READING AND STORAGE OF STAINED FILMS
HOW AND HOW LONG SHOULD A FILM BE EXAMINED
FOR THE PURPOSE OF DIAGNOSING MALARIA?
Though microscopic examination of thick and thin films is uniformly
accepted as the “Gold Standard” for the microscopic diagnosis of malaria
infection [57], there is no consensus as to the amount of time necessary or
the number of microscopic fields that should be examined. A review of the
literature reveals a broad variability in these parameters [15][28][45][51].
A scan of 10 to 100 microscopic fields is taken as the “Gold Standard” in
many studies; however, as may be logically expected, if the same samples are
re-examined by scanning 400 or 500 microscopic fields or the whole thick
film, many samples that tested negative with a shorter examination time will
actually turn out to be positive [31][58].
Moreover, although examination of thick film is theoretically more sensitive
than thin smear – given that a larger quantity of blood is examined in the
same amount of time – in practice it should be kept in mind that [18]:
- During dehemoglobinization and staining of a thick film a certain number
of parasites will be lost, depending on the plasmodium species and stage of
development; this loss may be accompanied by a macroscopic or microscopic
detachment of blood from the slide (thick film with too much blood and/or
not completely dry, slides that are not perfectly clean, incorrect washing of
the film).
- In the case of low parasitemia, the parasites may be unevenly distributed
and thus be present in some fields but absent from all the others.
- If the film is examined in a random way, i.e. without following a
well-defined sequence, it is likely that the same microscopic fields will be
examined more than once and if these happen to be in a part of the film that
is not correctly stained, identification of the parasites will be difficult.
- Contamination may occur among specimens: the parasites may be
transferred from one specimen to another, into the staining solution or in
the film that forms on top of it.
- In infections with a high level of parasitemia, the loss of parasites in thick
films is of little significance.
- In Africa malaria infections with low parasitemia are fairly common and
the loss of parasites in a thick film may thus be significant; in such cases,
if only 100 fields are examined, the detection of plasmodia may be purely
accidental.
It is also true, however, that low parasitemias have little or no clinical
relevance in highly endemic areas (where a large percentage of asymptomatic
individuals are carriers of circulating plasmodia), so it hardly makes sense to
require too high a sensitivity level.
By contrast, in areas with a low degree of endemicity and above all in the
cases of imported disease, any level of parasitemia may be clinically correlated
with malaria: in such cases, efforts must focus on identifying even very low
parasitemias and thus the examination time (number of fields to be scanned)
will necessarily be much higher.
In conclusion, the choice of parameters for thick film examination (number
of microscopic fields, time necessary) cannot be established by means of
a general rule and will primarily depend on the sensitivity and specificity
required in a given situation, the objective that is being pursued [50] (for
example, a clinical setting will differ from that of an epidemiological study)
and, not lastly, the experience and training of the personnel.