Experiment 1 - Differential Count

Importance
a. Differential counting is used for checking the accuracy of a white blood cell count.
b. It determines the percentage of each type of white blood cell present in the body.
c. Differential count can also detect the increase of a specific white blood cell that can help a
physician in making a proper diagnosis.

Prelaboratory Discussion

Differential count is a laboratory procedure used for the estimation of WBCs in the blood. It is
done as a double check for white blood cell count. Accurately identifying white blood cells is crucial to
differential count due to the cells not being properly distributed in a field. The smear used should be well
made with the tail being smooth. The widely used technique for smearing is the manual wedge technique.
An even, gentle pressure is used with a consistent angle. A poorly done smear will show increased
number of lymphocytes in the middle of the smear thereby giving the lymphocyte count a false decrease
and it also gives an increased amount for neutrophils, large abnormal cell, and monocytes at the tail of
the smear.

Procedure:

1. Inspect the smear under LPO. Locate the thin end of the smear where there is no overlapping of
erythrocytes.
2. Switch to HPO. Check that the white cells are evenly distributed.
3. Switch to oil immersion. Identify and count 100 consequtive leukocytes and record each cell type
separately on the differential counter. Begin at the thin end of the smear and count the white cells
observed as the slide is moved in a vertical direction. When near the edges of the smear, move
the slide horizontally for a distiance of about 2 fields, then proceed vertically back across the
smear. Continue this movement until 100 leukocytes have been counterd and classified.
4. If the WBC count is between 20,000 and 50,000 per cu mm of blood, count and classify 300
leukocytes. When the count is greater than 50,000 per cu mm of blood, count and classify 500
leukocytes.
5. The number of each type of leukocytes is expressed as a percent of the total number of white
cells counted. Absolute values may be counted by multiplying the percent value by the total
leukocyte count.

**Counting the leukocytes

6. Begin to count near the end of the smear, just where the red cells are beginning to overlap.
7. Examine the strip of the film moving from one field to the next systematically. Record the type of
leukocyte using the cell counter seen in each field.
8. Count a total of 100 leukocytes.

**Examination of the leukocytes

a. Note the shape of the leukocyte and its size in comparison with a red cell.
b. Note the shape of the nucleus and its size in relation to the total area of the cell;
Example: round, lobed, or indented
REFERENCE VALUE:

Post laboratory questions

a. Possible sources of error in Differential count?
- too thick smear
- improper visualization of a blood cell
- inability to identify the blood cell observed

b. Give the cause of the decrease and increase of monocytes.

c. Give the causes of the Increase and decrease of lymphocytes.

d. Why should the angle be lowered for patients with Polycythemia Vera?
- It is to make sure that the smear is not short and thick.

e. How can a too thin smear affect the differential count?

Experiment 2 Platelet Count- Direct method

Importance
A. to diagnose thrombocytopenia or thrombosis
B. Helps in the diagnosis of bleeding disorders,

Discussion

Procedure:

1.Diluting the Blood

a. The capillary bore of the RBC pipette is rinsed with Rees-Ecker diluting fluid. All excess
fluid should be removed from the pipette before blood is drawn into the pipette. This step
will ensure plateleet will not adhere on the walls of the capillary bore of the pipette.
b. Draw the blood into the pipette up to the 0.5 mark. If excess blood was drawn, adjust the
blood level to the 0.5 mark with NSS moistenedcotton. Clean the stem of the pipette first
before any adjustment will be made.
c. The blood should be dilulted immediately with the Rees-Ecker solution. Make a 1:200
dilution by filling the pipette with the solution up to 101 mark. Two pipettes should be
used simultaneously, and each should be used to charge one side of the counting
chamber.
d. After the Dilution, the pipettes should be shaken immediately for at least a minute. This
step will prevent platelet clumping and will ensure accurate counting.

2. Charging the counting chamber

a. After mixing, discard the first 5 drops from each pipette. (Note: Do not induce or blot the
tip of the pipette by any absorbent material to facilitate the process. The drop is induced
by gravity alone.). Each side of the hemocytometer should be charged with a different
pipette.
b. After charging the chambers, the hemocytometer should be kept in a covered container
for about 10-15 minutes with wet gauze or cotton pads beneath the hemocytometer to
prevent evaporation, this step will allow the platelets to settle aid in accurate counting
later on.

3. Counting the platelets

a. Carefully place the hemocytometer on the microscope stage so as not to disturb the
platelets. Count platelets in 5 RBC squares in the center of the counting chamber. Both
sides of the counting chamber should be mounted and results averaged. Using HPO
count all the platelets.
9

REFERENCE VALUE: 150-400 x 10 /uL

FORMULA: Platelet count= number of cells counted x Dilution Factor

Depth of counting chamber
POST LABORATORY QUESTIONS
 a. Why is the pipette inverted for 60 seconds?

b. Give 5 cause of thrombocytopenia

c. Give 5 causes of thrombosis

d. Compute for the platelet count

Number of cells counted=790
Dilution factor=0.2
Depth of the counting chamber= 0.1

e. What is the appearance of the platelets under the microscope?

Experiment 3- Platelet indirect Method

Importance
Discussion

Procedure:
1. Make a well-made blood smear and stain with Wright’s-Giemsa stain.
2. Focus the slide using LPO. Examine the smear for areas that is suitable for counting. Care must
be taken to stay in an appropriate area of the slide. The RBCs should barely be touching one
area of the estimate
3. Using the HPO check the area of the smear for suitability for counting the platelets.
4. Shift the objective to 100x magnification. Examine the strip of the film moving from one field to the
next systematically.
5. Count and record the platelets seen in ten consecutive fields