Automated imaging and analysis of the hemagglutination inhibition assay (HAI)
Inventors
Nguyen, Michael • Parkhill, Robert
Assignees
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Publication Number
US-10012643-B2
Publication Date
2018-07-03
Expiration Date
Abstract
A system and method provide for high through put determination of agglutination states. The system includes a rotating table and multiple plate tilting stations. The system also includes one or more optical paths positioned to image entire plate arrays in tilted and/or untilted configurations. The system preferably includes image analysis software to analyze an image of an array of test wells and determine an agglutination state of each well based on the image analysis.
Core Innovation
The disclosed invention relates to an automated high-throughput imaging and computer-vision analysis system for hemagglutination inhibition (HAI) assays. It addresses the subjectivity and throughput limits of manual RBC “streaming/foot” readouts by imaging whole 96-well plate arrays. The system uses imaging of plates at tilted and/or flat configurations rather than relying on manual visual interpretation.
The optical hardware includes telecentric, low-distortion optics and LED backlight trans-illumination to acquire images of the plate well arrays. A rotating table with plate holding stations moves multiple plates into alignment with an optical path, while a tilt motor adjusts a tilt angle for imaging. Documented implementations include imaging systems that tilt multiple plates simultaneously and a dual-mode approach that adds an optical train for 0° imaging.
For analysis, the system performs whole-plate well detection and color segmentation using an L*a*b* color space with a predefined RBC gate, producing a hemagglutination parameter (HA_P = L*B*Y). It classifies agglutination state for test wells and uses kinetic control wells to select an optimal time point, followed by titer assignment and outlier flagging. The system further provides visualization overlays and stores and exports results through a graphical user interface and a relational database.
Claims Coverage
The claim coverage centers on three core inventive aspects, with additional dependent features including illumination, tilt and timing constraints, an additional optical path for untilted plates, a CCD sensor specification, and computing-based agglutination-state determination.
Optical path with camera, lens, and mirror and rotating table alignment
The system includes an optical path comprising a camera, a lens, and a mirror, and a rotating table comprising a plurality of plate holding stations and a table motor, wherein the rotating table is rotated to move any of the plate holding stations into alignment with the optical path.
Plate holding station with plate array and tilt angle adjustment
Each plate holding station includes a plate holder for holding a plate comprising an array of test wells and a tilt motor for adjusting a tilt angle of the plate holder.
Controller-controlled imaging acquisition of plate images
A controller controls the table motor, rotation motor, and the camera to acquire at least one image of at least one plate.
Backlight plate illumination for imaging
A plate holding station further comprises a backlight to illuminate the plate.
Tilt to a predetermined angle and image at a predetermined time
The controller controls a tilt motor to tilt a plate holder to about 70° and controls the camera to acquire an image of the plate at a predetermined time after the plate holder is tilted.
Second optical path for imaging an untilted plate
The system further includes a second optical path with a camera, a lens, and a mirror arranged to capture an image of an untilted plate.
CCD camera sensor constraint for imaging
A CCD camera is included, wherein an image sensor has at least 5 megapixels.
Computing-based agglutination state determination for substantially all test wells
A computing element receives camera images, analyzes them, and determines an agglutination state for substantially all of the test wells of a plate.
The claims are directed to an imaging system for plate well arrays using an optical path and a rotating table that aligns plate holding stations into the optical path, combined with controller-controlled tilt adjustment to acquire plate images. Dependent claims add backlight illumination, tilt and timing constraints, an additional optical path for untilted plates, a CCD sensor specification, and computing-based determination of an agglutination state for substantially all test wells.
Stated Advantages
Overcomes subjectivity of manual RBC “streaming/foot” readouts.
Increases throughput relative to manual readouts.
Enables whole 96-well plate array imaging at tilted and/or flat configurations.
Provides an automated image analysis workflow including agglutination-state classification and titer assignment.
Supports quantitative continuous titer/dose-response interpretation using an HA-parameter fitting and IC50-like metrics.
Provides visualization overlays and data storage/export via a GUI and a relational database.
Documented Applications
Automated high-throughput imaging and computer-vision analysis for hemagglutination inhibition (HAI) assays with whole 96-well plate arrays.
Binding activity determination of an antibody by imaging an array of test wells on a plate.
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