vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Leucocytes Kathleen Tennant Clinical Lead, Diagnostic Laboratories

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

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Leucocyte measurement 3

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Leucocyte measurement Most methods use a mixture of impedence properties and optical scatter/ granularity to derive total numbers and a differential Fluorescence can give another way of differentiating cell types Information from the graphs can help you to understand how it has derived the differential…. 4

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What cell type will the blue ones be? 5

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What cell type? Neutrophils1. Lymphocytes2. Monocytes3. Eosinophils4. Basophils5. 6

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphocyte Lymphocytes are small compared to the other leucocytes and have round, non- complex nuclei 7

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk On this scatter plot Neutrophils Lymphocytes Monocytes Eosinophils Basophils are white Knowing what the cells look like, you can predict their position and see how cleanly the colours are grouped together 8

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Other measures such as granularity and lobularity allow a 5 part differential 9

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What leucocyte type will the green dots be? Neutrophils1. Lymphocytes2. Monocytes3. Eosinophils4. Basophils5. 10

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What leucocyte type will the green dots be? Neutrophils Lymphocytes Monocytes Eosinophils! Basophils are white 11

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Eosinnophils In dogs and cats the most obviously granular nucleated cells will be eosinophils 12

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Leucocytes - Neutrophils Combination of absolute numbers and morphology changes give the best chance of correctly interpreting. Mature neutrophils in three pools – in circulation, marginated and mature pool in the bone marrow

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Mature neutrophilia By redistribution (marrow to blood stream, marginated to free flowing) in response to inflammatory mediators or increased blood pressure Decreased loss to tissues in response to corticosteroids often with hypersegmentation

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Left shift Once the mature pool has been stripped from the marrow, if demand outstrips new neutrophil production, earlier precursors released: Bands, metamyelocytes, myelocytes (Unusual for promyelocytes/ blasts to be seen in peripheral blood) Presence indicates an acute inflammatory response that is overwhelming the ability of the marrow to cope

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Case 6: 3 y.o. Working Labrador with pyothorax – peripheral blood – what is this cell?

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk What is this cell? Lymphocyte, reactive1 Monocyte2 Band neutrophil3 Metamyelocyte4 Myelocyte5 17

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Left shifted neutrophil line The white arrow shows a myelocyte – reniform, and the earliest in the neutrophil maturation pool 18

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk ‘Toxic change’ In response to overwhelming demand, dysmature neutrophils released Organelles normally removed before the neutrophil is released persist, other organelles not fully matured Cytoplasmic basophilia, doehle bodies, vacuolation, persistent granules (toxic granulation), ring form nuclei Often seen alongside left shift

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Case 6 :Toxic change and left shift

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Case 6 : Toxic change

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lasercyte As immature neutrophils contain more RNA, they occupy a recognisable area of the scatter plot in this technology, but the severity of the left shift and toxic change are best evaluated on the smear 22

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Neutropenia Breed variations (Greyhounds/ sight hounds) and individual variation: if mild, track. May be secondary to decreased production with marrow disease/ suppression (esp chemo), increased utilisation with marked inflammation or immune mediated destruction With complete marrow destruction or suppression, neutrophils are the first cell line to decrease.

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Name the cell 24

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Name the cell Neutrophil1. Band neutrophil with toxic change2. Lymphocyte3. Monocyte4. 25

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Monocytes Have marginated and circulating pools Leave circulation to tissues, differentiate into macrophages with inflammatory cytokines. Larger than neutrophils, with a more open nuclear chromatin Cytoplasm blue – grey and may be vacolated

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Monocytosis Steroids may move them out of the marginated pool Many infectious causes: bacterial, fungal, protozoal Immune mediated disease, sepsis, necrosis, trauma ? systemic studies on prevalence of diagnostic categories with monocytosis as a finding – tend to be case reports/ case series

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphocytes Small, normal lymphocytes in circulation are 1 – 1.5 x red cell size with a thin rim of cytoplasm

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphocytes on the blood smear Reactive lymphocytes -Slightly larger -More basophilic -May have a pale perinuclear zone -Seen more frequently in young animals -May not reflect function

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphoblasts Large lymphoid cells, larger than neutrophils Their presence in circulation is always abnormal Look for nucleoli Generally lymphoma/ leukaemia in high number

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphocytosis Lymphocytosis in physiological response as well as immune stimulation with few diseases (Ehrlichia, Leishmania, Toxoplasma) Addison’s (5 – 10%) of cases NOT found as a response to vaccination in various studies (summarised in Avery and Avery (2007) Vet Clin Small Anim 37 p267 – 282) Marked lymphocytosis (> 20 x 10^9/L) raises concerns for neoplasia

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphopenia Lymphopaenia is a common finding Stressed and diseased animals Post steroids and some chemotherapeutics

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk 5 y.o. WHWT with cough and skin diease. Name that cell 33

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Name that cell Basophil1. Toxic neutrophil2. Eosinophil3. Macrophage4. 34

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Eosinophils - eosinophilia Commonest causes in dogs – pulmonary infiltrate with eosinophils and GI disease (eosinophilic I.B.D.) (Lilliehook and Tvedten, Vet Clin North Am Small Anim Pract Nov; 33 (6): Eosinophilic leukaemias and paraneoplastic increases also reported Hypereosinophilic syndrome (huskies) can give marked elevations of well differentiated cells

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Eosinophils - eosinopenia Usually corticosteroid related (endogenous or exogenous) Insulin administration Diurnal variation in humans may be reflected in dogs (fewer in the morning)

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Coughing 2 y.o. Springer Spaniel. Name that cell 37

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Name that cell Basophil1. Toxic neutrophil2. Neutrophil containing Ehrlichia3. Eosinophil4. Macrophage5. 38

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Basophils In hypersensitivity, parasitic and paraneoplastic responses Basophilic leukaemias reported but rare Increases in some myelodyplastic syndromes Dirofilaria in imported Decreases generally unrecognised

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Sample number y ME Bulldog Lethargy, inappetance, mild lymphadenomegaly, splenomegaly Biochemistry unremarkable

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Leucocytes through the machine 41 WBC50.30X 10^9/l5.50 – Neutrophils10.56X 10^9/l3.00 – Lymphocytes37.73X 10^9/l0.70 – 3.60 Monocytes2.01X 10^9/l0.10 – 1.50 Eosinophils0.00X 10^9/l0.20 – 1.40 Basophils0.00X 10^9/l0.00 – 0.10

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Which is the most likely pathology? Immune stimulation1. Chronic lymphocytic leukaemia2. Acute lymphoblastic leukaemia3. Stage 5 lymphoma4. 42

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Marked lymphocytosis – with other leucocytes unaffected 43 WBC50.30X 10^9/l5.50 – Neutrophils10.56X 10^9/l3.00 – Lymphocytes37.73X 10^9/l0.70 – 3.60 Monocytes2.01X 10^9/l0.10 – 1.50 Eosinophils0.00X 10^9/l0.20 – 1.40 Basophils0.00X 10^9/l0.00 – 0.10

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Lymphocytosis Cats can have lymphocytosis up to low twenties with immune stimulation Can not tell whether leukaemia or stage 5 lymphoma based on smear or bone marrow – needs clinical assessment and observation of progression Morphology on the smear needed to tell if acute (poorly differentiated/ large lymphoid cells) or chronic lymphocytic (small/ well differentiated) 44

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Large granular lymphocytes in very high numbers – suspicious for large granular lymphocyte leukaemia

vv | | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristolwww.langfordvets.co.uk Large granular lymphoid leukaemia can start in spleen as well as marrow These lymphocytes can be seen in health and in reactive processes, but not normally in these numbers High numbers of Howell – Jolly bodies (arrowed) – seen in red cell regeneration (not here!) and in some animals with splenic disease

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