A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

1. Sample Collection

Platelets can be easily activated through temperature, agitation or storage and therefore they should be handled with care. The use of vacutainers to collect blood for platelet preparation is unavoidable in most clinical situations, but should be carefully considered as suction can potentially cause platelet activation. Due to the clinical protocol used at our research hospital, our samples are carefully collected in sodium citrate vacutainers. We have not experienced any problems with premature platelet activation in this or our previous study ¹². We collect blood for platelet preparation from blood group O+ individuals to minimize nonspecific blood group antigen aggregation. Donors have also not taken medication in the previous 7-10 days as some drugs are known to influence platelet function.

1.1 Preparation of platelet-rich plasma (PRP)

1. Collect approximately 5 ml of whole blood from a malaria naive host or 2.5 ml of blood from CM or severe malaria (SM) patients in a sodium citrate vacutainer (BD product number 36276).
2. Centrifuge the whole blood at 250 x g for 10 min at room temperature. Do not centrifuge at 4 °C as low temperatures may cause the platelets to aggregate.
3. Carefully transfer the cloudy supernatant in a clean 15 ml tube. Platelets are easily activated by temperature variations and physical shocks and should therefore be handled with care. Avoid shaking or any jerky movements of the tube.
4. Use a Neubauer’s haematocytometer to count and adjust the platelet suspension to >300 x 10³ platelets/μl for healthy donors and <150 x 10³ platelets/μl from CM and SM patients using 1X phosphate buffer (pH 7.2-7.4; PBS). Make sure to choose a high dilution factor to ease the platelet count and ensure its accuracy.

Note: Malaria patients have a reduced platelet count compared to the healthy individuals ^16,17,18. Therefore platelet concentrations for CM and UM are adjusted according to the average platelet counts for patients within each malaria diagnostic group. The P. falciparum clumping phenotype is common in CM isolates and displays a strong binding affinity therefore the reduced platelet concentrations does not affect clump size or frequency since platelet concentration is standardized for all CM cases.

1.2 Preparation of platelet-poor plasma (PPP)

1. To obtain PPP, centrifuge a portion of the PRP obtained above at 1,500 x g for 10 min. The majority of platelets are pelleted at the bottom of the tube.
2. Both PRP and PPP can be stored at 4 °C for up to two weeks. Ideally, use fresh platelets for the clumping assay. After 8-10 days most of the platelets are likely to be inactive and probably aggregated.

2. Parasite Culture

1. Wash the pelleted pRBC three times with 5-10 ml RPMI 1640 by centrifugation at 370 x g for 5 min.
   Note: in this protocol all cultures started from approximately 1 ml packed cell volume (PCV) and maintained at 5% haematocrit. Cultures can be started with any PCV of pRBC and adjusted accordingly with uninfected RBC and media to reach desired haematocrit.
2. Place the pRBC in a culture flask and supplement with standard malaria culture medium of RPMI 1640 supplemented with 25 mM HEPES, 5% Albumax II or 10% serum and 40 μg/ml gentamycin to achieve a 5% haematocrit.
3. Permeate culture flasks with a mixture of 92.5% nitrogen, 2.5% oxygen and 5% carbon dioxide before sealing and incubating. Alternatively, the air-tight candle jar method of Trager-Jensen can be used.
4. For pRBC obtained directly from patients, incubate their culture flask for 24-36 hr in a 37 °C incubator in 5% CO₂ to get mature parasites.
   NOTE: Most laboratory and culture-adapted lines are very easy to maintain in culture under standard conditions. There are simple techniques described below that can be used to synchronise different parasite stages to retain growth rate.
5. Prepare a thin blood smear as described below and examine parasite maturation under a light microscope.

3. Thin Blood Film Slide Preparation

1. Place approximately 10-15 μl of blood on one end of a frosted glass slide resting on a flat surface.
2. Touch the drop of blood with the edge of a second slide until the blood is evenly spread across the edge of the second slide.
3. While holding the second slide at a 45° angle, quickly but gently, without exerting too much pressure on the first slide, slide the blood across the first slide to make a thin film of blood that is evenly spread. Air dry.
4. Dip the slide in methanol for 10 sec. Air dry.
5. Dip the slide in 2% Giemsa stain for at least 10 min. Rinse extensively until the water runs clear. Air dry.
6. Examine slide using a 90-100x oil emulsion lens on a light microscope

4. Purification and Synchronization of Asexual Stages

The purification of mature P. falciparum pRBC is a required step for the clumping assay. Indeed, only mature parasites are capable of binding to platelet receptors as it is at this life cycle stage that the relevant ligands are expressed and protrude from the surface of infected erythrocytes. There are several methods to purify and synchronize asexual stages of P. falciparum.: Late asexual stages can be enriched by Percoll gradient ¹⁹; magnetic cell sorting ²⁰ or using the gelatin sedimentation ²¹ protocol described here. Sorbitol-synchronization selects for ring stages ²², after which the rings are cultured for 24-26 hr to obtain mature stages (with no need to perform gelatin flotation).

4.1 Plasmagel Flotation

Plasmagel flotation uses gelatin-like solution to select for lower weight knobbed erythrocytes to remain in solution while the dense ring forms and rosettes sink to the bottom. This technique gives up to 90% purity of the mature stage-infected erythrocytes and can be used for both field and laboratory isolates. However, as previously mentioned, plasmagel flotation removes rosetting pRBC as well as immature stages which could result in a reduced clumping frequency in those samples with a high rosetting frequency. However, the absence of the rosetting pRBC after plasmagel flotation would not affect the clumping assay because rosetting is an interaction of pRBC and uninfected erythrocytes while clumping is pRBCs binding mediated by platelets. The ligand involved in these two traits is different; with mainly complement receptor CR-1 on uninfected erythrocytes mediating rosetting.

1. Prewarm Gelofusine solution and serum/Albumax II-free medium to 37 °C in a water bath.
2. Transfer culture to a clean sterile 15 ml tube and pellet cells by centrifugation at room temperature at 370 x g for 5 min.
3. Carefully aspirate supernatant so as not to disturb the pellet and resuspend cells in an equal volume of Gelofusine and protein-free medium.
4. Transfer mixture to a sterile 15 ml tube and allow to stand for 15-20 min in a 37 °C incubator or until a clear demarcation can be seen between an upper and lower level.
5. Carefully transfer the upper layer to a fresh sterile 15 ml tube and add 10 ml of fresh RPMI.
6. Centrifuge at 370 x g for 5 min and carefully discard supernatant.
7. Assess parasitaemia and developmental stage by thin blood smear and examine under a light microscope as described in section 4.

NOTE: Mature pRBC range between 60-90% of infected erythrocytes depending on the initial parasitaemia of the culture. For high percentage of mature parasites, use cultures with an initial parasitaemia of ≥10%.

5. Clumping Assay

1. Use a Neubauer’s haematocytometer to count and adjust the pRBC suspension obtained after plasmagel flotation to 1 x 10⁸ pRBC/μl.
2. In a fresh 1.5 ml tube, resuspend pRBC at 5% hematocrit, acridine orange at 20 μg/ml final concentration and PRP at 10% of the total volume.
   Instead of acridine orange, parasite cultures can be stained with 25 μg/ml ethidium bromide for 5 min before use.
   E.g. For a 200 μl reaction mix, add 10 μl mature pRBC, 20 μl of 300 x 10³ platelets/μl for PRP from healthy donors or 150 x 10³ platelets/μl from SM patients and 170 μl of protein-free medium.
   NOTE: The ratio of platelets:pRBC in the final reactions is 20:1. This is important as is allows the maximum clumping frequency of a sample to be determined. If fewer platelets are added in controls then not all clumping pRBCs might have the chance to clump.
3. In the same way prepare the following controls; uninfected red blood cells and PRP, and pRBC with PPP to ascertain the role of platelets in the pRBCs clumping.
4. Transfer the suspension to a 1.5-2.0 ml conical screw-cap vial.
5. Place the vial under gentle agitation by rolling at 10-12 rpm at room temperature.
6. Check for clump formation by a wet-slide prepared by pipetting 10 μl of pRBC ± platelet mix on a glass slide, cover with a glass slip and examine under fluorescence.
7. Sampling can be done at 15-20 min intervals for a total of 120 min. A clump is considered as an aggregate of three or more infected erythrocytes.