This is a descriptive study of all stages of the standardization and evaluation of a new health-related product that was designed to improve the teaching of malarial parasite morphology. This study was conducted in the Malaria Laboratory in the Infectious Diseases Clinic at Júlio Müller University Hospital from April 2012 to December 2013.
To standardize the preparation and staining of the blood smears on the transparent acetate sheets, blood from healthy laboratory staff members was used. Then, to analyse the quality of these blood smears for the diagnosis of malaria, blood samples were obtained from ten patients with malaria. The involvement of the individuals in this study was voluntary, and full consent was obtained. The study was approved by the Research Ethics Committee at Júlio Müller University Hospital as authorized by nº 171082 on 12 December, 2012.
PPC acetates obtained from different manufacturers and only one crystalline PVC brand name were tested with regard to their resistance in contact with methanol, which is used to fix blood smears. Small strips of each acetate sheet were immersed in test tubes filled with absolute methanol (Sigma-Aldrich, cat. M1775-1GA) and observed for 30 minutes. The acetate strip was then removed from the tube, and its texture and transparency were observed with the naked eye.
Preparation of blood smears
The acetate sheet was previously treated with gauze soaked in 96% absolute alcohol to guarantee the homogenous distribution of blood in the smear. First, an A4 paper sheet was printed with a grid table containing 273 uniform 1.2 sq cm cells. The acetate sheet was then overlapped the paper sheet, in order to guide the space to be filled by blood smear (Figures 1 and 2). Immediately after the blood was collected, thin and thick blood smears were prepared on glass slides and transparent acetate sheets.Using an automatic pipette, 4 μL of blood was dropped onto the acetate, at 1.2-cm intervals. With a medium-sized acrylic square, the blood was spread out vertically over the sheet to form a thin smear with approximately 3.6 cm length and 15.6 cm wide. This process was repeated in the remaining space on the acetate sheet (Figure 1). For the thick smear, 10 μL of blood was dropped onto the acetate at 1.2-cm intervals. The blood was then spread out using a rod to cover a larger area of acetate. Better homogenization of the thick smear was obtained when the blood spreading was limited to rectangles of approximately 50–90 sq cm (Figure 2). Only one patient sample was used per acetate sheet in order to prevent cross-contamination.
The sheets with the blood smears were kept in a dry environment and protected from dust and insects for 12–24 hours, after which the thin blood smears were fixed with methanol. Thick and thin blood smears were also prepared using the conventional method on glass slides [7] previously treated with 96% absolute alcohol as well.
Staining of blood smears
After drying, the thick blood smears were rapidly dehaemoglobinized with an aqueous solution of 0.4% methylene blue (Labsynth™, Labsynth Produtos Laboratório Ltda, São Paulo, Brazil), applied using a dispensing bottle, and then rinsed in buffered water. This rapid staining stage with methylene blue was added to increase the contrast of the parasites upon the debris of the haemolysed blood cells in the thick smear [5].
The acetate sheets were then placed on a plastic tray on two metal rods for support. This ensured that the entire length of the smear on the acetate was simultaneously stained. To stain the two acetate sheets, ie, the thick and thin smears, approximately 300 mL of 10% diluted Giemsa solution (Labsynth™, Labsynth Produtos Laboratório Ltda, São Paulo, Brazil) was used (Figure 3A and B). For acetate sheets and glass slides, the staining method was the same and was performed as recommended by WHO [7, 8]. An example of the final results of the stained smears on acetate is shown in Figure 4.Once stained, the acetate sheets were cut into rectangular strips and mounted on glass slides. A drop of immersion oil was used to fix the acetate strips to the glass slide according to the sequence shown in Figure 5.
Fixation of blood smears with finishing varnish and mounting on glass slides
After staining, the blood smears prepared on acetate were manually coated with a finishing varnish (DuPont®, São Paulo – SP, Brazil). Using a 5 ml syringe attached to a 27 gauge needle, a line of the varnish was manually applied to the edge of the acetate sheet. By sliding a glass rod over the sheet, the varnish was spread across all the smear (Figure 6). The blood smears were fixed to increase durability and protect them from possible damage or colonization by fungi and bacteria. The drying time of this varnish was 5–10 minutes.
After fixation, the acetate sheets were cut into small rectangular strips (1.2 sq cm for thick blood smears and 2.4 sq cm for thin blood smears). The acetate strip was then placed onto a glass slide atop a drop of immersion oil for fixation. No coverslips were used.
Evaluation of quality and durability of blood smears prepared on transparent acetate
Verifying the quality of blood smears prepared on acetate involves analysing the staining and morphology of parasites and blood cells under the microscope compared with blood smears mounted on glass slides. These images were photodocumented using a Zeiss-Scope-A1 microscope (Carl-Zeiss, Oberkochen, Germany). Moreover, all of the blood smears were analysed by three microscopists to estimate the parasitic density in each patient. The different blood stages of the parasites were identified from 200 microscopic fields (1,000×) and the parasitaemia was estimated by multiplying the amount recorded by a factor of 2.5 since this microscopic area is approximately 0.4 cu mm of blood [9]. The F-test and the Bartlett test were used to compare the variances between the parasite densities by the three microscopists with the significance level set at 95%.
The durability of the blood smears mounted on transparent acetate was evaluated based on the persistence of the quality characteristics evaluated above and the absence of fungal colonisation after the specimens were stored for a period of 12 months.
Possibility of reusing the smear mounted on acetate
Some glass slides were deliberately broken to simulate everyday accidents during malaria microscopy training sessions. The acetate strip containing the smear was recovered and reassembled onto a new glass slide. The preparation and staining quality of the recovered smear were then evaluated as described above.