|Basic Malaria Microscopy (part I and II) (WHO - OMS, 1991, 72 p.)|
By the end of this Unit you should be able to:
· name the diseases that can be transmitted by contaminated blood
· list the precautions that must be taken to prevent contamination
· list all the materials required for making blood films
· demonstrate under field conditions the preparation on the same slide of a thick and a thin blood film, each of good enough quality for malaria microscopy
· explain the reasons why a blood film should be correctly labelled
· demonstrate the correct labelling of blood films
· recognize and select thick and thin blood films of good quality
· identify the causes of common faults in both thick and thin blood films.
Diseases carried in the blood
Some people may carry a disease in their blood even if they do not appear to be ill. You cannot easily see the diseases in the blood, and sometimes the tests to demonstrate the diseases are very complicated. The principal diseases are:
· acquired immunodeficiency syndrome (AIDS)
The collection and handling of blood samples presents a potential risk of blood from a patient infected with one of these diseases accidentally contaminating another patient or a health worker. However, this risk can be reduced to a negligible minimum by taking the following precautions:
· Wear protective gloves when handling blood or taking blood samples.
· Avoid getting blood, including that from unstained slides, on your fingers or hands.
· Cover any cuts or abrasions on your hands with adhesive dressings.
· Take care not to prick yourself or others with any sharp instrument that has been in contact with blood.
· Never use disposable lancets more than once.
· Always wash your hands with soap and water after completing any task that involves the handling of blood.
· If blood does get on to your skin, wipe it off quickly with cotton wool dampened with alcohol and wash the affected area with soap and water as soon as possible.
· Any materials contaminated with blood, such as lancets, cotton swabs and discarded slides, should be boiled for 20 minutes, or placed in a solution of bleach or sodium hypochlorite (available chlorine level 10000 parts per million), then disposed of safely by burial or incineration.
Kinds of blood film
Two kinds of blood film - thick and thin - are used in malaria microscopy.
The thin film consists of a single layer of red blood cells and is used to assist in the identification of the malaria species, after the parasites have been seen in the thick film. It is also used as a label to identify the patient.
The thick film is made up of large numbers of dehaemoglobinized red blood cells. Any parasites present are concentrated in a smaller area than in the thin film and so are more quickly seen under the microscope.
Preparation of thick and thin blood films on the same
The following items are needed for preparation of blood films:
· cleaned and wrapped slides
· sterile lancets
· methylated spirit and water
· absorbent cotton wool
· slide box (or a cover to keep flies and dust off the slides)
· clean, lint-free cotton cloth
· record form or register.
The techniques for taking blood samples and preparing thick and thin blood films are detailed in Plate 1. Note that, whereas a thick and a thin film from the same patient may be made on the same slide, it is not recommended to make blood films from more than one individual on the same slide.
Plate 1. Preparation of thick and thin blood films on the same slide
After details about the patient have been recorded in the appropriate form or register, the blood films are made as follows:
1. Holding the patients left hand, palm upwards, select the third finger from the thumb. (The big toe can be used with infants. The thumb should never be used for adults or children).
Clean the finger with a piece of cotton wool lightly soaked in alcohol, using firm strokes to remove dirt and grease from the ball of the finger.
Dry the finger with a clean cotton cloth, using firm strokes to stimulate blood circulation.
2. Puncture the ball of the finger with a sterile lancet, using a quick rolling action.
Apply gentle pressure to the finger to express the first drop of blood and wipe it away with a dry piece of cotton wool. Make sure that no strands of cotton remain on the finger to be later mixed with the blood.
3. Working quickly and handling clean slides only by the edges, collect the blood as follows.
Apply gentle pressure to the finger and collect a single small drop of blood, about this size ·, on the middle of the slide. This is for the thin film.
Apply further pressure to express more blood and collect two or three larger drops, about this size ·, on the slide, about 1 cm from the drop intended for the thin film (see illustration).
Wipe the remaining blood away from the finger with a piece of cotton wool.
4. Thin film. Using a second clean slide as a spreader and, with the slide with the blood drops resting on a flat, firm surface, touch the small drop with the spreader and allow the blood to run along its edge. Firmly push the spreader along the slide, keeping the spreader at an angle of 45°. Make sure that the spreader; is in even contact with the surface of the slide all the time the blood is being spread.
5. Thick film. Always handle slides by the edges or by a corner to make the thick film as follows.
Using the corner of the spreader, quickly join the drops of
blood and spread them to make an even, thick film. The blood should not be
excessively stirred but can be spread in circular or rectangular form with 3 to
6 movements. The circular thick film should be about 1 cm (
inch) in diameter.
6. Label the dry thin film with a soft lead pencil by writing across the thicker portion of the film the patients name or number and the date. Do not use a ballpoint pen for labelling the slide. Allow the thick film to dry with the slide in a flat, level position, protected from flies, dust and extreme heat.
7. Wrap the dry slide in the patients record form and dispatch it to the laboratory as soon as possible.
8. The second slide used for spreading the blood films may now be used for the next patient and another clean slide from the pack will be used as a spreader.
Common faults in making blood films
A number of faults are common in making blood films. These can affect the labelling, the staining or the examination, and sometimes more than one of these.
Badly positioned blood films
Care should be taken that the blood films are correctly sited on the slide. If they are not, it may be difficult to examine the thick film. Also, portions of the films may even be rubbed off during the staining or drying process.
Too much blood
After staining films made with too much blood, the background to the thick film will be too blue. There will be too many white blood cells per thick film field, and these could obscure or cover up any malaria parasites that are present. If the thin film is too thick, red blood cells will be on top of one another and it will be impossible to examine them properly after fixation.
Too little blood
If too little blood is used to make the films, there will not be enough white cells in the thick film field and you will not examine enough blood in the standard examination. The thin film may be too small for use as a label.
Blood films spread on a greasy slide
The blood films will spread unevenly on a greasy slide, which makes examination very difficult. Some of the thick film will probably come off the slide during the staining process.
Edge of spreader slide chipped
When the edge of the spreader slide is chipped, the thin film spreads unevenly, is streaky and has many tails. The spreading of the thick film may also be affected.
Thin film too big, thick film In the wrong place
If the thin film is too large, the thick film will be out of place and may be so near the edge of the slide that it cannot be seen through the microscope. During staining or drying, portions of the thick film will probably be scraped off by the edges of the staining trough or drying rack. It may be very difficult, or impossible, to position the thick film on the microscope stage so that it can be examined.
Other common faults
Other faults that occur commonly in the preparation of blood films include the following:
· Flies, cockroaches or ants eat the dry blood and damage the films.
· Blood films are made on badly scratched slides.
· The thick film is allowed to dry unevenly.
· Autofixation of the thick film occurs with the passage of time or through exposure to heat, and staining then becomes difficult or unsatisfactory.
· Slides are wrapped together before all the thick films are properly dried, and the slides stick to one another.
Drying the blood films
Blood films must be stored correctly in order to allow the thick film to dry evenly. They should also be protected from flies and dust.
The best kind of box, both in the field and the laboratory, is that shown below:
Slides are stored horizontally, which allows the thick film to dry level and with even thickness. There is a door to keep out flies and dust and a handle for carrying the box. Only after 100 slides have been collected is it necessary to empty one side of the box before more slides can be collected. At the speed slides are usually taken in field collections, the first 50 will be dry before they need to be packed.
When the thick film is completely dry, the slides are stored front to back in the empty cardboard slide box previously used for the cleaned, wrapped slides. In warm humid climates, however, autofixation of unstained slides occurs quite rapidly and all slides should be stained as soon as possible, at the latest within 3 days of collection. When long storage is unavoidable, the slides can be kept in a desiccator to delay autofixation. Finally, it is important to ensure that the slides are packed correctly and that they are not put into strong sunlight or near to a source of heat (e.g. the exhaust pipe of a vehicle in the field).