David Duncan 1015710 Haematology LAB 2 Due: Oct-11-2019

Title: Manual White Blood Cell Count

Aim: To enumerate the total number leukocyte in a given blood sample

Principle:
Whole blood is diluted with a dilution fluid which contains 3% acetic acid solution and this
results in hemolysis of mature RBCs and which leaves the WBCs to be counted unhindered. The
dilution is mixed well and left to stand for 5 minutes then placed on a hemacytometer. The cells
are allowed to settle and then are counted in specific areas of the hemacytometer chamber under
the microscope. The number of leukocytes are calculated per µL (x 109/L) of blood.

Clinical Significance:

A high WBc count is known as leukocytosis which may be as a result of acute infections,
especially of a bacterial nature. An increase is also observed during pregnancy and cancers such
as leukemia. A below average count is known as leukopenia which is indicative of viral
infections, malaria pernicious anemia and many other conditions. A high WBCs count may also
be as a result of stress, exercise or anxiety.

Sample requirements: anticoagulated whole blood use this for reference

Materials:

 Hemocytometer chamber

 Light microscope

 WBC pipette

 WBC diluting fluid

 Capillary tubes

 Gauze

 70% alcohol

Procedure:
1. 380ml of WBC diluting solution was measured and placed into a clean test tube

2. 20ml of EDTA was mixed well and added to the WBC diluting fluid
David Duncan 1015710 Haematology LAB 2 Due: Oct-11-2019

3. The diluted blood sample was allowed to stand for 5 minutes while the counting chamber was
assembled

4. The diluted blood sample was remixed after the 5 minutes had expired

5. The counting chamber was filled with a capillary tube

6. The white blood cells were allowed to settle to settle by leaving the counting chamber
undisturbed for 2 minutes

7. The counting chamber was scanned using the x10 objective and ensuring that the cells had
no more than a 10 cell variation ( checked for even distribution).

8. All the white blood cells were counted

Only cells that touched the left and upper outside lines were counted while the ones that touched
the right and lower outside border were ignored.

9. The total white blood cell count was calculated using the formula

Total WBC count/mm3 = # of wbc counted x dilution factor

Chamber depth x area of counted chamber

Where the dilution factor= 20

Camber depth = 0.1mm

Area of counted chamber = 4mm

Normal range: 4.5 to 11.3 × 109/L.

Quality Control:

Periodic running of commercial quality control materials with defined control limits should be
done. The workload of the laboratory will determine how frequent this is done.

Limitations:

Cannot be performed on blood that has been diluted for more than 3 hours

Is liable to human error and biases

A prolonged stay in the hemocytometer will cause drying out

Should not be done in areas of low humidity for an extended period of time without moisture
support surrounding it.
David Duncan 1015710 Haematology LAB 2 Due: Oct-11-2019

The test is not independent of physiological occurrences of the patient. Eg Someone who does
physical exercise will have a high WBC although the patient does not have a disease

Sources of error:

Excessive trauma to tissue while collecting blood results in cell clumping and a false low count
will ensue

If nucleated RBCs are present a falsely high WBC count can be a consequence since the
nucleated RBCs can be mistaken for WBCs.

Dirty slides with microscopic debris can appear as WBCs and even impede the counting process
by overlapping true WBCs.

Overcharging and undercharging the hemocytometer (i.e providing too much or too little sample)
can affect readings

Too much light during microscopy will result in poor visibility of the sample

Calculations: Total WBC count/mm3 = 100 x 20/0.1X4 =5.0X109/mm3

References

Turgeon ML, Ringsrud KM, Linné JJ. Linne & Ringsrud’s clinical laboratory science: the basics
and routine techniques. 6th ed. Maryland Heights, MO: Mosby Elsevier; 2012. 659 p.

Manual cell counting V.S. Automated cell counting [Internet]. Chemometec - the science of cell
counting using Automatic Cellcounters. 2015 [cited 2019 Oct 11]. Available from:
https://chemometec.com/manual-cell-counting/

ch07LEUK.pdf [Internet]. [cited 2019 Oct 11]. Available from:

http://wps.prenhall.com/wps/media/objects/684/700987/ch07LEUK.pdf
David Duncan 1015710 Haematology LAB 2 Due: Oct-11-2019