v Leucocytes Kathleen Tennant Clinical Lead Diagnostic Laboratories

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

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

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

Leucocyte measurement • Most methods use a mixture of impedence properties and optical scatter/ granularity to derive total numbers and a differential v • 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

What cell type? • • • 6 | Neutrophils Lymphocytes Monocytes Eosinophils Basophils www. langfordvets. co. uk | 1. 2. 3. 4. 5. v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Lymphocyte • Lymphocytes are small compared to the other leucocytes and have round, noncomplex nuclei 7 | www. langfordvets. co. uk | v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

What leucocyte type will the green dots be? • • • 10 | Neutrophils Lymphocytes Monocytes Eosinophils Basophils www. langfordvets. co. uk | 1. 2. 3. 4. 5. v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

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

Left shift • Once the mature pool has been stripped from the marrow, if demand outstrips new neutrophil production, earlier precursors released: v • 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

What is this cell? • • • 17 | Lymphocyte, reactive Monocyte Band neutrophil Metamyelocyte Myelocyte www. langfordvets. co. uk | 1 2 3 v 4 5 Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

‘Toxic change’ • In response to overwhelming demand, dysmature neutrophils released • Organelles normally removed before the v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

Neutropenia • Breed variations (Greyhounds/ sight hounds) and individual variation: if mild, track. • May be secondary to decreased production v 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. | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Name the cell • Neutrophil 1. • Band neutrophil with toxic change 2. • Lymphocyte 3. • Monocyte 4. 25 | www. langfordvets. co. uk | v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Monocytosis • Steroids may move them out of the marginated pool • Many infectious causes: bacterial, fungal, v 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 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Lymphocytes • Small, normal lymphocytes in circulation are 1 – 1. 5 x red cell size with a thin rim of cytoplasm | www. langfordvets. co. uk | v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

Lymphocytosis • Lymphocytosis in physiological response as well as immune stimulation with few diseases (Ehrlichia, Leishmania, Toxoplasma) • Addison’s (5 – 10%) of v cases • NOT found as a response to vaccination in various studies (summarised in Avery and Avery (2007) Vet Clin Small Anim 37 p 267 – 282) • Marked lymphocytosis (> 20 x 10^9/L) raises concerns for neoplasia | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

Name that cell • • 34 | Basophil Toxic neutrophil Eosinophil Macrophage www. langfordvets. co. uk | 1. 2. 3. 4. v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Eosinophils - eosinophilia • Commonest causes in dogs – pulmonary infiltrate with eosinophils and GI disease (eosinophilic I. B. D. ) v (Lilliehook and Tvedten, Vet Clin North Am Small Anim Pract. 2003 Nov; 33 (6): 1359 -78 • Eosinophilic leukaemias and paraneoplastic increases also reported • Hypereosinophilic syndrome (huskies) can give marked elevations of well differentiated cells | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

Name that cell • Basophil 1. • Toxic neutrophil 2. • Neutrophil containing Ehrlichia 3. • Eosinophil 4. • Macrophage 5. 38 | www. langfordvets. co. uk | v Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Sample number 3235 • 12 y ME Bulldog • Lethargy, inappetance, mild lymphadenomegaly, vsplenomegaly • Biochemistry unremarkable | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Leucocytes through the machine 41 WBC 50. 30 X 10^9/l 5. 50 – 17. 00 Neutrophils 10. 56 X 10^9/l 3. 00 – 11. 50 v Lymphocytes 37. 73 X 10^9/l 0. 70 – 3. 60 Monocytes 2. 01 X 10^9/l 0. 10 – 1. 50 Eosinophils 0. 00 X 10^9/l 0. 20 – 1. 40 Basophils 0. 00 X 10^9/l 0. 00 – 0. 10 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

Which is the most likely pathology? • • 42 | Immune stimulation Chronic lymphocytic leukaemia Acute lymphoblastic vleukaemia Stage 5 lymphoma www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol 1. 2. 3. 4.

Marked lymphocytosis – with other leucocytes unaffected 43 WBC 50. 30 X 10^9/l 5. 50 – 17. 00 Neutrophils 10. 56 X 10^9/l 3. 00 – 11. 50 v Lymphocytes 37. 73 X 10^9/l 0. 70 – 3. 60 Monocytes 2. 01 X 10^9/l 0. 10 – 1. 50 Eosinophils 0. 00 X 10^9/l 0. 20 – 1. 40 Basophils 0. 00 X 10^9/l 0. 00 – 0. 10 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

Large granular lymphoid leukaemia can start in spleen as well as marrow v | www. langfordvets. co. uk | • These lymphocytes can be seen in health and in reactive processes, but 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 Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

v 47 | www. langfordvets. co. uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol