Paper-Based Microchip Electrophoresis Enabled First Point-of-Care Diagnostic Test for Beta-Thalassemia

Introduction: Hemoglobin (Hb) disorders are among the world's most common monogenic diseases. Nearly 7% of the world's population carry Hb gene variants. Among various Hb disorders, approximately 1.5% of the world population carry β-thalassemia, with around 40,000 affected newborns every year. Half of babies born with β-thalassemia are dependent on blood transfusions. Three clinical and hematological conditions of increasing severity are recognized, including β-thalassemia intermedia, β-thalassemia major, and the β-thalassemia carrier state (also known as β-thalassemia trait). Early screening and timely diagnosis is essential in β-thalassemia patients for prevention and management of later clinical complications. The current centralized laboratory tests used for newborn screening for β-thalassemia carriers are high performance liquid chromatography (HPLC) and isoelectric focusing. However, in Africa to Southern Europe, Middle East, and Southeast Asia, where β-thalassemia are most prevalent, diagnosis and screening of β-thalassemia are still of challenge due to the cost and logistical burden of laboratory diagnostic tests. As a result, there is a need for affordable, portable, easy-to-use, accurate, point-of-care (POC) tests to facilitate decentralized hemoglobin testing in low-resource settings to enable national screening programs for β-thalassemia.

Here, we present the first POC diagnostic test for β-thalassemia. We have leveraged the World Health Organization (WHO) listed (WHO Essential In Vitro Diagnostics) Hb electrophoresis test and developed a POC electrophoresis microchip system, Gazelle (Fig. 1A). Gazelle-Multispectral enables sensitive detection and accurate identification of Hb types under ultraviolet (UV 410nm) light illumination. We hypothesized that the high absorbance of Hb around 410 nm wavelength would enhance the limit of detection and allow the accurate quantification of Hb types in blood that are at both higher concentration (e.g., Hb A, Hb F) and lower concentration (e.g., HbA₂).

Methods: To test this hypothesis, we have developed customized learning-based algorithm for accurate quantification of HbA₂ in addition to our existing algorithm for accurate quantification of HbA, HbF, and HbS. Briefly, a space-time plot is generated to extract the information contained in the 8-minutes long electrophoresis test (Fig. 1B). Specific frames are then selected according to the space-time plot for quantifying HbA₂ based on the band total intensity in that frame (Fig. 1B, dashed redline). Other Hb variants including HbA, HbF, and HbS are quantified and reported (Fig. 1C). In this study, we have conducted clinical testing over 372 subjects under age range of 4 - 63 years at Apple Diagnostics lab in Ghatkopar, Mumbai, India, under local IRB-approved study protocol. Additionally, 30 blood samples were prepared to mimic β-thalassemia intermediate and β-thalassemia major samples. In total, 402 tests were conducted in this study (372 patient samples and 30 prepared blood samples).

Results and Discussion: The relative percentage of HbA₂ was obtained by comparing to other dominant Hb variants. Subjects with 6-40% HbA, 60-80% HbF, and 0-10% HbA₂ were categorized to have β-thalassemia intermedia; subjects with 0-20% HbA, 80-100% HbF, 0-9% HbA₂ were categorized to have β-thalassemia major or intermedia; and subjects with 90-100% HbA and >3.5% HbA₂ were categorized to have β-thalassemia trait. Following the Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines, 393 out of 402 (97.8%) tests were recognized as 'Valid', while 9 out of 402 (2.2%) tests were recognized as 'Inconclusive'. Within the 393 valid tests, subjects with β-thalassemia major or intermedia were identified with 100% sensitivity and specificity against healthy subjects and subjects with β-thalassemia. Subjects with β-thalassemia trait were identified with 100% sensitivity and 98.0% specificity with respect to healthy subjects (Fig. 1 Table).

Conclusion: In summary, Gazelle-Multispectral algorithm allowed accurate detection and quantification of HbA₂ in addition to other Hb types including HbA, HbF, and HbS, enabling accurate detection of β-thalassemia carriers. These results suggest that Gazelle is the first accessible, accurate POC diagnostic test for affordable (~$2/test) and rapid (<8 minutes/test) diagnosis of β-thalassemia.

Avanaki:Hemex Health: Current Employment. Thota:Hemex Health: Current Employment. Nemade:Hemex Health: Consultancy. Mehta:Hemex Health: Consultancy. Gurkan:Dx Now Inc.: Patents & Royalties; Xatek Inc.: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Biochip Labs: Patents & Royalties.