High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.
Fanconi anemia (FA) is caused by mutations of DNA repair genes. The risk of oral squamous cell carcinoma (OSCC) among FA patients is 800-folds higher than in the general population. Early detection of OSCC, preferably at it precursor stage, is critical in FA patients to improve their survival. In an ongoing clinical trial, we are evaluating the effectiveness of the programmable bio-nanochip (p-BNC)-based oral cytology test in diagnosing oral potentially malignant disorders (OPMD) in non-FA patients. We used this test to compare cytomorphometric and molecular biomarkers in OSCC cell lines derived from FA and non-FA patients to brush biopsy samples of a FA patient with OPMD and normal mucosa of healthy volunteers. Our data showed that expression patterns of molecular biomarkers were not notably different between sporadic and FA-OSCC cell lines. The p-BNC assay revealed significant differences in cytometric parameters and biomarker MCM2 expression between cytobrush samples of the FA patient and cytobrush samples of normal oral mucosa obtained from healthy volunteers. Microscopic examination of the FA patient's OPMD confirmed the presence of dysplasia. Our pilot data suggests that the p-BNC brush biopsy test recognized dysplastic oral epithelial cells in a brush biopsy sample of a FA patient.
Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a "cytology-on-a-chip" (COC)–based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients' OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients' OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.
This project encompasses the design of a pretreatment protocol for blood serum and adaption of that protocol to a microfluidic environment in order to optimize key sample characteristics, namely pH, conductivity, and viscosity, to enable on-chip electrokinetic separations. The two major parts of this project include (1) designing a pretreatment protocol to optimize key parameters of the sample solution within a target range and (2) designing /fabricating a microchip that will effectively combine the sample solution with the appropriate buffers to replicate the same bench-scale protocol on the micro-scale.
Abstract Fanconi Anemia (FA) is an autosomal recessive disorder caused by mutations of DNA repair genes. The risk of oral cancer (OC) among FA patients is 800-times higher than in the general population, occurring at younger ages. Hematopoietic stem cell transplantation (HSCT), which greatly extends the life-expectancy in FA patients further increases the risk of OC in these patients. Patients with FA cannot tolerate chemo-and radiation therapy; hence, early detection of OC is critical to improve survival. Objective: To investigate the role of a numerical index for monitoring OC progression applied to FA patients in combination with a microfluidic “cytology-on-chip” assay and autofluorescence visualization (AFV) using VELscope (Visually Enhanced Lesion scope). Methods: Patients attending the Meeting for Adults with FA, held in Baltimore, Maryland in March 2014 underwent a conventional oral examination followed by AFV. Transepithelial brush samples of mucosal lesions suspected of being oral potentially malignant disorders (OPMD) were obtained using Orcellex® brush (Rovers Medical Devices, Oss, The Netherlands) and transported in ThinPrep® CytoLyt® for cytology-on-chip assays. A numerical OC risk index (0-100) developed and validated on 506 prospectively recruited non-FA patients with OPMD was applied to site-matched brush biopsy samples of FA patients. Results: A total of 28 FA patients (Age range: 18-61 yrs., M = 9, F = 19) participated in this study, of whom 13 have had HSCT. The majority of these lesions (89%) revealed loss of fluorescence (LOF+ve) with AFV. Compared to a cohort of prospectively recruited non-FA patients with OPMD (mean = 36.71, SD = 33.60), the mean OC risk index score for FA patients was 50.66 (SD = 32.83). Additionally, HSCT-recipient FA patients exhibited higher risk index scores (mean = 58.69, SD = 32.16) compared to non -HSCT-recipient FA patients (mean = 40.34, SD = 33.08). The frequency of nuclear aberrations such as micronuclei, bi- and poly-nucleated cells was significantly higher in FA patient cells than healthy controls and significantly higher in LOF+ve compared to LOV-negative OPMD. Two samples exhibited significantly higher WBC (white blood cell) counts, an indicator of inflammation; both were from LOF+ve OPMD found in HSCT-recipient FA patients. Conclusion: FA patients who are HSCT recipients have significantly higher OC risk index scores as well as increased prevalence of LOF+ve OPMD that demonstrate higher incidence of nuclear aberrations and inflammation. These findings are consistent with recent reports that HSCT in FA patients increases their risk for oral cancer. Therefore, use of the OC numerical index and cytology-on-chip assay in combination with AFV may improve the efficacy of conventional oral examination for long-term surveillance of OC in this high-risk patient population, while minimizing unwarranted scalpel biopsies. Citation Format: Timothy J. Abram, Pierre N. Floriano, Rameez Raja, Nancy Bass, Anne Gillenwater, Nadarajah Vigneswaran, John T. McDevitt. Minimally-invasive “cytology-on-chip” assay combined with continuous risk index for screening and surveillance of oral cancer in patients with Fanconi anemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-248.
The lack of standard tools and methodologies and the absence of a streamlined multimarker approval process have hindered the translation rate of new biomarkers into clinical practice for a variety of diseases afflicting humankind. Advanced novel technologies with superior analytical performance and reduced reagent costs, like the programmable bio-nano-chip system featured in this article, have potential to change the delivery of healthcare. This universal platform system has the capacity to digitize biology, resulting in a sensor modality with a capacity to learn. With well-planned device design, development, and distribution plans, there is an opportunity to translate benchtop discoveries in the genomics, proteomics, metabolomics, and glycomics fields by transforming the information content of key biomarkers into actionable signatures that can empower physicians and patients for a better management of healthcare. While the process is complicated and will take some time, showcased here are three application areas for this flexible platform that combines biomarker content with minimally invasive or non-invasive sampling, such as brush biopsy for oral cancer risk assessment; serum, plasma, and small volumes of blood for the assessment of cardiac risk and wellness; and oral fluid sampling for drugs of abuse testing at the point of need.
To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.
