Cryogenic enrichment of Plasmodium falciparum gametocytes from spiked whole blood

Abstract

Each individual cell type typically requires a unique set of conditions for optimal cryopreservation outcome, which relates to its specific response to cryoprotective agent (CPA) toxicity, osmotic behavior and sensitivity to ice crystallization. Cryopreservation of heterogenous cell populations is therefore exceedingly difficult as it requires separate and often conflicting conditions for each cell type. Conversely, these contrasting conditions could be utilized to favor cryogenic preference of a single cell population within a heterogenous sample, leading to its enrichment by elimination of remaining cells. To establish proof-of-concept for this overall approach, a protocol was developed for the cryogenic enrichment of Plasmodium falciparum gametocytes from whole blood. To accomplish this goal, we evaluated the effects of CPAs and cooling conditions during cryopreservation of whole blood samples spiked with P. falciparum gametocytes. We identified that cooling to -80 °C at a rate of -1 °C/min in the presence of 11 % glycerol selectively favors recovery of gametocytes. This protocol eliminates 95.3 ± 1.7 % of total blood cells and recovers 43.2 ± 6.5 % of parasites, leading to a 19-fold enrichment as assessed by microscopic examination of blood smears. This protocol is tunable, where gametocyte enrichment 900-fold may be feasible, however there is an apparent tradeoff in overall parasite recovery. Although translation of this protocol for point-of-care testing for malaria presents many challenges, the overall approach of cryogenic purification may prove useful for alternative diagnostic applications.

Keywords: Cryogenic; Enrichment; Gametocyte; Heterogenous sample; Plasmodium.

Figure 1.. Schematic of cryogenic enrichment approach.

A heterogenous sample of RBCs, WBCs, and gametocyte-infected RBCs is frozen and thawed, after which the uninfected RBCs and WBCs are significantly reduced, leading to a relative increase in gametocyte concentration. Image created with BioRender.com.

Figure 2.. Effects of glycerol concentration on cell recovery following cryopreservation.

A) Loss of >99% RBCs occurs at glycerol concentrations <15% (n=3). B) WBC recovery is minimally affected by glycerol concentration but is slightly decreased at lower concentrations (n=3). C) Gametocyte recovery is nearly maximized at 7% glycerol (n=6). The range of glycerol concentration of ~5–15% (shaded bar) leads to simultaneous maximum RBC and WBC lysis and maximum gametocyte recovery. Stepwise CPA loading and unloading was performed at 37 °C for all experiments as indicated in Tables S1 and S2.

Figure 3.. Optimization of cooling rate and temperature for differential recovery of RBCs and gametocytes using 11% glycerol.

A) RBC recovery is reduced as a function of lower temperatures, where optimal lysis is observed during freezing at a rate of −1 °C/min between −50 and −80 °C. Incorporation of a 1 h holding time following temperature equilibration further increases lysis at temperatures above −50 °C and below −20 °C. B) Comparison of RBC and gametocyte recovery in response to cooling rate shows that optimal conditions, where low RBC recovery with simultaneous maximal gametocyte recovery is observed when a −1 °C/min cooling rate is employed. CPA 2-step loading and 3-step unloading was performed at 37 °C for all experiments. Samples were thawed immediately following equilibration to the endpoint temperature, specifically −80 °C for cooling rates ranging −0.5 and −70 °C/min, and −196 °C for cooling rate of −1000 °C/min.

Figure 4.. Optimization of CPA parameters to improve gametocyte enrichment using streamlined experimental design.

Percent recovery of A) gametocytes (n=5) from gametocyte culture, and B) RBCs (n=4) and C) WBCs (n=4) from whole blood using each respective protocol. CPA loading and unloading was performed at 37 °C for all experiments. D) Summary of prioritized experimental protocols shown in A, including the CPA cocktail composition, sample HCT, and CPA addition and removal method with a corresponding hypothetical gametocyte enrichment value calculated as a ratio of average gametocyte recovery to average RBC or WBCs recovery (gam:RBC and gam:WBC) E) Giemsa-stained thin blood smears of the gametocyte culture before and after enrichment using condition 3 imaged at 1000x magnification; scale bar: 20μm.

Figure 5.. Gametocyte detection improves with cryogenic enrichment.

A) Heatmap of detectable trials (%, n=5) of gametocytes in spiked blood samples with parasite density ranging from 0.2 to 1000 p/μL in 10 μL sample volume. B) To enable a more quantitative measure of gametocyte recovery, gametocytes were counted in 600 nL volume using a hemocytometer before and after cryogenic enrichment in blood samples spiked with gametocytes at densities ranging from 0.2 to 1000 p/μL (n=4). C) An example of Giemsa-stained blood smears from whole blood spiked with parasites at 5000 p/μL before (0.32% gametocytemia) and after (6.1% gametocytemia) cryogenic enrichment imaged at 400x and 1000x magnification; scale bar: 20μm. CPA loading and unloading was performed at 37 °C for all experiments.