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HEMATOLOGY SECTION - ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 1  |  Page : 108-112
Comparative study of peripheral blood smear, quantitative buffy coat and modified centrifuged blood smear in malaria diagnosis


1 Department of Pathology, Kasturba Medical College, Mangalore, India
2 Department of Community Medicine, Kasturba Medical College, Mangalore, India
3 Head of Pathology, A J Institute of Medical Science, Mangalore, India

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   Abstract 

The present study was aimed at modifying the centrifuged blood smear (modified centrifuged blood smear or MCBS), to make it a feasible and standardized procedure. The results obtained were compared with the current diagnostic methods - peripheral blood smear (PBS) and quantitative buffy coat (QBC). Blood samples collected from 100 suspected malaria patients were subjected to all three tests. It was found that PBS had 86.79% sensitivity and was absolutely specific. QBC was 96.22% sensitive and 93.61% specific. The majority of variations occurred in PBS negative cases; cases with parasite count <++ and with Plasmodium falciparum. It was seen that by the addition of centrifugation to the conventional smear technique (MCBS) improved its sensitivity from 86.79% to near 100%. QBC and MCBS were found superior to PBS. Since MCBS combines principles of both QBC and PBS, it is as sensitive as QBC, as specific as PBS, and above all, easily performed and affordable.

Keywords: Malaria, peripheral blood smear, quantitative buffy coat

How to cite this article:
Bhandari P L, Raghuveer C V, Rajeev A, Bhandari P D. Comparative study of peripheral blood smear, quantitative buffy coat and modified centrifuged blood smear in malaria diagnosis. Indian J Pathol Microbiol 2008;51:108-12

How to cite this URL:
Bhandari P L, Raghuveer C V, Rajeev A, Bhandari P D. Comparative study of peripheral blood smear, quantitative buffy coat and modified centrifuged blood smear in malaria diagnosis. Indian J Pathol Microbiol [serial online] 2008 [cited 2014 Nov 27];51:108-12. Available from: http://www.ijpmonline.org/text.asp?2008/51/1/108/40419



   Introduction Top


Malaria occurs throughout the tropics causing over 100 million cases and over 1 million deaths every year. [1] The earliest symptoms of malaria are very nonspecific and variable such as fever, headache, body ache, malaise, fatigue and abdominal discomfort. Hence, there is difficulty to clinically diagnose malaria but the treatment has to be started immediately in order to avoid complications. [2] The nonspecific nature of the clinical presentation of malaria may lead to over-treatment of malaria in malaria endemic areas and missing the diagnosis of malaria in low-transmission areas. In malaria endemic areas, this may lead to misdiagnosis and nontreatment of other diseases. The early diagnosis of malaria not only mitigates the sufferings but also reduces the transmission of the parasite in the community. [3] Therefore precise laboratory diagnosis and species identification is very essential.

The laboratory diagnosis of malaria is made by different techniques such as the conventional thin and thick peripheral blood smears (PBS), concentration techniques such as buffy coat smears and fluorescent (QBC) technique, Serologic tests such as the detection of parasite-specific proteins (Dipstick) [4] and PCR. These techniques have their own advantages and disadvantages with respect to sensitivity, specificity, time consumption, cost effectiveness, ease of procedure etc. It would be of great help if a new technique that utilizes most of the advantages, while eliminating most of the disadvantages of the above techniques, is developed and standardized.

The thin smear technique has no false positivity but it is not very sensitive. Hence, false negativity is frequent and requires two or more repetitions that may not be well appreciated by patients. The thick blood smear has a better rate of parasite detection; however, species identification becomes difficult sometimes. Dehemoglobinization is required if Leishman's stain is to be used. Jaswant Singh and Bhattacharya (JSB) and other field stains are not easily available to all laboratories. On the other hand, the fluorescent (QBC) technique, even though provides fast diagnosis with high sensitivity has disadvantages such as over-diagnosis (glowing particles other than actual malarial parasite are identified as positive), thereby leading to a risk of false positivity. Species identification is also difficult and leads to frequent errors, thereby tempting the technician to present a report of mixed infections (both Plasmodium vivax and Plasmodium falciparum ). In fact, the percentage of mixed positivity reported in this area is many times more than the national average. The cost factor is also considerably high, which is not affordable to many poor patients.

The centrifuged buffy coat smear involves the collection of 2 ml of venous blood into anticoagulant bottles, filling the Wintrobe's tube and centrifugation for 20-30 min, finally obtaining a buffy coat layer onto a slide is a very cumbersome procedure. Although capillary tubes were used in the past, it was unacceptable due to problematic procedure and lack of standardization. [5]

The present study aims at modification of the centrifuged buffy coat smear (MCBS) by the simplification of procedure as follows:

  1. Use of heparinized micro PCV tubes - this reduces the volume of the blood that can be obtained even with finger prick.
  2. High-speed centrifugation - this reduces the centrifugation time.
  3. Tube were cut with a special instrument in order to avoid shards of glass pieces
  4. The standardization of the technique to obtain good smears that are almost similar to thin smear in consistency


The results are compared with those obtained by PBS (thin and thick smears) and QBC with respect to sensitivity, specificity, speed of diagnosis, procedural convenience and cost effectiveness.


   Materials and Methods Top


Blood samples were collected from 100 patients attending Malaria Clinics at Govt. Wenlock Hospital, Mangalore, into heparinized capillary (Hematocrit) tubes and one end of the tubes was immediately sealed using bees wax. Simultaneously, direct thin blood smears were made and blood samples were also collected into QBC fluorescent tubes. The direct smears were stained with conventional Leishman's stain. The QBC tubes were centrifuged in the microcentrifuge at 6000 rpm for 5 min. The samples collected in the capillary tubes were centrifuged at 6000 rpm for 5 min. The capillary tube was cut by Adam's plier at the required junction, and the sediment column of blood was pushed by a steel wire onto a slide and a smear was prepared [Figure - 1],[Figure - 2],[Figure - 3].

These smears were air dried and stained by Leishman's stain and Sorensen's buffered distilled water at pH 7.2 as recommended by Dacie and Lewis. This renders the better identification of parasites and Schuffner's dots. [6]

The most important part of this technique was to obtain a good thin smear that contained a good concentration of parasitized RBC. The following methods were attempted:

A. The tube was cut just at the junction of buffy coat and RBC column by Adam's plier. The RBC column was pushed onto a slide with a steel wire and smears were prepared. These smears showed clumped RBCs making it difficult to identify the parasites [Figure - 4].

B. The tube was cut at the junction of plasma and buffy coat and smears were prepared from the buffy coat and sub buffy coat RBC column. The results were almost similar to those of technique A except for some RBCs between the WBC clumps that were clearly visible and parasite identification in these RBCs was easier.

C. Technique A was attempted by mixing a small drop of saline to sub buffy coat RBC column; however, the drawn blood film was not uniform.

D. The tube was cut just above the buffy coat layer, saving a small column of plasma. The retained plasma column, the buffy coat and an equal quantity of sub buffy coat RBC column were pushed on to the slide; they were gently and uniformly mixed, and smears were drawn. These smears had a thickness equivalent to peripheral blood smears, providing an advantage of including the concentration of parasitized RBCs in a thin smear [Figure - 4],[Figure - 5],[Figure - 6].

For PBS, two observers examined the smear under 100x (oil immersion magnification) for a maximum period of 10 min each, before calling the smear negative. One observer examined the QBC tube under fluorescent microscope for 10 min before presenting the negative report. This time included the setting of the QBC tube onto the stage and obtaining the proper focus. For MCBS, one observer examined the smear for a maximum period of 5 min before presenting the negative report. The smears that were positive by MCBS were submitted to parasite counting according to the "+" system.

The cost of PBS was Rs. 5 per test, that of QBC was Rs. 70 per test, while that of MCBS was found to be Rs. 12 per test.


   Results and Discussion Top


Justification for using the MCBS as gold standard

This technique combines the advantages of PBS and QBC. The parasites are observed directly without any aid and hence this technique is unquestionable as far as diagnosis goes. Tube centrifugation adds to the sensitivity of PBS and makes it similar to QBC. Hence, the two tests were matched against the new technique.

Part I: Validity of the tests

The validity of PBS and QBC were obtained by plotting a 2 x 2 table as shown in [Table - 1],[Table - 2], respectively.

[Table - 1] shows that PBS when compared with MCBS had a sensitivity of 86.79% and was absolutely specific. The positive and negative predictive values were 100% and 87.03%, respectively. As expected, the sensitivity of PBS was low when compared to MCBS. However, the smear test proved its specificity to be 100%, underlining its paramount importance in clinical medicine.

[Table - 2] shows that QBC with respect to MCBS was 96.22% sensitive, 93.61% specific with a positive predictive value of 94.44% and negative predictive value of 95.65%. As always suspected, the QBC technique did not have the specificity of PBS. Positive predictive value also did not reveal the absolute certainty of diagnosis. However, the claim of near 100% sensitivity was proven again in this study.

From [Table - 3], it is clear that QBC is superior to PBS by detecting 9.69% more cases than PBS. QBC has got a high specificity of 93.61%; however, it does give the remainder 6.39% (i. e. 3 out of 47, refer [Table - 2]) false positive results. On the other hand, PBS was highly specific but had a lesser sensitivity of 86.53%.

Part II: Distribution of cases

The three diagnostic tests gave varied results for the 100 blood samples as given in [Table - 4].

[Table - 5] shows the following points:

  1. By adding the process of centrifugation to the conventional smear technique, 7 more cases could be detected, improving its sensitivity to a great extent from 86.79% to near 100% - a total of 13.21% extra cases could be detected
  2. Among PBS positive cases, QBC had an exceptional sensitivity of 97.8% and was absolutely specific.
  3. Among PBS negative cases, however, QBC could detect 6 out of 7 cases (85.7%), but it over-detected 3 cases among 47. This false positivity of 7.3% may still be accepted due to its ability to detect a very high proportion (85.7%) of cases, which could not be detected by conventional PBS. This underlines the necessity of using QBC test in suspected malaria cases who are negative by PBS on one or more occasions.
  4. 85.7% of PBS negative patients were detected by QBC, who constitute 6% in the total sample population, are the ones who would be greatly benefited by undergoing the QBC tests. At the same time 89% of sample were not affected by QBC tests, while 3% were over-diagnosed and 2% missed. Thus, QBC is of great importance only in PBS negative cases and should be preferably used as a final diagnostic test and not as a screening test or first-line investigation considering its high cost and tendency to report false positives.
  5. Ten out of the 11 variations occurred in PBS negative cases, which implies that the variation is usually seen when the parasitemia is less than that required for PBS to detect. Variations can still occur in PBS positive cases; although rare, there was only one case in the present study.


As seen in [Table - 6], among the 11 variations, 3 were due to the false positive report by QBC (parasite count is not possible). The remaining 8 cases occurred when the parasite count was less than ++, i.e., less than 99 parasites per 100 oil immersion fields. This implies that when there is high parasitemia, all the tests are positive and only when the parasite count falls there are variations.

[Table - 7] shows the distribution of cases reported as falciparum by the tests. Report of MCBS was taken as standard. It shows that 10 out of 11 variations occurred in case of Plasmodium falciparum and only 1 variation in case of P. vivax. Hence, it is the more important group of falciparum malaria which is at the risk of variation.

Among falciparum cases with low parasitemia (PBS negative), QBC faired poorly with a sensitivity of 57.14% and a positive predictive value of 60%. It misdiagnosed 2 out of 7 PBS negative P. falciparum cases as P. vivax, while missing one completely. This observation is clinically very importance since the treatment regimen and the complications of the two types of malaria are quite different.


   Conclusion Top


It was found that QBC and MCBS were found superior to PBS. The new technique MCBS is as sensitive as QBC, as specific as PBS and eliminates false positives of QBC; it is also easy to perform and cost effective. However, more studies are required before we can establish that MCBS is a new gold standard in malaria diagnosis.

Suggestions

At the end of the study, the following suggestions were considered as appropriate.

Guidelines for rational use of QBC

a) QBC should be used in suspected malaria cases that are negative by PBS on one or more occasions.

b) QBC is of great importance only in PBS negative cases and should be preferably used as a final diagnostic test and not as a screening test or first line investigation considering its high cost and tendency to report false positives.

Establishment of facilities for MCBS

Most of India's population is rural and is covered by Primary Health Centers (PHCs) and sub-centers. Most of the equipments required for MCBS (e.g., Microscope, slide, stains, etc.) are already established in the PHCs and Malaria clinics. Providing other materials (capillary tubes, centrifuge, tube cutter and steel wire) will enable these centers to perform the MCBS technique, which is superior to QBC. The addition of centrifugation will increase the sensitivity of the smear technique from 86.79% to near 100%. This will detect 13.21% of extra cases in the community who would otherwise escape detection.


   Acknowledgement Top


It is with sincere gratitude that the investigator acknowledges the help extended by the following

  • Director General, ICMR for the award of short-term research studentship in the year 2005 to author 1.
  • District Surgeon and Superintendent, Govt. Wenlock Hospital, Mangalore for permission to use the facilities of the "Malaria Clinic."
  • Dr. Chandrayya Achari I/C Pathology Lab and his staff at "Malaria Clinic," Govt. Wenlock Hospital, Mangalore, for providing adequate clinical material.


 
   References Top

1.Park K. Malaria. In : Park's textbook of social and preventive medicine. 18 th ed; Banarsidas Bhanot: Jabalpur; 2004. p. 201-3,687.  Back to cited text no. 1    
2.White NJ, Breman JG. Malaria and babesiosis diseases caused by RBC parasites. In : Harrison's principles of internal medicine. Kasper DL, et al , editors. 16 th ed (Vol. 1); McGraw Hill: New Delhi; 2005. p. 1224-6.  Back to cited text no. 2    
3.Govt of Karnataka; Circular No NVBDCP: HE: 27:04-05; Ministry Of Health and Family Welfare: 2004. p. 1-4.  Back to cited text no. 3    
4.Todd WT, Lockwood DN, Nye FJ, Wilkins EG, Carey PB. Infection and immune failure. In : Davidson's principles and practice of medicine. Haslett C, et al , editors. 19 th ed; Churchill Livingstone: London; 2002. p. 54.  Back to cited text no. 4    
5.Woo PT, Hauck L. The haematocrit centrifuge smear technique for the detection of mammalian Plasmodium . Trans Royal Soc Trop Med Hyg 1987;81:727-8.  Back to cited text no. 5    
6.Bain BJ, Lewis SM. Preparation and staining methods for blood and bone marrow films. In : Dacie and Lewis Practical Hematology. Lewis SM, et al , editors. 9 th ed; Churchill Livingstone: London; 2001. p. 49.  Back to cited text no. 6    

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Correspondence Address:
P L Bhandari
"Mangala", Chilimbi Hilldale Road, Ladyhill, P.O. Ashoknagar, Mangalore - 575 006, Karnataka
India
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DOI: 10.4103/0377-4929.40419

PMID: 18417878

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    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7]

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