Peripheral

Blood SmearDR. ANWAR H. SIDDIQUI

Compound Microscope 1

Parts of the Compound

Microscope Tube

It is cylindrical tube through which the light from an illuminated object traverses enroute its formation of an image. It consists of an outer body tube and an inner draw tube which can slide inside the outer, eye piece and lower part carrying a revolving nose piece

Eye Piece

It comprises two co-axial lenses fixed to the ends of a cylindrical tube, which can slide into the upper open end of the draw tube. It is usually available in magnifications of 12.5X and 10X.

Nose Piece

It is a plate pivoted to the lower end of the tube. It is fitted with objectives, any of which can at a time be brought in line with the optical axis by rotating the nose piece. The objectives are so fixed that the magnification will increase as the user turns the revolving nose piece clockwise.

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Objectives

These are cylindrical in shape and fitted with coaxial lenses. Three objectives are

provided:

(a) Low power objective (magnification 10X)

(b) High power objective (magnification 40X)

(c) Oil immersion objective (magnification 100X)

Adjustment Knobs

for coarse and fine adjustment.

Mirror

it is plane on one side and curved on other. The concave side is used to reflect light from a point source while the plane side is used in diffuse light

Iris Diaphragm

It controls the amount of light reaching the condenser.

Condenser

It is system of lenses which focuses light from the source on the object. It is raised fully while using the oil immersion lens and lowered fully while using the low power objective.

Arm

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Image Formation in the

Compound Microscope 5

PREPARATION OF BLOOD FILMS

Blood films can be made from anticoagulated, or finger-prick blood.

PROCEDURES

Take 3 or 4 clean glass slides,the surface of which should be even and smooth and place it on your work-table,.

Prick your finger and allow a medium-sized drop of blood to form on the finger-tip.

Lift a slide from the table, holding it along its long edges. Then touch its center, about 1 cm from the narrow end, to the blood drop.

Now grasp the long edges of a second slide, the “spreader”, between thumb and fingers of your right hand,.

Place the narrow edge of the spreader on the first slide, at an angle of 40°, just in front of the blood drop. Pull the spreader back gently so that it touches the front of the blood drop. Hold it there, (or move it a little from side to side) till the blood, moving along the junction of the two thus distributing the blood evenly across its width.

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Steady the first slide with your left hand, and maintaining a

light but even pressure and 40° angle ,move the spreader

forwards to the left in a single, smooth, fairly fast gliding

motion, pulling the blood behind it in the form of a thin smear.

Make as many trials as possible to get acceptable films,

keeping in mind the features of an ideal blood smear, as

described below.

Dry the film by waving the slide in the air (Do not try to blot-dry

the film).

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Features of an Ideal Blood Film The blood film should occupy the middle two-thirds

(about 5 cm) of the slide, with a clear margin of about 2

mm on either side.

It should be tongue-shaped, i.e. broad at the head

(starting point), and taper towards the other end, but

without any ‘tails’.

It should be translucent, uniformly thick throughout, with

no vacant areas, striations (longitudinal or transverse), or

‘granular’ areas.

It should be neither very thick nor very thin (this can be

learned only with practice). A thin film looks faintly pink

against a white surface, while

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Fixing and Staining of Blood

Films Fixation is the process that makes the blood film and its cells

adhere to the glass slide. It also preserves the shape and chemistry of blood cells as near living cells as possible.

Staining is the process that stains (colors) the nuclei and cytoplasm of the cells. Both these purposes are achieved by the Leishman’s stain.

LEISHMAN STAIN

It contains a compound dye—eosinate of methylene-blue dissolved in acetone-free methyl alcohol.

i. Eosin. It is an acidic dye (negatively charged) and stains basic (positive) particles—granules of eosinophils, and RBCs a pink color.

ii. Methylene-blue. It is a basic dye (positively charged) and stains acidic (negatively charged) granules in the cytoplasm, nuclei of leukocytes, especially the granules of basophils, a blue-violet color.

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iii. Acetone-free and water-free absolute methyl alcohol. The methyl alcohol is a fixative and must be free from acetone and water. It serves two functions:

a. It fixes the blood smear to the glass slide. The alcohol precipitates the plasma proteins, which then act as a ‘glue’ which attaches (fixes) the blood cells to the slide so that they are not washed away during staining.

b. The alcohol preserves the morphology and chemical status of the cells.

The alcohol must be free from acetone because acetone being a very strong lipid solvent, will, if present, cause crenation, shrinkage, or even destruction of cell membranes. This will make the identification of the cells difficult. (If acetone is present, the stain deteriorates quickly).

The alcohol must be free from water since the latter may result in rouleaux formation and even hemolysis. The water may even wash away the blood film from the slide.

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Procedure of Fixing And

Staining Pour Leishman’s stain on slide enough to cover the

smear. Leave it about for 2 minute.

Now add few drops of buffer solution. Mix the stain and

buffer slowly by gently blowing air intermittently. Now

leave for 10 minutes(in summers put it under moist

chamber).

Wash the slide with tap water gently till the film has a

pinkish tinge.

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Assessment of Stained Blood

Smears Take an assessment of all the blood films. Examine with

naked eye first, and then under low and high

magnifications.

Choose the best stained films for cell counting.

Make sure that you can identify all the cells with

certainty.

Ensure that you are examining the blood smear side of

the slide. Hold the slide in bright light and tilt it this way

and that to see if there are any reflections. The clean side

shows reflections, while the side which has the blood

smear appears dull and does not show any reflections.

(You may also scratch the margin of the smear with a pin).

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Features of a Well-stained

Blood SmearNaked eye appearance.

The smear appears translucent and bluish-pink when

seen against a white surface, its thickness being uniform

throughout. (It is assumed that an ideal blood film has

been stained).

Under low magnifications (100 x).

The red cells appear as dots, uniformly spread-out in a

single layer. The WBCs cannot be differentiated.

Under high magnification (450 x or 400 x).

The red cells are stained dull orange-pink and show a

central pallor (due to biconcavity) which, if wide, may give the appearance of rings. The WBCs, with their nuclei

deep blue-violet, lie unevenly here and there among the

red cells. The platelets occur in small groups.

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Naked eye appearance

Under low power of compound microscope

Under high power of compound microscope

Identification of Various Blood

Cells1. The red cells. (RBC)

Stained orange-pink, the red cells appear as numerous,

evenly spread out, non-nucleated, biconcave discs of

uniform size of 7.2–7.8 μm. Normally, the centralpaleness

occupies the middle third of the cells but is wider in

anemias.

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2. The leukocytes.(WBC)

Five main types of WBCs are commonly seen in blood films.

They are all larger than the red cells, nucleated, and

unevenly distributed here and there among the red cells.

They include 3 types of granulocytes (Neutrophil.

Easionophill & Basophill), and 2 types of agranulocytes

(monocytes and lymphocytes).

There are fewer and poorly-stained WBCs in the head

end and the extreme tail; and some of these may be distorted.

There appear to be more monocytes in the tail end,

probably dragged there by the spreader because of

their larger size.

Population-wise, neutrophils are the most numerous

leukocytes, then come the lymphocytes, monocytes,

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