Automation of slide staining for large tissue sections.

Staining is a critical step in tissue analysis as it enhances the visibility and contrast of tissue structures for microscopic examination. Large tissue sections such as the human brain, heart, and liver are becoming increasingly important in studying complex tissue structures, providing critical information about the tissue's normal or abnormal development, function, and disease processes. Manual staining methods are still widely used and are prone to inconsistencies and inaccuracies, leading to unreliable results. Commercially available automated staining systems offer a more efficient alternative, but currently, these systems are only available for smaller 1" x 3" slides which are ill-suited for examining larger tissue sections. To address this challenge, we present a custom-designed Large format Automated Slide Stainer that can handle various glass slides, from the standard 1" x 3" slides to the custom-sized 2" x 3", 5" x 7", and 6" x 8" glass slides. The system uses a Cartesian robotic arm to stain the slides and has a user-friendly and intuitive interface for creating and modifying custom staining protocols. Safety features include chemical isolation, a ventilation system, an emergency shutdown, and a protective shield to minimize hazards from handling chemicals and biological materials. The automated stainer showed little variability in positioning with a mean offset error of 1.65 ± 0.65 mm and 1.73 ± 0.76 mm in the X and Y axes, respectively. In addition, the automated staining process showed better uniformity than manual staining. A pairwise distance was used to evaluate how well image histograms matched within a batch. The automated staining had a mean pairwise distance of 0.0070 ± 0.0017 (Nissl) and 0.0060 ± 0.0003 (Hematoxylin and Eosin(H&E)), which were far superior to the manual staining distances (Nissl: 0.0173 ± 0.0107 and H&E: 0.0185 ± 0.0067). This system represents a substantial advancement in tissue staining and has the potential to improve the reliability of tissue analysis significantly.Clinical relevance - Automated system for providing accurate, reproducible, and high-throughput staining of large tissue sections for use in histopathology and research.

Automation of slide coverslipping for large tissue sections.

Coverslipping is the process of placing a cover glass or coverslip over a glass slide mounted with a stained tissue specimen without forming air bubbles, which can negatively impact the microscopic examination. While manual coverslipping is still widely used, automated systems have made the process easier and more consistent. Commercially available automated cover-slippers are limited to handling only slides that are 1" x 3", suitable for processing smaller tissue specimens. However, for larger tissue specimens sectioned from organs like the brain, liver, etc., slides can reach sizes up to 6" x 8", exceeding the capabilities of these systems. We present SLIDE PROTEKT, a fully automated large format coverslipping system designed to efficiently coverslip large format slides. This system has multiple zones for slide and coverslip transportation, dispensing of mounting medium, and precise placement of the coverslip without air bubbles. The ability of the system to output quality coverslipped slides was validated by processing 50 large-format brain tissue slides. The results were found to be comparable to manual coverslipping. The system achieved a coverslip placement accuracy of 80% with a mean positional offset that was within a tolerance of ±3 millimeters. Additionally, 75% of the slides had no air bubbles, while the remaining slides had air bubbles that were less than 120 micrometers in size. These results demonstrate the potential impact of SLIDE PROTEKT in the field of histology.

iQuant Auto: Automated Rapid Test Platform for Immunodiagnostics.

Point-of-care diagnostic devices aid in the early and rapid detection of immunological markers leading to better medical outcomes. Lateral flow immunoassays (LFIA) are fast assays that provide both qualitative and semi-quantitative results on site. Sample preparation for LFIA tests is usually done manually with multiple mixing steps and is prone to errors. The iQuant Auto is an entirely automated system that can handle sample preparation, effective transfer to lateral flow test membrane, and subsequent result estimation. The system uses a pneumatic system for fluid handling and sample preparation, and an image-based fluorescence reader for reaction visualization. The device works with test specific milli-fluidic lateral flow kit called iQPrep Kit, which has integrated reagent storage areas and valves for fluid control. The test kit can be manufactured using standard manufacturing techniques and can be produced at scale cheaply. The iQPrep Kit can be modified to test for various markers like HbA1c, Vitamin D, and TSH, while the hardware for sample preparation and the fluorescence reader remains the same. The device has a minimalist and intelligible graphical interface aiding smooth operation by less skilled people at resource-limited settings. Standard reference cartridges of different volume ratios were used to validate the functionality of the instrument. The intra- instrument coefficient of variation (CoV) of the mobile kit reader was found to be less than 0.62%. The positional accuracy of the system to ensure precise kit engagement with the other auxiliary systems was checked, and the CoV was 0.16%. The fluid handling capabilities were tested, and it was found that an average fluid loss of 10 µl results due to the liquid adherence to fluid channel walls and valve interfaces. The iQuant Auto is an easily operable, total analysis system for immunodiagnostics.

iQuant™ Analyser: A rapid quantitative immunoassay reader.

Lateral flow immunoassays (LFIA) used in rapid quantitative point of care testing require an accurate, reliable and easy to operate instrument to read the LFIA kit and calculate the quantitative result value. We present iQuant® Analyser, an immunoassay reader designed for reading the Quanti® range of LFIA test kits for key markers such as HbA1C, Vitamin D, TSH etc. The instrument utilizes a laser based confocal optics system to capture the test and control lines from the LFIA kit, digitizes the fluorescent signal with high spatiotemporal resolution, computes necessary peak area ratios, applies calibration curves and declares the final result in an automated manner with minimal operator input. The instrument uses kit specific calibration information embedded on each LFIA test kit, to compute the final clinical parameter without using any external calibration chip. An intuitive icon based interface enables easy operation with minimal key presses, suited for point of care applications. The technology is designed in a modular manner to enable the instrument to perform tests on various parameters such as HbA1C, TSH, and Vitamin D etc without any hardware changes, using test-specific LFIA kits. The functional performance of the iQuant Analyser was verified over the range of expected area ratio values with standard reference cartridges that provided stable fluorescent lines. Repeatability of the instrument was found to be excellent with coefficient of variation (CoV) of area ratios found to be less than 1%. The inter-instrument reproducibility was also found to be good with CoV less than 4 %. Tests using blood samples with Quanti LFIA kits verified the accuracy of HbA1C results to be acceptable as per international standards with errors <; 4 %. The iQuant Analyser is a portable, easy to use rapid quantitative immunoassay reader best suited for point of care applications.

ImQuant - An image based fluorescence reader for quantitative lateral flow immunoassays.

Fluorescence immunoassays (FIA) have strong potential in enabling rapid quantitative immunological testing at point of care. Existing state of the art instruments employ laser scanning of FIA test kits, which require the use of electromechanical systems, making the instrument bulky, heavy and less reliable in the long term. There is a growing need for an extremely compact and reliable, motion-free, automated reader for accurate quantification of immunoassays. Here, we present ImQuant, an image based FIA reader with integrated monochromatic excitation source, optical filtering and real time image capture, assisted with an intuitive graphical user interface. A 12bit dynamic range RAW ouput CMOS camera with 1/3" sensor format and a sharp cut off wideband filter were utilized to ensure improved accuracy. A laboratory prototype was constructed and tested using a set of stable reference cartridges that provided repeatable fluorescent lines. The ImQuant could capture the lines and measure area ratios with coefficient of variation (CV) less than 2%. Further, tests with blood samples on Quanti® HbA1C test kits also demonstrated good repeatability with CV<;2%. The ImQuant is a novel platform of motion-free fluorescent scanning of FIA immunoassays and is being used for developing a variety of test kits and instruments.