled water.

Slide Examination

Using a ×40 objective lens requires a coverslip to prevent cells appearing slightly blurred. Slide examination starts with a low power (×4–10) scan, followed by sequentially higher powered examination. Much can be performed at ×40, or even ×10 or 20 with experience, with ×100 only needed for specific detailed examination. Examination of cell morphology must take place in the mono-layer, just behind the feathered edge (red cells are just touching but not overlapping - see hatched area in Figure 1). The following is one systematic way of examining a blood smear.

1.  Low power, check the feathered edge for platelet clumps and rarely abnormal cells or parasites. Check the sides for any atypical or abnormal cells.

2.  At higher power (×40 and/or ×100), look at the red cells, checking numbers, cell shape and colour. Look for inclusions, check for clumping.

3.  Repeat step 2 for white cells with a differential count too.

4.  At high power (×100) estimate platelet numbers, check their size and shape. Check for inclusions.

5.  Check the background colour and appearance.

Any changes noted, such as variation in size, should be subjectively quantified to allow accurate serial monitoring and detailed clinical interpretation. Use a standardised consistent scheme such as 'occasional, mild, moderate, marked'.

Platelets

In a good smear, at ×100, every platelet seen roughly equates to a count of 15 × 10⁹/l; count for five ×100 fields, then average. Healthy animals typically have 10–30 platelets per field. Any automated thrombocytopenia must be checked in this way. Artefactually low counts are the commonest cause of thrombocytopenia in cats. Thrombocytosis is non-specific but can be seen with neoplasia. Thrombocytopenia can be seen paraneoplastically or with myelophthisis. With chemotherapy, counts below 50 are generally considered contraindicative.

Red Cells

The first recognisable in the marrow is an erythroblast. This matures into a prorubricyte, to a rubricyte, then metarubricyte. Here, cell division stops, the nucleus is lost and visible haemoglobinisation occurs. This creates a polychromatic anucleate reticulocyte that in 24–48 hours matures into an erythrocyte.

The maturation process can be seen in the circulation, but should occur in an expected fashion, so reticulocytes dominate, with reducing numbers of earlier stages. If not seen, the anaemia is inappropriately regenerative and often means neoplastic bone marrow or possibly splenic disease.

With neoplasia there may be alterations in size (anisocytosis) or shape (poikilocytosis):

 Anisocytosis:

 Microcytic - seen as a low mean corpuscular volume (MCV) or in the smear earlier as small erythrocytes. Support hepatic or gastrointestinal disease that may be neoplastic.

 Macrocytic - seen as an elevated MCV or in the smear earlier as larger normochromic. Support marrow disease, more so in cats and associated with feline leukaemia virus (FeLV) (latent) infection potentially.

 Poikilocytosis. This is abnormal erythrocyte shape and has many forms with neoplasia which are nonspecific. They include:

 Acanthocyte ('spur cell') - rounded, irregular, uneven projections. Can be seen with vascular organ neoplasia such as splenic or hepatic haemangiosarcoma so with maturation (and storage) there should be many more mature cells than immature in the marrow.

 Schistocyte - red cell fragments, smaller than erythrocytes and very irregular in shape. Seen with microvascular neoplastic diseases

 Inclusions. Seen inside erythrocytes and are also non-specific but can be significant:

 Howell-Jolly bodies - small, eccentric, dark purple singular circular nuclear remnant. If regeneration is not seen, they can be a feature of splenic or bone marrow neoplasia, especially if larger and multiple

 Heinz bodies - are small circular blebs which protrude from, or indent, the cell surface. Reported with certain neoplasias such as lymphoma.

Most commonly cancer can be associated with a mild to moderate non-regenerative anaemia with some of the above features. If there is bleeding from the tumour or paraneoplastic haemolysis, polychromatic regeneration may also be seen.

Rouleaux are erythrocytes that are clumped together in a chain-like a stack of coins. Increased rouleaux formation can be seen when globulins increase such as monoclonal gammopathies, often with a pale blue background in the smear.

White Cells / Leucocytes

White cells derive from two cells of origin - lymphoid and myeloid, myeloid stem cells also generating erythroid cells. Lymphoid maturation is very different. The other cell lines differentiate into their precursors in a varied fashion. Granulocytes mature similarly, the earliest being a myeloblast, dividing/maturing into a promyelocyte. Here, the three lines split, with their distinctive secondary granules seen at the next myelocyte stage. Metamyelocytes are next, then band forms and finally the mature leucocyte. Cell division occurs until the myelocyte stage, so with maturation (and storage) there should be many more mature cells than immature in the marrow.

Inflammation / Infection or Leukaemia?

 Cytosis. With inflammation or infection, one feature can be an increase in the number of certain white cells. This increase is very variable but can be marked and appear leukaemoid. Changes in other cell lines can help distinguish but the cell morphology is often crucial and is not provided by your analyser. Toxic changes in neutrophils most commonly indicate inflammation/infection not leukaemia. Mixed reactive changes in lymphocytes support this. An increase, often marked, in one cell line with no other cell lines affected in numbers or morphology may be more likely leukaemic

 Cytopenia. Reduction in numbers can also be inflammatory or neoplastic. In the former, it is often transient and often associated with toxicity or reactive morphology. In the latter it persists with apparently unremarkable morphology or dysplastic cells

 Leukaemia. Diagnosing acute leukaemia can be very easy when cells are obviously immature, especially if in very high numbers. The lineage though can be very difficult or impossible from the morphology alone, requiring further testing like immunophenotyping or special stains. Early or developing leukaemia can be very difficult to diagnose without bone marrow samples. (Chronic) Leukaemia can involve mature cells that are identical to those seen in health. The key feature is a monomorphic population of ('atypical') cells present inappropriately or in excessive numbers. Diagnosis relies on a fresh blood smear, cell counts and potentially bone marrow examination. Nuclear features are critical, but cytoplasmic features may help identify the lineage. With chronic leukaemias, the diagnosis can rely on serial monitoring for increasing numbers and/ or exclusion of inflammatory/infectious diseases, such as gastrointestinal ulceration, that produce a similar (leukaemoid) response.