v Haematology cases red cells Kathleen Tennant Clinical

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

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

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

Red cell measurements • Of the three measures of red cells mass (HCT, RBC, Hb) haemoglobin is the ultimate determinant of oxygen v 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…) | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

MCV - Microcytic • Red cell haemoglobin concentration determines when division stops – iron deficiency allows one more division: smaller red cells. v • 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Microcytosis • Artefactual machine change in hyponatraemia – sudden normalisation of the osmotic levels in the diluent used causes v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Macrocytosis • Artefactual in hypernatraemic animals: up to 10. 4 f. L in a cat with Na+ of 186 mmol/L – Boisvert et al (1999) Vet Clin path 28 (3) v p 91 - 96 • Artefactual in hyperglycaemia (Holt et al {1982} Am J Clin Path 77 (5) 561 - 7 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Normochromic/hypochromic • Measures of haemoglobin concentration in the red cell mass • MCHC / MCH on panel – MCHC preferable v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Hypochromic v | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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 v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Polychromatophils v | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Reticulocytes • Some directly measured relying on increased cell size and more RNA content • New Methylene blue smears (equal volumes v 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) | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Absolute reticulocyte count – accounts for degree of anaemia 9/l) = Absolute reticulocyte count (x 10 v observed % retics X RBC count (x 1012/l) X 10 Regenerative if > 50 x 109/l Strongly regenerative if > 200 x 109/l | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

Parameter Patient value Units Reference interval Hb 3. 66 g/dl 12. 00 – 18. 00 HCT 12. 0 % 35 - 55 RBC 1. 28 X 10^12/l 5. 40 – 8. 00 MCV 93. 8 fl MCH 28. 6 pg 22. 0 – 25. 0 MCHC 30. 5 g/dl 34. 0 – 37. 0 Plt 213 X 10^9/l 170 - 500 WBC Corrected 65. 20 X 10^9/l 5. 50 – 17. 00 v 65. 0 – 75. 0 Nucleated 3. 9 23 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol red cells

Which is more likely? • Regenerative, because of macrocytosis and hypochromasia 1. • Non – regenerative because of v macrocytosis and severity – possible bone marrow disorder 2. • Iron deficiency because of marked hypochromasia 3. 24 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Which cell type? • • • 26 | Macroplatelet Neutrophil v Polychromatophil (immature red) Adult erythrocyte Spherocyte www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol 1. 2. 3. 4. 5.

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

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

What is the cell type? • Microcyte, associated with iron deficiency 1. • Red cell shear injury product, as might be seen in a dog with haemangiosarcoma 2. v • Spherocyte, as might be seen in immune mediated hemolytic anaemia 3. • Adult, normochromic, normocytic red cell 4. 29 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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 v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

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

Case 96072 Parameter Patient value Units Reference interval MCH 23. 1 pg 22. 0 – 25. 0 MCHC 32. 2 g/dl 34. 0 – 37. 0 Plt 353 X 10^9/l 170 - 500 • 15 year old FE Crossbred dog Hb 14. 40 g/dl 12. 00 – 18. 00 • 2 week 44. 6 history of lethargy 35 - 55 HCT % v • Spleen 6. 22 feels large and irregular on RBC X 10^12/l 5. 40 – 8. 00 MCVpalpation 71. 7 fl 65. 0 – 75. 0 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

What is this? • Acanthocyte – associated with shear injury to the red cell/ liver disease/ lipid disorders 1. • Schistocyte – associated with shear injury to the v red cell, as might be seen with some tumours, D. I. C. , endocarditis 2. • Crenated red cell/ echinocyte – seen in slow drying smears as an artefact and in some glomerulonephritis cases 3. 38 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Acanthocyte v 39 | www. langfordvets. co. uk | • 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 Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

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

What is the red cell abnormality? • Howell – Jolly bodies • Haemoplasmas v • Heinz bodies 44 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol 1. 2. 3.

Heinz bodies in a dog frequently fed pickled onions! v 45 | www. langfordvets. co. uk | • 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 Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

What is arrowed • • • 48 | Howell Jolly body Small lymphocyte Neoplastic lymphoidvcell Nucleated red cell Immature platelet www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol 1. 2. 3. 4. 5.

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 v | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

What is/ are these? • • 51 | Haemoplasmas (Mycoplasma hemofelis) Basophilic stippling from lead poisoning Howell Jolly Bodies v Stain precipitate www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol 1. 2. 3. 4.

Mycoplasma hemofelis • • v • • • 52 | www. langfordvets. co. uk | 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 q. PCR – can also speciate M hemofelis and M turicensis always clinically significant M haemominutum can be carried asymptomatically and become significant with immunosuppression/ other illness Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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