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Point-Of-Care Device to Diagnose and Monitor Neonatal Jaundice in Low Point-of-care device to diagnose and monitor neonatal PNAS PLUS jaundice in low-resource settings Pelham A. Keaheya, Mathieu L. Simerala, Kristofer J. Schrodera, Meaghan M. Bonda, Prince J. Mtenthaonngab, Robert H. Mirosc, Queen Dubeb, and Rebecca R. Richards-Kortuma,1 aDepartment of Bioengineering, Rice University, Houston, TX 77030; bDepartment of Pediatrics, Queen Elizabeth Central Hospital, Blantyre, Malawi; and c3rd Stone Design, San Rafael, CA 94901 Contributed by Rebecca Richards-Kortum, October 31, 2017 (sent for review August 23, 2017; reviewed by John C. Carrano and Muhammad H. Zaman) Newborns are at increased risk of jaundice, a condition in which (TSB); these tools have proved too expensive and complex to excess bilirubin accumulates in blood. Left untreated, jaundice implement in under-resourced settings (2). For example, spec- can lead to neurological impairment and death. Jaundice result- trophotometric methods to measure TSB require a centrifuge ing from unconjugated hyperbilirubinemia is easily treated with to separate plasma from whole blood and prevent hemoglobin exposure to blue light, and phototherapy systems have been interference, as well as a spectrophotometer—tools that can developed for low-resource settings; however, there are no appro- cost thousands of dollars and are often not available in dis- priate solutions to diagnose and monitor jaundice in these set- trict hospital or health center laboratories (4). Bilirubin levels tings. To address this need we present BiliSpec, a low-cost reader can also be measured using chemical approaches such as the and disposable lateral flow card designed to measure the concen- diazo method, enzymatic determination, or high-performance tration of total bilirubin from several drops of blood at the point liquid chromatography. The expensive reagents and laboratory of care. We evaluated the performance of BiliSpec, using blood analyzers needed to perform these tests are not available in from normal volunteers spiked with varying amounts of bilirubin; the majority of low-resource settings (5, 6). Alternatively, tran- results measured using BiliSpec correlated well with a reference scutaneous measurement of TSB does not require blood col- laboratory bilirubinometer (r = 0.996). We then performed a pilot lection and has been proposed as an appropriate solution for clinical study using BiliSpec to measure total bilirubin in neonates low-resource settings. However, transcutaneous readers are at risk for jaundice at Queen Elizabeth Central Hospital in Blan- expensive and some require a costly disposable calibration stan- tyre, Malawi. Concentrations measured using BiliSpec correlated dard. Even if cost could be reduced, studies have shown the well with those measured using a laboratory reference standard accuracy of transcutaneous assessment of TSB is lower in babies in 94 patient samples ranging from 1.1 mg/dL to 23.0 mg/dL in with darker skin or who are premature. Moreover, transcuta- concentration (r = 0.973). The mean difference between bilirubin neous measurement can only be used for initial measurement levels measured with BiliSpec and the reference standard was of TSB and does not accurately monitor response to blue-light 0.3 mg/dL (95% CI: −1.7–2.2 mg/dL). phototherapy (7–10). As a result, jaundice is diagnosed clini- cally in most low-resource settings based on visual assessment neonatal jaundice j point-of-care j lateral flow j low-resource setting of yellowing of the skin or sclera. Unfortunately, visual assess- ment is subjective and accuracy is poor compared with labo- ratory measurement of TSB (11). Thus, there is an important very year, 24 million newborns develop jaundice. Jaundice resulting from hyperbilirubinemia is especially common in E Significance preterm babies, who lack sufficient liver function to excrete excess bilirubin. Neonatal jaundice is easily treated using blue- light phototherapy, exploiting the strong optical absorbance of Neonatal jaundice, a condition caused by the accumulation bilirubin at 460 nm to photodecompose bilirubin to a form that of bilirubin in the bloodstream, affects approximately half of can be excreted. In extreme cases, exchange transfusions can be all newborns. In high-resource settings, babies with elevated performed to quickly lower the concentration of bilirubin in the serum bilirubin levels are identified through routine hospital blood. In high-resource settings, morbidity and mortality from laboratory testing. When identified, jaundice is easily treated jaundice are extremely rare due to the widespread availability using blue-light phototherapy. Low-cost, rugged photother- of tools to measure serum bilirubin levels and treat newborns apy lights have been developed and shown to be effective suffering from jaundice. In contrast, jaundice remains a signifi- in low-resource settings. However, jaundice regularly goes cant source of neonatal morbidity and mortality in low-resource undetected in these settings due to a lack of diagnostic tools to measure bilirubin levels. Left untreated, jaundice can lead settings. Globally, 120,000 babies still die each year from jaun- MEDICAL SCIENCES dice and many more suffer permanent neurological damage (ker- to permanent neurological damage and mortality, the vast nicterus). The vast majority of these deaths occur in low-income majority of which currently occurs in low-resource settings. countries in sub-Saharan Africa and South Asia (1). In this paper, we present a low-cost method to measure total Severe jaundice may not present until several days after birth, bilirubin at the point of care in low-resource settings. and thus early monitoring of bilirubin is critical, particularly Author contributions: P.A.K., M.L.S., K.J.S., M.M.B., R.H.M., and R.R.K. designed research; in premature babies who are at greater risk of death and dis- P.A.K., M.L.S., K.J.S., M.M.B., and P.J.M. performed research; P.A.K., M.L.S., K.J.S., and ability due to jaundice (2). World Health Organization guide- M.M.B. contributed new reagents/analytic tools; P.A.K., M.L.S., K.J.S., M.M.B., Q.D., and ENGINEERING lines for managing hyperbilirubinemia using serum bilirubin con- R.R.K. analyzed data; and P.A.K., M.L.S., K.J.S., M.M.B., R.H.M., Q.D., and R.R.K. wrote the centration shift based on age and prematurity (Table S1) (3). paper. While several low-cost phototherapy systems have recently been Reviewers: J.C.C., Paratus Diagnostics; and M.H.Z., Boston University. developed (D-rev; Brilliance, Design that Matters; Firefly) for The authors declare no conflict of interest. use in low-resource settings, there is still a lack of low-cost This open access article is distributed under Creative Commons Attribution- diagnostic tools to measure bilirubin levels to identify babies NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). who require treatment and monitor their response to ther- 1To whom correspondence should be addressed. Email: [email protected]. apy. In high-resource settings a number of laboratory devices This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and methods are available to measure total serum bilirubin 1073/pnas.1714020114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1714020114 PNAS j Published online December 4, 2017 j E10965–E10971 Downloaded by guest on October 2, 2021 need for improved methods to measure TSB in low-resource band absorption peak at 420 nm which overlaps with the bilirubin settings. absorption peak at 460 nm. The lateral flow cards are designed To meet this need, we developed BiliSpec, a low-cost, battery- to be operated by visual cues alone. Users are instructed to visu- powered reader designed to rapidly quantify serum bilirubin lev- ally fill the blood collection pad, seal the device, and then insert els from small drops of whole blood applied to a lateral flow card. the device into the reader once the plasma has reached the end BiliSpec consists of two components: (i) a lateral-flow separation of the nitrocellulose strip. The blood collection pad is sized to card to collect blood from a heel prick, separate plasma, and sta- appear visually full following application of 40–50 µL of blood bilize the sample and (ii) a reader to measure light transmitted (typically two to three drops of blood). The target window (Fig. through the plasma on the card and display a digital report of the 1A) consists of a clear piece of acetate covering the area illumi- concentration of TSB. Here, we describe laboratory experiments nated in the reader to measure plasma absorbance, preventing to optimize the design of the lateral flow card and to character- exposure to the air and reducing drying after sample collection. ize accuracy of the system compared with a laboratory reference The target window is made from acetate not coated with adhe- standard using blood from normal volunteers spiked with varying sive to avoid interference of glue with flow of plasma along the levels of bilirubin. We then describe results from a pilot study to strip. The leak-proofing bar and overflow channels are designed evaluate the accuracy of BiliSpec to measure TSB in neonates at to prevent excess blood from leaking into the target area from a risk for jaundice at Queen Elizabeth Central Hospital in Blan- saturated collection pad or out of the lateral flow card if the card tyre, Malawi. is squeezed or overfilled. After the blood collection pad has been visibly filled, the device can be immediately sealed. Separation of Results plasma takes ∼1–2 min (Movie S1). When the plasma reaches Design of Lateral Flow Cards and Reader. The lateral flow cards are the end of the nitrocellulose strip, the card can be inserted into designed to accept drops of whole blood obtained directly from the reader and analyzed (Fig. 1B). Representative photographs a heel or finger prick, separate plasma from whole blood within of lateral flow cards spotted with blood containing normal or ele- 1–2 min, and preserve the sample so that bilirubin concentra- vated TSB levels ready to be measured are shown in Fig.
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