Enumeration of White Blood Cell(WBC) count

 

 

Aim: To determine the total number of White Blood Cell in the given sample of blood.

Apparatus: Neubauer's counting chamber, WBC pipette, WBC diluting fluid (Turk's fluid), Spirit, Needle/Lancet, Microscope.

Principle: Sample of blood is diluted with a diluting fluid(Turk's Fluid), which contains Glacial acetic acid 2%( destroys hemolysis RBCs), Gentian violet 1% (stains the nucleus of WBC), the cells are then counted in a  counting chamber and their number in undiluted blood reported as leukocytes /mm3.

    

WBC diluting fluid: Turk's Fluid

         Composition: Glacial acetic acid 2% ( destroys hemolysis RBCs), Gentian violet 1% (stains the nucleus of WBC) distilled water100%.

Procedure:

• Place the counting chamber on the microscope stage. Adjust the illumination and focus the right upper group of 16 WBC squares.
• Observing all the aseptic precaution Clean the fingertip with spirit and  make a deep prick.

              # Draw blood up to 0.5 mark.

              # Then draw WBC diluting fluid up to the mark 11.

              # Mix the blood thoroughly by rolling pipette horizontally with the palms.

              # Discard 2-3 drops.

              # Clean the counting chamber and charge the chamber with prepared solution. Air  bubbles should be avoided.

               #Focus the field under low power objective and count the WBC in all the corner squares, so that the WBC in 64 squares are counted. knowing this value, the total number of WBC in 1 cubic mm of blood can be calculated.

Calculation:

Dilution: The volume of blood taken in WBC pipette is 10(11-1=10). When blood is taken to the mark 0.5, followed by diluting it into 11, the volume of diluted to the mark 11, the volume of the blood is now 10, which contains 0.5-part blood and 9.5-part diluting fluid. This gives dilution of  0.5 in 10 or 1.0 in 20 (so the blood will be diluted to 20 times).

counting of cells: 

                                 W1= 

                                 W2=

                                 W3=

                                 W4=

Number of WBC cell counted: W1+ W2+ W3+ W4 =

The number of WBC /mm3 of undiluted blood=  [( No. of WBCs counted x dilution) / ( Areas counted x  depth of fluid) ]

                              Dilution = 20

                              Area counted = 4 x 1 sq.mm ;

                              Depth of fluid = 0.1 mm

         Value = no. WBCs counted x 50

Result: 

     Number of White cells /cu mm of whole blood = __________.

Normal value: The normal range of WBC count is 4500 - 11000/mm3 of blood.

Pathological variation: 

              Increase in number of WBC is called leucocytosis.

              Decrease in number of WBC is called leucopenia.

Leucocytosis:

Physiological causes of leucocytosis:

1. After exercise.
2. Severe pain.
3. Excitation.
4. Injection of adrenaline.
5. In infants, during pregnancy and during menstruation.
6. Due to cold and exposure to UV radiations.
7. High temperature.

Pathological cause of  leucocytosis:

1. Leukemia.
2. Hemorrhage.
3. Inflammatory conditions.
4. Acute and chronic infections.
5. Allergic conditions.

Leukopenia:

1. Typhoid.
2. Viral diseases.