Body r4a45

HEMOCYTOMETER

HEMOCYTOMETER Hemo: blood Cyto: cell Meter: measurement Thus, it is an instrument used to count the blood cells.

It includes: a) Neubauer’s slide b) Diluting pipette c) Cover glass

Hemocytometer Chamber

The four corner squares are further divided into sixteen smaller squares and are used for WBC counting.

Four corner squares are meant for WBC counting. Total = 64 small squares

• Central square is divided into 25 medium sized square and are separated by triple line • The medium sized square are further divided into 16 small square(tiny) • The four corner and central square are used for platelet and RBC count.

Counting Rule • Do not count cells touching

▫ Bottom line ▫ Right line  This is to avoid double counting.

Thomas pipette • Consists of graduated capillary tube, mixing bulb with glass bead and aspirating tube • Parts: stem, bulb, rubber tube • Thomas pipette are: WBC pipette and RBC pipette

DIFFERENCES BETWEEN RBC AND WBC PIPETTE RBC pipette

WBC pipette

1)

It has a red bead

It has a white bead

2)

It has graduations upto mark 101

It has graduations upto mark 11

3)

Size of bulb is larger

Size of bulb is smaller

RBC PIPETTE

WBC PIPETTE

For WBC counting 0.5 part of blood is mixed in 10 parts of fluid So, 1 part of blood is in 20 parts of fluid Thus, dilution factor for WBC counting is

20.

FOCUSING • 4X to see the general formation of slide. • 10X for WBC counting • 40X for RBC/Plt. counting

Calculation Cell count=N x dilution factor x depth factor/area counted

Total leukocyte count

White cell count (WBC) • White cell count (WBC) is the total number of leukocytes in a volume of blood, expressed as thousands/µl. • WBC can be done by manual methods or by automated cell counters. • Normal Values: • • • • • •

Newborn 9.0-30.0 x 103/μl 1 week 5.0-21.0 x 103/μl 1 month 5.0-19.5 x 103/μl 6-12 months 6.0-17.5 x 103/μl 2 years 6.2-17.0 x 103/μl Child/adult 4.8-10.8 x 103/μl

Principle of WBCs count test ▫ Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent) at a specific volume in the thoma pipette. ▫ The diluent lyses the erythrocytes but preserves leukocytes and stains the nuceli. ▫ The diluted blood is added to the hemacytometer chamber.

•

Specimen: ▫

•

EDTA- anticoagulated blood

Reagents, supplies and equipment: ▫

White blood cells count diluting fluid Turks' solution which is formed of: ▫ Glacial acetic acid 3 ml ▫ Crystal violet 1 ml ▫ 100 ml distilled water.

Equipment 1.

White blood cells count diluting fluid 2. Thoma white pipette 3. Hemocytometer and cover slip 4. Microscope

haemocytometer chamber

Thoma white pipette

Rubber sucking tube

Hemocytometer

Procedure 1. 2. 3.

4.

Draw the blood up to 0.5 mark in the thoma pipette. Wipe the outside of the capillary pipette to remove excess blood that would interfere with the dilution factor. Holding the pipette almost vertical place into the fluid. Draw the diluting fluid into the pipette slowly until the mixture reaches the 11 mark, while gently rotating the pipette to ensure a proper amount of mixing. Place the pipette in a horizontal position and firmly hold the index finger of either hand over the opening in the tip of the pipette, detach the aspirator from the other end of the pipette now the dilution of the blood is completed

5. 6. 7.

Mix the sample for at least 3 minutes to facilitate hemolysis of RBCs. Clean the hemocytometer and its cover slip with an alcohol pad and then dry with a wipe. Before filling the chamber, discard the first four to five drops of the mixture on apiece of gauze to expel the diluent from the stem.

8. Carefully charge hemocytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

Procedure for counting WBC’s 1. 2.

3.

Under 10 x magnifications, scan to ensure even distribution. Leukocytes are counted in all 4 large squares of counting chamber. Count cells starting in the upper left large corner square. Move to the upper right corner square, bottom right corner square, bottom left corner square and end in the middle square. Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.

Calculations Dilution factor 20 Volume= Area x depth TLC/uL= No of WBCx Correction for Volume X dilution No of large squares (4) = N x 20 x10 4 = N x 50