1. Differential WBC Count
(Differential Leucocyte Count
Aim: To make a blood smear and to count the different types of leucocytes
present in a stained blood smear and express their relative
counts in percentage
Principle
The blood contains various types of white blood cells. The number of different types may deviate from normal acceptable range. The percentage deviation from normal may indicate certain disease state of the patient such as allergic and drug reactions; parasitic infection and other types of infection. It can also identify various stages of leukemia.
Cell morphology and number will also differ among species.
We will use 2 blood samples (from man and chicken).

2. Differential WBC Count
Requirements:
Glass slides
Leishman stain
Microscopes
Disposable needles
Vials containing anticoagulants
Methylated-spirit
Staining rack

3. Differential WBC Count
Procedure
Preparation of blood smear
Staining of the smear
Examination of the stained blood smear
Identification and counting of the cells
Method of recording and expression of the count
Note: Different staining methods are possible. (Wright’s stain, Giemsa stain, Diff Quick stain; all these are known as Romanovsky-type stains).

4. Differential WBC Count
Preparation of the smear
Use the blood from the finger tip prick (man) or a sample of blood mixed with anticoagulant (collection site depends on the animal used, refer to previous lab instruction)
Place a drop of blood on a glass slide about 2 cm from the end of the slide
Hold the slide with thumb and other fingers of left hand firmly.
Hold the spreader slide by its long sides with thumb and other fingers of right hand and apply it over the slide by its short edge in front of the blood drop at an angle of 45.
Pull the spreader gently backwards till it touches the blood drop. The blood drop spreads along the short edge of the spreader.
Move the spreader slowly and steadily to the other end of the slide applying uniform pressure and maintaining the angle at 45.
Wave the slide in the air for quick drying of the smear.
A good film - fairly uniform with a single cell thickness without uneven streaks or gaps.

5. Preparation of the blood smear: Wedge method 12
Once the slide is in contact with the blood, pause movement of the slide to allow the blood to spread laterally. Then propel the spreader slide forward with a gentle, smooth motion to spread the blood along the slide.
Place a drop of blood towards the end of a slide (on a fl at surface). Then reverse the spreader slide into the drop of blood.

6. Staining (Leishman stain)
(Leishman stain powder g +
100 ml acetone free methyl alcohol)
1.     Place fix air-dried smear across the staining rack
Drop a number of Leishman stain to cover the entire blood smear
Allow to stand for 2 min. (Do not allow the stain to dry up)
4.     Poor the double the number of drops of DW on the slide.
Do not allow the stain-DW mix to overflow.
Blow air gently on to the slide to ensure proper mixing of stain with DW.
Allow to remain for 10 min.
After 10 min, pour stain water mix into the tray and wash the slide under running tap water.
Water should not fall directly over the smear.
Wipe the back side of the slide with dry cotton. Dry the slide keeping in a slanting position. At times wave the slide in the air.     

7. Counting
1.    Place the slide on the microscope stage and observe under low power. If the smear is not good enough for counting, repeat the whole process.
Focus the smear under oil immersion lens.
(a) In the first phase, looking from the side raise the stage until the oil immersion lens touches the oil drop placed on the slide.
(b) In the second phase, looking from the eye-piece raise the stage slowly using coarse knob till the cells appear vaugely.
(c) In the third phase, focus the cells clearly by using fine knob,
Identify and count different types of the cells. Use the following characteristics.

8. Count the cells from the body areas of the smear in a zig-zag manner up to 100 cells.
(a) Prepare a large square and divide it into 100 small squares.
(b) Enter the identified cells into the squares as “N” for neutrophil, “B” for basophil, so on till the last small square is filled.
(c) Count each type of cells separately and express it as percentage.

9. Differential WBC Count
Characteristics of different types of the white cells
Neutrophils nucleus……… dark blue
                   cytoplasm....………………….pale pink
Eosinophils    nucleus …………………. blue
                   cytoplasm.....…………………blue
                   granules red to red/orange
Basophils      nucleus …………………purple or dark blue
                   granules …………….….. dark purple/black
Monocytes    nucleus(lobated)………….…violet
                   cytoplasm ……...……...sky blue

10. Monocyte
Thrombocyte

11.  Sources of error
Film preparation
Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
Holes in film: Slide contaminated with fat or grease
Irregular leucocyte and platelet distribution, especially in tail: poor film-making technique
Film too short and too thick: spreader held at incorrect angle
Film extending to end of slide: blood drop too large
Short thin film: blood drop too small
Film extends to edge of slide: spreader too wide or not positioned correctly
Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water

12. Avian normal blood values (Gallus domesticus and man)
Parameter
Chicken
Man
RBC (106/mm3)
4.9 – 5.5
PCV (%)
22 – 35
Hemoglobin (g/dL)
7 – 13
14 – 18
MCV
90 – 140 fL
78 – 95 mm3
MCH
33 – 47%
28 – 33 pg/cell
MCHC
26 – 35%
32-36 g/dL
WBC (cells/mm3)
12000 – 30000
× 103/mm3
  heterophils
3000 – 6000 (25 -
45 – 74 %
   lymphocytes
7000 – 17500
16 – 45 %
   basophils
rare
0 – 2%
   eosinophils
0 – 1000
0 – 7%
   monocytes
150 – 2000
4 – 10%
   buffy coat
1% or less
>1%
Plasma color
clear or pale yellow
T.P. (total protein)
gm/dl

13. Arneth count
In human: neutrophil counts based on the lobes of its nucleus
Same blood smear can be used
100 neutrophils are counted and categorized in 5 groups
The number of nuclear lobe increased with age
Normal pattern
Stage 1 N 1 = singled lobed nucleus = 0.5%
Stage 2 N 2 = 2 lobes = 30%
Stage 3 N 3 = 3 lobes = 45%
Stage 4 N 4 = 4 lobes = 18%
Stage N 5 = 5 lobes = 2%
Shift to the left: an increase in the number of first three stages by more than 80% (acute pyogenic infection, haemorrhage)
Shift to the right: an increase in the number of later three stages by more than 20% (pernicious anaemia, vitamin deficiency, aplastic anaemia)

14. Estimation of RBC count and hemoglobin value
Find PCV of your blood sample.
- Use PCV values to estimate the RBC count and amount of Hb in the blood samples. 
   To estimate RBC count
- divide the PCV by 6 and record as value x 106 cells/µl.  
- e.g. If PCV is 42, the RBC count would be 7 x 106 cells/µl
   To estimate hemoglobin value
- divide the PCV by 3 and record as g/dl
      - e.g. if PCV is 42, the hemoglobin value would be 14 g/dl

15. Calculate MCV (mean corpuscular volume)
An average volume of SINGLE RBC expressed in mm3
Use the following formula
PCV per 100 ml of blood
MCV =  10
RBC counts in million/mm3
Normocyte = normal RBC
Macrocyte = increased MCV
Microcyte = decreased MCV

16. Summary take blood samples - 3 ml blood from a volunteer student
- 3 ml blood from the chicken
Make blood smears from each blood samples
Count WBCs
Find PCV of each blood sample
Estimate RBC counts
Calculate MCV

17. Format of the report DVT 1073 Fisiologi Haiwan II Lab work no. ____
Name: ____________ No. Matrik: ______________ Date: ________
Title of the laboratory work.
Aim
Requirements
Procedures
Observation and findings
Discussion/Comments/conclusion

18. Blood smear from an African gray showing nucleated avian
erythrocytes; three heterophils, one eosinophil and two thrombocytes.