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

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

• 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

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

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

• 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 v
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?
• Neutrophils
• Lymphocytes
• Monocytes
• Eosinophils
• Basophils
1.
2.
3 .
4.
5.
v
10 | 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?
• Neutrophils
• Lymphocytes
• Monocytes
• Eosinophils!
• Basophils are white v
11 | www.langfordvets.co.uk
| Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

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

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

• 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

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

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

• In response to overwhelming demand, dysmature neutrophils released
• Organelles normally removed before the 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

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

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

• As immature neutrophils contain more RNA, they occupy a recognisable area of v 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
| Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

• Breed variations (Greyhounds/ sight hounds) and individual variation: if mild, track.
• May be secondary to decreased production 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

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

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

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

• Steroids may move them out of the marginated pool
• Many infectious causes: bacterial, fungal, protozoal v
• 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

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

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

• Lymphocytosis in physiological response as well as immune stimulation with few diseases
(Ehrlichia, Leishmania, Toxoplasma)
• 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
| www.langfordvets.co.uk
| Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

•
Lymphopaenia is a common finding
•
Stressed and diseased animals
•
Post steroids and some chemotherapeutics v
| www.langfordvets.co.uk
| 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

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

• Commonest causes in dogs – pulmonary infiltrate with eosinophils and GI disease
(eosinophilic I.B.D.)
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

•
Usually corticosteroid related (endogenous or exogenous)
•
Insulin administration
•
Diurnal variation in humans may be reflected in dogs (fewer in the morning) v
| www.langfordvets.co.uk
| 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

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

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

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

Leucocytes through the machine
WBC 50.30
Neutrophils 10.56
Lymphocytes 37.73
Monocytes 2.01
Eosinophils 0.00
Basophils 0.00
X 10^9/l
X 10^9/l
X 10^9/l v
X 10^9/l
X 10^9/l
X 10^9/l
5.50 – 17.00
3.00 – 11.50
0.70 – 3.60
0.10 – 1.50
0.20 – 1.40
0.00 – 0.10
41 | www.langfordvets.co.uk
| Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

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

Marked lymphocytosis – with other leucocytes unaffected
WBC 50.30 X 10^9/l 5.50 – 17.00
Neutrophils 10.56
Lymphocytes 37.73
Monocytes 2.01
Eosinophils 0.00
Basophils 0.00
X 10^9/l
X 10^9/l v
X 10^9/l
X 10^9/l
X 10^9/l
3.00 – 11.50
0.70 – 3.60
0.10 – 1.50
0.20 – 1.40
0.00 – 0.10
43 | www.langfordvets.co.uk
| Langford Veterinary Services Ltd is a wholly owned subsidiary of the University of Bristol

• 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 | 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
• 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
| www.langfordvets.co.uk
| 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