Haematology

• Suitable samples


  • For cell counts use EDTA-anticoagulated blood and make a fresh smear
  • For clotting times use citrate-anticoagulated blood
• Making a blood smear

  Take a sample out of the EDTA tube, put a drop on the slide and then smear
• Important to do a smear to cross-check blood counts from a machine
• Reduce artefact


  • Respect the amount of blood required for the tube (do not under or overfill)
  • Mix gently but thoroughly immediately after collection
  • Perform clean venipuncture
  • Large veins if possible (unless known/suspected bleeding disorder)
  • If sending externally, separate the plasma ASAP and freeze
• Common artefacts that interfere with machine complete blood counts (CBC)

  • Clots
  • Platelet clumps
  • RBC agglutination
  • WBC agglutination
• Mistaken identity might alter the cell count

  • Macroplatelets
  • Nucleated RBCs – machine will often think this is a WBC
  • Heinz bodies (clumps of damaged Hb in RBCs)
  • Atypical cells
• Other artefacts


  • Lipaemia – sample becomes more turgid due to accumulation of lipoprotein particles. This is often caused by not fasting before the blood sample
  • Haemolysos – destruction of RBCs due to mishandling of blood sample
  • Cell swelling during storage
• • philia


  Increase in granulocyte cell numbers
• • cytosis


  Increase in cell numbers for all other cells
• • penia


  Decrease in cell numbers
• RBCs


  • Production: 5 days to produce an immature RBC -> reticulocyte
  • Removal: Senescent RBCs are removed by the spleen or by haemolysis
• Main ways to evaluate RBCs

  • Complete Blood Count – done in an analyser
  • Peripheral Blood Smear exam – done under a microscope
  • Packed Cell Volume – done manually in a centrifuge
• Complete blood count parameters

  • Haemoglobin
  • Red blood cell count (RBC)
  • Mean cell volume (MCV)
  • Haematocrit (HCT)
  • Mean corpuscular haemoglobin (MCH)
  • Mean corpuscular haemoglobin concentration (MCHC)
  • Red cell distribution width (RDW)
  • Reticulocyte count/percentage/indices
• Peripheral blood smear


  • Allows you to assess RBC morphology
  • Helps to verify findings given by analyser
• Packed cell volume


  • Allow you to determine: % of RBCs in a volume of blood, Buffy coat assessment, Plasma colour evaluation, Plasma total protein measurement
  • Technique: Centrifuge whole anti-coagulated blood, RBC is read a % of a column
• Main parts of a blood smear

  • Base or head
  • Monolayer
  • Feathered edge
• Reticulocytes


  Anucleated cells with an increased reticulum
• Counting reticulocytes


  1. Manually, using vital stain for reticulum (New methylene blue, Brilliant cresyl violet)

  2. Using an analyser
• Types of WBC


  • Neutrophils
  • Lymphocytes (T cells, B cells)
  • Monocytes
  • Eosinophils
  • Basophils
• Evaluating WBCs


  1. Haematology analyser (Total WBC count, White cell differential count)

  2. Blood film examination (Cell morphology, Can also be used to estimate WBC count)
• Tests that can be performed alongside a WBC test

  • Acute phase proteins
  • PCT and serology for infectious agents
  • Bone marrow aspiration/biopsy
  • PCR for Antigen Receptor Rearrangements (PARR) test (lymphoproliferative disease)
  • Flow cytometry (classification of leukaemias)
  • Anti-neutrophil antibody
• Factors influencing total WBC numbers

  • Dynamic equilibrium (Peripheral demand, Bone marrow production)
  • Position of the leukocyte within the blood vessel (Marginated or circulating)
  • Availability for sampling (Sequestered in the tissue)
• Haemostasis


  • Ability to stop bleeding
  • A set of mechanisms that maintain an equilibrium between defective haemostasis (haemorrhage) and excessive haemostasis (thrombotic events)
• Main stages of haemostasis

  • Primary haemostasis (Formation of a platelet plug)
  • Secondary haemostasis (Formation of fibrin mesh)
  • Tertiary haemostasis (Breaking down of the clot and return to normal vascular flow)
• Limitations of using an automated counting system to measure the number of platelets

  • RBCs and platelets are similar in size so the machine can over (goats) or under (cats) measure platelet numbers
  • Platelets clump together (Low platelet count)
  • Blood film should be reviewed in all cases
• Species differences in RBC half-life

  • Dogs – 10 days
  • Cats – 70 days
  • Horses – 145 days
  • Cattle – 160 days
• Birds, reptiles, amphibians and fish RBCs are nucleated and their platelet equivalent is also nucleated (thrombocyte). Machine counting cannot be performed in these species as RBCs and thrombocytes cannot be distinguished from WBCs.
• Regenerative anaemia


  Loss of RBC
• Non-regenerative anaemia


  Bone marrow fails to produce new RBCs or produces not enough
• Reticulocyte response in different species

  • Dogs and cats: Expect reticulocyte (immature RBCs) response
  • Horses: Reticulocytes not seen as retained in bone marrow
  • Cattle/sheep: Reticulocytotis only seen with severe anaemia due to variable release
• Types of reticulocytes in cats

  • Aggregate (early reticulocytes, look like canine reticulocytes, released in response to anaemia, mature into punctuate reticulocytes after 12-24 hours)
  • Punctuate (more mature, have only 2-6 dots of reticulum dots, can remain in the blood stream for up to 4 weeks after anaemia has resolved)
• In horses, cattle and sheep, changes in a leukogram are less helpful and need to combine haematology and serum acute phase proteins to detect inflammation.
• Low RBC count


  Indicates anaemia, with clinical signs including mucous membrane pallor and lethargy
• High RBC count

  Indicates polycythaemia/erythrocytosis, with clinical signs including hyperaemic mucous membrane, sneezing, nosebleeds, and neurological signs
• Types of anaemia


  • Regenerative
  • Non-regenerative
• How the body regenerates RBCs

  1. Cells in kidneys respond to low O2 levels by releasing erythropoietin (EPO)

  2. EPO stimulates bone marrow to increase RBC production

  3. This is characteristic by increased immature RBCs (reticulocytes)
• How to tell what type of anaemia is present

  • Severity (mild, moderate, severe)
  • Regenerative vs non-regenerative
  • Colour (hypochromic, normochromic)
  • Cell size (macrocytic, microcytic)
• WBC morphology changes to look for in a blood smear

  • Left shift (immature band neutrophils)
  • Toxic change
  • Reactive lymphocytes
  • Atypical cells (likely cancerous)
  • Infectious agents
• Adrenaline response effect on WBCs

  • Leukocytes from the marginated pool -> circulating pool
  • Increase in mature neutrophils and lymphocytes (short duration)
• Chronic stress/steroid leukogram effect on WBCs

  • Increase in mature neutrophils and monocytes
  • Decrease in lymphocytes and eosinophils