PERIPHERAL BLOOD FILM/SMEAR

1) CLEAN the slides. Dirty or greasy slides results in uneven smear formation.

Select the spreader among the slides given to you. It should have smooth edges. Hemocytometer

coverslip can be used as a spreader.

Once cleaned do not touch the surface of slides

Keep slides on the new sheet of blotting paper

2) DISINFECT the finger to be pricked, by cotton, moist with 70% isopropyl alcohol also known as

surgical spirit or rubbing alcohol. To sterilise rub the distal(terminal) phalanx with cotton, starting from

centre and moving towards periphery (scrubbing increases local blood flow)

Allow the area to dry because:

i) Disinfection by alcohol is effective only after it has dried

ii) Blood drop does not form properly on the fingertip if is not dry. Blood spread sideways along

with spirit.

iii) Blood cells are hemolyzed when they come in contact with alcohol

3) PRICK the finger with 20/21G (gauge) sterile disposable needle (lancets with thin and shallow points

shouldn’t be used as epithelial cells tends to block the puncture)

SITE: ring finger of non-

dominant hand >> middle finger, index

finger on its palmar surface about 3-5

mm lateral from the nail bed. NEVER

PRICK THUMB AND LITTLE FINGER. Ear

lobes in adults and heels in infants can

be used to draw capillary blood.

Tenosynovitis of the Synovial Sheaths of the Flexor Tendons: Tenosynovitis is an infection of a synovial

sheath. It most commonly results from the introduction of bacteria into a sheath through a small

penetrating wound, such as that made by the point of a needle or thorn. Anatomically, the digital

sheath of the index finger is related to the thenar space, whereas that of the ring finger is related to

the mid palmar space. The sheath for the middle finger is related to both the thenar and mid palmar

spaces. These relationships explain how infection can extend from the digital synovial sheaths and

involve the palmar fascial spaces. In the case of infection of the digital sheaths of the little finger and

thumb, the ulnar and radial bursa are quickly involved. Should such an infection be neglected, pus may

burst through the proximal ends of these bursae and enter the fascial space of the forearm between

the flexor digitorum profundus anteriorly and the pronator quadratus and the interosseous membrane

posteriorly. This fascial space in the forearm is commonly referred to clinically as the space of Parona.

(Reference: Snell’s clinical anatomy by regions 9ed pg. no. 399-400)

DEPTH: 2-3 mm deep prick should be done. Blood drop should form freely on the fingertip which

requires deep puncture. If superficial prick has been done then one has to squeeze the finger.

Squeezing causes exudation of tissue fluid which dilutes the blood resulting in false lowering of cell

count. One deep puncture will result in free flowing blood drop formation and is less painful then

several superficial pricks.

Apply gentle pressure on one side of terminal phalanx

(which is to be pricked) with the help of the thumb (of

the same hand to which finger to be pricked belongs).

Hold needle in between thumb and fingers of your

dominant (working) hand and prick with one gentle

movement.

PRECAUTIONS:

i) The puncture should be at the middle of the tip, not too far down on the finger nor too close to the

nail.

ii) Keep the needle in its cover as soon as the puncture/prick of fingertip has been done

iii) Do not squeeze the fingertip for blood drop formation

Wipe off the first drop of blood with DRY cotton, as usually it is mixed with the tissue fluid, epithelial

cells and endothelial cells. Use successive blood drop for spreading.

4) PLACE THE BLOOD DROP under the surface of the slide, about 1cm from one end of the slide.

The blood drop should not be placed on the

slide, as one cannot see the amount of blood placed

on the slide.

Do not touch the slide with fingertip as

moisture/oil from the skin may spoil the smear.

5) SPREAD (wedge method) the blood drop to make the film:

a) Put the specimen slide (on which blood drop has been placed) on the white sheet of paper placed

on the flat surface of the table, with the blood drop to the right side.

b) Support the left end of the slide with your thumb and fingers of your left hand. Now grasp the long

edges of the spreader between thumb and fingers of your right hand. Place the smooth clean edge

of the spreader slide on the specimen slide just in front of the blood drop, making an angle of 30°-

45° (if angle is increased thin smear will be formed, and angle is decreased thick smear will be

formed : C.L. Ghai 8ed pg. 33) from the horizontal level.

c) Pull the spreader back gently so that it just touches the blood drop, the blood should evenly spread

along the edge of the spreader (the spreader can be slightly shaken sideways to facilitate the spread

of blood along the edge of the spreader.

d) Now PUSH the spreader to the left in a single, smooth, fairly fast gliding motion (smear becomes

thick if made very fast or thin if made slow), maintaining constant pressure and angle (30°- 45°);

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

All blood should be completely used in making the smear, otherwise distribution of cells

becomes unequal

Never use the same edge of a spreader for spreading twice.

e) Dry the smears quickly by waving them in air. Delay in drying causes cells to shrink and they appear

distorted, also rouleaux formation may occur.

6) IDEAL SMEAR: Make as many trials as possible to get acceptable films, keeping in mind the features

of an ideal blood smear, which are:

a) Smear should cover middle 2/3rd of the slide.

b) Tongue shaped (broad at the head/starting end and gradually tapers towards the tail/finishing

end).

c) Uniformly spread: no vacant areas, no striations, and no thick and thin patches.

d) Neither too thick nor too thin: too thick film appears translucent red when placed on the white

background, too thin smears will appear pale/faint pink. Ideal smear appears as “buff” coloured.

STAINING THE BLOOD FILM

1. Place the slides, smear side up, on a ‘staining rack’ assembled over a sink (two metal rods placed across

the sink). Ensure the horizontal placement of slides.

2. Pour 8–10 drops of the Leishman’s stain on each unfixed slide by dripping it from a drop bottle (This

amount of stain usually covers the entire surface and “stands up” from the edges of the slides without

running off). CAUTION: do not shake the bottle containing stain.

3. Allow the stain to remain undisturbed for 1–2 minutes. During this time FIXATION of the film occurs

NOT the STAINING.

Acetone-free and water-free absolute methyl alcohol present in

Leishman’s stain acts as a fixative, also all types of Romanowsky stains (Leishman, Giemsa, Wright) are

water-insoluble but can be dissolved in methyl alcohol. 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.

Acetone is a very strong lipid solvent, causes crenation, shrinkage, or even destruction of cell

membranes. This will make the identification of the cells difficult.

Water result in the rouleaux formation and even hemolysis. The water may even wash away the

blood film from the slide.

Note: during this time watch stain carefully, it should not get dry at anywhere on the slide. If the

stain dries, it will precipitate on the blood film and appear as round, blue granules. This can be

prevented by pouring more stain on the slides as required.

4. Pour an equal number of drops of buffered water (or double the drops of distilled water if the buffered

water in not available; tap water should be avoided because of its unknown pH which might prevent the

ionisation of eosin and methylene blue dye, also it contains impurities which may show up as artefacts).

Buffered water is phosphate buffer in which the pH is adjusted at 6.8. At this pH, there is optimal

ionisation of the stain particles so that they can penetrate the cells better. The buffer is prepared

by dissolving 3.7 g of disodium hydrogen phosphate and 2.1 g of potassium dihydrogen phosphate

in distilled water made to 1000 ml.

CAUTION: do not let the content (stain + buffer) over slide to spill from edges

5. Allow the buffer + stain to remain on the slide for 6-8 minutes (STAINING TIME). Occasionally gently

blow air from the side, to mix the content. Usually, glossy greenish scum (layer) floating on the surface

of the diluted stain shows that the staining has been done properly.

6. Wash off the stain under running tap water for approximately 30 seconds. Place your thumb at the head

end of the slide and let water stream to gently trickle down on the smear through you thumb nail.

Never pour off the stain as the precipitate (greenish scum) will get deposited on the smear (Bright light

and heat oxidise the stain, especially when in aqueous solution and will cause precipitation of insoluble

precipitates e.g. of methylene violet).

7. Wipe the back side of the slide with a clean and dry filter paper/cloth. Keep it in an inclined position to

dry (stained sides facing downwards, to prevent dust particles settling on them).

8. Examine with naked eye first and then under low power objective and then in high power.

LEISHMAN’S COMPOSITION:

1) Eosin: acidic dye, stains the basic parts of the cell: granules of eosinophil and neutrophil, and

Haemoglobin of RBCs.

2) Methylene blue: basic dye, stains the acidic part of the cell: nuclei (DNA + RNA) and acidic granules

in the cytoplasm of WBCs (e.g.: neutrophil and basophil).

3) Methyl alcohol: acetone and water free, works as a fixative.

*(neutrophil granules are slightly basic and stain weakly with the azurophilic component: Wintrobe’s

13ed pg. no.9)

DIFFERENCES BETWEEN CAPILLARY AND VENOUS BLOOD

Venous blood and capillary blood are not quite the same. Blood from a skin puncture is a mixture of

blood from arterioles, veins and capillaries, and it contains some interstitial and intracellular fluid.

Although some studies have suggested that there are negligible differences when a free flow of blood

has been obtained, others have shown definite differences in composition between skin puncture and

venous blood samples in neonates, children and adults. The differences may be exaggerated by cold

with resulting slow capillary blood flow. The packed cell volume (PCV), red cell count (RBC) and

haemoglobin concentration (Hb) of capillary blood are slightly greater than in venous blood. The total

leucocyte and neutrophil counts are higher by about 8%, the monocyte count by about 12%, and in

some cases by as much as 100%, especially in children. Conversely, the platelet count appears to be

higher in venous than in capillary blood; this is on average by about 9% and in some cases by as much

as 32%. This may be due to adhesion of platelets to the site of the skin puncture.