Page 316 - The Veterinary Laboratory and Field Manual 3rd Edition

Haematology 285

by filtering out foreign material. There are two differential white blood cell count
populations of lymphocytes, ‘T’ and ‘B’ types (diff. Wbc)
named after the thymus and bursa derived
populations in chickens. It is not possible to dis- To perform a differential white cell count a thin,
tinguish the two types in blood or tissue smears. even blood film should be prepared and either
In simple terms, the B-lymphocytes produce air dried and placed in a covered slide container
antibodies to foreign material (antigens) and or, if there will be a delay of greater than 24 h,
the T-lymphocytes are important in cell medi- fixed in methanol. The slide can then be stained
ated immunity (see Chapter 6 for more details). with Giemsa (or an alternative Romanowsky
Monocytes are often present in large numbers stain) as outlined previously. In a well-prepared
in chronic inflammation. The peroxidase test blood smear the white cells should be distrib-
(below) can be used to help distinguish between uted evenly. However, even in well-made smears
monocytes and lymphocytes. there is potential for a large degree of error. If
smears are too thick it is not possible to accu-
rately differentiate cells, especially monocytes
Peroxidase reaction and lymphocytes. If smears are too thin there
is an unrepresentative distribution of cells
This staining system may assist in the identifi- towards the edges and tail of the smear. Equally,
cation of cell types in blood smears from some an uneven edged spreader will give a mislead-
species. ing representation of different cells throughout
the smear. For cell counts, the blood film should
1 Fix air dried blood films in 9 parts ethanol initially be scanned under low power both 200×
(95%) and 1 part formalin for 5 s. (20× objective) and 400× (40× objective), then
2 Wash the slide in tap water for 1 min. high power (oil immersion 1000×) to evaluate
3 Stain the slide in 10% Giemsa stain for the shape, size and staining characteristics of the
30 min. red and white cells in the film. The presence of
4 Pour off the stain and flood with 0.5% aque- any blood parasites (for example, Trypanosoma
ous copper sulphate (CuSO .5H O) for 5 s. sp., Babesia sp., Theileria sp. and Rickettsia, see
4 2
5 Prepare a 0.1% aqueous solution of benzidine Chapter 3, section 3.6) should also be noted.
and add 2 drops of 3% hydrogen peroxide Depending on the parasite you will find them
(H O ) per 100 ml. either on scanning on the lower magnification,
2 2
6 Pour the copper sulphate off the slide and for example, Dirofilaria, or on high magnifica-
place the slide in the benzidine solution for tion, for example, Babesia, Ehrlichia and so on.
2 min. One of the most popular counting methods is
7 Wash the slide in tap water and air dry. the Battlement method (Figure 5.3).
8 Examine under oil immersion (1000×). The Battlement method involves examining
Blue granules are present in developing and the slide field by field along a horizontal line for
mature neutrophils, a few granules are usu- three fields, then for two fields up, three fields
ally seen in monocytes and no granules are horizontally, then a further two fields down to the
seen in lymphocytes. edge of the film. Counting is continued until 100
white cells have been counted on each section of
the film. For routine purposes, at least 200 white
cells should be counted and then each cell type
can be expressed as a percentage of the total.

Vet Lab.indb 285 26/03/2019 10:25