WBC manual count using

hemocytometer
 Introduction
 Blood represent about 8%
of total body weight
 It consist of 3 type of
specialized cellular
elements:
 Erythrocytes RBCs
 Leukocytes WBCs

 Platelets (thrombocytes)

 These cells suspended

in complex liquid plasma
 Introduction
 Blood perform two major
function:
1. Transport through the body
 O2 & CO2
 Food molecules (Glucose, lipid, aa)
 Wastes (ex. Urea)
 Hormones
 heat

2. Defense of the body against

infections & other foreign
materials, all WBCs participate
in these defenses
 Introduction

 All various types

of blood cells
are produced in
the
bone marrow
 Introduction
 WBCs (leukocytes)
 Have nuclei
 Consist of:
 lymphocytes with
relatively clear
cytoplasm
 And 3 types of
granulocytes whose
cytoplasm contain
granules
 Introduction

 WBCs count is the count of leukocytes

in a volume of blood
 Expressed as WBCs/mm³
Principle
 The # of WBCs is very large, so its
practical to dilute a sample with
diluting solution (2% Glacial acetic
acid with methylene blue)
 This solution will lyses cytoplasmic
membrane, and leave the nuclei of
WBCs.
Material
1. Blood sample (EDTA anticoagulated
blood or capillary blood)
2. WBCs diluting pipette
3. Diluting solution (2% AA with
methylene blue)
4. Hemocytometer
5. microscope
 The Hemacytometer

 contains 2
Neubauer counting
chamber
 Each chamber
contains:
 *4 WBC counting
squares
 *Each contains 16
squares
The Hemacytometer
The Hemacytometer

0.25mm]

0.20mm]
 Methodology
 With a safety bulb draw blood up to
0.5 marks on WBCs pipette and
complete to 11 with WBCs diluting
solution.
 Mix for 2-3 minute.
 Charge hemacytometer
 Discard the first 4-5
drops
 Place tip of the
pipette at the edge
central platform
 Then let the
hemacytometer to
stand on the bench
for 3-5 minute.
:Count and calculate
:Count and calculate
 Calculation
 Number of cells/mm³=
counted cells in 4 large square x
diluting factor x
volume correction factor.
 Calculation
 The dilution factor=
total volume/sample volume
= 11-1/0.5 =20
 Volume correction factor=
Desired volume/counted volume
1 mm³/ counted volume
 Calculation
 counted volume =
The total volume of the 4 large squares=
= Volume x number of large squares
= (width x length x depth )x 4
= (1mm x 1mm x 1/10 mm) x 4
=0.4mm³
 Volume correction factor=

1 mm³/ counted volume = 1 /0.4 = 2.5

 Calculation
 Number of cells/mm³=
counted cells in 4 large square x
diluting factor x
volume correction factor.

 Number of cells/mm³= n x20 x 2.5

= n x 50
:Example

 If total # of WBCs in 4 squares is 120

 Then the # of WBCs in 1mm³=
120 x 50 =6000
:Normal values of WBCs

 Newborn 9000-30000 cell/mm³

 Adult 4000-11000 cell/mm³
Significance of the test
 Leukocytosis
 Occur as an indicator of body defense
against foreign materials (bacteria,
parasites, toxins)
 Metabolic disorders

 Chemical and drug poisoning

 Acute hemorrhage
Significance of the test
 Leukopenia
 Result from X-ray therapy
 Alcoholism
 Antibiotic therapy
 Typhoid infection
 Measls
 Infectious hepatitis
 TB
 And cirrhosis of the liver
Sources of error

1. Flooding of chamber with excess

sample
2. Failing to count all the cells in the
squares or conversely including
artifacts in the count.