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 pipette3. Diluting solution (2% AA with

methylene blue)4. Hemocytometer5. 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.