Introduction to haematology

Elizabeth Villiers

doi:10.22233/9781910443255.3

British Small Animal Veterinary Association , 27 (2016); https://doi.org/10.22233/9781910443255.3

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Introduction to haematology

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Author: Elizabeth Villiers
From: BSAVA Manual of Canine and Feline Clinical Pathology
Item: Chapter 3, pp 27 - 37
DOI: 10.22233/9781910443255.3
Copyright: © 2016 British Small Animal Veterinary Association
Publication Date: March 2016

Abstract

The complete blood count (CBC) is an integral part of the diagnostic investigation of any systemic disease process. It consists of two components: quantitive examination and qualitive examination.This chapter deals with blood sampling, blood quantification techniques, automated cell counts, red blood cell parameters, white cell counts, platelet counts and blood films.

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3.1

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3.1 A blood clot in an EDTA sample is detected by wiping a wooden stick around the inner surface of the tube. Clotted samples should be discarded.

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3.2

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3.2 (a) A sample of plasma that is haemolysed. (b) A sample of plasma that is both haemolysed and lipaemic.

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3.3

Diagrammatic representation of a microhaematocrit tube following centrifugation. The PCV is calculated by dividing the length of the packed red cells (B) by the total length of the packed red cells, buffy coat and plasma (B + C + D), using either a sliding measuring device (haematocrit reader) or a microhaematocrit capillary tube reader. © 2016 British Small Animal Veterinary Association

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3.3 Diagrammatic representation of a microhaematocrit tube following centrifugation. The PCV is calculated by dividing the length of the packed red cells (B) by the total length of the packed red cells, buffy coat and plasma (B + C + D), using either a sliding measuring device (haematocrit reader) or a microhaematocrit capillary tube reader.

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3.4

Measuring plasma protein using a refractometer. (a) The microhaematocrit tube is scored just above the buffy coat, using a diamond writer or razor blade. (b) The tube is broken at the scored line. (c) The plasma is expelled from the tube on to the refractometer prism by swiftly flicking the tube downwards towards the prism, taking care not to touch the prism with the tube. The prism cover is then replaced and the plasma protein read from the internal scale. © 2016 British Small Animal Veterinary Association

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3.4 Measuring plasma protein using a refractometer. (a) The microhaematocrit tube is scored just above the buffy coat, using a diamond writer or razor blade. (b) The tube is broken at the scored line. (c) The plasma is expelled from the tube on to the refractometer prism by swiftly flicking the tube downwards towards the prism, taking care not to touch the prism with the tube. The prism cover is then replaced and the plasma protein read from the internal scale.

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3.5

Diagrammatic representation of an impedance counting chamber. Cells within an electrically conducting fluid pass through an aperture; in so doing they impede the flow of electricity through the aperture, creating a pulse. © 2016 British Small Animal Veterinary Association

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3.5 Diagrammatic representation of an impedance counting chamber. Cells within an electrically conducting fluid pass through an aperture; in so doing they impede the flow of electricity through the aperture, creating a pulse.

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3.7

Histogram plots produced by the Cell-Dyn 3500 analyser showing separation of platelets and red cells on the basis of size. Plot (a), from a dog, shows good separation with a well defined peak of platelets to the left and red cells to the right. However on plot (b), from a cat, the two peaks are not well defined. The analyser gave a platelet count of 1481 × 109/l. Inspection of the blood film suggested the count was much lower than this, with approximately 34 platelets per high power field, equivalent to a count of 510 × 109/l. The MCV was low, and some of the microcytic red cells were being counted as platelets. © 2016 British Small Animal Veterinary Association

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3.7 Histogram plots produced by the Cell-Dyn 3500 analyser showing separation of platelets and red cells on the basis of size. Plot (a), from a dog, shows good separation with a well defined peak of platelets to the left and red cells to the right. However on plot (b), from a cat, the two peaks are not well defined. The analyser gave a platelet count of 1481 × 109/l. Inspection of the blood film suggested the count was much lower than this, with approximately 34 platelets per high power field, equivalent to a count of 510 × 109/l. The MCV was low, and some of the microcytic red cells were being counted as platelets.

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3.8

(a) Scatter plot from a normal dog obtained using the ProCyte Dx® analyser. Neutrophils are shown in lilac, monocytes in red, lymphocytes in blue, eosinophils in green and basophils in turquoise. (b) Scatter plot from a dog with leukaemia obtained using the ProCyte Dx® analyser. There is a marked increase in the number of white cells and an obviously abnormal dot plot presentation with overlapping ‘clouds’ (clusters), making misclassification highly likely. In such cases a blood film examination should be performed and reviewed by a cytologist. (Courtesy of G Bilborough, Idexx) © 2016 British Small Animal Veterinary Association

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3.8 (a) Scatter plot from a normal dog obtained using the ProCyte Dx® analyser. Neutrophils are shown in lilac, monocytes in red, lymphocytes in blue, eosinophils in green and basophils in turquoise. (b) Scatter plot from a dog with leukaemia obtained using the ProCyte Dx® analyser. There is a marked increase in the number of white cells and an obviously abnormal dot plot presentation with overlapping ‘clouds’ (clusters), making misclassification highly likely. In such cases a blood film examination should be performed and reviewed by a cytologist. (Courtesy of G Bilborough, Idexx)

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3.9

Diagrammatic representation of the buffy coat, expanded by a cylindrical float, and a histogram showing the amount of fluorescence emitted by DNA (in nucleated cells) and RNA/lipoprotein (in reticulocytes, platelets (PLT) and eosinophils (EOS)) in the various layers of the buffy coat. GRANS = granulocytes; L/M = lymphocytes and monocytes. (Courtesy of Idexx Laboratories, Wetherby) © 2016 British Small Animal Veterinary Association

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3.9 Diagrammatic representation of the buffy coat, expanded by a cylindrical float, and a histogram showing the amount of fluorescence emitted by DNA (in nucleated cells) and RNA/lipoprotein (in reticulocytes, platelets (PLT) and eosinophils (EOS)) in the various layers of the buffy coat. GRANS = granulocytes; L/M = lymphocytes and monocytes. (Courtesy of Idexx Laboratories, Wetherby)

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3.10

Using the Cell-Dyn analyser the red cell distribution width (RDW) is calculated from the frequency distribution plot of red cell size (size on x-axis, frequency on y-axis). The RDW is the coefficient of variation of red cell size and is expressed as a percentage. (a) Healthy dog (RDW = 15%). (b) A dog with a markedly regenerative anaemia (RDW = 25%): the red cell curve is much wider and extends further to the right, reflecting the numerous larger red cells present. © 2016 British Small Animal Veterinary Association

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3.10 Using the Cell-Dyn analyser the red cell distribution width (RDW) is calculated from the frequency distribution plot of red cell size (size on x-axis, frequency on y-axis). The RDW is the coefficient of variation of red cell size and is expressed as a percentage. (a) Healthy dog (RDW = 15%). (b) A dog with a markedly regenerative anaemia (RDW = 25%): the red cell curve is much wider and extends further to the right, reflecting the numerous larger red cells present.

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3.11

A platelet clump seen at the tail of a blood smear from a dog. Platelets are round with slightly grainy cytoplasm but no nucleus. (May–Grünwald–Giemsa stain; original magnification X1000) © 2016 British Small Animal Veterinary Association

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3.11 A platelet clump seen at the tail of a blood smear from a dog. Platelets are round with slightly grainy cytoplasm but no nucleus. (May–Grünwald–Giemsa stain; original magnification X1000)

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3.12

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3.12 Preparation of blood films.

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3.14

Examples of blood smears. (a) This smear has a ragged feathered edge due to uneven contact of the spreader with the slide, possibly because the spreader was dirty or roughened. (b) Holes in a smear, which may be due to grease on the slide or lipaemia. (c) This smear is too long because too much blood has been applied to the slide. Blood in front of the spreader has resulted in a dense line at the tail. (d) This smear is too short, possibly because insufficient blood was applied to the slide or the spreader was moved too rapidly. (e) A good smear with an even ‘square’ end. © 2016 British Small Animal Veterinary Association

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3.14 Examples of blood smears. (a) This smear has a ragged feathered edge due to uneven contact of the spreader with the slide, possibly because the spreader was dirty or roughened. (b) Holes in a smear, which may be due to grease on the slide or lipaemia. (c) This smear is too long because too much blood has been applied to the slide. Blood in front of the spreader has resulted in a dense line at the tail. (d) This smear is too short, possibly because insufficient blood was applied to the slide or the spreader was moved too rapidly. (e) A good smear with an even ‘square’ end.

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3.15

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3.15 Diagrammatic representation of a blood film showing the feathered edge and the monolayer (where cells should be examined). (Courtesy of P Monti)

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3.16

Atlas of red cells and leucocytes. (a, i) Normal canine red cells with central pallor. (a, ii) Normal feline red cells are smaller than canine cells and do not have obvious central pallor. (b, i) Normal neutrophil with segmented nucleus and light, clear cytoplasm. (b, ii) In females a proportion of neutrophils have a Barr body (arrowed), a small protuberance at one end of the nucleus which is the site of the X chromosome. (b, iii) Band neutrophil containing a nucleus with parallel sides (may have a shallow indentation). (c, i) Normal monocytes are larger than neutrophils, have variably shaped nuclei and basophilic cytoplasm containing several vacuoles. (c, ii) The monocyte nucleus (upper left) may be band-shaped but is wider than the neutrophil nucleus (lower right) and has more open, stippled chromatin. (d, i) Small lymphocyte with dense round nucleus and cytoplasm only visible at the top. (d, ii) Larger lymphocyte with more abundant cytoplasm. (d, iii) Large granular lymphocyte containing several large pink granules. (e, i) Feline eosinophil with rod-shaped cytoplasmic granules. The neutrophil above it is smaller and has clear cytoplasm. (e, ii) Canine eosinophil with larger round granules which are unevenly distributed in the cytoplasm. (e, iii) Eosinophil with vacuolated cytoplasm, from a Greyhound. (f, i) Canine basophil with an elongated ribbon-like nucleus and indistinct lilac granules. (f, ii) Feline basophil with lilac and purple granules. (a–e = May–Grünwald–Giemsa stain, f = Rapi-Diff® stain; original magnification X1000) (f, Courtesy of L Blackwood) © 2016 British Small Animal Veterinary Association

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3.16 Atlas of red cells and leucocytes. (a, i) Normal canine red cells with central pallor. (a, ii) Normal feline red cells are smaller than canine cells and do not have obvious central pallor. (b, i) Normal neutrophil with segmented nucleus and light, clear cytoplasm. (b, ii) In females a proportion of neutrophils have a Barr body (arrowed), a small protuberance at one end of the nucleus which is the site of the X chromosome. (b, iii) Band neutrophil containing a nucleus with parallel sides (may have a shallow indentation). (c, i) Normal monocytes are larger than neutrophils, have variably shaped nuclei and basophilic cytoplasm containing several vacuoles. (c, ii) The monocyte nucleus (upper left) may be band-shaped but is wider than the neutrophil nucleus (lower right) and has more open, stippled chromatin. (d, i) Small lymphocyte with dense round nucleus and cytoplasm only visible at the top. (d, ii) Larger lymphocyte with more abundant cytoplasm. (d, iii) Large granular lymphocyte containing several large pink granules. (e, i) Feline eosinophil with rod-shaped cytoplasmic granules. The neutrophil above it is smaller and has clear cytoplasm. (e, ii) Canine eosinophil with larger round granules which are unevenly distributed in the cytoplasm. (e, iii) Eosinophil with vacuolated cytoplasm, from a Greyhound. (f, i) Canine basophil with an elongated ribbon-like nucleus and indistinct lilac granules. (f, ii) Feline basophil with lilac and purple granules. (a–e = May–Grünwald–Giemsa stain, f = Rapi-Diff® stain; original magnification X1000) (f, Courtesy of L Blackwood)

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3.17

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3.17 Battlement meander track for performing a differential white cell count.

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3.19

Performing an estimated platelet count. The blood film is examined using the X100 oil immersion lens in the examination area. The number of platelets per field is counted and a mean value for five fields is calculated. This value is multiplied by 15 to produce the count × 109/l. © 2016 British Small Animal Veterinary Association

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3.19 Performing an estimated platelet count. The blood film is examined using the X100 oil immersion lens in the examination area. The number of platelets per field is counted and a mean value for five fields is calculated. This value is multiplied by 15 to produce the count × 109/l.