Thick Blood smear/films for examining microfilariae (Wuchereria spp.)

Standard method for diagnosing active infection –by-identification of microfilariae in a blood smear by microscopic examination. The microfilariae that cause lymphatic filariasis circulate in the blood at night (called nocturnal periodicity). Blood collection should be done at night to coincide with the appearance of the microfilariae, and a thick smear should be made and stained with Giemsa or hematoxylin and eosin. For increased sensitivity, concentration techniques can be used.

Sites of blood collection: •

Finger prick,

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Earlobe or

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Venous blood (using EDTA anticoagulant)

Preparation of thick blood film • Clean slide with an alcohol swab to remove lint and oil residue. • Label the slide. • With the patient’s left hand palm upwards, select the third or fourth finger. • Use cotton wool lightly soaked in ethanol to clean the finger – using firm strokes to remove dirt and grease from the ball of the finger. Dry the finger with a clean piece of cotton wool or lint. • With a sterile lancet, puncture the internal side of the finger using a quick rolling action. • By applying gentle pressure to the finger, express the first drop of blood and wipe it away with dry cotton wool. • Make sure that no strands of cotton wool remain on the finger. Discard the lancet into a sharp’s container.

Working quickly and handling clean slides only by the edges, collect the blood as follows: •

Apply gentle pressure to the finger and collect 60 μl of blood into a blood collection tube or calibrated capillary tube.

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Try not to get air bubbles into the capillary tube. If you do, fill the blood past the line to compensate.

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Wipe the remaining blood away with cotton wool.

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Ask the patient to hold the cotton wool firmly on the finger until it stops bleeding.

Film preparation: •

Using the micropipettor or capillary tube prepare three parallel lines of blood (20μl each) along the length of the slide.

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Air dry the blood film thoroughly for 24–72 hours.

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Carefully, load the slides into the staining racks.

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Dehaemoglobinize the blood film for approximately 5 minutes in tap water, distilled water or normal saline.

Dehaemoglobinization is necessary to clear the red blood cells so that the microfilariae can be more easily visualized, and is complete when the smear turns an opaque greyish-white. Caution must be exercised at this time because the smear is fragile, and rough washing or agitation can result in its floating off the slide. •

Air dry. This can be done in the staining racks.

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Fix in methanol 3–5 minutes.

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Stain with Giemsa (dilute the stain 1 in 20)- pour the stain about 5 ml (totally cover film) and kept for 30-45 minutes. Flush the slide with tap water gently.

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As a rule of thumb, if the white blood cell nuclei are properly stained, microfilariae should also be adequately stained.

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It should be noted that for Giemsa staining of films to be examined for microfilariae, unlike those to be examined for malaria parasites, the pH of the staining solution is not critical.

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Air dry.

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Examine the preparation under the microscope. Use the x 10 objective first to locate the microfi lariae; then identify the fi larial species using the x 40 and x 100 objectives.

References World health organization global programme to eliminate Lymphatic filariasis, 2011. Monitoring and epidemiological assessment of mass drug administration. a manual for national elimination programmes, WHO.