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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  References

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Year : 2001  |  Volume : 19  |  Issue : 4  |  Page : 219-221
 

Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria


Department of Microbiology, Goa medical college, Bambolim, Goa - 403 202, India

Correspondence Address:
Department of Microbiology, Goa medical college, Bambolim, Goa - 403 202, India

 ~ Abstract 

A rapid test for diagnosis of malaria based on acridine orange staining of centrifuged blood samples in a microhematocrit tube (QBC) was compared with thick and thin peripheral blood smears in 2274 samples. Malaria was diagnosed in 239 (10.5%) patients by Leishman's staining technique and QBC method. The QBC method allowed detection of an additional 89 (3.9%) cases. Thus the prevalence rate of malaria during the study was 14.4%. In 1946 patients who were negative by the QBC technique, the Leishman's stained smears did not provide any help in malaria diagnosis. Analysis of the relative quantity of parasites in the specimens, in the QBC method, revealed that 80 out of 89 QBC positive but smear negative cases, had a very low parasite number (less than 10 parasites per QBC field). Although QBC method was superior to the smear for malarial parasite detection, species identification was not possible in 26 (7.9%) cases by this technique. In 95.7% (n = 314) QBC positive cases, the buffy coat in the QBC tube appeared pigmented (gray to black). The colour of the buffy coat was therefore considered by us as a predictor of positivity and could be taken as an indicator for a careful and more prolonged search for the parasites. Thus, the QBC technique has its advantages in terms of speed, sensitivity and ease, especially in an endemic area as ours, where the level of parasitaemia is low and more than 70 to 80 smears need to be examined per day. However, the age old Romanowsky stains still appear superior for species identification.

How to cite this article:
Pinto M J, Rodrigues S R, Desouza R, Verenkar M P. Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria. Indian J Med Microbiol 2001;19:219-21


How to cite this URL:
Pinto M J, Rodrigues S R, Desouza R, Verenkar M P. Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria. Indian J Med Microbiol [serial online] 2001 [cited 2019 May 21];19:219-21. Available from: http://www.ijmm.org/text.asp?2001/19/4/219/8196


The commonly employed method for diagnosis of malaria involves the microscopic examination of Romanowsky stained blood films.[1] An expert microscopist can detect 20 - 40 parasites per microlitre on standard blood films. Although a thick blood smear allows identification of the Plasmodial species and stages, the technique is laborious, time consuming and requires a well trained microscopist for accurate identification.
In the recent years, numerous quick and new techniques for malaria diagnosis have been developed, one such being the QBC (quantitative buffy coat) technique. Malarial parasite, unlike their red blood cell host, contain nucleic acid that stains with fluorescent dyes, such as acridine orange. As the parasites within erythrocyte mature, they reduce the buoyant density of infected erythrocyte. These two properties are exploited in QBC technique for Malaria diagnosis.[2]
Due to continually escalating malaria cases in our locality, it becomes imperative to employ a diagnostic test that is sensitive yet specific, rapid and cheap.
We therefore evaluated the QBC method for malaria diagnosis and attempted to correlate it with the Romanowsky stained thick and thin smear microscopy, in terms of sensitivity, reliability, speed and cost effectiveness.

 ~ Materials and Methods Top

This prospective study was conducted on 2274 blood samples from patients presenting with pyrexia and/or atypical presentations, attending the various Out Patient Departments of Goa Medical College.
Blood samples were subjected to thick and thin smear examination after staining with Leishman's stain. Approximately 80 to 100 fields were examined over 7 to 10 minutes by the same Microscopist who visualised the QBC system.
In the QBC technique, approximately, 50 microlitres of whole blood was taken into a capillary tube coated with acridine orange and fitted with a cap. A plastic float was inserted inside the tube and then spun in the QBC microhaematocrit centrifuge at 12000 rpm for 5 minutes. The tube was then mounted on a small plastic holder and examined through an ordinary light microscope with customised fluorescence (Paralens attachment).
The principle of QBC is based on the fact that on centrifugation at a high speed, the whole blood separates into plasma, buffy coat and packed red cell layer. The float gets buoyed by the packed blood cells and is automatically positioned within the buffy coat layer. Blood cells in the buffy coat layer separate according to their densities, forming visibly discrete bands; platelets remaining at the top, lymphocytes and monocytes within the middle layer and granulocytes at the bottom.
Due to acridine orange, the malarial parasite stains green (DNA; nucleus) and orange (RNA; cytoplasm). The tube is examined in the region between the red blood cells and granulocytes and within the granulocytes and mononuclear cell layer, where parasites are most abundant.
An attempt was made to estimate the relative quantity of parasites in the specimen, using the plus system.
+ (1+) - 1 parasite per QBC field
++ (2+) - 1-10 parasites per QBC field
+++ (3+) - 11-100 parasites per QBC field
++++ (4+) - 100 parasites per QBC field
Parasite count was however not attempted with the Romanowsky technique.
It has been observed by us earlier (unpublished data) that the buffy coat in the QBC tube appears pigmented gray or black in colour in positive samples. Based on this observation, an attempt was made to analyse the correlation of the buffy coat colour and positivity.

 ~ Results Top

Two thousand two hundred and seventy four blood samples were evaluated by both thick and thin Leishman stained smears and QBC tube.
Malarial parasite was detected in 239 (10.5%) cases by smear examination. These cases were also positive by the QBC method. An additional 89 (3.9%) cases were diagnosed as malaria by QBC technique [Table - 1]. Thus the prevalence rate of malaria was 14.4%. All patients (n=1946) who were malarial parasite negative by QBC method were also smear negative. The species of malarial parasites encountered by smear examination were Pasmodium falciparum (77;32.2%) and P. vivax (102; 42.7%). Mixed infections (falciparum and vivax malaria) accounted for 25.1% (n=60) cases. By the QBC method, species identification was not possible in 26 (7.9%) cases. We observed that smear examination required on an average 10 to 12 minutes in contrast to 1 to 2 minutes to report a QBC tube.
QBC tube method allowed detection of as few as two parasites per QBC field. An analysis of the relative quantity of parasites in the specimen by this technique revealed that 80 out of 89 QBC positive but smear negative cases had a very low parasite load of less than 10 parasites per QBC field. On the contrary, all blood samples positive by both methods had high parasitaemia.
Gray to black colour of the buffy coat in the QBC tube was noted before it was examined under the microscope. In 314 (95.7%) malaria parasite positive tubes the buffy coat appeared pigmented and thus served to predict positivity.

 ~ Discussion Top

The potential uses of diagnostic assays range from the clinical and/or research laboratory to epidemiological surveys, in a field environment.
Our results demonstrated a higher sensitivity and greater rapidity of QBC tube method as compared to Leishman stained thick and thin blood films, confirming the results of other field studies.[3] The speed of this method in detecting malarial parasites, especially with low parasitaemia is a definite advantage in laboratories like ours that screen large number of blood samples. In addition, low levels of parasitaemia can be easily detected as more blood is being used per sample (55 microlitres). However, in a Phillipine study, the sensitivity of QBC for malaria diagnosis was only 70% when compared with Giemsa stained thick blood films.[4]
Another advantage of QBC is its ease of interpretation and it being technically easy to perform. A technician can be taught to carry out the QBC test and detect malarial parasite accurately, in less than a day, in contrast to the smear examination and interpretation, which takes weeks. We therefore preferred this technique because of its ease, reliability, speed and fewer opportunities for error.
Concern over the ability of the QBC method to enable species identification has been noted, with success claims ranging from 75% to 93%.[5] In our study, species identification was not possible in 7.9% cases. This difficulty is encountered as the morphology of the infected erythrocyte which aides in identification of malarial parasite species remains occult.
Possible drawbacks of the QBC method are that the quantitation of parasitaemia relies on a subjective grading system. Further, expense is an important factor especially for developing countries. The events of blood filled QBC tubes breaking or leaking in the centrifuge machine are other drawbacks.
In 95.7% of the cases, macroscopic visualization of pigmentation of the buffy coat enabled us to predict a positive QBC result. We therefore presume that the colour of the buffy coat, if pigmented, can be considered as a predictor of positivity. However, this is our independent finding and the experience of other workers in this respect will have to be taken into account before arriving at a definite conclusion.
We believe that QBC method provides a reliable, quick, easily mastered, accurate method for diagnosis of malaria in our setting and may be especially useful in evaluating large populations for fever in malarious areas, when the technical staff is inexperienced. The QBC system can well be used in diagnosis of other parasitic diseases from blood (Filariasis). 

 ~ References Top

1.Bruce-Chwatt LJ. DNA probes for malaria diagnosis. Lancet 1984; 1:795.  Back to cited text no. 1    
2.Kevube RA, Wardlaw SC, Patton CL. Detection of haemoparasites using quantitative buffy coat analysis tubes. Parasitology Today 1989; 5:34.  Back to cited text no. 2    
3.Gay F, Traore B, Zanoni J, Danis M, Fribourg-Blanc A. Direct acridine orange fluorescence examination of blood slides compared to current technique for malaria diagnosis. Transactions of Royal Society of Tropical Medicine and Hygiene 1996; 90:516-518.  Back to cited text no. 3    
4.Levine LS, Long GW, Oberst R et al. Rapid diagnosis of malaria by acridine orange staining of centrifuged parasites. Lancet 1989;1:68-71.  Back to cited text no. 4    
5.Long GW, Jones TR, Rickman LS, Fries L, Egan J, Wellde B et al. Acridine orange diagnosis of Plasmodium falciparum; evaluation after experimental infection. The American Society of Tropical Medicine and Hygiene 1994; 51(5):613-616.  Back to cited text no. 5    
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