The mechanical retention of rigid erythrocytes in the spleen is central in major hematological diseases such as hereditary spherocytosis, sickle-cell disease and malaria. Here, we describe the use of microsphiltration (microsphere filtration) to assess erythrocyte deformability in hundreds to thousands of samples in parallel, by filtering them through microsphere layers in 384-well plates adapted for the discovery of compounds that stiffen Plasmodium falciparum gametocytes, with the aim of interrupting malaria transmission. Compound-exposed gametocytes are loaded into microsphiltration plates, filtered and then transferred to imaging plates for analysis. High-content imaging detects viable gametocytes upstream and downstream from filters and quantifies spleen-like retention. This screening assay takes 3–4 d. Unlike currently available methods used to assess red blood cell (RBC) deformability, microsphiltration enables high-throughput pharmacological screening (tens of thousands of compounds tested in a matter of months) and involves a cell mechanical challenge that induces a physiologically relevant dumbbell-shape deformation. It therefore directly assesses the ability of RBCs to cross inter-endothelial splenic slits in vivo. This protocol has potential applications in quality control for transfusion and in determination of phenotypic markers of erythrocytes in hematological diseases.
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We thank the Bill & Melinda Gates Foundation (BMGF; grant OPP1123683), GlaxoSmithKline's (GSK's) Tres Cantos and the GSK Tres Cantos Open Lab Foundation (TCOLF) for funding this project. We gratefully acknowledge the Human Resources Department of INTS, including C. Pille, for the administrative support provided. We are indebted to O. Vandal from BMGF for the constant trust he placed in this HTS project. We express our sincere gratitude to S. Ruchaud for writing support. We thank HRA Pharma Paris, as well as V.M. Avery and J.P. Holleran, for their initial involvement in the microsphiltration project.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Retention rates of P. falciparum ring asexual stages in microsphere filters when exposed to 0.05% DMSO or 250 nM cipargamin (NITD609) over 2 h.
Quantification of retention rates using flow cytometry involved the staining of ring-infected RBC samples with Sybr Green I and mitotracker deep red FM before proceeding to microsphiltration. To quantify retention rates using high content imaging, samples were stained with Syto 40 blue, mitotracker deep red FM and Cellmask orange. Cipargamin 250 nM increases the retention of rings up to an average of 60%, what may define a hit threshold for screening activities. Screening on rings would then use glutaraldehyde-fixed RBC as positive control to define the highest retention rate of the assay.
Supplementary Figure 2 High-content imaging of mature gametocyte-infected RBCs from control wells in upstream imaging plates (= aspect before filtration; 60× objective).
i-ii/ Elongated and metabolically active gametocytes in DMSO and calyculin wells. iii/ Swollen mature gametocytes in cipargamin (NITD609, KAE609) wells. iv/ Dead gametocytes in Gentian Violet wells (Mitotracker signal low or absent).
Supplementary Figure 3 Testing different batches of microspheres is essential to setting up a microsphiltration assay.
Retention rates of mature gametocytes exposed to DMSO 0.05% or calyculin A 50-nM during 2-hours and tested in microsphiltration using various sphere batches from two international microsphere providers. Here, IPS (Industrie des Poudres Sphériques, France) sphere batch #1 was selected because it produced the highest retention differential between DMSO and calyculin A wells, with optimal repetitivity.
a = manifold base; b = square well collar; c = polyethylene block; d = reservoir plate; e = microsphiltration plate.
A soldering iron is used to quickly squeeze the director nozzle of deficient filter wells where a microsphere leak is detected. Those wells are identified by the presence of microspheres in the director nozzle.
RBC from 11 donors were tested using microplate-based microsphiltration. a/ Unstained RBC from each donor were mixed in CFSE-stained RBC from a reference donor in a 5/95 ratio, then filtered using microsphiltration plate. Quantification of the proportion of unstained / stained RBC was performed by flow cytometry, enabling the determination of retention rates. Retention values are positive when retention was observed (like with glutaraldehyde-fixed RBC, positive control), while negative values correspond to an enrichment of test RBC in the downstream sample, suggesting that RBC from donors were mildly to moderately more deformable than control RBC. Negative control corresponds to unstained RBC from the reference donor diluted in stained RBC from this same donor. The deformability of red blood cells was determined from 4 replicates and 12 replicates per donor in Experiment 1 and Experiment 2, respectively. b/ Significant inter-individual differences were observed in a global test involving all donors (double-tailed Kruskall-Walis test p < 0.0001). When paired comparisons (each involving 2 donors) were performed, 23 of 30 comparisons showed a significant difference (double-tailed Kruskall-Walis test < 0.03). In this preliminary set of results, Donor 1 was tested twice in 2 experiments performed one month apart, and showed stable retention rates (Mean retention = -6.99 versus -1.65%, no significant difference with p = 0.1429 [Mann Whitney test]).
Supplementary Figure 7 Gating strategy used to quantify retention rates of Plasmodium falciparum asexual stage–infected RBCs stained with Sybr Green I using flow cytometry.
As an example, samples of RBC containing 2-5% of ring-stage infected RBC were filtered across microsphiltration plates. Filtered samples (Downstream sample) and their associated control (Upstream sample; unfiltered cell population) were then immediately stained using Sybr green I at 1X final concentration and placed into the incubator for 15 minutes for subsequent analysis with BD Accuri C6 flow cytometer (BD Biosciences) using a 488nm laser. The RBC selection involved the exclusion of debris using the forward scatter and side scatter (FSC-A/SSC-A) profile (Plot 1). A second round of selection using the FSC-A/FSC-H profile was used to exclude RBC doublets (Plot 2). Finally, the SSC-A/FL1-Sybr green I profile (Plot 3) was used to select (Gate G3) and quantify the population of parasitized RBC stained with Sybr green I, thereby enabling the quantification of retention rates. The gate G3 was defined using an uninfected RBC sample stained with Sybr green I (negative control).
Supplementary Figure 8 Gating strategy used to quantify retention rates of CFSE-stained RBCs with flow cytometry.
Samples of RBC containing 2-5% of uninfected RBC (normal, heat-stiffened or glutaraldehyde-fixed) stained with CFSE were filtered across microsphiltration plates. Filtered samples (Downstream sample) and their associated control (Upstream sample; unfiltered cell population) were then immediately analysed with BD Accuri C6 flow cytometer (BD Biosciences) using a 488nm laser. The RBC selection involved the exclusion of debris using the forward scatter and side scatter (FSC-A/SSC-A) profile (Plot 1). A second round of selection using the FSC-A/FSC-H profile was used to exclude RBC doublets (Plot 2). Finally, the SSC-A/FL1-CFSE profile (Plot 3) was used to select (Gate P3) and quantify the population of CFSE-stained RBC, thereby enabling the quantification of retention rates. The gate P3 was defined using unstained RBC sample (negative control).
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Duez, J., Carucci, M., Garcia-Barbazan, I. et al. High-throughput microsphiltration to assess red blood cell deformability and screen for malaria transmission–blocking drugs. Nat Protoc 13, 1362–1376 (2018). https://doi.org/10.1038/nprot.2018.035
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