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Introduction: Sickle cell disease (SCD) is a common inherited blood disorder caused by sickle hemoglobin (HbS) which, unlike normal adult hemoglobin (HbA), becomes insoluble and polymerizes under hypoxic conditions. Patients with SCD experience chronic hemolytic anemia, episodic pain crises and abnormal blood flow to critical organs that cumulatively result in significant illness and shortened lifespans for many. The severity of SCD varies significantly between patients, but for individuals the rate of adverse events is strongly correlated with intraerythrocytic concentration of HbS (%HbS). High per test costs and long turnaround times make conventional laboratory methods (e.g. Hb electrophoresis, HPLC, IEF) impractical for quantifying %HbS in real-time (e.g. during transfusion therapy). The objective of this study was to demonstrate that %HbS in blood could be quantified using our recently developed rapid, low-cost paper-based SCD assay [1]. Methods: Blood samples were obtained from SCD and sickle cell trait (SCT) patients at the Texas Children’s Hematology Center (Houston, TX). To perform the SCD assay a 20μL droplet of blood mixed with Hb solubility buffer (1:10 by volume) was dropped on chromatography paper. The resulting blood stain was digitized with a flatbed scanner (Canon USA Inc, Melville, NY) and analyzed using a custom image analysis code (The MathWorks Inc, Natick, MA). Conventional Hb electrophoresis was performed with the semi-automated Sebia Hydrasys 2 Scan system (Sebia Inc, Norcross, GA). Results: The difference in transport of Hb through the paper produced a blood stain with two parts: the area of the initial drop where polymerized HbS is retained (center spot) and the area where soluble Hb is wicked laterally (peripheral ring). The relative color intensity of the center spot and peripheral ring is related to the blood sample %HbS (Fig. 1). The image analysis code automatically isolates and calculates the ratio of the average color intensities of each area (S-index). A series of reconstituted blood samples with artificially adjusted %HbS from 0 to 60% was used to calibrate the assay so that %HbS could be estimated based on blood stain color intensities (Fig. 2a). The values of %HbS estimated for patient samples using our paper-based SCD assay and actual values measured using conventional Hb electrophoresis were highly correlated with R2 = 0.898 (Fig. 2b). The estimated and actual %HbS values also showed strong agreement with the standard deviation of the difference between the two measurements = 5.5 %HbS (Fig. 2c). The majority of the differences between actual and estimated %HbS (96.67%) are within 2 standard deviations of the mean of the differences. The assay could be performed in under 35 minutes and multiple assays could be performed and analyzed in parallel. The cost of consumable materials and reagents for the paper-based SCD assay is less than $0.03. Conclusions: This study demonstrates the feasibility of using our recently developed paper-based assay to quantify %HbS in blood samples in real-time. The ability to rapidly, inexpensively measure %HbS will be particularly useful for monitoring the effectiveness of chronic transfusion or hydroxyurea therapy for long-term control of HbS content in blood of SCD patients. The ability to measure %HbS in real-time could also potentially facilitate more aggressive prophylactic therapy to intervene rapidly and significantly reduce the rate of life-threatening complications in SCD patients, including stroke. Figure 1: Figure 1:. Figure 2: Figure 2:. Acknowledgments: This work was supported in part by a 2012 NIH Director's Transformative Research Award (NHLBI R01HL117329, PI: SSS). References: [1] Yang X, et al. Lab Chip, 2013, 13, 1464-1467. Disclosures Piety: Tulane University: PCT/US2012/064856 Patents & Royalties. Yang:Tulane University: PCT/US2012/064856 Patents & Royalties. Shevkoplyas:Tulane University: PCT/US2012/064856 Patents & Royalties; Halcyon Biomedical Incorporated: Equity Ownership.

Introduction: Global health research is hampered by the lack of inexpensive and reliable assays to annotate clinical study cohorts for geographically localized endemic genetic disorders. The most prevalent modifier in Africa is sickle cell disease (SCD, HbSS). In SCD, sickling of erythrocytes by polymerization of hemoglobin S (HbS) causes vaso-occlusion and hemolysis, leading to significant morbidity/mortality and constituting a major healthcare and economic burden. The inhomogeneity bias introduced into the clinical data of patient cohorts by the changes associated to undiagnosed SCD is a significant challenge. Thus, our aims were to: a) optimize a paper-based screening test for SCD for its application in suboptimal and resource-limited conditions, b) demonstrate the feasibility of retrospective SCD annotation of a large cohort of children enrolled in a global health research study, and c) extract novel evidence on potential differences in clinical presentation between febrile children with and without SCD enrolled in this cohort. Materials & Methods: First, we adapted and simplified a paper-based SCD assay (Piety et al. 2017) for suboptimal sample conditions (frozen EDTA-whole blood) and limited-resource settings. Five microlitres of thawed EDTA-whole blood was solubilized (high phosphate buffer with saponin/sodium hydrosulfite), and blotted on standard blotting paper. Dots formed by capillarity, and their aspect depended on HbS%. A high-resolution scanner and two smartphone brands were compared for image capture. An algorithm for visual calling of the presence an HbS allele was developed, and the freeware ImageJ determined peripheral/central pixel intensities. An HbS dosimetric curve was determined after identification of optimal parameter correlations by scatter plot matrix. Second, blood samples from 1103 children aged 9 to 35 months enrolled in a randomized open prospective non-inferiority pediatric cohort study on algorithm-based management of febrile illnesses in Tanzania (ePOCT Trial, 2014-6, NCT02225769, Swiss/Tanzanian IRB approval, Keitel et al.2017) were assayed with the optimized paper test. In parallel, confirmation by gold-standard alkaline Hb electrophoresis (Helena Biosciences, UK) was performed. Third, clinical data from the newly SCD-annotated ePOCT database was analyzed (STATA software suite) to identify clinical features associated with febrile illnesses in the HbAA/AS/SS groups. Results: All EDTA-blood samples frozen onsite in Tanzania (shipped and then stored for >1 year) could be assayed. The test was rapid (5'), inexpensive (0.05 USD/test) and accessible (widely available reagents, no requirement for costly equipment, open-source freeware). Determination of HbS status (homozygous HbSS/AA or heterozygous HbSA) was effective both visually and by informatic means. Smartphone cameras yielded identical results (R2=0.96 & 0.97) when compared to a high-definition scanner. Local prevalence was at 1.6% and 13.6% for HbSS and HbAS respectively, in line with previously published data. By visual sorting of blood dots, the assay was 100% sensitive and 75.8% specific for the determination of HbSS-SCD and 97.6% sensitive and 87.5% specific for the identification of an HbS allele (in an hetero- or homozygous state). After analysis of the annotated ePOCT multiparametric clinical dataset, we observed that cough as the main complaint was more prevalent in children with HbSS (OR 3.1, 95% CI: 1.1-8.3). HbSS children were more severely malnourished (MUAC/age average z-scores -0.7 vs 0.0, p=0.01) and slightly more anemic (Hb 9.1 vs 9.8 g/dL, p=0.06) when compared to HbAA counterparts. Interestingly, children with HbAS had a better nutrition status than those with HbAA (MUAC/age average z-score 0.14, p Conclusions: We demonstrated that the retrospective annotation of clinical cohorts for SCD with an optimized paper-based assay is feasible, straightforward and inexpensive. Our approach has the potential to eliminate the interpretation bias associated with SCD, and thus facilitate the downstream analysis of valuable datasets in other studies. As a proof of principle, we examined a properly annotated dataset highlighting novel features of febrile illness in children with SCD and HbAS carriers in Africa. Disclosures No relevant conflicts of interest to declare.

To the Editor: To provide samples for neonatal mass screening, a few drops of blood are routinely collected from the heel of the newborn infant and allowed to dry on a thick filter paper called the Guthrie card.[1][1] Guthrie cards represent a valuable and comprehensive genetic repository because

Abstract 3479 Poster Board III-416 Introduction ELISA assays have been proposed as a complement to the Bethesda assay for detection and follow-up of factor VIII (FVIII) antibodies in subjects with hemophilia. The Bethesda assay has several drawbacks. Most importantly, the Bethesda assay would not detect non-inhibitory antibodies, even though these could be responsible for low in vivo recovery of FVIII and /or a pharmacokinetic pattern suggestive of rapid clearance of FVIII. ELISA assays for detecting antibodies to FVIII are not routinely performed in most hospital laboratories and as such, specimens need to be transported to specialized laboratories for processing. Transportation of plasma specimens frozen on dry ice, however, is both costly and cumbersome. Room temperature storage of whole blood and serum specimens adsorbed and dried onto filter paper has been shown to be a convenient and reliable alternative to frozen whole blood and serum samples for various analyses. This study was undertaken to assess whether FVIII antibodies from haemophilic plasma would display similar stability when stored adsorbed onto filter paper at room temperature for 1 month or on plasma specimens stored frozen. Stability was assessed by comparing results of ELISA assays. Methods two hundred and thirteen frozen (at -80°C) plasma samples from patients with or without known FVIII inhibitors were tested. Samples came from 97 congenital haemophilic subjects and 5 subjects with acquired haemophilia. The samples were tested with a previously reported ELISA assay (Haemophilia 2009; 15: 374-6) with minor modifications. The coating antigens were two therapeutic preparations of recombinant FVIII: the full length FVIII preparation Helixate® FS, and the B-domain deleted FVIII Xyntha®. Five dilutions of plasma from a subject with congenital severe hemophilia A known to have a high Bethesda titer was put on all plates as positive control. Six normal plasmas were used on all plates as negative controls. Mean and standard deviation of optical densities were calculated for negative controls. Mean was subtracted from optical density of each test plasma. The resulting value was divided by the value of one standard deviation of negative controls to generate the number of standard deviations for test plasmas. All plasmas were assayed in duplicate. The numbers of standard deviations were compared between frozen samples and samples dried on filter papers. Results The correlation between the numbers of standard deviations obtained with frozen specimens and with specimens dried on filter paper was excellent for both therapeutic preparations of recombinant FVIII, going from R= 0.91 to R= 0.99. Conclusion FVIII antibodies tested by ELISA using plasma dried on filter paper for one month at room temperature or plasma stored at -80°C give comparable results. Disclosures: No relevant conflicts of interest to declare.

[Graphic][1] In many parts of the world, diagnosis and monitoring of CML patients is limited by the availability and cost of molecular testing. In countries without molecular diagnostic capabilities, blood samples can be shipped to central labs, but this is both hampered by sample degradation, and the high costs of shipping. This study explores the method of directly spotting peripheral blood onto a paper template (dried blood spots), with subsequent shipping, RNA extraction, and BCR-ABL testing. Methods: Blood Spots and Shipment. We received dried blood spots from Australia and African countries by mail or courier, and blood from CML patients from our institution were also used for these experiments. 200μL of blood (PB) was pipetted onto Whatman 503 Protein Saver Cards (PSC; Sigma-Aldrich), where each card contains four 50μL spots. Cards were allowed to dry for at least 24 hours at room temperature. For mailing, PSCs were sealed into glassine envelopes with a packet of desiccant, and then placed inside a mailing envelope following DOT and IATA regulation for shipping non-regulated, exempt human specimens. RNA Extraction from Cards and %BCR-ABL determination. Blood spots were incubated with proteinase K followed by RNA isolation using RNeasy Mini Kits (Qiagen). Extracted RNA was quantified using a NanoDrop spectrometer (Thermo Scientific). %BCR-ABL was determined using the automated Cepheid GeneXpert platform or manual two-step quantitative RT-PCR on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Results: Bench top time course: To test for effects of long transit times on RNA quality, we performed a time course study of cards at room temperature (RT) with 5 samples. For each sample, multiple cards were spotted with PB. The cards were then allowed to sit at RT for predetermined amounts of time, up to 42 days, before extracting RNA. We measured RNA integrity for one of the specimens (CML # 5) and found rapid degradation with the RIN number going from 8.7 for the fresh blood to 2.8 after 28 days on the card. However the amplification for both BCR-ABL and ABL differed less than one cycle between the fresh blood and the last time point by manual qRT-PCR (BCR-ABL Ct = 23.63 for fresh blood and 24.06 for day 28 PSC; ABL Ct = 26.69 for fresh blood and 27.64 for day 28 PSC). [Figure 1][2] shows the results of the time course experiment for the 5 samples as a plot of ΔCt versus time in days. BCR-ABL qRT-PCR concordance studies: We compared the %BCR-ABL results obtained in fresh specimen at the institution sending the sample with the %BCR-ABL results we obtained from RNA extracted from PSC using the Cepheid GeneXpert. Paired evaluable results were available for 9 samples with a median WBC = 9.8 x 109/L (range: 3.37x109/L – 85.5x109/L). Samples were 8 to 49 days old at the time of extraction. The amount of RNA input into the GeneXpert reaction ranged from 38.75ng to 1μg. The %BCR-ABL detected ranged from 0.37% to 27% (see Table). The mean absolute difference between fresh blood and PSC BCR-ABL% is 2%; the relative mean percent change for BCR-ABL, using fresh blood as the reference is 13.1% (S.D., 31.2), P = 0.24. Conclusions and future directions: Dried blood spots are relatively inexpensive method to transport blood that preserves enough RNA stability to allow highly accurate BCR-ABL detection, when compared to results performed on an identical platform using fresh peripheral blood samples. Further studies are undergoing to accurately determine the sensitivity of this method and the feasibility of using regular mail for inexpensive transport of specimens. | ID | WBC (1000/μL) | Sample Age at Spotting (Days) | Sample Age at RNA extraction (Days) | RNA ng/μl | Volume GeneXPert (μL) | Paper %BCR-ABL (IS) GeneXpert | Fresh Blood % BCR-ABL (IS) GeneXpert | | --- | ------------- | ----------------------------- | ----------------------------------- | --------- | --------------------- | ------------------------------------ | ------------------------------------ | | I1 | na | | 10 | 426 | 3 | 49 | na | | I2 | 24.1 | | 13 | 110 | 9 | 27 | 45 | | I3 | 80 | | 9 | 18 | 15 | 44 | na | | I4 | 7.4 | 2 | 8 | 5 | 10 | 2.4* | 3.1 | | I5 | 5.5 | | 49 | 5 | 24 | 1.9 | 2 | | I6 | 3.6 | 1 | 30 | 7.42 | 25 | 9 | 12 | | I7 | 85.5 | 1 | 30 | 102 | 10 | 24 | 39 | | I8 | 12.2 | 1 | 29 | 12.4 | 15 | 12 | 8.8 | | I9 | na | 1 | 28 | 1.5 | 25 | 0.37* | 0.71 | | I10 | 3.37 | | 27 | 3 | 25 | 7.8 | 5.7 | | I11 | 15.9 | 1 | 27 | 31 | 10 | 23 | 25 | | I12 | 6.6 | 1 | 27 | 14.4 | 15 | na | 2.3 | Table 1 *%BCR-ABL was manually calculated due to late ABL Cts because of low starting material. ![Figure 1][3] Figure 1 Disclosures No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif [2]: #F1 [3]: pending:yes

Newborn screening for sickle cell disease (SCD) in developing countries is limited by the cost and technical complexity of current screening methodologies and the delayed availability of screening results. We have recently developed a rapid diagnostic test for SCD that can quickly and inexpensively identify blood samples containing hemoglobin S. We hypothesized that our rapid test would be practical for use in a resource-limited setting in Cabinda, Angola, and that screening mothers or neonates for the presence of hemoglobin S in blood samples would be an effective means of identifying neonates at high risk of having sickle cell disease prior to more definitive testing. After informed consent, we collected blood samples heel-stick from neonates and by finger-stick from mothers at the primary obstetric hospital in Cabinda. We then tested these samples by the rapid SCD test and scored them by visual assessment of staining patterns. Neonates were scored as positive (HbS detected) or negative (no HbS detected) and mothers as AA, AS (sickle trait), or SS (sickle cell disease). Neonatal samples were subsequently tested by isoelectric focusing (IEF) electrophoresis to determine exact sickle cell status. In a cohort of 133 mother-neonate pairs, we used rapid testing on maternal samples to categorize neonates as high-risk (mother positive for HbS) or low-risk (mother negative for HbS). The rapid test was highly accurate in identifying neonates who could be excluded from IEF testing, with a negative predictive value of 93% (Figure 1). In a cohort of 95 neonates similarly triaged by rapid testing on neonatal samples, the negative predictive value of the test was 96% (Figure 2). In both cohorts, the one neonate with HbSS disease was successfully triaged into the high-risk group. Maternal screening with the rapid test would have reduced the proportion of neonates requiring confirmatory IEF testing to 19%, while neonatal screening would have reduced this proportion to 26%. These results indicate the potential utility of the rapid diagnostic test as a screening tool prior to more definitive testing. Used in combination with confirmatory IEF, our rapid test could significantly decrease the cost of newborn screening for SCD and increase its clinical utility by permitting more rapid identification of affected infants. Disclosures Piety: Halcyon Biomedical: Patents & Royalties: Mr. Piety is a co-inventor on a utility PCT application, "Paper-based diagnostic test" (PCT/US2012/064856, 11/13/2012), claiming priority benefit of U.S. 61/692,994 (8/24/2012) and U.S. 61/558,009 (11/10/2011). . Shevkoplyas:Halcyon Biomedical: Equity Ownership, Patents & Royalties: Co-inventor on a utility PCT application, "Paper-based diagnostic test" (PCT/US2012/064856, 11/13/2012), claiming priority benefit of U.S. 61/692,994 (8/24/2012) and U.S. 61/558,009 (11/10/2011). Part-owner of Halcyon Biomedical Inc.,.

Abstract 245 Introduction: Sickle cell disease (SCD) is the most common inherited blood disorder caused by a mutation in the beta-globin chain of the hemoglobin (Hb) molecule. The result of this mutation, Hb S, polymerizes when deoxygenated, giving the red blood cells a characteristic, sickled shape. This polymerization causes in vivo vaso-occlusion and disrupts the vascular endothelium causing multi-organ complications in affected individuals. Nearly 70% of all affected births occur in Africa, where conservative estimates of SCD prevalence suggest a 10.68/1000 rate at birth (compared to 0.49/1000 in the U.S.). Although SCD causes significant morbidity and premature mortality, most affected persons born in the U.S. are able to survive into adulthood (primarily due to the implementation of universal newborn screening). In contrast, most affected individuals born in low-income countries die before the age of 5 years due to lack of early intervention. Current technology for diagnosing SCD relies on hemoglobin electrophoresis, isoelectric focusing or high performance liquid chromatography. This type of testing remains prohibitively expensive to low-income countries, where SCD is most prevalent and newborn screening is unavailable. Thus, many affected individuals are not diagnosed before severe complications or death can occur. The urgent need to develop a low-cost diagnostic test for SCD has been recently recognized as a priority by the World Health Organization. In this study, we describe the implementation of a paper-based hemoglobin solubility assay as a simple, low-cost and rapid test for definitive diagnosis of SCD. Materials and Methods: Normal human venous blood (Hb AA) was collected and compared to blood from patients with SCD (Hb SS) and SCT (Hb AS). To perform the test, a 20uL droplet of whole blood mixed with the components of the Hb solubility assay (SickleDex™, Streck) was deposited onto a paper-fluidic device fabricated using a previously published method. Polymerized deoxy-Hb S remained in the center of the blood stain entrapped by the paper substrate, while other forms of Hb remained soluble and were transported laterally to the periphery of the stain by capillary action. The resulting blood stain was digitized with a portable scanner and analyzed automatically. The red color intensity profiles were normalized by the total area under the curve to account for the differences in hematocrit among subjects. The SCD index was defined as the normalized color intensity 5 mm from the center of the blood stain. Data were presented as results of individual experiments and as mean ± SD. The values of the SCD index for samples from each Hb genotype were compared using a paired two-tailed t-test. Results: The entanglement of polymerized deoxy-Hb S by the fibers of the paper substrate resulted in the formation of a dark red spot in the center of the blood stain, while the wicking of the soluble forms of Hb from the center produced a pink ring on the periphery of the stain. The patterns of the blood stains produced on paper by normal (Hb AA), SCT (Hb AS) and SCD (Hb SS) samples were significantly different (Fig. A). The tight clustering of the normalized color intensity profiles for different subjects with the same Hb genotype enabled the use of the SCD index as a quantitative metric for distinguishing between the three types of samples (Fig. B). The difference between the SCD index measured for Hb AA, Hb AS and Hb SS samples was highly significant, with p Conclusions: This proof-of-concept study demonstrated the feasibility of using the paper-based Hb solubility assay as a simple, low-cost, point-of-care diagnostic test for SCD. The ability to diagnose SCD quickly and inexpensively will be particularly useful for universal SCD screening in resource-limited settings, such as Africa. This test will also be very useful in the emergency room setting in high-income countries to enable healthcare professionals to objectively confirm suspected SCD at the bedside. Disclosures: No relevant conflicts of interest to declare.

Two-dimensional paper chromatography was used to partition the acid-soluble phosphates in the erythrocytes of normal newborn infants, infants with erythroblastosis fetalis and adults. The inorganic phosphate concentration was higher in the fresh red cells of normal and erythroblastotic infants and rose more during incubation for four hours than in the cells of adults. The amount of erythrocyte adenosine-5’-triphosphate in the adults exceeded the quantities found in the cells of the two groups of babies. The 2,3-diphosphoglycerate level was greater in the erythrocytes of adults and infants with erythroblastosis than it was in the normal infants and incubation for four hours resulted in a sharper decrease of this compound in cells of the normal and erythroblastotic infants than in the adults.