This procedure offers some standardized guidelines to perform platelet mediated clumping assays of clinical or laboratory plasmodium false parem isolates. First, collect blood from an infected patient and harvest parasitized erythrocytes by centrifugation, then culture and purify the asexual mature stages. Next, collect blood from a healthy donor in order to prepare and standardize the platelet rich plasma by centrifugation.
Combine both the platelets and the parasitized erythrocytes for the clumping assay. Say results from immunofluorescence microscopy can show P Fal sperum clumps mediated by platelets. This technique is specifically designed to measure the clumping phenotype of plasmodium sra.
Clinical isolates in a resource poor setting. Minimal variations in procedures have been reportedly to increase influence in the outcome of clumping acid in the field. A clear and easy to follow protocol adapted to low resource settings to standardize across malaria endemic field sites was therefore needed In a sodium citrate, vacutainer collect approximately five milliliters of whole blood from a malaria naive host or 2.5 milliliters of blood from CM or SM malaria patients.
Centrifuge the whole blood at 250 Gs for 10 minutes at room temperature. Then carefully transfer the cloudy supernatant to a clean 15 milliliter tube. Remember that platelets are easily activated by temperature, variations, and physical shocks.
Enumerate the PRP suspension using annoy bower's, hemo cytometer and adjust to desired concentration with PBS to obtain platelet poor plasma pellet, the majority of platelets from a portion of the PRP fraction by centrifugation at 1500 GS for 10 minutes. Store PRP and PPP fractions at four degrees Celsius for up to eight to 10 days. Ideally using fresh platelets for the clumping assay.
Wash the pelleted peripheral red blood cells three times with five to 10 milliliters of RPMI 1640 in a culture flask Supplement PRBC with standard malaria culture, medium and fresh uninfected red blood cells to achieve a 5%hematocrit. Permeate the culture flasks with a mixture of 92.5%nitrogen, 2.5%oxygen, and 5%carbon dioxide. Then seal and culture for mature parasites from patient pbcs.
Incubate the culture flask for 24 to 36 hours in a 37 degrees Celsius incubator at 5%carbon dioxide. Place approximately 10 to 15 microliters of blood on one end of a frosted glass slide resting on a flat surface. 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 while holding the second slide at a 45 degree angle.
Quickly but gently without exerting too much pressure on the first slide evenly spread the blood across the first slide. Then air dry the thin film of blood, dehydrate the specimen area with methanol for 10 seconds. Then air dry next, immerse the specimen with 2%keem.
Sustain for at least 10 minutes. Rinse extensively until the water runs clear and air dry. Examine the samples under a 100 x oil emulsion lens on a light microscope.
Next, purify and synchronize the mature PFS parum, PRBC as described in the text protocol. All transfer the culture to a clean, sterile 15 milliliter tube. Pellet the cells by centrifugation at room temperature at 370 GS for five minutes.
Now carefully aspirate the supernatant without disturbing the pellet. Then resuspend the cells in such a way that one half of the total volume is jello fusing. One third of the second half is protein free medium and two thirds of the second half is pbcs.
Transfer the suspension to a sterile 15 milliliter tube and incubate for 15 to 20 minutes in a 37 degrees Celsius incubator until a clear demarcation forms between upper and lower levels. Carefully transfer the upper layer to a fresh sterile 15 milliliter tube and add 10 milliliters of fresh RPMI centrifuge at 370 to 430 GS for five minutes. Then carefully discard the supernatant.
Now assess parsit and developmental stage by thin blood smear and examine under a light microscope as described earlier. Adjust the P RBC suspension obtained after plasma gel flotation to one times 10 to the eighth P RBCs per microliter in a fresh 1.5 milliliter tube. We suspend pbcs at 5%hematocrit aine orange or atherium bromide at 20 micrograms per milliliter, final concentration and PRP at 10%of the total volume.
Similarly, prepare the following controls uninfected red blood cells and PRP and PRBC with PPP to ascertain the role of platelets in the PBC's clumping. Now transfer the suspension to a two milliliter screw cap vial. Place the vial under gentle agitation by rolling at 10 to 12 RRP at room temperature sampling at 15 to 20 minute intervals for a total of 120 minutes to check for clump formation.
Pipette 10 microliters of the sample on a glass slide and cover with a glass slip to examine under fluorescence, A clump is considered as an aggregate of three or more infected erythrocytes. This experiment assayed approximately 70%mature stages of p FALs parem after enrichment by plasma gel flotation clumps are easily spotted under the microscope as under normal light. They appear as cell aggregates and under fluorescence as spots of green or red.
When stained with acro and orange or atherium bromide dies respectively. The clump size significantly increases to an average of greater than 15 pbcs per clump after incubation for 120 minutes with some clumps containing numerous pbcs per clump that could not be visually counted. The frequency of the clumping phenotype is calculated as the number of infected cells present in clumps per 1000 infected cells counted in duplicate assays.
After watching this video, you should now have a clear understanding of how to perform a plasmodium falciparum, platelet mediated clumping assay. Be careful not to activate the platelets with jerky moves and temperature variations. This assay can also be applied to measure the platelet mediated clumping assay in severe malaria.
