Microscopy-based Methods for Rosetting Assay in Malaria Research

[Abstract] In malaria, rosetting phenomenon is a condition where a Plasmodium -infected erythrocyte stably adheres to at least an uninfected erythrocyte. This phenomenon that occurs in all species of human malaria parasite is likely to be an immune escape mechanism for the parasite. However, it has been associated with malaria pathogenesis, possibly by facilitating microvasculature occlusion along with direct endothelial cytoadherence by the infected erythrocytes. There are different microscopy-based techniques to visualize rosettes but neither of these techniques has yet to qualify as the official “gold standard” method. We have found that these techniques can be used interchangeably, provided that the conditions of the experiments are properly controlled. Here, we presented three methods as options for rosetting assay, i.e. , the unstained wet mount technique, acridine orange based-fluorescence microscopy technique and Giemsa stained wet mount method, with preparation steps that enable consistent performance in rosetting experiments.

3. Prepare an aliquot of the culture suspension, and process with either of the three methods (Steps 4-6), according to the feasibility of the techniques in the laboratory. 4. Stain the suspension with Giemsa (5% working concentration volume by volume) for 15 min prior to wet mount preparation for rosetting assay.
5. Alternatively, add Acridine Orange (working concentration 2 µg/ml) into the suspension and allow staining for 15 min prior to wet mount preparation for rosetting. 6. The third method is to use the suspension for wet mount preparation without staining.
7. Wet mount is prepared by pipetting small volume of the suspension above onto a clean glass slide, and cover the droplet with a clean glass coverslip by landing the coverslip slowly to minimize trapment of air bubbles.
8. The Giemsa-stained and unstained wet mount can be viewed with light microscope (of any model), whereas the Acridine Orange-stained wet mount can only be viewed with fluorescence microscope (of any model). 9. Determine the rosetting rates by recruiting 200 infected erythrocytes. Rosetting rate is the percentage of infected erythrocytes that adhere to uninfected erythrocytes.

Data analysis
Detailed information of data analyses are available in research article (Lee et al., 2020), where the rosetting rates of P. falciparum were collected using the three rosetting assay methods described above, and no significant difference were found among the methods (Supplementary file 7. Method comparison for rosetting assay).

Notes
1. Serum is needed to allow rosette formation.
2. It is important to type-match the ABO blood group of the erythrocytes used and the sera used for enrichment of culture media. This is to avoid agglutination of the cells (Sazama, 1990;Williamson et al., 1999). If the ABO blood group cannot be determined, use only AB serum. 4. Try to avoid using EDTA-vacutainer to collect blood samples for rosetting assay. All anticoagulants hamper rosette formation. However, we noticed that the rosette-inhibitory effect by EDTA is more difficult to be restored after removal of the anticoagulant, which is similar to an earlier report on another chelating agent, EGTA (Chotivanich et al., 1998).  8. The unstained wet mount is the simplest way forward for rosetting assay. However, one will need to adjust the light intensity and play with the contrast enhancing features of the microscope to allow clear visualization of the unstained parasites.
9. Resuspend the cell suspension prior to wet mount preparation to ensure the correct hematocrit of cell suspension. 10. The volume of cell suspension for wet mount-making depends on the size of the glass coverslip used. If the volume is too small for the coverslip, the suspension will be "smeared" and "look dried", which become unreadable for rosetting assay. As the wet mount is examined under 5 www.bio-protocol.org/e3665 11. If the volume is too big for a small coverslip, the cells will "move" rapidly and it hampers the examination.
13. Never read a wet mount for too long. Crenation of cells starts to happen minutes after the wet mount is placed on the microscope stage, lit by strong bright light (due to evaporation). The cells around the rim/edge of the glass coverslips are affected first, and the effect will spread inward.
Once the cells are crenated, the wet mount is no longer suitable for rosetting assay and new wet mount should be prepared.
14. Clean glass slides (oil/grease-free) should be used for the wet mount preparation.
15. Rosetting is a phenomenon that is seen in the late stages. If the parasite suspension used is not stage-synchronised, the worker should not include the ring stages into their rosetting rate counting.

Giemsa stock preparation
a. Filter the dye stock solution with 0.45 µm filter before use. This is to ensure clear and clean visualization of parasites in wet mount.
b. For the dye intended for wet mount staining, the dye filtering should be done once per month.
c. Alternately, filter paper can be used for the filtration. Nevertheless, do not filter a large volume of dye and keep it for too long.
d. The stock should not be exposed to direct sun light.

Media
Enrich the plain medium with 20% type-matching heat-inactivated human serum. For instance, 10 ml of human serum is added to 40 ml of plain medium to prepare 50 ml of medium needed.
RPMI1640 medium is used. However, McCoy's 5A medium that is used for P. vivax maturation assay can be used for rosetting assays on P. vivax as well.

Notes:
a. Fresh media should be used for the experiment.
b. Pay attention to the color of the media. Old media that are usually more intense pinkish (alkaline) may affect the staining efficiency of the dyes.