Overview of the paper-based SCA diagnostic test.

<p>(<b>a</b>) To perform the test, a 20 μL droplet of blood mixed 1:10 (by volume) with Hb solubility buffer–a concentrated phosphate buffer (2.49M) containing a hemolytic (saponin) and a reducing agent (sodium hydrosulfite)–was deposited on paper. Differential transport of polymerized HbS and soluble forms of Hb in the paper substrate produced blood stains with characteristic patterns. (<b>b</b>) Representative images of the blood stain patterns produced by HbAA, HbAS and HbSS samples.</p

Diagnosis of other forms of sickle cell disease.

<p>(<b>a</b>) Representative images of blood stains produced in paper by HbSC samples and by HbAS samples, for comparison. (<b>b</b>) Representative images of blood stains produced in paper by HbSβ⁺-thalassemia samples and by HbSS samples, for comparison. (<b>c</b>) Classification of HbSC and HbSβ⁺-thalassemia samples in the S-index domain. The values of the S-index for (○) HbAA, HbAS and HbSS samples (n = 55), (■) HbSC (n = 16) and (♦) HbSβ⁺-thalassemia (n = 3) are shown on Y-axis. The location along the X-axis of each data point for HbAS, HbSS, HbSC and HbSβ⁺-thalassemia samples corresponds to their %HbS measured with Hb electrophoresis by an off-site clinical laboratory. HbAA samples (n = 18) contained no HbS; hence the random lateral spread of the data points representing these samples on the plot.</p

Automated analysis of blood stains in paper.

<p>(<b>a</b>) A custom image analysis algorithm automatically detected the center of each blood stain (dashed crosshair) and extracted the RGB values for all pixels contained within the dark red center spot (smaller dashed circle) and within the pink peripheral ring (area between the smaller and the larger dashed circles). The S-index was defined as the quotient of the mean red color intensity of the center spot and that of the peripheral ring of the blood stain. (<b>b</b>) The values of the S-index for (●) HbAA (n = 18), (♦) HbAS (n = 17) and (■) HbSS (n = 20) samples obtained from healthy subjects and patients in New Orleans, LA. (<b>c</b>) Receiver operating characteristic (ROC) curves for the use of S-index to identify HbAA, HbAS and HbSS samples. The area under the curve (AUC) for discriminating HbAA from HbAS and HbSS was 1.00, the AUC for discriminating HbSS from HbAA and HbAS was 0.9986 and the AUC for discriminating HbSS from HbAS was 0.9971.</p

Validation of the paper-based SCA test in a research laboratory and in a resource-limited clinical setting (Cabinda, Angola).

<p>(<b>a</b>) Aggregate confusion matrix for the diagnoses of blood samples collected from normal volunteers and patients of Pediatric Hematology-Oncology Clinic at Tulane University Hospital and of the Sickle Cell Center of Southern Louisiana (New Orleans, LA) performed via visual evaluation of the blood stains by human scorers (n = 5). Rows correspond to true genotypes (diagnosed by hemoglobin electrophoresis) and columns correspond to predicted genotypes (diagnosed by the paper-based test). Shaded cells along the diagonal contain numbers of correct diagnoses. (<b>b</b>) Confusion matrix for the diagnoses of blood samples collected at the Primero de Maio obstetric hospital from postnatal females with unknown SCA status. The rapid test was performed and interpreted via visual evaluation by healthcare workers at the newborn screening laboratory of the Clinica de Celulas Falciformes at the Dispensario Materno Infantil (Cabinda, Angola). Rows correspond to true genotypes (diagnosed by isoelectric focusing) and columns correspond to predicted genotypes (diagnosed by the paper-based test). Shaded cells along the diagonal contain numbers of correct diagnoses.</p

Measuring Binding of Protein to Gel-Bound Ligands Using Magnetic Levitation

This paper describes the use of magnetic levitation (MagLev) to measure the association of proteins and ligands. The method starts with diamagnetic gel beads that are functionalized covalently with small molecules (putative ligands). Binding of protein to the ligands within the bead causes a change in the density of the bead. When these beads are suspended in a paramagnetic aqueous buffer and placed between the poles of two NbFeB magnets with like poles facing, the changes in the density of the bead on binding of protein result in changes in the levitation height of the bead that can be used to quantify the amount of protein bound. This paper uses a reaction–diffusion model to examine the physical principles that determine the values of rate and equilibrium constants measured by this system, using the well-defined model system of carbonic anhydrase and aryl sulfonamides. By tuning the experimental protocol, the method is capable of quantifying either the concentration of protein in a solution, or the binding affinities of a protein to several resin-bound small molecules simultaneously. Since this method requires no electricity and only a single piece of inexpensive equipment, it may find use in situations where portability and low cost are important, such as in bioanalysis in resource-limited settings, point-of-care diagnosis, veterinary medicine, and plant pathology. It still has several practical disadvantages. Most notably, the method requires relatively long assay times and cannot be applied to large proteins (>70 kDa), including antibodies. The design and synthesis of beads with improved characteristics (e.g., larger pore size) has the potential to resolve these problems

A portable system for processing donated whole blood into high quality components without centrifugation

<div><p>Background</p><p>The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of ‘whole blood’ into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor.</p><p>Methods and findings</p><p>In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2–3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01).</p><p>Conclusion</p><p>This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation—particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.</p></div

Schematic illustration of the design of the dual arm, split unit study comparing the quality of blood components produced using the new passive separation system versus conventional centrifugation.

<p>Schematic illustration of the design of the dual arm, split unit study comparing the quality of blood components produced using the new passive separation system versus conventional centrifugation.</p

Activation of platelets due to processing of a typical WB unit into components using the two separation methods.

<p>Each panel shows i) the level of platelet activation (primary y-axis: all platelets, green; secondary y-axis: platelet aggregates, blue), and ii) the associated formation of platelet aggregates, for (<b>a</b>) the WB unit prior to processing, and the two PC units produced (<b>b</b>) via conventional centrifugation, and (<b>c</b>) using the passive separation system. Platelets were labelled with CD42b-PerCP, and activated platelets were identified using CD62p (P-selectin)-FITC. The threshold of positive P-selectin expression was defined using an isotype control (IgG-FITC). A manual gate was applied to SSC vs. CD42b scatter plots to demarcate the presence of any (typically highly activated) platelet aggregates, which would be observed in the form of a characteristic ‘comet tail’ near the upper-right edge of a platelet distribution.</p

The microfluidic platelet concentrator (MPC) of passive WB separation system (Stage 2).

<p>(<b>a</b>) Components of the MPC: (i) top layer, containing five inlet channel branches for distributing PRP throughout the device, and two outlet channels for collecting the streams of separated PC and PPP; (ii) two device layers each consisting of ten individual CIF microfluidic devices in parallel, which perform the separation; (iii) a flat bottom layer for sealing the device. Inset shows a single CIF microdevice schematically; arrows indicate the direction of flow. (<b>b</b>) Photograph of an assembled MPC device. A one cent U.S. coin is shown for size reference. (<b>c</b>) Photograph of PRP being expressed from the sedimentation bag, through the MPC module, separated into PC and PPP.</p

The microfluidic platelet concentrator (MPC) of passive WB separation system (Stage 2).

<p>(<b>a</b>) Components of the MPC: (i) top layer, containing five inlet channel branches for distributing PRP throughout the device, and two outlet channels for collecting the streams of separated PC and PPP; (ii) two device layers each consisting of ten individual CIF microfluidic devices in parallel, which perform the separation; (iii) a flat bottom layer for sealing the device. Inset shows a single CIF microdevice schematically; arrows indicate the direction of flow. (<b>b</b>) Photograph of an assembled MPC device. A one cent U.S. coin is shown for size reference. (<b>c</b>) Photograph of PRP being expressed from the sedimentation bag, through the MPC module, separated into PC and PPP.</p