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).

Differential WBC Count(Differential Leucocyte Count

Requirements:

Differential WBC Count

• Glass slides • Leishman stain• Microscopes • Disposable needles• Vials containing anticoagulants• Methylated-spirit• Staining rack

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

Differential WBC Count

Note: Different staining methods are possible. (Wright’s stain, Giemsa stain, Diff Quick stain; all these are known as Romanovsky-type stains).

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.

Differential WBC Count

Preparation of the blood smear: Wedge method 1

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.

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.

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1.     Place fix air-dried smear across the staining rack2. Drop a number of Leishman stain to cover the entire blood smear3. 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.5.     Do not allow the stain-DW mix to overflow.6. Blow air gently on to the slide to ensure proper mixing of stain with DW.7. Allow to remain for 10 min.8. After 10 min, pour stain water mix into the tray and wash the slide under 

running tap water.9. Water should not fall directly over the smear.10. 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.     

Staining (Leishman stain)(Leishman stain powder  0.15 g

 + 100 ml acetone free methyl alcohol)

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.

2. 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, 

3. Identify and count different types of the cells. Use the following  characteristics.

4. 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.

Neutrophils nucleus………....................dark blue cytoplasm....………………….pale pinkEosinophils nucleus........…………………. blue cytoplasm.....…………………blue granules........................... red to red/orangeBasophils nucleus.........…………………purple or dark blue granules........…………….…..dark purple/blackMonocytes nucleus(lobated)………….…violet cytoplasm........……...……...sky blue

Differential WBC Count

Characteristics of different types of the white cells

Monocyte

Thrombocyte

 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

Parameter Chicken Man

RBC (106/mm3) 2.5 - 3.5  4.9 – 5.5PCV (%) 22 – 35 42 - 52Hemoglobin (g/dL) 7 – 13  14 – 18 MCV 90 – 140 fL 78 – 95 mm3

MCH  33 – 47% 28 – 33 pg/cellMCHC  26 – 35% 32-36 g/dLWBC (cells/mm3) 12000 – 30000   4.3-10.8 × 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 yellowT.P. (total protein) 2.5 - 5.5  gm/dl 

Avian normal blood values (Gallus domesticus and man)

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 5 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)

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

Calculate MCV (mean corpuscular volume)

-  An average volume of  SINGLE RBC expressed in mm3

-  Use the following formula

             PCV per 100 ml of bloodMCV = --------------------------------       10             RBC counts in million/mm3 

Normocyte = normal RBCMacrocyte = increased MCVMicrocyte = decreased MCV

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

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

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

thrombocytes.