white blood cell count & differential count.doc

WHITE BLOOD CELL COUNT & DIFFERENTIAL COUNT

Objectives :-

At the end of the practical the student should be able to,1. Identify the apparatus used in counting WBCs.2. Determine the total white cell count using the Neubauer counting chamber.3. Prepare a peripheral blood film4. Identify the different types of white blood cells.5. Describe how to obtain a differential white cell count.6. List the normal values of,

i. Total white cell countii. Neutrophils

iii. Eosinophilsiv. Basophilsv. Lymphocytes &

vi. MonocytesAnd the conditions where these values are abnormal.

Calculation of the Total White Cell Count

Apparatus and materials :-

- The haemacytometer set which contains, Neubauer counting chamber White cell pipette Suction apparatus Cover glasses

- Microscope- Sterile lancet. Surgical spirit and cotton swabs.- Diluting fluid Glacial acetic acid (2%) 1.5 ml

C malachite green 0.5 mlGentian violet aqueous 0.5 mlDistilled water 98 ml

(For this, the blood is diluted with a fluid which lyses red blood cells, but spares white blood cells and nucleated red cells.)

Method :-

0.5 ml

Column of blood

11 ml1 ml

White Bead

Practical No : 04

- Wipe a volunteer's finger with surgical spirit and allow to dry.

- Stab the pulp of the finger with a sterile lancet and wipe away the first drop of blood (do not rub or squeeze finger to assist flow of blood).

- When a big enough drop of blood forms, apply the tip of the pipette to the drop and suck blood upto the 0.5ml mark.

- Draw up the diluting fluid till the 11 mark - till glass bubble is full. Dilution would be 1 : 20.

- Hold the pipette horizontally and rotate it vigorously between finger and thumb to mix.

- Let out the fluid contained within the stem of the pipette.

- Take the Neubauer counting chamber, place it on the stage of the microscope and put the cover glass over it.

- Touch the pipette to the side of the counting chamber, where it projects beyond the cover glass. Known as "charging the chamber".

- The chamber will fill with the diluted blood.

- Now wait two minutes to allow for the cells to settle.

- Observe under low power.

- As WBCs are larger cells and are found in less amounts than RBCs, the larger squares of the counting chamber are used.

Calculation :-

Depth = 1/10 mm

1mm

A B

CD 1mm

1mm

A B

CD 1mm

The volume in one large square (eg. A) = 1 x 1 x 1/10 mm3

= 1/10 mm3

The volume in 4 large squares = 4 x 1/10 mm3

= 4/10 mm3

No. of WBCs in one square = YApprox. No. of WBC in 4 squares = 4Y = Z concentration of WBC = Z / 4/10Blood was diluted 20 times No. of WBC in 1mm3 of undiluted blood = 10Z x 20 / 4

= 50Z

Preparation of a peripheral blood film

- Clean fingertip with surgical spirit and allow to dry- Stab with a sterile lancet and wait for blood droplet to form.- Place the drop of blood on a clean slide.

- Take another clean dry slide and place it vertically, touching the drop of blood.

- Drag this slide slightly backwards and then slide it smoothly forward to spread the drop of blood evenly.

- Allow the slide to air dry for around one minute

- When dry, add 8 - 10 drops of Leishmann stain to the slide and allow to stain for 1 minute. (stain is in alcohol medium).

- Next, add double the amount of drops of water to the slide and mix slightly- Allow to air dry.

- Add a drop of oil and view under oil immersion lens of the microscope

The Differential Leukocyte Count

- The differential count is obtained by counting the number of white cells on the peripheral blood film

- The is done using a chart as follows, this chart has 10 rows.

field N E B L M12345678910

- A field is chosen under the microscope, in the 'body' area of the blood film- 10 white cells are counted, in a field, while filling the above chart according to the

different numbers of white cells contained in the total 10 cellseg. 6 neutrophils, 3 lymphocytes, 1 monocyte

- A similar count is done in 9 more fields, bringing the total number of cells counted, to 100.

- The total number of each type of white cell is then taken as the percentage.

- To obtain the absolute count, these percentages are multiplied by the total white cell count

Head

Body

Tail

Neutrophils

Absolute Count : 2000 - 7500

Percentage : 50 - 70 %

Description : Numerous, small, purple granules

Large, round well defined cell

Lobulated Nucleus (3 – 5 lobules)

Lobules are linked by chromatin strands

Abundant cytoplasm

Increased in :

Bacterial infections

Leukaemias

Severe inflammatory conditions and tissue necrosis

Decreased in :

Viral infections

Pancytopaenia

Typhoid fever

The White Blood Cells

Abundant cytoplasm

Lobulatednucleus (3-5) Pink/blue

granules

Neutrophils

Absolute Count : 2000 - 7500


Description : Numerous, small, purple granules

Large, round well defined cell

Lobulated Nucleus (3 – 5 lobules)

Lobules are linked by chromatin strands

Abundant cytoplasm

Increased in :

Bacterial infections

Leukaemias

Severe inflammatory conditions and tissue necrosis

Decreased in :

Viral infections

Pancytopaenia

Typhoid fever

The White Blood Cells

Abundant cytoplasm


granules

Abundant cytoplasm


granules

Eosinophils

Coarse Red Granules

Red, BilobedNucleus

Size : Equal to Neutrophils

Absolute Count: 40 – 400 /µl

Percentage : 1 – 4 %

Description :

Numerous, closely packed, orange red granules

Large, round cell

Characteristic, bilobed nucleus

Increased in :

Parasitic Infections

- Amoebiasis

- Tropical pulmonary eosinophilia

- Filariasis

- Hook worm infection

Allergic Disorders

- Bronchial Asthma

- Hay fever

and on recovery from acute infection

Size : much smaller than Neutrophils

Absolute Count: 20 - 100 /µl

Percentage : 0.4 %

Description : Very large, deep purple granules in the cytoplasm

Densely arranged

Nucleus is kidney shaped, covered in granules and is difficult to see.

Increased in :

Asthma

Anaphylaxis

Chronic Myelocytic Leukaemia

Decreased in :

Pancytopaenia

Elderly

Basophils Lymphocytes



Percentage : 0.4 %

Description : Very large, deep purple granules in the cytoplasm

Densely arranged

Nucleus is kidney shaped, covered in granules and is difficult to see.

Increased in :

Asthma

Anaphylaxis

Chronic Myelocytic Leukaemia

Decreased in :

Pancytopaenia

Elderly

Basophils Lymphocytes

Large Nucleus

Less cytoplasm




Made in : Bone Marrow

Lymphoid tissueTonsilsSpleenThymusLymph nodes

Description : Very scanty cytoplasm (hardly seen)

Nucleus is large and occupies most of the cell

Increased in :

Chronic infections (Tuberculosis / Syphilis)

Viral infections (HIV / Hepatitis)

Decreased in :

Pancytopaenia

Elderly

Nucleus with a constriction in the center

Granules (Dark)

Contains Histamine, Heparin

Monocytes

Size : equal to lymphocytes



Formed in : Bone Marrow

Description : More cytoplasm than in lymphocytes

Contains very fine, dark granules

Nucleus is kidney shaped

Increased in :

Tuberculosis

Malaria

Typhoid

Has phagocytic properties

Kidney shaped nucleus

Cytoplasm (blue) –ground glass appearance

white blood cell count & differential count.doc

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