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Manual White Blood Cell (WBC) Count: Unit Intended Learning Outcomes

This document provides instructions for performing a manual white blood cell count. Key steps include: 1) Making a 1:20 dilution of blood sample in diluting fluid. 2) Charging a hemocytometer, allowing cells to settle for 10 minutes, and counting cells under the microscope. 3) Averaging cell counts from both sides of the hemocytometer and using the dilution factor and area counted to calculate the WBC count in cells/mm3.

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482 views

Manual White Blood Cell (WBC) Count: Unit Intended Learning Outcomes

This document provides instructions for performing a manual white blood cell count. Key steps include: 1) Making a 1:20 dilution of blood sample in diluting fluid. 2) Charging a hemocytometer, allowing cells to settle for 10 minutes, and counting cells under the microscope. 3) Averaging cell counts from both sides of the hemocytometer and using the dilution factor and area counted to calculate the WBC count in cells/mm3.

Uploaded by

Maria Clara
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ACTIVITY 11

MANUAL WHITE BLOOD CELL (WBC) COUNT

Unit Intended Learning Outcomes

At the end of the experiment, the students (should be able/are expected to):

Disinfect properly the different areas of the laboratory.


Wear complete and proper personal protective equipment.
Perform correctly the dilution used for WBC count.
Make a moist chamber properly.
Charge diluted blood correctly.
Count accurately the number of WBCs in the correct ruled area.
Calculate accurately the WBC count expressed in conventional and SI unit.
Estimate correctly the WBC count from a blood smear.
Perform correctly the proper waste segregation and disposal.

Pre-laboratory Discussion

Normal white blood cells (WBC) or leukocytes perform important physiological functions. They function
mainly as the body’s line of defense, thus imparting immunity to the system. Therefore, the decrease or
increase in circulating WBC may indicate physiological condition in the body. Hence, the amount of
circulating WBCs, as determined by leukocyte count, is useful in diagnosis and monitoring infections and
other diseases.

Diluting Fluid
Whole blood is in an acid reagent which hemolyzes the red cells (but not the nucleus of nucleated red
cells), leaving the white blood cells to be counted.

1. Turk’s solution
2. Glacial acetic acid

CORRECTED LEUKOCYTE COUNT


WBC count should be corrected if more than 10% of nucleated red cells are present upon examining a
stained blood film.
𝑈𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑋 100
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
𝑁𝑜. 𝑜𝑓 𝑁𝑅𝐵𝐶 + 100

Where, the number of NRBC is the number of nucleated red cells which are counted during the
enumeration of 100 leucocytes in the differential count

LEUKOCYTOSIS
An abnormally high WBC count (above 11,000 cells/mm3) which may indicate acute infections, metabolic
disorders, leukemia, hemorrhage, poisoning by drugs or chemicals, stress, menstruation, and strenuous
exercise.

LEUKOPENIA
A decrease in the white cell number (below 4,000 cells/mm3) may indicate viral infections, typhoid fever,
measles, infectious hepatitis or cirrhosis, tuberculosis, excessive antibiotic or X-ray therapy, bone marrow
failure (as in aplastic anemia), and anaphylactic shock. A person with leukopenia lacks the usual protective
mechanisms we would expect to find in the immune system.

Teaching and Learning Activities

• Pre-Analytical Phase

1. Disinfect the working area using 10% sodium hypochlorite.


2. Wear complete and proper PPE.

• Analytical Phase

Reagents, Supplies and Equipment

1. EDTA anticoagulated blood


2. Hemocytometer (Improved Neubauer counting chamber and cover slip)
3. WBC pipette
4. Aspirator
5. Diluting fluid
6. Pipette rotator
7. Beaker
8. Microhematocrit tube
9. Microscope
10. 70% isopropyl alcohol
11. Filter paper
12. Petri dish
13. Applicator stick
14. Timer
15. Tally counter
Procedure

MANUAL WHITE BLOOD CELL (WBC) COUNT


A. Preparation of Diluted Blood
1
1. Make a 1:20 (20) dilution of blood and diluting fluid.
a. Aspirate a well-mixed blood or blood from a freely flowing capillary puncture to the
“0.5” mark of the WBC pipette. Wipe off excess blood using a clean piece of tissue
paper. Make sure that the tissue paper does not touch the tip of the pipette bore.
b. Place the diluting fluid in a beaker. Aspirate diluting fluid up to the "11" mark.
2. Hold the pipette horizontally and role it with both hands between finger and thumb to
ensure proper mixing of blood and diluting fluid.
3. Allow the dilution to sit for 10 minutes.

Note:
100 cells is a reasonable and practical figure for visual counts. When the leucocyte count
is low (below 4.0 × 103/mm3), it is advisable for greater accuracy to use a 1:10 dilution,
i.e., take blood to the “1” mark of the pipette and diluting fluid to the “11” mark.

B. Charging
1. Clean hemocytometer with alcohol and dry well; place coverslip on top of the
hemocytometer.
2. Mix the diluted sample by inversion and discard a few drops of the sample.
3. Fill a plain microhematocrit tube with the sample.
4. Charge both sides of the hemacytometer by holding the microhematocrit tube at a 45-
degree angle and touching the tip to the coverslip edge where it meets the chamber floor.
5. After charging the counting chamber, place it in a moist chamber for 10 minutes before
counting the cells to give them time to settle. Care should be taken not to disturb the
coverslip.
a. A moist chamber may be made by placing a piece of damp filter paper in the bottom
of a Petri dish. An applicator stick broken in half can serve as a support for the
chamber.
6. While keeping the hemacytometer in a horizontal position, place it on the microscope
stage.

C. Manual WBC Count


1. Locate the ruled area for WBC counting (4 large corner squares) by focusing the
microscope under LPO. Once located, shift to HPO for actual counting.
2. Count all the cells in the 4 large corner squares. Cells that touch the top and left lines
should be counted; cells that touch the bottom and right lines should be ignored. Record
the count on each large corner.
3. Repeat the count on the other side of the counting chamber. The difference between
totals for the two sides should be less than 10%. A greater variation could indicate an
uneven distribution, which requires that the procedure be repeated.

D. Computation
1. Average the two sides. Using the average, calculate the WBC count.

𝑐𝑒𝑙𝑙𝑠 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑋 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟


𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 ( )=
𝑚𝑚3 𝑎𝑟𝑒𝑎 (𝑚𝑚2) 𝑋 𝑑𝑒𝑝𝑡ℎ (0.1 𝑚𝑚)
or
𝑐𝑒𝑙𝑙𝑠 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑋 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑋 10
𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 ( )=
𝑚𝑚3 𝑎𝑟𝑒𝑎 (𝑚𝑚2)
or
𝑐𝑒𝑙𝑙𝑠 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑋 20
𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 ( )=
𝑚𝑚3 (4 𝑚𝑚2)(0.1)
or
𝑐𝑒𝑙𝑙𝑠
𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 ( ) = 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑋 50
𝑚𝑚3

Where, 10 is the reciprocal of the depth (0.1 mm)


20 is the dilution factor

WHITE BLOOD CELL (WBC) COUNT ESTIMATE


1. Choose a portion of the blood smear where there is only slight overlapping of the RBCs.
2. Count 10 fields under HPO (40X).
3. Take the total number of white cells and divide by 10 to get the average.
4. Multiply the average number of white cells and multiplying by the leukocyte estimate
factor 2,000.
𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑊𝐵𝐶 𝐶𝑜𝑢𝑛𝑡 = ( ) 𝑋 2,000
10 𝑓𝑖𝑒𝑙𝑑𝑠

• Post-Analytical Phase

1. Segregate and dispose all infectious and biohazardous waste in their proper waste bins.
2. Disinfect the working area using 10% sodium hypochlorite.
3. Wash laboratory equipment before storage.
4. Remove all used PPE after the activity.

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