A well prepared blood smear is necessary for microscopic examination of blood. Blood smears are used to determine leukocyte differentials, to evaluate erythrocyte, platelet and leukocyte morphology, and, if necessary, to estimate platelet and leukocyte counts.
Desirable qualities of a blood smear include:
i. Sufficient reading area
ii. Acceptable morphology within the working area
iii. Even distribution of leukocytes
The Clinical Pathology Laboratory uses the wedge technique for preparation of blood smears. This method produces a gradual decrease in thickness of the blood from thick to thin ends with the smear terminating in a feathered edge approximately 2 mm long.
The smear is greater than 25 mm long and the feathered edge stops approximately 10 mm from the end of the slide. The blood film occupies the central portion of the slide and has definite margins on all sides that are accessible to examination by oil immersion.
The thin end of the film becomes thinner gradually and does not have grainy streaks, troughs, ridges, waves or holes – features that can result in an uneven distribution of leukocytes. In preparations from normal patients, the thin section of the smear occupies approximately 1/3 of the total area and, within that area; erythrocytes are distributed in a monolayer.
The thickness of the spread is influenced by the angle of spreader slide (the greater the angle, the thicker and shorter the blood smear), the size of the drop of blood and the speed of spreading. Glass cover slips are mounted on all blood smears to prevent damage to smear during examination, cleaning, handling and storage.
Preparation of Blood Smear:
Collection of Sample:
1. Finger Prick or
2. E.D.T.A. blood (within 1 hr. of collection)
Preparation of Blood Film:
The slide should be clean. Place a small drop of blood, or one side about 1-2 cm from one end. Without delay place a spreader at an angle of 45° from the slide and move it back to make contact with the drop. The drop should spread out quickly along the line of contact of spreader with the slide.
The moment this occurs, spread the film by rapid smooth forward movement of the spreader. The film should be 3-4 cm in length. The ideal thickness is such that there is some overlap of R.B.C. throughout most of its length with proper separation and lack of distortion of RBC’s. the end from where the spread had ended is called tail end.
The ideal zone to examine the blood film is the areas between tail and body. If the film is made too thin or if a rough edged spread is used many leucocytes accumulate in edges and at tail. DLC should not be attempt on such a slide.
Characteristics of an Ideal Blood Smear:
1. It should be in the central 2/3 of slide.
2. It should have straight lateral border and short tongue shaped tail.
Precautions to be taken during preparation:
1. Angle should be maintained at 45°.
2. Blood drop should be of proper size.
3. Spreader’s edges should be smooth and it should be smaller than the slide on which smear is being made.
4. Pressure applied should be proper.
5. Drop should be pulled with spreader not pushed with it.
Process of Staining:
The slide is covered with leishman stain for 2 mins. This much time is required for fixation. After 2 mins it is diluted with double the volume of buffer water. On adding buffer water a metallic shin will be formed, if the stain is dry. Allow this to stand for 15 min. after min, flood the slide with water to remove stain. Then wash under tap water wiping the back of slide with finger or cotton. Dry in air.
Precautions Suring Staining:
1. Time: Initial time 2 minutes, is important. After dilution increase of 1-2 minutes, does not alter staining.
2. Never let the stain dry on the slide otherwise stain deposits will make it impossible to count leucocytes (DLC).
3. Staining should be deposit free.
4. For washing the smear – let the water stream replace the stain. Don not throws the stain first.
Technique of Differential Leucocyte Counting:
D.L.C. is done is oil immersion, with wide open diaphragm and high up condenser. D.L.C. is best done in area where RBC morphology is good. There tends to be somewhat unequal distribution of WBC is normal blood film.
The smaller cells (like small lymphocytes) being in greater relative number in central thicker portions and the larger cells (monocytes eosinophils) being in greater relative number- along the edges and the tail. So the D.L.C. on a smear depends upon the area of the slide used for counting. To overcome this difficulty both area are included in counting.
Two methods are employed in counting:
1. Going back and forth lengthwise including both body and tail.
2. Going back and forth sidewise including both edges and centre. Usually 100 cells are counted.
Thick Blood Film:
It is used for malaria and microfilaria.
Sample is taken at or just after height of fever with shivering.
Sample is collected at midnight. Drop of blood is taken on slide which is spread evenly, air dried and then de-hemoglobinised by placing film in distilled water for 5-10 min. Leishman’s stain is mostly used for malaria.
A buffy coat preparation facilitates the search for immature and abnormal white blood cells, particularly when the peripheral blood count is low, and in identification of bacteria or yeast in septic and immune-compromised or immunosuppressed patients.
Micro-hematocrit centrifugation of specimen. Concentration of WBC’s; Wright-Giemsa Stain.
Specimen Requirements: Collect:
Routine venipuncture, EDTA (Lavender-top tube)
Stable at Room Temperature for 24 hours.
Stable Refrigerated for 36 hours.
Causes for Rejection:
Clotted, QNS, old specimen, identification errors, labeling errors, inappropriate anticoagulants
Common abnormalities seen on blood smear and their significance: