Miguel Ossandon

Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis

A Lab-on-a-chip (LOC) was designed, fabricated and tested for the in vitro detection of botulinum neurotoxin serotype A (BoNT-A) activity using an assay that measures cleavage of a fluorophore-tagged peptide substrate specific for BoNT-A (SNAP-25) by the toxin light chain (LcA). LcA cleavage was detected by Förster Resonance Energy Transfer (FRET) fluorescence. FRET fluorescence was measured by a newly developed portable charge-coupled device (CCD) fluorescent detector equipped with multi-wavelength light-emitting diodes (LED) illumination. An eight V-junction microchannel device for BoNTs activity assays was constructed using Laminated Object Manufacturing (LOM) technology. The six-layer device was fabricated with a Poly(methyl methacrylate (PMMA) core and five polycarbonate (PC) layers micromachined by CO2 laser. The LOC is operated by syringe and is equipped with reagents, sample wells, reaction wells, diffusion traps (to avoid cross contamination among channels) and waste reservoirs. The system was detected LcA at concentrations as low as 0.5 nM, which is the reported sensitivity of the SNAP-25 in vitro cleavage assay. Combined with our CCD detector, the simple point of care system enables the detection of BoNTs activity and may be useful for the performance of other complex medical assays without a laboratory. This approach may realize the potential to enhance the quality of health care delivery for underserved populations.

Low-cost technologies for medical diagnostics in low-resource settings

INTRODUCTION Medical diagnostics is a critical element of effective medical treatment. However, many modern and emerging diagnostic technologies are not affordable or compatible with the needs and conditions found in low- and middle-income countries. Resource-poor countries require low-cost, robust, easy-to-use, and portable diagnostic devices compatible with telemedicine that can be adapted to meet diverse medical needs. AREAS COVERED The most suitable devices are likely those that will be based on optical technologies, which are used for many types of biological analyses. This manuscript describes several prototypes of low-cost optical technologies and their application developed at the FDAs Office of Science and Engineering laboratories including a webcam-based multiwavelength fluorescence plate reader, a webcam-based fluorescence microscope demonstrated for colonic mucosa tissue pathology analysis, a lens-free optical detector used for the detection of Botulinum A neurotoxin activity, and a lab-on-a-chip which enables the performance of enzyme-linked immunosorbent assay and other immunological or enzymatic assays without the need of dedicated laboratories and complex equipment demonstrated for the detection of the toxin staphylococcal enterotoxin B. EXPERT OPINION Sensitive and effective optical detection devices can be developed using readily available consumer electronics components such as webcams, charge-coupled device cameras, and LEDs. There are challenges in developing devices with sufficient sensitivity and specificity. Several optical and computational approaches were developed to overcome these challenges to create optical detectors that can serve as low-cost medical diagnostics in resource-poor settings.

Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health

Resource-poor countries and regions require effective, low-cost diagnostic devices for accurate identification and diagnosis of health conditions. Optical detection technologies used for many types of biological and clinical analysis can play a significant role in addressing this need, but must be sufficiently affordable and portable for use in global health settings. Most current clinical optical imaging technologies are accurate and sensitive, but also expensive and difficult to adapt for use in these settings. These challenges can be mitigated by taking advantage of affordable consumer electronics mobile devices such as webcams, mobile phones, charge-coupled device (CCD) cameras, lasers, and LEDs. Low-cost, portable multi-wavelength fluorescence plate readers have been developed for many applications including detection of microbial toxins such as C. Botulinum A neurotoxin, Shiga toxin, and S. aureus enterotoxin B (SEB), and flow cytometry has been used to detect very low cell concentrations. However, the relatively low sensitivities of these devices limit their clinical utility. We have developed several approaches to improve their sensitivity presented here for webcam based fluorescence detectors, including (1) image stacking to improve signal-to-noise ratios; (2) lasers to enable fluorescence excitation for flow cytometry; and (3) streak imaging to capture the trajectory of a single cell, enabling imaging sensors with high noise levels to detect rare cell events. These approaches can also help to overcome some of the limitations of other low-cost optical detection technologies such as CCD or phone-based detectors (like high noise levels or low sensitivities), and provide for their use in low-cost medical diagnostics in resource-poor settings.

The National Institutes of Health Affordable Cancer Technologies Program: Improving Access to Resource-Appropriate Technologies for Cancer Detection, Diagnosis, Monitoring, and Treatment in Low- and Middle-Income Countries

Point-of-care (POC) technologies have proved valuable in cancer detection, diagnosis, monitoring, and treatment in the developed world, and have shown promise in low-and-middle-income countries (LMIC) as well. Despite this promise, the unique design constraints presented in low-resource settings, coupled with the variety of country-specific regulatory and institutional dynamics, have made it difficult for investigators to translate successful POC cancer interventions to the LMIC markets. In response to this need, the National Cancer Institute has partnered with the National Institute of Biomedical Imaging and Bioengineering to create the National Institutes of Health Affordable Cancer Technologies (ACTs) program. This program seeks to simplify the pathway to market by funding multidisciplinary investigative teams to adapt and validate the existing technologies for cancer detection, diagnosis, and treatment in LMIC settings. The various projects under ACTs range from microfluidic cancer diagnostic tools to novel treatment devices, each geared for successful clinical adaptation to LMIC settings. Via progression through this program, each POC innovation will be uniquely leveraged for successful clinical translation to LMICs in a way not before seen in this arena.Point-of-care (POC) technologies have proved valuable in cancer detection, diagnosis, monitoring, and treatment in the developed world, and have shown promise in low-and-middle-income countries (LMIC) as well. Despite this promise, the unique design constraints presented in low-resource settings, coupled with the variety of country-specific regulatory and institutional dynamics, have made it difficult for investigators to translate successful POC cancer interventions to the LMIC markets. In response to this need, the National Cancer Institute has partnered with the National Institute of Biomedical Imaging and Bioengineering to create the National Institutes of Health Affordable Cancer Technologies (ACTs) program. This program seeks to simplify the pathway to market by funding multidisciplinary investigative teams to adapt and validate the existing technologies for cancer detection, diagnosis, and treatment in LMIC settings. The various projects under ACTs range from microfluidic cancer diagnostic tools to novel treatment devices, each geared for successful clinical adaptation to LMIC settings. Via progression through this program, each POC innovation will be uniquely leveraged for successful clinical translation to LMICs in a way not before seen in this arena.

The Role of Affordable, Point-of-Care Technologies for Cancer Care in Low- and Middle-Income Countries: A Review and Commentary

As the burden of non-communicable diseases such as cancer continues to rise in low- and middle-income countries (LMICs), it is essential to identify and invest in promising solutions for cancer control and treatment. Point-of-care technologies (POCTs) have played critical roles in curbing infectious disease epidemics in both high- and low-income settings, and their successes can serve as a model for transforming cancer care in LMICs, where access to traditional clinical resources is often limited. The versatility, cost-effectiveness, and simplicity of POCTs warrant attention for their potential to revolutionize cancer detection, diagnosis, and treatment. This paper reviews the landscape of affordable POCTs for cancer care in LMICs with a focus on imaging tools, in vitro diagnostics, and treatment technologies and aspires to encourage innovation and further investment in this space.

Evaluation of a Methodology for Automated Cell Counting for Streak ModeImaging Flow Cytometry

Identification of Circulating Tumor Cells (CTCs) has shown promising clinical applications, but since CTCs are found in very low concentration in blood large sample volumes are needed for meaningful enumeration. This issue impedes the analysis of CTCs using standard flow cytometry due to its low throughput. To address this issue, a high throughput microfluidic cytometer was recently developed using a wide field flow- flow cell instead of the conventional narrow hydrodynamic focusing cells (used in traditional flow cytometry) enabling analysis of large volumes at lower flow rate. This wide-field flow cytometer adopts a technique known as “streak photography” where exposure times and flow velocities are set such that the particles are imaged as short “streaks”. Since streaks are imaged with large number of pixels, they are easily distinguished from the noise which appears as “speckles” increasing the detection capabilities of the device, making it more suitable for analysis using current low sensitivity, high noise webcams or mobile phone cameras. The non-stationary nature of the high noisy background found in streak cytometry introduces additional challenges for automated cell counting methods using traditional cell detection techniques such TLC, CellProfiler, CellTracker and other tools based in traditional edge detection (e.g., Canny based filters) or manual thresholding. In order to address this issue, we developed a new automated enumeration approach that does not rely on edge detection or manual thresholding of individual cells, rather is based in image quantizing, morphological operations, 2D order-statistic filtering and decisions rules that take into account knowledge of the structure and expected location of the streaks in consecutive frames. We evaluated our approach comparing it with two current methods representing the major computational modalities for cell detection: CellTrack (based in edge detection) and MTrack2 (based in manual thresholding). Samples of 1 cell/mL nominal concentration were analyzed in batch size of 30 mL at flow rate of 10 mL/min and imaged at 4 frames per second (fps), the files were saved in uncompressed AVI format files. The cells were annotated and the signal to noise ratio (SNR) was calculated. For samples with average SNR greater than 4.4 dB, our method achieved a sensitivity of 91% compared to CellTrack (60%) and MTrack2 (71%). The True Positive Rate (TPR) of cells detected was 0.93 for our method compared with 0.80 for Mtrack2 and 0.29 for CellTrack. This demonstrated the ability of the algorithm to count rare cells with high accuracy for concentrations of 1 cell/mL with SNR greater than 4.4 dB. This cell counting capability will enable to automate low cost imaging flow cytometry based on CCD detector and the expansion of cell-based clinical diagnostics in resource-poor settings.

The Program for Cancer Detection, Diagnosis, and Treatment Technologies for Global Health: A Pathway for the Translation of Affordable, Minimally-Invasive Point-of-Care (POC) Technologies to Less-Resourced Settings

Abstract 9Cancer kills more people worldwide than HIV/AIDS, tuberculosis, and malaria combined, and low- and middle-income countries (LMICs) bear the majority of this burden. While success in detection, diagnosis, and treatment has been reported in LMICs through the use of low-cost, point-of-care (POC) technologies, this area has been largely overlooked by the medical device industry and venture capital communities, as low-cost solutions offer less financial incentive for investment. The program presented here aims to simplify the pathway to market by funding investigation teams to adapt and validate existing technologies in low-resource settings. This program specifically supports the translation of these technologies, prioritizing patient outcomes in a manner not typically seen.This program, currently in its second year, will soon support 15 technologies for cancer detection, diagnosis, and treatment (e.g., in vitro assays, imaging devices, ablation devices). It is anticipated that by year seven of the ...

Abstract 1428: The program for cancer detection, diagnosis, and treatment technologies for global health: Translating affordable, minimally invasive point-of-care technologies to less-resourced settings

Cancer kills more people worldwide than HIV/AIDS, tuberculosis and malaria combined, and low-and-middle income countries (LMICs) bear the majority of this burden. Success in detection, diagnosis and treatment has been reported in LMICs through the use of low-cost point-of-care (POC) technologies, and the program presented offers a unique pathway to this POC market by funding multidisciplinary investigative teams to adapt and clinically validate existing technologies for cancer detection, diagnosis and treatment in low-resource settings. Each project consists of an adaptation phase (2 years:

Lensless CCD-based fluorometer using a micromachined optical Söller collimator.

500k total costs/year) and validation phase (3 years:

Modeling and design of micromachined optical Söller collimators for lensless CCD-based fluorometry

1M total costs/year). Projects are selected through NIH peer review process by a carefully-selected special emphasis panel briefed on the goals of the program. Projects are competitively vetted for Phase II funding based on completion of Phase I milestones. The program currently supports seven technologies for cancer detection, diagnosis and treatment, each of which is progressing towards experimental and clinical validation. The first project is an LED-based photodynamic therapy device for oral cancer, that has similar efficacy in vivo and ex vivo as existing laser phototherapy. Another supported project is an automated high resolution microendoscope for cervical cancer detection, with an impressive histological concordance in detecting CIN2/3 (90%+ for CIN3). Two cervical cancer cryotherapy projects are funded: a cryopen, that can achieve an approximately 4.0 mm depth of necrosis (>90% of disease) for cervical cancer treatment, and an efficient cryopop device that consumes less than 10% of CO2, compared to commercially-available devices and exhibits comparable therapeutic efficacy in ballistic gel studies. The program is also supporting two POC tests, a HPV test and a Hepatitis C viral antigen level and viral load detection. Additionally, a breast cancer triaging device/algorithm, with 95% sensitivity and capabilities to reduce false positive detection rate by 40%, is also being supported. Each project has its own detailed outline for Phase I and Phase II studies, which will be highlighting in our presentation. The program is in the process of adding another six projects, and it is anticipated that by year seven of the program, at least nine projects will have progressed through optimization, clinical validation, and business planning for commercialization and field/clinic dissemination. Through these process, we will uniquely accelerate these technologies for success in clinical translation. Citation Format: Michael Gwede, Paul Pearlman, Pushpa Tandon, Miguel Ossandon, Lokesh Agrawal, Houston Baker, Vinay Pai, Tiffani Lash. The program for cancer detection, diagnosis, and treatment technologies for global health: Translating affordable, minimally invasive point-of-care technologies to less-resourced settings. [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 1428.