Substituting Sodium Hydrosulfite with Sodium Metabisulfite Improves Long-Term Stability of a Distributable Paper-Based Test Kit for Point-of-Care Screening for Sickle Cell Anemia

Abstract

Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users (n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD).

Keywords: paper-based diagnostics; point-of-care screening; sickle cell anemia.

Figure 1

Overview of the distributable paper-based SCA diagnostic test kit. (a) Photograph of the sickle cell kit with all components necessary to perform the test: (i) patterned chromatography paper; (ii) reagent tube containing reagents; (iii) reagent dropper; (iv) blood dropper; and (v) foil pouch. (b) Schematic illustration showing the steps required to perform the paper-based test: (i) ~20 μL of whole blood is collected via finger-stick using the blood dropper and deposited in the reagent tube; (ii) the blood is mixed with a preset volume of buffer (containing either sodium hydrosulfite or sodium metabisulfite) via manual agitation; and (iii) after 10 min, a drop (~20 μL) of the mixture is deposited on the chromatography paper using the reagent dropper and allowed to dry for up to 25 min before being evaluated visually. (c) Representative bloodstains produced by the metabisulfite (top) and hydrosulfite (bottom) versions of the paper-based test for samples with various sickle hemoglobin (HbS) concentrations. The limit of detection (LOD) for each version is indicated by a dashed line. Typical HbS concentration ranges for adults and children older than six months of age with different genotypes (normal—HbAA; SCT—HbAS; SCA—HbSS) are marked below the stains.

Figure 2

Diagnostic accuracy of the paper-based SCA screening test kit. (a) Aggregate confusion matrix for screening of blood samples (n = 185) with characteristic HbS concentrations via visual interpretation (inexperienced users; n = 3) of the blood stains produced on paper by the metabisulfite (MS) formulation of the test (185 samples × 3 users = 555 total scores). (b) Aggregate confusion matrix for the screening of blood samples (n = 143) with characteristic HbS concentrations via visual interpretation (inexperienced users; n = 3) of the blood stains produced on paper by the hydrosulfite (HS) formulation of the test (143 samples × 3 users = 429 total scores). Rows correspond to characteristic genotypes (based on HbS concentration measured using conventional high-performance liquid chromatography—HPLC) and columns correspond to predicted genotypes (diagnosed by the paper-based test). Shaded cells contain the numbers of correct diagnoses.

Figure 2

Diagnostic accuracy of the paper-based SCA screening test kit. (a) Aggregate confusion matrix for screening of blood samples (n = 185) with characteristic HbS concentrations via visual interpretation (inexperienced users; n = 3) of the blood stains produced on paper by the metabisulfite (MS) formulation of the test (185 samples × 3 users = 555 total scores). (b) Aggregate confusion matrix for the screening of blood samples (n = 143) with characteristic HbS concentrations via visual interpretation (inexperienced users; n = 3) of the blood stains produced on paper by the hydrosulfite (HS) formulation of the test (143 samples × 3 users = 429 total scores). Rows correspond to characteristic genotypes (based on HbS concentration measured using conventional high-performance liquid chromatography—HPLC) and columns correspond to predicted genotypes (diagnosed by the paper-based test). Shaded cells contain the numbers of correct diagnoses.

Figure 3

Representative images of blood stains demonstrating the reagent stability of the metabisulfite (MS) and hydrosulfite (HS) formulations of the paper-based SCA screening test under wet and dry storage conditions. (left) Blood stain images for samples with 0% HbS (control) produced by the MS and HS versions of the test using freshly prepared reagents. (right) Blood stain images for samples with 20% HbS (limit of detection of HS version of test) produced by the MS and HS versions of the test using reagents stored under wet (MS powder mixed with aqueous buffer components before storage) or dry (MS powder stored separately from aqueous buffer components) conditions. The limit of stability (LOS)—i.e., the maximum amount of time reagents can be stored before the difference in pattern between samples with 0% HbS and samples with HbS concentrations greater than, or equal to, the limit of detection of tests performed using fresh reagents becomes statistically insignificant—is indicated by a dashed line.