1. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Haematology cases – red cells Kathleen Tennant Clinical Lead, Diagnostic Laboratories

2. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 2

3. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Machine measurements Machine methods – impedence and optical Sample is mixed with diluent/ sheath fluid and red cells streamed through laser or electrical field Number and size of cells measured Agglutination/ lipaemia may affect results

4. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Flourescence methods (Lasercyte) These methods can distinguish the adult red cell population from reticulocytes using size and increased nucleic acid content to allow automated counting 4

5. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Red cell measurements Of the three measures of red cells mass (HCT, RBC, Hb) haemoglobin is the ultimate determinant of oxygen carrying capacity (A cat with a haematocrit or PCV of 5% is given haemoglobin in the form of ‘oxyglobin’…the haematocrit doesn’t change, but the cat looks better…)

6. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Cell size Cell numbers platelets mean cell volume

7. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Cell size Cell numbers platelets Red cell distribution width (RDW)

8. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk MCV - Normocytic Erythrocytes formed correctly Mild to moderate non - regenerative anaemia of chronic disease Acute haemorrhage Renal disease Decreased production of marrow origin

9. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk MCV - Microcytic Red cell haemoglobin concentration determines when division stops – iron deficiency allows one more division: smaller red cells. External blood loss, portosystemic shunts, liver disease, dietary Fe deficiency Early iron deficiency both macrocytic polychromatophils and microcytes may be present – MCV depends on dominant cell type

10. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Microcytosis and macrocytosis together

11. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Microcytosis Artefactual machine change in hyponatraemia – sudden normalisation of the osmotic levels in the diluent used causes shrinkage of the adapted red cells Anaemia of chronic/ inflammatory disease Goodfellow et al. (2008) Res Vet Sci June, pp 490-493 showed storage of microcytic samples for 24 hours could lead to misclassification as normocytic

12. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk MCV Macrocytic - in regeneration – polychromatophils larger than mature reds, also in some FeLV affected cats, in myelodysplasia and as a common artefact in stored (usually posted) blood

13. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Macrocytosis Artefactual in hypernatraemic animals: up to 10.4 fL in a cat with Na+ of 186 mmol/L – Boisvert et al (1999) Vet Clin path 28 (3) p91 - 96 Artefactual in hyperglycaemia (Holt et al {1982} Am J Clin Path 77 (5) 561 - 7

14. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Normochromic/hypochromic Measures of haemoglobin concentration in the red cell mass MCHC / MCH on panel – MCHC preferable as takes MCV into account Decreased in iron deficiency/ poor iron incorporation (usually with microcytosis) and in regenerative anaemias (immature reds not fully haemoglobinised) Hyperchromic not possible: haemolysis

15. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Hypochromic

16. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Iron deficiency anaemia Weiser and O’Grady (1983) Vet Pathol Mar;20 (2):230-41. 12 dogs with iron deficiency Moderate to marked reticulocytosis, decreased mean corpuscular volume, and decreased mean corpuscular hemoglobin concentration Only 4/12 hypoproteinaemic Therapy resulted in rapid production of normochromic, normocytic cells ?Situation in cats

17. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Assessing regeneration

18. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Polychromatophils

19. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Reticulocytes Some directly measured relying on increased cell size and more RNA content New Methylene blue smears (equal volumes EDTA blood and filtered New Methylene blue for 15 – 20 minutes), then smear Count 500 cells and derive % (New Methylene Blue smears also very good for seeing Heinz bodies in red cells)

20. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Absolute reticulocyte count – accounts for degree of anaemia Absolute reticulocyte count (x10 9 /l) = observed % retics X RBC count (x10 12 /l) X 10 Regenerative if > 50 x10 9 /l Strongly regenerative if > 200 x10 9 /l

21. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Reticulocytes (white arrow punctate, black arrow aggregate)

22. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Case 3296 4 y.o FN Old English Sheepdog Sudden onset lethargy, dyspnoea, pallor

23. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 23 Parameter Patient value UnitsReference interval Hb3.66g/dl12.00 – 18.00 HCT12.0%35 - 55 RBC1.28X 10^12/l5.40 – 8.00 MCV93.8fl65.0 – 75.0 MCH28.6pg22.0 – 25.0 MCHC30.5g/dl34.0 – 37.0 Plt213X 10^9/l170 - 500 WBC Corrected 65.20X 10^9/l5.50 – 17.00 Nucleated red cells 3.9

24. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Which is more likely? Regenerative, because of macrocytosis and hypochromasia1. Non – regenerative because of macrocytosis and severity – possible bone marrow disorder2. Iron deficiency because of marked hypochromasia3. 24

25. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 1000x field – which cell type is arrowed?

26. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Which cell type? Macroplatelet1. Neutrophil2. Polychromatophil (immature red)3. Adult erythrocyte4. Spherocyte5. 26

27. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Polychromatophil – larger and with less hemoglobin (so increased MCV and decreased MCH and MHC) - regeneration

28. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Same blood film, different area – what is the cell type?

29. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is the cell type? Microcyte, associated with iron deficiency1. Red cell shear injury product, as might be seen in a dog with haemangiosarcoma2. Spherocyte, as might be seen in immune mediated hemolytic anaemia3. Adult, normochromic, normocytic red cell4. 29

30. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Spherocyte – small, perfectly round, no central pallor, slightly darker staining than normal red cells

31. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Immune mediated hemolytic anaemia Classic presentation of severe, rapidly developing, strongly regenerative anaemia The immune mediated destruction usually provokes a marked neutrophilia, with or without left shift and toxic change Spherocytes may be seen with extravascular destruction. Difficult to see in cats Ghost cells (red cell membranes) may be seen in intravascular hemolysis, along with possible INCREASES in MCH and MCHC because of free hemoglobin 31

32. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Ghost cell – intravascular haemolysis 32

33. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Neutrophils – left shift only – bands and metamyelocytes present

34. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Autoimmune haemolytic anaemia Don’t get so carried away with the red cell and leucocyte changes that you forget to check the platelet values!

35. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk In immune mediated hemolytic anaemia, make a point of checking platelets are alright

36. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Case 96072 15 year old FE Crossbred dog 2 week history of lethargy Spleen feels large and irregular on palpation ParameterPatient valueUnitsReference interval Hb14.40g/dl12.00 – 18.00 HCT44.6%35 - 55 RBC6.22X 10^12/l5.40 – 8.00 MCV71.7fl65.0 – 75.0 MCH23.1pg22.0 – 25.0 MCHC32.2g/dl34.0 – 37.0 Plt353X 10^9/l170 - 500

37. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is this?

38. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is this? Acanthocyte – associated with shear injury to the red cell/ liver disease/ lipid disorders1. Schistocyte – associated with shear injury to the red cell, as might be seen with some tumours, D.I.C., endocarditis2. Crenated red cell/ echinocyte – seen in slow drying smears as an artefact and in some glomerulonephritis cases3. 38

39. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Acanthocyte Red cells with large blunt ended projections May be seen in lipid disorders, liver disease Often where red cells have ‘shear injury’ from being forced through small vessels in tumours or through strands of fibrin when there are clots in vessels: schistocytes, blister cells, keratocytes all share this origin 39

40. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Schistocytes – red cell fragments which are thin and tapered

41. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Shear injury – schistocytes/ keratocytes/ blister cells

42. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Echinocytes/ crenated red cells Small, spiky cytoplasmic projections. If these are artefactual (most common) they may be unevenly distributed across the smear 42

43. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 5 y.o. Griffon with intermittent exercise intolerance/ pallor – what is the red cell abnormality? 43

44. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is the red cell abnormality? Howell – Jolly bodies1. Haemoplasmas 2. Heinz bodies3. 44

45. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Heinz bodies in a dog frequently fed pickled onions! A result of oxidative damage, cats are more vulnerable (up to 25% of red cells affected in clinically normal cats) Paracetomol (cats), alliums, diabetic ketoacidosis Easier to see and enumerate using New Methylene blue stain 45

46. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Heinz bodies with New Methylene blue stain 46

47. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is arrowed?

48. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is arrowed Howell Jolly body1. Small lymphocyte2. Neoplastic lymphoid cell3. Nucleated red cell4. Immature platelet5. 48

49. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Nucleated red cell – seen in red cell regeneration and also more with splenic disease/ splenectomy There is also a Howell Jolly body (white arrow), a nuclear fragment also seen more with red cell regeneration

50. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is/ are these? 50

51. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is/ are these? Haemoplasmas (Mycoplasma hemofelis)1. Basophilic stippling from lead poisoning2. Howell Jolly Bodies3. Stain precipitate4. 51

52. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Mycoplasma hemofelis These are VERY infrequently seen on ordinary blood smears – epierythrocytic position Most times we’re asked for a second opinion, we find stain precipitate or Howell Jolly bodies (yellow arrow) Much more sensitive to test by qPCR – can also speciate M hemofelis and M turicensis always clinically significant M haemominutum can be carried asymptomatically and become significant with immunosuppression/ other illness 52

53. vv | www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 53