‘In-house’ quantitative buffy coat technique in the diagnosis of malarial parasites: a cost-effective method

Pragati Chigurupati; Murthy K Srinivas

doi:10.4103/1687-7942.192991

ORIGINAL ARTICLE

Year : 2016 | Volume : 9 | Issue : 1 | Page : 55-58

‘In-house’ quantitative buffy coat technique in the diagnosis of malarial parasites: a cost-effective method

Pragati Chigurupati¹, Murthy K Srinivas²
¹ Department of Microbiology, GSL Medical College, Rajahmundry, Andhra Pradesh, India
² Dhanavantari Blood Bank, Rajahmundry, Andhra Pradesh, India

Date of Submission	26-Jan-2016
Date of Acceptance	11-May-2016
Date of Web Publication	25-Oct-2016

Correspondence Address:
Pragati Chigurupati
80-26-10, A-3, Heritage Residency, A.V.A. Road, Rajahmundry 533103, Andhra Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1687-7942.192991

Abstract

Background
Microscopic examination of blood films is accepted as the universal ‘gold-standard’ for the diagnosis of malaria. However, low parasitemia accounting for false-negative results and time consumption for declaring results are limiting factors for smear microscopy. The quantitative buffy coat (QBC) technique is a more rapid test and can detect malarial parasites (MP) in the condition of low parasitemia.
Aim
The aim of this study was to show the usefulness of an ‘in-house’ QBC method in the diagnosis of MP.
Patients and methods
One hundred positive smear samples of patients diagnosed with malaria were subjected to analysis by both the ‘in-house’ QBC and the commercial QBC technique. Two hundred samples negative for malaria on microscopic smear examination were also tested using the ‘in-house’ QBC method.
Results
The 100 smear positive samples tested positive for MP with both commercial QBC testing and the ‘in-house’ QBC technique. Of the 200 samples that tested negative on smear examination, 16 were positive for malaria on testing with the ‘in-house’ QBC technique.
Conclusion
The described ‘in-house’ QBC technique provides an additional advantage of being cost-effective in comparison with the available commercial QBC kits.

Keywords: malaria, quantitative buffy coat, smear examination

How to cite this article:
Chigurupati P, Srinivas MK. ‘In-house’ quantitative buffy coat technique in the diagnosis of malarial parasites: a cost-effective method. Parasitol United J 2016;9:55-8

How to cite this URL:
Chigurupati P, Srinivas MK. ‘In-house’ quantitative buffy coat technique in the diagnosis of malarial parasites: a cost-effective method. Parasitol United J [serial online] 2016 [cited 2023 Mar 21];9:55-8. Available from: http://www.new.puj.eg.net/text.asp?2016/9/1/55/192991

Introduction

Laboratory diagnosis of malarial infection by smear examination has been the gold-standard technique for over 100 years in expert hands [1]. The sensitivity of microscopy is 20–30 parasites/µl of blood and 100 parasites/µl in field conditions. Low parasitemia accounts for false-negative results. This indicates the need for a method with better sensitivity, for example, quantitative buffy coat (QBC) examination of blood samples [2]. In recent years, commercial QBC methods have been used in the diagnostic field to detect malarial parasites (MP) that are low in number (low parasitemia) in blood samples [3]. The constraint is the cost, and to address this problem, we developed an ‘in-house’ cost-effective QBC technique.

Patients and methods

This descriptive analytical research was carried out at GSL General Hospital and Anand Diagnostic Centre, Rajahmundry, Andhra Pradesh, India. We selected 100 blood samples from patients proven to be positive for malaria infection by stained smears using Field’s technique [4], subjected them to both commercial QBC (Becton and Dickenson Inc., Franklin Lakes, New Jersey, USA) and in-house QBC methods, and analyzed the results. Patients were admitted and treatment consent was taken as per standard protocol which included investigations, hence no separate consent form.

From each patient, 1.2 ml blood sample was collected in an ethylene diamine tetra acetic acid (EDTA) solution under sterile conditions. A precoated QBC capillary tube [coated with EDTA, acridine orange (AO) solution] was dipped in blood and filled to the provided mark by capillary suction. One end of the tube was sealed with closure and the other end with a floater, ensuring that the floater did not touch the column of blood. The tubes were labeled, centrifuged at 12000 rpm for 5–7 min, adjusted to the QBC viewing device, and examined under an ultraviolet (UV) microscope using oil immersion and a ×60 fluorescent objective. For the in-house QBC technique, uncoated QBC tubes were filled by capillary action with 100 µl known malaria-positive EDTA blood, mixed with 10 µl 0.01% AO solution up to the provided mark, and similarly closed at both ends as the coated commercial tubes. Centrifugation was performed at a reduced rate of 3500 rpm for 20 min, followed by examination using immersion oil applied to ×60 or ×45 fluorescent objectives. Once the ‘in-house’ QBC was standardized, the sensitivity of QBC over the routine blood smear examination was studied. In addition, 200 blood samples that proved negative for MP on smear examination were tested using the ‘in-house’ QBC method.

Results

Both the commercial QBC and the ‘in-house’ QBC technique successfully detected MP in the 100 positive samples. Furthermore, the ‘in-house’ QBC technique detected additional 16 samples of the 200 samples that tested negative on smear examination. Plasmodium vivax and Plasmodium falciparum were diagnosed in 10 and 4 samples, respectively, whereas the ‘in-house’ QBC failed to identify the causative Plasmodium spp. in only two samples. Out of the 16 positive samples, grades 1+ (<1/QBC field) and 2+ (1–10/QBC field) were detected in 10 and 6 cases, respectively. The results are shown in [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5].

Figure 1 Plasmodium vivax intra-erythrocytic ring forms viewed by quantitative buffy coat.

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Figure 2 Plasmodium falciparum intra-erythrocytic accole forms viewed by quantitative buffy coat.

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Figure 3 Plasmodium falciparum intra-erythrocytic ring forms viewed by quantitative buffy coat.

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Figure 4 Plasmodium intra-erythrocytic trophozoite forms viewed by quantitative buffy coat.

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Figure 5 Plasmodium intra-erythrocytic schizont forms viewed by quantitative buffy coat.

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Discussion

Malaria presents a diagnostic challenge to laboratories in most countries. The urgency and importance of obtaining results quickly from the examination of blood samples from patients suspected of having acute malaria render some of the more sensitive methods for malaria diagnosis impractical for routine laboratory use. The majority of malaria cases are found in countries where cost-effectiveness is an important factor and ease of diagnostic test performance and training of personnel are also major considerations.

The accepted laboratory practice for the diagnosis of malaria is the preparation and microscopic examination of blood films stained with Giemsa, Wright’s, or Field’s stain [4]. Both thick and thin blood films should be prepared. The expected sensitivity that can be achieved by an experienced microscopist for the examination of the thick blood film procedure is about 50 parasites/µl of blood [assuming a total red blood cell (RBC) count of 5×10⁶/µl of blood], which is equivalent to 0.001% of RBC infected [5]. Warhurst and Williams [6] reported that examination of thin blood films is only 1/10 as sensitive as examination of thick blood films for the quantification of MP, although morphological identification of the Plasmodium spp. present is much easier using thin films. Therefore, most laboratories involved in the quantification and identification of MP by microscopy produce both thick and thin blood films. It is highly recommended that both thick and thin films be prepared and examined each time blood film examinations for parasites are requested. The staining process may take up to 60 min of preparation time to produce a stained thin or thick film and is labor intensive. Interpretation requires considerable expertise, particularly at low levels of parasitemia.

In an attempt to enhance the detection of MP in blood films, alternative methods have been introduced. Certain fluorescent dyes have an affinity for the nucleic acid in the parasite nucleus and will attach to the nuclei. When excited by UV light at an appropriate wavelength, the nucleus will fluoresce strongly. AO is one such dye that shows excitation at 490 nm and shows apple green or yellow fluorescence. Several methods have been published in which AO has been used either as a direct-staining technique or combined with a concentration method such as a thick blood film [7]. The QBC test is one such rapid method for identifying the MP in the peripheral blood [8]. It involves staining of the centrifuged and compressed red cell layer with AO and its examination under a UV light source. It is fast, easy, and considered to be more sensitive than the traditional thick smear examination [9],[10],[11]. The key feature of the method is centrifugation and thereby the concentration of the RBCs in a predictable area of the QBC tube, making detection easy and fast. Red cells containing plasmodia are less dense than normal ones and concentrate just below the leukocytes at the top of the erythrocyte column [12]. The float forces all the surrounding red cells into the 40 micron space between its outside circumference and the inside of the tube. As the parasites contain DNA that takes up the AO stain, they appear as bright specks of light among the nonfluorescing red cells [13]. Almost all of the parasites found in the 60 μl of blood can be visualized by rotating the tube under the microscope. A negative test can be reported within 1 min and a positive result within minutes. Studies that have compared the QBC with the peripheral smear report that the test is as sensitive as the smear and more specific. However, identification of the species and quantification of parasitemia are difficult with the QBC technique [14]. The sensitivity of AO staining for the detection of MP in infections with parasite levels of <100 parasites/µl (0.002% parasitemia) has been reported to range from 41 to 93% [15]. The specificity for infections with P. falciparum is excellent (>93%) [16], with most observers able to identify the small ring forms. With experience, workers using methods involving fluorochrome compounds can detect parasites rapidly and accurately. However, an important limitation of the QBC method is the ability to easily differentiate among Plasmodium spp. With experience, the various stages of malaria observed in the peripheral blood may be located in defined areas of the centrifuged buffy coat, but specific identification of species remains difficult without examination of the RBC morphology and parasite inclusions. Despite some reservations on the use of fluorescence methods for malaria detection [17], including the requirement of special training and expensive equipment and supplies, fluorescence microscopy remains a viable and rapid alternative to Romanowsky staining.

In developing countries such as India, where one has to account for the cost-effectiveness of a test, the ‘in-house’ QBC technique scores over the commercial QBC method. Our estimate for the commercial QBC method is: costly instrument (2.5–2.7 lakh rupees), costly centrifuge (12 000 rupees), and cost per test comes to 40 rupees. In contrast, for the in-house method, the instrument is less costly (1.5–1.7 lakh rupees), the centrifuge costs 3500 rupees, and the cost per test comes to 10 rupees.

Conclusion

The ‘in-house’ QBC is more cost-effective than the commercial QBC method. An extra 8% samples were detected to be positive by ‘in-house’ QBC over routine smear examination. Species identification of the MP is not possible by QBC. It is advisable that all blood smear-negative samples be tested by QBC before declaring them negative for MP.

Author contributions

Both authors shared equally in the research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	World Health Organization New perspectives in malaria diagnosis. Geneva, Switzerland: World Health Organization; 2000.
2.	Baird JK, Purnomo , Jones TR. Diagnosis of malaria in the field by fluorescence microscopy of QBC capillary tubes. Trans R Soc Trop Med Hyg 1992; 86:3–5. [PUBMED]
3.	Benito A, Roche J, Molina RA, Amela C, Altar J. Application and evaluation of QBC® malaria diagnosis in a holoendemic area. Appl Parasitol 1994; 35:266–272.
4.	Gilles H. Diagnostic methods in malaria. In: Gilles HM, Warrell DA, editors. Essential malariology. 3rd ed. London, UK. Edward Arnold; 1993.
5.	Milne LM, Kyi MS, Chiodini PL, Warhurst DC. Accuracy of routine laboratory diagnosis of malaria in the United Kingdom. J Clin Pathol 1994; 47:740–742.
6.	Warhurst DC, Williams JE. Laboratory diagnosis of malaria. J Clin Pathol 1996; 49:533–538.
7.	Hänscheid T. Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab Haematol 1999; 21:235–245.
8.	Gay F, Traoré B, Zanoni J, Danis M, Fribourg-Blanc A. Direct acridine orange fluorescence examination of blood slides compared to current techniques for malaria diagnosis. Trans R Soc Trop Med Hyg 1996; 90:516–518.
9.	Kong HH, Chung DI. Comparison of acridine orange and Giemsa stains for malaria diagnosis. Korean J Parasitol 1995; 33:391–394.
10.	Lowe BS, Jeffa NK, New L, Pedersen C, Engbaek K, Marsh K. Acridine orange fluorescence techniques as alternatives to traditional Giemsa staining for the diagnosis of malaria in developing countries. Trans R Soc Trop Med Hyg 1996; 90:34–36.
11.	Adeoye GO, Nga IC. Comparison of quantitative buffy coat technique (QBC) with Giemsa-stained thick film (GTF) for diagnosis of malaria. Parasitol Int 2007; 56:308–312.
12.	Cabezos J, Bada JL. The diagnosis of malaria by the thick film and the QBC: a comparative study of both techniques. Med Clin (Barc) 1993; 101:91–94.
13.	Pinto MJ, Rodrigues SR, Desouza R, Verenkar MP. Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria. Indian J Med Microbiol 2001; 19:219–221. [PUBMED]
14.	Wang X, Zhu S, Liu Q, Hu A, Zan Z, Yu Q, Yin Q. Field evaluation of the QBC technique for rapid diagnosis of vivax malaria. Bull World Health Organ 1996; 74:599–603.
15.	Wongsrichanalai C, Pornsilapatip J, Namsiripongpun V, Webster HK, Luccini A, Pansamdang P et al. Acridine orange fluorescent microscopy and the detection of malaria in populations with low-density parasitemia. Am J Trop Med Hyg 1991; 44:17–20.
16.	Gaye O, Diouf M, Diallo S. A comparison of thick films, QBC malaria, PCR and PATH falciparum malaria test strip in Plasmodium falciparum diagnosis. Parasite 1991; 6:273–275.
17.	Delacollett D, van der Stuyft P. Direct acridine orange staining is not a ‘miracle’ solution to the problems of malaria diagnosis in the field. Trans R Soc Trop Med Hyg 1994; 88:187–188.