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

Leucocytes

Kathleen Tennant

Clinical Lead, Diagnostic Laboratories

http://www.langfordvets.co.uk/

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| www.langfordvets.co.uk | Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol2


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Leucocyte measurement

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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….

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What cell type will the blue ones be?

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What cell type?

• Neutrophils1.

• Lymphocytes2.

• Monocytes3.

• Eosinophils4.

• Basophils5.

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Lymphocyte

• Lymphocytes are small compared to the other leucocytes and have round, non- complex nuclei

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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

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Other measures such as granularity and lobularity allow a 5 part differential

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What leucocyte type will the green dots be?

• Neutrophils1.

• Lymphocytes2.

• Monocytes3.

• Eosinophils4.

• Basophils5.

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What leucocyte type will the green dots be?

• Neutrophils• Lymphocytes• Monocytes• Eosinophils!• Basophils are white

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Eosinnophils

• In dogs and cats the most obviously granular nucleated cells will be eosinophils

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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


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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


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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


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Case 6: 3 y.o. Working Labrador with pyothorax – peripheral blood – what is this cell?


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What is this cell?

• Lymphocyte, reactive 1• Monocyte

2• Band neutrophil

3• Metamyelocyte

4• Myelocyte

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Left shifted neutrophil line

• The white arrow shows a myelocyte – reniform, and the earliest in the neutrophil maturation pool

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‘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


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Case 6 :Toxic change and left shift


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Case 6 : Toxic change


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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

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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.


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Name the cell

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Name the cell

• Neutrophil1.

• Band neutrophil with toxic change

2.• Lymphocyte

3.• Monocyte

4.

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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


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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


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Lymphocytes

• Small, normal lymphocytes in circulation are 1 – 1.5 x red cell size with a thin rim of cytoplasm


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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


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Lymphoblasts

• Large lymphoid cells, larger than neutrophils

• Their presence in circulation is always abnormal

• Look for nucleoli• Generally

lymphoma/ leukaemia in high number


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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


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Lymphopenia

• Lymphopaenia is a common finding

• Stressed and diseased animals

• Post steroids and some chemotherapeutics


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5 y.o. WHWT with cough and skin diease. Name that cell

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Name that cell

• Basophil1.

• Toxic neutrophil 2.• Eosinophil

3.• Macrophage

4.

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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. 2003 Nov; 33 (6):1359-78

• Eosinophilic leukaemias and paraneoplastic increases also reported

• Hypereosinophilic syndrome (huskies) can give marked elevations of well differentiated cells


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Eosinophils - eosinopenia• Usually corticosteroid

related (endogenous or exogenous)

• Insulin administration• Diurnal variation in

humans may be reflected in dogs (fewer in the morning)


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Coughing 2 y.o. Springer Spaniel. Name that cell

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Name that cell

• Basophil1.

• Toxic neutrophil 2.• Neutrophil containing

Ehrlichia3.

• Eosinophil4.

• Macrophage5.

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Basophils

• In hypersensitivity, parasitic and paraneoplastic responses

• Basophilic leukaemias reported but rare

• Increases in some myelodyplastic syndromes

• Dirofilaria in imported• Decreases generally

unrecognised


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Sample number 3235

• 12 y ME Bulldog• Lethargy, inappetance, mild

lymphadenomegaly, splenomegaly• Biochemistry unremarkable


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Leucocytes through the machine

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WBC 50.30 X 10^9/l 5.50 – 17.00

Neutrophils 10.56 X 10^9/l 3.00 – 11.50

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


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Which is the most likely pathology?

• Immune stimulation1.

• Chronic lymphocytic leukaemia2.

• Acute lymphoblastic leukaemia3.

• Stage 5 lymphoma4.

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Marked lymphocytosis – with other leucocytes unaffected

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WBC 50.30 X 10^9/l 5.50 – 17.00

Neutrophils 10.56 X 10^9/l 3.00 – 11.50

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


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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)

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Large granular lymphocytes in very high numbers – suspicious for large granular lymphocyte leukaemia


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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


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eosinophils12 v

hypersegmentation v

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complex nuclei7 v