A rapid paper‐based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease

Abstract

Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 μL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R2 = 0.86) and strong agreement (standard deviation of difference = 7 %HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6 %HbS, n = 4). The paper-based assay can be completed in less than 35 minutes and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10–97%) and hemoglobin concentrations (5.6–12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.

A Paper-Based Test for Screening Newborns for Sickle Cell Disease

“…1a)131415. When evaluated visually, the limit of detection (LOD) for the adult test was about 15% HbS15, which was insufficient for detecting the very low percentages of HbS typically found in newborn samples −6.5 ± 2.8% for SCT (HbFAS) and 10.2 ± 3.9% for SCD (HbFS)16.…”

“…The reason for the relatively high LOD was the faint center spot in the center of the stain formed by cellular debris of lysed red blood cells even in the complete absence of any HbS (Fig. 1b, inset)14.…”

“…We have previously described the development and clinical validation of a rapid, low-cost paper-based diagnostic test for SCD that permits the diagnosis of adults and children older than 6 months of age131415. We now describe a test based on the same principles which is optimized to detect the low levels of sickle hemoglobin (HbS) present in the blood of newborns, allowing the direct screening of newborns for SCD and sickle cell trait (SCT; HbAS).…”

Emerging point-of-care technologies for sickle cell disease screening and monitoring

“…In addition, the test relies on naked-eye detection which may render it prone to operator error, which can be overcome by pairing the test with an automated image processing algorithm. Moreover, the validation results show that this test cannot accurately distinguish between HbSC and HbAS even when used with automated image processing 179, 180 . Finally, high levels of HbF, especially present in newborns, prevent the polymerization and precipitation of HbS, and consequently hinder the application of the test to newborn screening 171 .…”

Time to Invest in Sickle Cell Anemia as a Global Health Priority

“…There are a number of prototypes that are currently being developed and validated, and it appears that POC testing for SCA will soon become widely available. [11][12][13][14][15][16] Once simple and inexpensive POC tests become available, they will be highly useful for both NBS and public awareness campaigns to empower adolescents and young adults with both the knowledge and implications of their "sickle status" to make informed reproductive decisions. Although it is somewhat controversial in many geographic regions, genetic counseling should also be a priority, as neonatal screening programs may be able to reduce morbidity and mortality associated with SCA, but it cannot reduce the actual number of SCA births.…”