Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

LABORATORY TECHNIQUES - BLOOD FILM EXAMINATION The first key point is what sort of sample to use. The best smears are prepared from freshly drawn blood before it has a chance to clot. This can be fiddly to achieve and the results are just as good from samples taken into an EDTA tube which is then carefully mixed and a smear prepared, preferably within a few minutes. The next key step is in preparing a good quality blood smear. Select two clean, grease free microscope slides with ground edges to help spreading. If the corners are removed from the spreader slide, this makes it much easier to use. Write the case reference number indelibly on the object slide (pencil or diamond stylus). Thoroughly mix the blood sample and place a drop of blood centrally at one end of the slide using a disposable stick or capillary tube. Hold the spreader slide at about 40 to the sample slide, with the sample slide on a flat clean surface. Draw the spreader slide backwards to contact the blood drop. The blood then spreads along the trailing edge of the slide. Push the spreader slide along the sample slide in one continuous motion. Examine the resulting smear to ensure that there is a feathered tail and two edges, suitable for examination. Allow the smear to air dry.

Once we have a good quality air-dried smear we must select a staining method suitable for the practice setting. In most cases this means a rapid Romanowsky stain such as Diff-Quik. In a commercial laboratory we use a different type of Romanowsky stain called Leishmans stain. Slides are fixed in a separate methanol solution in the case of Diff-Quik or in the methanol contained in the Leishmans stain solution. All Romanowsky stains include both a basic dye and an acid dye. In Diff-Quik these dye solutions are separate but in Leishman's stain they are combined in the same solution. This gives rise to a coloured salt that is also involved in staining cell components.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Romanowsky stains give fair nuclear staining and excellent cytoplasmic staining, and are dependant upon pH, temperature, buffer, dye concentration and fixation. Some experimentation is usually required to optimise the staining procedure in a laboratory. Rapid Romanowsky staining using rapi-diff Solution A Solution B Solution C Solution D 1. 2. 3. 4. 5. Fixing solution Acid dye Basic dye Buffer Methanol Eosin Methylene blue pH 6.8 phosphate buffer

Prepare a blood smear and air dry. Fix by immersing in Solution A for 30 seconds. Transfer, without rinsing or drying to Solution B and stain for 15-30 seconds. Gently agitate during this period. Without rinsing transfer the slide to Solution C and stain for 15-30 seconds. Gently agitate during this period. Rinse briefly in buffered water pH 6.8 and allow to dry.

Leishmans stain using staining racks Solution A Solution B 1. 2. 3. 4. 5. 6. Leishmans stain pH 6.8 phosphate buffered water

Prepare a blood smear and air dry. Place on staining rack. Flood slide with 1-2 ml of Leishmans Stain, leave for 1-2 minutes. Add 2-4ml of pH 6.8 buffer, this may need mixing using a Pasteur pipette, leave for 10-15 minutes. Rinse off with tap water. Rinse briefly in buffered water pH 6.8, wipe the back of the slide to remove any stain deposit and allow to dry.

This method can be adapted for use in staining jars. The blood film examination should always follow the same procedure to ensure that all the cellular blood components are examined and assessed. Orientate the slide: Always orientate the blood smear the same way, usually with the feathered edge of the smear to the left. Scan the slide: Preferably at x400 magnification to gain an impression of the overall cell populations and to check for platelet clumping. Examine RBC morphology and platelets: Select the area of the slide behind the feathered edge where RBCs are arranged in a monolayer.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Examine these cells under oil immersion (x1000) to assess RBC morphology and platelet numbers. Perform a 100 cell differential WBC count: There are various methods published for performing a differential WBC count on a blood smear. The general principal is to track down or across the slide to take account of the uneven distribution of WBCs in a smear. The easiest technique having examined the RBCs is to revert to x400 (high power dry objective) and to track down the slide longitudinally. When the smear becomes too dense for accurate morphological assessment then track back up the smear towards the feathered edge. Repeat this until 100 WBCs have been counted. An alternative is to track backwards and forwards across the smear from side to side. The battlement technique is a refinement of these techniques but requires practice. In addition to counting the WBCs present, nucleated red cells should be counted. The number seen during the differential WBC count is expressed as the number of nucleated RBCs per 100 WBCs. If this number exceeds 5 / 100 WBCs then the total WBC count should be corrected: Corrected WBC count = 100 x measured WBC count 100 + nucleated RBC count

Assess WBC morphology: Any abnormalities in the WBCs will have been mentally logged during the differential WBC count. The smear can be examined using oil immersion (x1000) to further assess WBC morphology. Calculate the absolute differential WBC counts: These are calculated for each cell type by multiplying the total WBC count by the percentage of that cell type. Assess erythroid regeneration on all anaemic animals.

Red Blood Cell Morphology Changes in Size (i) Anisocytosis: This describes variation in RBC size and is often classified as mild, moderate or marked. Mild anisocytosis is a normal feature of cat RBCs. More marked anisocytosis most commonly accompanies erythroid regeneration. (ii) Microcytosis: This describes populations of RBCs that are smaller than normal for that species. Such RBCs often also have a reduced staining intensity (hypochromasia).
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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

(iii) Spherocytosis: Spherocytes are RBCs of about 2/3 normal diameter but with increased haemoglobin saturation. Typically they lack central pallor. They arise due to stripping of the outer cell membrane by the action of cells in the mononuclear phagocytic system and are closely associated with immune mediated haemolytic anaemia in the dog. Spherocytes are an important diagnostic feature in dogs but they are very hard to recognise in cats. Changes in Shape Poikilocytosis is a catchall term used to describe any abnormality in RBC shape. It is only used when a more exact descriptive term does not apply. Many abnormalities in shape are described and more than a few are carried over from medical haematology. Relatively few are seen routinely in cat and dog blood films. (i) Spiculated erythrocytes: A common abnormality is the presence of RBCs with multiple small spiky projections from the cell membrane. Most commonly this is an artifact of drying and ageing and is described as crenation. Less commonly such cells are described as either echinocytes or Burr cells and are characterised by having 10 to 30 evenly distributed spicules. These cells are associated with renal disease, chemotherapy and are also seen in cases of lymphoma. (ii) Acanthocytes: Typically with 2 to 20 irregularly distributed spicules, the projections seen in acanthocytes are thicker and more blunt. They arise due to alterations in the phospholipid and cholesterol content of the RBC membranes and are associated with liver disease and splenic haemangiosarcoma in dogs. (iii) Schistocytes: Schistocytes (schizocytes) are RBC fragments with 2 to 4 angular projections and arise due to mechanical damage. This may be caused by intravascular fibrin strands in disseminated intravascular coagulation or by abnormal microvasculature e.g. in splenic haemangiosarcoma. They are also seen in dogs and cats with immune mediated haemolytic anaemia, thrombosis, glomerulonephritis, congestive heart failure, valvular heart disease and myelofibrosis. Changes in Staining Intensity (i) Hypochromasia: Hypochromic RBCs have a reduced intensity of staining, particularly in the central area, due to reduced haemoglobin content of the cells. (ii) Polychromasia: Polychromatic RBCs are relatively immature RBCs that are released into the peripheral blood during erythroid regeneration. They can be up to 2 x the diameter of mature RBCs and have a blue to blue-red cytoplasm due to the presence of residual RNA.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

(iii) Target cells (codocytes): This is a commonly recognised abnormality in which RBCs have a central round out-folding of cell membrane. The RBC has a target-like appearance. Moderate to large numbers are often associated with liver disease and may also be seen in iron deficient anaemia. Arrangement of RBCs Rouleaux formation describes an organised stacking of RBCs and is only seen microscopically. This is a common feature in feline blood smears. It is not common in dog blood smears but may be seen wherever there is a moderate to marked hyperglobulinaemia. In contrast, autoagglutination is often appreciated macroscopically and is an immune mediated event. Rouleaux formation must be distinguished from autoagglutination but this is relatively simple to do. 1. 2. 3. 4. 5. Add one drop of blood to two drops of physiological saline on a slide Mix gently by swirling the slide View against a white background for macroscopic agglutination Add a cover slip and view at x100 magnification for microscopic agglutination. Rouleaux will disperse in physiological saline whilst autoagglutination will persist.

Autoagglutination is considered pathognomonic for immune mediated haemolytic anaemia in the dog. RBC Inclusions (i) Howell-Jolly bodies: These are found in small numbers of RBCs in healthy dogs and cats (particularly cats). They are nuclear remnants and form regular, small (1mm diameter), round dark staining bodies in RBCs. They occur individually in cells and are present in large numbers during erythroid regeneration. Numbers may also be increased in any animal with reduced splenic function. They are relatively easy to recognise. (ii) Heinz bodies: Heinz bodies are recognised as pale, unstained, punched out areas in RBCs in Romanowsky stained smears. They represent haemoglobin that has been denatured due to oxidative damage. The composition of feline haemoglobin with its increased number of reactive sulphhydryl groups compared to the dog makes feline RBCs much more susceptible to oxidative damage. Small numbers of Heinz bodies (<10%) are seen in blood smears from asymptomatic cats. Supravital stains such as new methylene blue will stain Heinz bodies blue making them much easier to recognise.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

(iii) Haemobartonella felis: This is the only significant indigenous haemoparasite of cats in the UK. Bodies are seen as individual small cocci, chains of cocci, rods and occasional annular or ring forms. Even with the benefit of special stains such as May-Grunwald Giemsa, these are notoriously difficult to identify. The main hazards are low parasitaemias and the presence of smear artefacts such as stain precipitate and refractile bodies. Stain precipitate is blue staining but is present both on RBCs and in the spaces between RBCs. Refractile bodies tend to be associated with the RBCs but unlike the parasite are refractile when the microscope is focussed up and down. They also vary more in size. Platelets Platelets look similar in cats and dogs. They are typically 2 to 4 mm in diameter, anucleate, and light blue staining cells. The appearance of giant or shift platelets in the peripheral blood is an indication of platelet regeneration. Shift platelets are > 5mm in diameter. For convenience they can be thought of as being equal to or greater than the size of a RBC. Small numbers of shift platelets are however a common feature in cat blood and are therefore a less reliable indicator of platelet regeneration than in the dog. Platelets that become activated during collection have a tendency to clump, especially in the feathered end or edges of a blood smear. Platelet numbers can be estimated from a blood smear. Estimated platelet count Select a stained blood smear for estimation of the platelet count. First survey the edges and tail of the smear using the x10 and x40 dry objectives for any evidence of platelet clumping. The presence of platelet clumps and small clots in samples will have an affect on all methods of platelet counting/estimation. Locate the RBC monolayer in the body of the smear. Using oil immersion (x1000), count the number of platelets present in each of 10 fields tracking laterally across the smear. Calculate the mean number of platelets per x1000 microscope field.

Published papers indicate that the each platelet seen in an oil immersion field approximates to a platelet count of 15 20 x 109/l. As a guide there should be approximately 10-25 platelets per oil immersion field in the body of the smear. Using this method platelet counts can be recorded as low, normal or elevated. It is very important that the original EDTA sample is also checked for any evidence of clotting.
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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

White Blood Cells Neutrophils Neutrophils are the commonest white blood cells in the peripheral blood of dogs and cats. They are typically 10 to 12 mm in diameter and vary little in size. They tend to get used as a yardstick therefore to measure other cells, particularly in cytology. The nucleus is typically segmented with between 3 and 5 lobes. The nuclear chromatin consists of dark condensed areas with lighter areas in between. The colour of the cytoplasm varies with the stain used but is usually a pale blue. Nuclear appearance is used to determine the presence of a left or right shift. Changes and inclusions in the cytoplasm are generally used to identify neutrophil toxicity. Neutrophils have a typical generation time of about 7 days. This comprises 3 to 5 days in the marrow followed by as little as 7 to 14 hours in the circulation before random distribution into the tissues and a further life span of 2 to 3 days. The significance of a short generation time is that a neutropenia can develop relatively rapidly in response to an excessive demand for neutrophils and/or bone marrow toxicity. Lymphocytes Lymphocytes are the second most abundant WBC in dogs and cats. Typically these cells are slightly smaller than neutrophils and round with a round to oval nucleus. The chromatin pattern is fairly homogenous being smooth and dark with areas that are more condensed. There is usually a thin crescent of light blue cytoplasm. Because neutrophils and lymphocytes are much more numerous than other cell types, whenever you detect a low white blood cell count (leucopenia) or an elevated white blood cell count (leucocytosis) you should look to these two cell types to explain the abnormal cell count. In many cases a visual smear assessment will suggest which cell series is affected. Monocytes Monocytes are usually present in low numbers only. They are 1.5 to 2x the size of neutrophils (typically 15 - 20 mm). The nucleus is described as amoeboid which means it can vary in shape from oval to oval with a small indentation to multiple lobules and indentations.

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

The nuclear chromatin is however almost uniformly finely granular or reticular with far fewer areas of condensation than are seen in neutrophils. The cytoplasm is often described as slate grey and may contain vacuoles. Eosinophils Eosinophils are also only present in low numbers normally. They may be absent from a 100 to 200-cell differential white cell count. As they follow an identical maturation series to neutrophils the mature cells are a similar size. The nuclei are not as markedly segmented. The cytoplasm is a pale blue usually but this is rarely noticed due to the prominent reddish-orange granules present in the cytoplasm. This is one respect in which the dog and cat differ. The granules in dog eosinophils are round and vary in size and number. Cat eosinophil granules in contrast are rod shaped and tend to fill the cell. Basophils The final cell type that is occasionally seen is the basophil. Basophils are absent from most differential counts. Again they follow the same maturation series as neutrophils and eosinophils and mature cells are therefore of a similar size. The cytoplasm is light purple. The nucleus is poorly segmented, more like the eosinophil than the neutrophil in this respect. Granules are generally small and are purple in the dog and lavender in the cat. Erythroid regeneration Healthy bone marrow can respond to anaemia by an increase of up to 10 x the normal rate of erythroid regeneration. There are two simple methods of determining the degree of erythroid regeneration in dogs and cats. A useful initial assessment of regeneration is to assess the proportion of polychromatic RBCs. These immature RBCs are distinguished by their slightly larger size and slightly basophilic cytoplasm (Romanowsky stains). These are young, immature but anucleate erythrocytes. Increased numbers of polychromatic RBCs are a reliable indicator of erythroid regeneration in both the dog and the cat, but absence or low numbers of polychromatic RBCs may underestimate the degree of erythroid regeneration. In dogs only the youngest reticulocytes stain polychromatic with Romanowsky stains. In cats, immature RBCs do not appear polychromatic when rapid Romanowsky Stains such as Diff-Quik stain are used. PCV <15% 15-20% >20% No. polychromatic cells per x1000 oil immersion field indicative of erythroid regeneration 1-3 3-4 >4

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

Reticulocytes are juvenile red cells; they contain remnants of ribosomes and ribosomal RNA that were present in larger amounts in the nucleated precursor cells from which they were derived. The ribosomes and RNA react with certain dyes such as new methylene blue or brilliant cresyl blue to form a coloured precipitate within the red cells. All reticulocytes should be counted in dogs but in cats the story is slightly different. Two distinct populations of reticulocytes are recognised. Aggregate reticulocytes are the more immature and typically take 12 hours to mature to punctate reticulocytes. Punctate reticulocytes are much longer lived and may take 10 days to develop into mature erythrocytes. Because of this relatively long life span, punctate reticulocytes are not a reliable indicator of current RBC regeneration. In cats therefore we count aggregate reticulocytes only. Even then this may underestimate the degree of the regenerative response in mild anaemia as in this circumstance, aggregate reticulocytes are often retained in the bone marrow until they mature to punctate reticulocytes. Reticulocyte count 1. Write the case reference number indelibly on the tube to be used. Add 3-4 drops of well-mixed blood to dried reticulocyte stain in a tube or mix 2-4 drops of EDTA blood with 2-4 drops of liquid reticulocyte stain. Mix well but gently Incubate at 37oC 1oC for 15-30 minutes in a water bath Mix the sample and then make a wedge smear (same technique as for blood smear). Allow to air dry. Examine under a x100 oil immersion lens. Reticulocytes are red cells that contain blue staining material; they may be either punctate (tiny dots evenly throughout the cell) or aggregate (large clumps of reticular material). Locate the monolayer area of the smear behind the tail and count the approximate number of RBCs in one oil immersion field (x1000). Estimate how many fields must be examined such that approximately 1000 RBCs are examined. Tracking sideways across the smear, count the number of reticulocytes in the determined number of fields. Calculate the % reticulocyte count Number of reticulocytes counted x 100 % Number of RBCs per field x number of fields

2. 3. 4. 5.

6.

7.

% reticulocyte count =

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Tim Jagger BVM&S MSc DipRCPath MRCVS BVNA August 2002

The reticulocyte count is expressed as a percentage of erythrocytes, usually counting a minimum of 1000 red cells. This is a crude measure of erythroid regeneration, as it does not take into account the degree of anaemia and the effect that this has on the speed of maturation of immature RBCs. The most commonly quoted and useful parameter is the absolute reticulocyte count. Absolute retic count (x 109/l) = RBC count (x1012/l) x reticulocyte count (%) 100

Regeneration None Slight Moderate Marked

Absolute Retic. Count (Dog) <60 150 300 >500

Absolute Aggregate Retic. Count (Cat) <15 50 100 >200

Tim Jagger BVM&S MSc DipRCPath MRCVS Leeds Veterinary Laboratories 2002

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