Dr Piyush Tailor

Purpose of examination

A number of screening tests based on immunological methods have been developed for the detection of blood parasites especially malaria, but the essential method for a definitive diagnosis remains the finding of parasites in the blood film and the identification of species by morphology. The stained smears are examined light microscopically and screened for blood parasites.

Principle of examination

A manual preparation of blood smear is done either from finger prick samples or from whole blood samples. Usually smears are prepared from samples of anticoagulated blood remaining from automated hematological analysis. In addition to standard thin films, thick films are also prepared which are extremely useful when the parasites are scanty. Species identification is much easier in thin films as compared with thick films. Mixed infections may be missed on thick films.

Performance specifications

Not applicable.

Sample type required

Anticoagulated whole blood (EDTA).

Slide with thick and thin smear.

Preservatives needed

Sample collected in EDTA bulb.

Reagents required

Giemsa stain

Methanol (acetone free)

Calibration method

Not applicable

Detailed work bench instruction / programming steps:

In accordance with good laboratory practice, all samples should be considered potentially infectious and should be handled with care. The personnel should use protective laboratory coats, gloves, eye glasses & MASK.

Preparation of thin smear

• Take a clean & dry slide
• Transfer a small drop of blood near the edge of the slide
• Place a spreader slide at angle of 30 degree; pull back the spreader until it touches the drop of blood. Let the blood run along the edge of the spreader.
• Push the spreader forward to the end of the slide with a smooth movement.
• Dry the blood smear at room temperature.
• By using a lead pencil, write ID number on slide

Preparation of Thick smear:

• Place a small drop of blood on the slide
• Spread the drop out with another slide to cover an area

About four times its original area

The correct thickness for a satisfactory thick film will have been achieved if, with the slide placed on a piece of newspaper, small print is just visible.

Allow the film to dry at least 30 minutes at 37 degree C before staining

Fixation on thin smear;

• Fix the thin smear by dipping in methyl alcohol for 1-2 second
• Dry the film after fixing in methyl alcohol

Preparation of Giemsa stain

• Take 5 gm of Giemsa powder and crush it properly, then mix it in 500 ml of glycerol and keep it for 48 hours.
• Add & well mix 500 ml of absolute methanol in the above mentioned solution and keep at room temperature for one week.
• After filtering it is stored in amber colored bottle at room temperature

Staining procedure

• Add buffer( 6.8 ph) in the filtered stain in 1:1 dilution
• Arrange thick/ thin smears on double rod stand.
• Pour the stain-buffer mixture on the arranged slides. Leave it for 10-15 minutes
• Stain over the slides should be mixed by blowing with pipette.
• After 10-15 minutes, wash the slides in slowly running tap water.
• Dry the washed slides in air at room temperature.

Examination of the blood smear

• Using a light microscope the smear is first examined under 10 x objectives to assess the adequacy of cellular distribution and staining.
• The slide is examined under 40 x and scanned for hemoparasites.
• Examine the slide under oil immersion to study the species morphology.

Quality control procedure:

QUALITY CONTROL

• Maintain integrity of the Giemsa Wright stain by proper storage and frequent preparation.
• Check the stain for degenerative changes and stain deposits
• Filter prior to use.
• Check staining for every new lot prepared.
• Frequent checks of staining buffer pH
• Check for background staining. In these cases ensure proper preparation of the stain with emphasis on grinding with glycerol.

• Internal Quality Control: Weekly one random PSMP slide is checked internally by three different observer and records of comparison are kept in control C\Records\File\9\Records of internal quality control records.
• External quality control: Monthly one random sample for malaria examination is sent to other laboratory and records of comparison are kept in C\Records\File\2\Results of EQA and interlaboratory comparison.

For any nonconformance, Procedure for identification, correction and prevention of nonconformities is activated and records are filed in C\Records\File\12\Records of Nonconformities

Interferences

• Individual variation in interpretation.

• Slide must be clean and free of grease, lint and dust .Failure to observe these precautions may prevent adequate spreading of blood specimen over the entire surface and thus introduces morphological artifacts.

• Stains must be free of water even small amount can cause marked red cell artifacts.

Calculation of results

For malarial parasite s, at least 100 oil immersion fields should be screened before reporting negative.

Malaria grading:

Biological reference interval

No parasites seen

Reportable interval for examination results:

Negative for malarial parasite to ++++

Critical values:

Plasmodium falciparum ++++

Interpretation by the laboratory

Malaria: The most common and prevalent disease in India is malaria (Plasmodium).

The various species of plasmodium are P.falciparum, P.vivax, P.ovale and P.malariae.

Potential sources of variability

Individual variation in interpretation

Artefactual changes

Presence of contaminants in commercial dye powders