preparation of peripheral blood flim

MADE BY: MADHUR KR SEJWAL ROLL NO 41

Value of blood films:

• Examination of thin blood films isimportant in the investigation andmanagement of infections and parasiticdiseases like malaria .

• A blood film report can provide rapidlyand at low cost, useful information abouta patient’s condition.

Three basic steps to make blood film:

1. Preparation of blood smear.

2. Fixation of blood smear.

3. Staining of blood smear.

Two methods may be used to make blood smears:1. The cover glass smear.

2. The wedge smear.

• Specimen:

Smears should be made within 1 hour ofblood collection to avoid distortion of cellmorphology

Blood smears can also be made fromfinger stick blood directly onto slide.

• Equipment

•Spreaders• Clean slides

• Blood capillary tube or micropipette 10 µL

1. Place a drop of blood, about 2 mm in diameterapproximately an inch from the frosted area of theslide.

2. Place the slide on a flat surface, and hold the narrowside of the non frosted edge between your left thumband forefinger.

3. With your right hand, place the smooth clean edge of asecond (spreader) slide on the specimen slide, just infront of the blood drop.

4. Hold the spreader slide at a 30° angle, and draw it backagainst the drop of blood.

6. Allow the blood to spread almost to the edges ofthe slide.

7. Push the spread forward with one light, smooth,and fluid motion. A thin film of blood in the shapeof a bullet with a feathered edge will remain on theslide.

8. Label the frosted edge with patient name, ID# anddate.

9. Allow the blood film to air-dry completely beforestaining. (Do not blow to dry. The moisture fromyour breath will cause RBC artifact.)

Two types of blood film

eg : for malaria parasites

Thick Blood Smear – use to determine if parasite is present

Thin Blood Smear – use to confirm the Plasmodium species present

Romanowsky stains are universally employed for routine staining of blood films.

It depends on two components, namely, azure B(trimethylthionin) and eosine Y(tetrabromo-fluorescein).

The original Romanowsky combination was polychrome methylene blue and eosine.

The presence of basic grouping on the haemoglobin molecule results in its affinity for acidic dyes and its staining by eosin. The nucleic acids uptake of the basic dye azure B.

LEISHMAN’S STAIN

• Leishman staining is used for staining blood in microscopy and

its purpose is to both identify and differentiate trypanosomas,

leucocytes and malaria parasites.

GIEMSA STAIN

• The most dependable stain for blood parasites, particularly in

thick films, is Giemsa stain containing azure B

COMPONENTS:

1.Methanol : fixes cells to slide

2.methylene blue stains RNA,DNA

blue-grey color

3.Eosin stains hemoglobin, eosin granules

orange-red color

pH value of phosphate buffer is very important

COMPONENTS:

1. METHYLENE BLUE

2. AZURE B

3. SODIUM SALT OF EOSIN Y

1. A good blood film preparation will be thick atthe drop end and thin at the opposite end.

2. The blood smear should occupy the centralportion of the slide.

3. The blood smear should not touch the edges.except for point of application.

4. Should be margin free

Is determined by:

1. The angle of the spreaderslide. (the greater the angle,the thicker and shorter thesmear).

2. Size of the blood drop.

3. Speed of spreading

1. Drop of blood too large or too small.

2. Spreader slide pushed across the slide in a jerkymanner.

3. Failure to keep the entire edge of the spreaderslide against the slide while making the smear.

4. Failure to keep the spreader slide at a 30° anglewith the slide.

5. Failure to push the spreader slide completelyacross the slide.

6. Irregular spread with ridges and long tail: Edge ofspreader dirty or chipped; dusty slide.