TOTAL LEUKOCYTE
COUNT
Dr. Karee Angu
Senior Resident
Learning Objectives:
1. Describe the relevance of doing white cell count.
2. Identify the WBC pipette and fill it with blood and diluents in a single
attempt.
3. List the composition of diluting fluid and function of each constituent.
4. Charge the improved Neubauer’s counting chamber with WBC pipette
and count WBCs accurately.
5. Calculate WBC in the sample of blood.
6. Interpret results.
7. Enumerate sources of error and precautions taken during the practical
Principle
A measured quantity of blood is
mixed with a diluting fluid, which
is isotonic with blood.
The diluting fluid further destroys
the RBCs and also stains the nuclei
of WBCs
Diluting the blood → known
degree of diluting fluid → then
counting the cells (N) in the
diluted blood contained in a
known volume
Final result(count/unit volume)=
(N x dilution factor) ÷ Volume
Apparatus required:
• Microscope
• Hemocytometer box (Improved Neubauer’s
chamber, WBC pipette)
• Lancet
• Diluting fluid (Turk’s)
• Cover slip.
Turk’s Fluid
• Glacial acetic acid (3ml): Destroys RBCs,
work as fixative, impart refraction to the
cells
• 1% Gentian violet stain or aqueous
methylene blue (1ml): Stain nuclei of WBC
• Distilled water up to 100ml
Procedure
1. Keep all equipment ready
2. Filling the capillary pipette
3. Charging the chamber
Keeping the equipment ready
• Ensure that improved Neubauer’s chamber, cover slip and lenses of
the microscopic are clean.
• The lines in the improved Neubauer’s chamber should be clearly
visible under microscope.
• WBC pipette should be dry and patent.
• Take about 3-5 ml Turk’s fluid in a watch glass
Filling the capillary pipette
• Prick under all aseptic condition → discard the first drop
• Pipette between lips & keeping pipette → 40֯ to horizontal → tip within the
edge of the drop
• Gently suck & draw blood up to 0.5 mark
• Remove the pipette → clean the outer surface with a cotton swab by wiping
it towards the tip. Do not touch the bore at the tip or blood will be pulled out
• Immerse the tip in diluting fluid → suck up to 11 mark.
• Keep the pipette horizontal → knot at the rubber tube → gently mix the
blood and diluting fluid by rolling it between palms of the hands
• Charge the chamber after discarding the fluid from the stem of the pipe
Charging the chamber
Sample Introduction points
Repeat the procedure in case of Overcharging and Undercharging
After charging the chamber correctly → allow the fluid to settle for a period of three
minutes →focus the chamber under the Low power of microscope
• WBCs appear to be regular
round shape, dark stained
nuclei (black dots) and
refractile bodies (halo around). WBC WBC
WBC WBC
Rules of counting:
The white blood cells present on the left and upper lines of a WBC square as inside of that
square and those on right and lower line as out of that square
Among the triple lines central line should be considered as main line/boundary for counting
of cells.
Volume accommodated in
counting squares -WBC
1mm
1mm
Volume accommodated in WBC
squares=
(1mm2 x 4)x1/10 mm= 0.4 mm3
Dept = 0.1mm
3mm
Dilution factor
Dilution of blood occurs in bulb only
WBC Pipette:
• Volume of bulb = 11-1=10
• 10 volume bulb has 0.5 volume blood
• Dilution factor= Volume of bulb/ volume of blood = 10/0.5 = 20
Calculation
Volume of 4 WBC Square = 0.4 mm3
• Let number of cell in 0.4 mm3 of diluted blood = N
• Let number of cell in 1 mm3 of diluted blood = N / 0.4
• Let number of cell in 1 mm3 of undiluted blood = N / 0.4 × 20
= N ×50 cells
Sources of Error
• Field error – variation in the distribution of cells on the counting chamber
• Chamber error – due to inaccuracy of calibration and of the method of charging
the chamber
• Pipette error - due to inaccuracy of calibration and of the method of filling the
pipette and mixing.
Precautions
• Apparatus should be dry and thoroughly cleaned.
• Give bold prick under aseptic conditions and do not squeeze.
• Wipe off the first drop.
• Take blood exactly up to 0.5 mark.
• Pipette should be free of air bubbles.
• Discard the first two drops from the pipette before charging.
• Do not overcharge or under charge, after charging the chamber allows the
cells to settle for 1-2 min.
• Avoid counting the same cells twice.
• If cells are not uniformly distributed, recharge the chamber.
Significance:
Normal count:
• Adults: 4000-11000 /mm3
• Newborns: 10000-25000 /mm3
• Infants: 6000-18000 /mm3
• Children: 5000-15000 /mm3
Increase above normal is called leukocytosis (>11000 /mm3) and below
normal (<4000 /mm3) is called leukopenia.
Leukocytosis (>11000 mm3)
PHYSIOLOGICAL CAUSES: PATHOLOGICAL CAUSES:
• Normal infants • Acute infection with
• Food intake and digestion pyogenic bacteria
• Physical exercise • Post operative
• Pregnancy • Myocardial infarction
• Burns
• Amebic hepatitis.
• Malignancies
Leukopenia <4000 mm3
PHYSIOLOGICAL CAUSES:
• Exposure to extreme cold
PHYSIOLOGICAL CAUSES:
• Infection with non-pyogenic organisms: Typhoid and paratyphoid fevers, and sometimes in
protozoal infection like malaria.
• Viral infections: Influenza, mumps, smallpox, AIDS (Acquired immunodeficiency syndrome).
• Drugs: Chloramphenicol, sulphonamides, aspirin, penicillins, cyclosporins, phenytoin, etc.
• Repeated exposures to X-rays and radium
• Chemical poisons that depress bone marrow: Arsenic, dinitrophenol, antimony
• Malnutrition Preleukemic stage of leukemias may show leukopenia.
Leukocytosis is an increase in Leukemia is a cancerous disease Leukemoid reaction: It is an
TLC count above 11,000/mm3, of the blood forming tissues. extreme elevation of TLC
irrespective of the types of Uncontrolled proliferation of one above 50,000/mm3.
cells involved. or more of the various The causes include severe
• The count usually does not hematopoietic cells occurs and chronic infections, especially in
exceed 20–25,000/mm3 these progressively displace the children, severe hemolysis,
• There are no immature cells normal cellular elements. malignant growths (cancer of
in the circulation. breast, lung, kidney).
Leukemia results in appearance
of abnormal and immature cells
visible as a very high
leukocytosis (40–50,000/mm3 or
even a few lacs). in the
peripheral blood
Questions:
1. Give normal range of total leucocyte count?
2. Write the causes for increase and decrease leucocyte count?
3. Differentiate leukocytosis, leukemoid reaction and leukemia?
4. What is clinical significance of this practical?
THANK YOU