Multispectral imaging for MicroChip electrophoresis enables point-of-care newborn hemoglobin variant screening.

Hemoglobin (Hb) disorders affect nearly 7% of the world's population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 265 newborns with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-Multispectral detected levels of Hb A, Hb F, Hb S, and Hb C/E/A2, demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.93, and 0.95. Gazelle-Multispectral demonstrated accuracy of 96.8% in subjects of 0-3 days, and 96.9% in newborns. The ability to obtain accurate results on newborn samples suggest that Gazelle-Multispectral can be suitable for large-scale newborn screening and for diagnosis of SCD in low resource settings.

Performance of a sensitive haemozoin-based malaria diagnostic test validated for vivax malaria diagnosis in Brazilian Amazon.

BACKGROUND: Vivax malaria diagnosis remains a challenge in malaria elimination, with current point of care rapid diagnostic tests (RDT) missing many clinically significant infections because of usually lower peripheral parasitaemia. Haemozoin-detecting assays have been suggested as an alternative to immunoassay platforms but to date have not reached successful field deployment. Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites and is present in the food vacuole of malaria parasite-infected erythrocytes. This study aimed to compare the diagnostic capability of a new haemozoin-detecting platform, the Gazelle™ device with optical microscopy, RDT and PCR in a vivax malaria-endemic region. METHODS: A comparative, double-blind study evaluating symptomatic malaria patients seeking medical care was conducted at an infectious diseases reference hospital in the western Brazilian Amazon. Optical microscopy, PCR, RDT, and Gazelle™ were used to analyse blood samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Kappa values were calculated. RESULTS: Out of 300 patients, 24 test results were excluded from the final analysis due to protocol violation (6) and inconclusive and/or irretrievable results (18). Gazelle™ sensitivity was 96.1 % (91.3-98.3) and 72.1 % (65.0-78.3) when compared to optical microscopy and PCR, respectively whereas it was 83.9 % and 62.8 % for RDTs. The platform presented specificity of 100 % (97.4-100), and 99.0 % (94.8-99.9) when compared to optical microscopy, and PCR, respectively, which  was the same for RDTs. Its correct classification rate was 98.2 % when compared to optical microscopy and 82.3 % for PCR; the test's accuracy when compared to optical microscopy was 98.1 % (96.4-99.7), when compared to RDT was 95.2 % (93.0-97.5), and when compared to PCR was 85.6 % (82.1-89.1). Kappa (95 % CI) values for Gazelle™ were 96.4 (93.2-99.5), 88.2 (82.6-93.8) and 65.3 (57.0-73.6) for optical microscopy, RDT and PCR, respectively. CONCLUSIONS: The Gazelle™ device was shown to have faster, easier, good sensitivity, specificity, and accuracy when compared to microscopy and was superior to RDT, demonstrating to be an alternative for vivax malaria screening particularly in areas where malaria is concomitant with other febrile infections (including dengue fever, zika, chikungunya, Chagas, yellow fever, babesiosis).

Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.

Identification, Quantification, and System Analysis of Protein N-ε Lysine Methylation in Anucleate Blood Platelets.

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N-ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl-lysine (meK) proteome of anucleate blood platelets is characterized. With high-resolution, multiplex MS methods, 190 mono-, di-, and tri-meK modifications are identified on 150 different platelet proteins-including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin-like protein (DBNL, Hip-55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.

Pilot study of novel lab methodology and testing of platelet function in adolescent women with heavy menstrual bleeding.

BackgroundApproximately 40% of adolescent women experience heavy menstrual bleeding (HMB), and 10-62% of them have an underlying bleeding disorder (BD). Diagnosing a BD remains challenging because of limitations of available clinical platelet function assays. The aim of this study was to characterize platelet function in a population of adolescent women with HMB using small-volume whole-blood assays.MethodsAnticoagulated whole blood was used to assess platelet GPIIbIIIa activation, α-granule secretion, and aggregation in response to multiple agonists. Platelet adhesion on collagen or von Willebrand Factor (VWF) under static and shear flow was also assessed.ResultsFifteen participants with HMB were included in the study, of which eight were diagnosed with a clinically identifiable BD. Platelet activation was blunted in response to calcium ionophore in participants without a BD diagnosis compared with that in all other participants. Impaired GPIIbIIIa activation was observed in response to all GPCR agonists, except adenosine diphosphate (ADP), in participants with qualitative platelet disorders. Our assays detected platelet aggregation in the majority of participants with a BD in response to ADP, collagen-related peptide (CRP), thrombin receptor activator 6 (TRAP-6), or U46619. Platelet adhesion and aggregation on collagen and VWF was decreased for participants with VWD.ConclusionParticipants with and without BD exhibited aberrant platelet function in several assays in response to select agonists.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach.

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

Comparison of human and mouse E-selectin binding to Sialyl-Lewis(x).

BACKGROUND: During inflammation, leukocytes are captured by the selectin family of adhesion receptors lining blood vessels to facilitate exit from the bloodstream. E-selectin is upregulated on stimulated endothelial cells and binds to several ligands on the surface of leukocytes. Selectin:ligand interactions are mediated in part by the interaction between the lectin domain and Sialyl-Lewis x (sLe(x)), a tetrasaccharide common to selectin ligands. There is a high degree of homology between selectins of various species: about 72 and 60 % in the lectin and EGF domains, respectively. In this study, molecular dynamics, docking, and steered molecular dynamics simulations were used to compare the binding and dissociation mechanisms of sLe(x) with mouse and human E-selectin. First, a mouse E-selectin homology model was generated using the human E-selectin crystal structure as a template. RESULTS: Mouse E-selectin was found to have a greater interdomain angle, which has been previously shown to correlate with stronger binding among selectins. sLe(x) was docked onto human and mouse E-selectin, and the mouse complex was found to have a higher free energy of binding and a lower dissociation constant, suggesting stronger binding. The mouse complex had higher flexibility in a few key residues. Finally, steered molecular dynamics was used to dissociate the complexes at force loading rates of 2000-5000 pm/ps(2). The mouse complex took longer to dissociate at every force loading rate and the difference was statistically significant at 3000 pm/ps(2). When sLe(x)-coated microspheres were perfused through microtubes coated with human or mouse E-selectin, the particles rolled more slowly on mouse E-selectin. CONCLUSIONS: Both molecular dynamics simulations and microsphere adhesion experiments show that mouse E-selectin protein binds more strongly to sialyl Lewis x ligand than human E-selectin. This difference was explained by a greater interdomain angle for mouse E-selectin, and greater flexibility in key residues. Future work could introduce similar amino acid substitutions into the human E-selectin sequence to further modulate adhesion behavior.

Effect of Pseudopod Extensions on Neutrophil Hemodynamic Transport Near a Wall.

During inflammation, circulating neutrophils roll on, and eventually tether to, the endothelial lining of blood vessels, allowing them to exit the bloodstream and enter the surrounding tissue to target pathogens. This process is mediated by the selectin family of adhesion proteins expressed by endothelial cells. Interestingly, only 10% of activated, migrating neutrophils transmigrate into the extravascular space; the other 90% detach from the wall and rejoin the blood flow. Neutrophils extrude pseudopods during the adhesion cascade; however, the transport behavior of this unique cell geometry has not been previously addressed. In this study, a three-dimensional computational model was applied to neutrophils with pseudopodial extensions to study the effect of cell shape on the hydrodynamic transport of neutrophils. The collision time, contact area, contact force, and collision frequency were analyzed as a function of pseudopod length. It was found that neutrophils experience more frequent collisions compared to prolate spheroids of equal volume and length. Longer pseudopods and lower shear rates increase the collision time integral contact area, a predictor of binding potential. Our results indicate that contact between the neutrophil and the vessel wall was found to be focused predominantly on the pseudopod tip.

Simulation and Analysis of Tethering Behavior of Neutrophils with Pseudopods.

P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play important roles in mediating the inflammatory cascade. Selectin kinetics, together with neutrophil hydrodynamics, regulate the fundamental adhesion cascade of cell tethering and rolling on the endothelium. The current study uses the Multiscale Adhesive Dynamics computational model to simulate, for the first time, the tethering and rolling behavior of pseudopod-containing neutrophils as mediated by P-selectin/PSGL-1 bonds. This paper looks at the effect of including P-selectin/PSGL-1 adhesion kinetics. The parameters examined included the shear rate, adhesion on-rate, initial neutrophil position, and receptor number sensitivity. The outcomes analyzed included types of adhesive behavior observed, tether rolling distance and time, number of bonds formed during an adhesive event, contact area, and contact time. In contrast to the hydrodynamic model, P-selectin/PSGL-1 binding slows the neutrophil's translation in the direction of flow and causes the neutrophil to swing around perpendicular to flow. Several behaviors were observed during the simulations, including tethering without firm adhesion, tethering with downstream firm adhesion, and firm adhesion upon first contact with the endothelium. These behaviors were qualitatively consistent with in vivo data of murine neutrophils with pseudopods. In the simulations, increasing shear rate, receptor count, and bond formation rate increased the incidence of firm adhesion upon first contact with the endothelium. Tethering was conserved across a range of physiological shear rates and was resistant to fluctuations in the number of surface PSGL-1 molecules. In simulations where bonding occurred, interaction with the side of the pseudopod, rather than the tip, afforded more surface area and greater contact time with the endothelial wall.