Introduction to haematological diagnostic techniques

image of Introduction to haematological diagnostic techniques
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The major innovations in small animal haematology in the last few years have been the introduction and wide application of laser-based counting systems, flow cytometry, immunolabelling and digital imaging. This chapter looks at blood sampling; basic quantification techniques; automated quantification techniques; blood cell morphology and interpretation; interpreting and haematological profile.

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1.1 Jugular venepuncture in a dog, showing the restraint, positioning and site of access. (Courtesy of Jonathon Bray)
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1.2 Reader with a sliding parabolic scale to facilitate measurement of the packed cell volume or microhaematocrit from a capillary tube.
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1.3 Buffy coat profile from a 1-year-old neutered male Labrador Retriever with pyrexia of unknown origin. The automated analysis reported significant eosinophilia (with no flags). Blood smear examination by a haematologist revealed significant lymphocytosis; the majority of these cells were granular lymphocytes that the analyser had misclassified as eosinophils (‘Grans’) owing to their granularity. The dog had an idiosyncratic and eventually self-resolving reaction to a booster vaccination.
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1.4 Impedance histograms for red cells (RBC) and platelets (PLT) in a healthy dog; the number of cells ( axis) is plotted against cell volume (× axis). There is good discrimination, the smallest red cells being just visible as a line over the 30 μm mark.
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1.5 Dog with iron deficiency anaemia caused by gastrointestinal blood loss. The combination of microcytosis and thrombocytosis with macroplatelets prevents accurate automated differentiation because the platelet histogram (PLT) merges into the red cells on the right side, the set discrimination threshold (dotted line) not being applicable in this instance.
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1.6 Graphical display of white blood cells (WBCs) from a laser flow cytometer showing peroxidase staining (Perox; × axis) versus cell size ( axis). Each dot represents a cell, and the staggered lines show set thresholds for different cell types. Graph showing particles after phthalic acid surfactant lysis for basophil (Baso) counting, showing nuclear configuration ( axis) and cell/nuclear size ( axis). While basophils are poorly identified in cats and dogs, the complexity of bare nuclei is also shown: mononuclear cells to the left, segmented cells to the right.
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1.7 Impedance red blood cell (RBC) histogram of a dog with iron deficient regenerative anaemia (black line); a histogram from a healthy dog is also shown (dotted red line). The peaks are similar, and therefore the mean cell volume (MCV) was within reference limits, but the histogram clearly shows increased numbers of small cells (microcytosis) as well as macrocytosis caused by regeneration with polychromasia (reticulocytosis).
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1.8 Red blood cell (RBC V/HC) scattergram from a resolving haemorrhagic anaemia, with haemoglobin concentration ( axis) plotted against red cell volume ( axis); the grid lines show set thresholds. In health, the majority of red cells fall in the central square, as seen here. Macrocytes would show in the top three rectangles and microcytes in the bottom three rectangles. Hypochromic red cells appear in the left three rectangles. Here, a slight shift up and to the left is evident, indicating immature red cells and a residual regenerative response. Prolonged effects of EDTA can mimic this pattern.
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1.9 Several blood smears: the well made smear on the far left will allow accurate and reproducible examination and counting. Those to the far right can be used, but the cells may be altered and unevenly distributed, precluding accurate analysis.
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1.10 A good fresh blood smear: the hatched rectangular area shows the monolayer for examination of cell morphology and differential counts.
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1.11 High power view of the monolayer area for accurate and reproducible morphological examination (original magnification ×500, modified Wright’s stain).
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1.12 Healthy normochromic normocytic erythrocytes, canine above, feline below (original magnification ×500, modified Wright’s stain).
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1.13 Anisocytosis, with normal, larger (macrocytic) and smaller (microcytic) red blood cells seen during regeneration with chronic haemorrhage (original magnification ×500, modified Wright’s stain).
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1.14 High power view showing a normochromic macrocyte (top left) in relation to a mature segmented neutrophil (bottom right), with the top of a polychromatic reticulocyte (bottom centre) (original magnification ×1000, modified Wright’s stain).
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1.15 Microcytic hypochromic anaemia with polychromasia and anisocytosis in a dog with chronic gastrointestinal haemorrhage (original magnification ×500, modified Wright’s stain).
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1.16 Poikilocytosis with several different non-circular and non-discoid shapes seen, including blister cells, pyknocytes, keratocytes and elliptocytes; no one specific form dominates (original magnification ×500, modified Wright’s stain).
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1.17 High power view showing an anaemic dog with anisocytosis and polychromasia. The three nucleated cells, clockwise from top left, are a monocyte, a metamyelocyte and a metarubricyte (original magnification ×500, modified Wright’s stain).
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1.18 Reactive lymphocyte with expanded amoeboid smooth dark blue cytoplasm. Plasmacytoid lymphocyte with very dark blue cytoplasm and whiter perinuclear clearing. Lymphoblast with irregular clumped chromatin, two prominent nucleoli and a small quantity of blue cytoplasm (All are ×1000 original magnification; modified Wright’s stain).
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1.19 Two platelets, top and bottom, with an irregular outline and fine pink irregular grainy cytoplasm (original magnification ×500, modified Wright’s). Four macroplatelets, similar in size or larger than red cells, from a Cavalier King Charles Spaniel (original magnification ×500, modified Wright’s stain).
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1.20 High power view showing mild red cell anisocytosis, a macroplatelet (top right), mature neutrophil (centre lower left) and four azurophilic granular leukaemic lymphoblasts in an acute lymphoid leukaemia (original magnification ×500, modified Wright’s stain).
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Erythrocyte morphology index (EMI) Erythrocyte morphology index (EMI)
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Leucocyte morphology index (LMI) Leucocyte morphology index (LMI)
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