Methods for quantitative assessment of muscle fibers in muscular dystrophy

Abstract

The disclosure concerns a method for assessing muscular dystrophy-linked protein expression in muscle fibers using digital image analysis of tissue. The method relates to assessing disease severity in individuals with muscular dystrophy. Muscle tissue samples are obtained from patients submitted for evaluation and processed to produce tissue sections mounted on glass slides which have been stained for a muscular dystrophy-linked protein. Digital images of the stained tissue sections are generated and analyzed by applying an algorithm process implemented by a computer to the images. The algorithm process extracts the morphometric and staining features of the muscular dystrophy-linked protein staining in the tissue, and parameters relating to these features are used to score the disease status for each patient submitted for evaluation. The score of disease status is ultimately used to infer disease severity, monitor the efficacy of a therapeutic approach, or select patients as candidates for a therapeutic approach.

What is claimed is: 
     
         1 . A method comprising:
 capturing at least one digital image of at least one stained muscle tissue section;   extracting at least one image analysis feature from each muscle fiber in the at least one digital image, wherein the at least one image analysis feature is selected from the group consisting of staining features and morphometric features;   combining at least one staining and morphometric feature to derive a score of disease status; and   interpreting the score of disease status to draw inferences associated with the severity of disease.   
     
     
         2 . The method of  claim 1 , wherein the at least one tissue section is stained for at least one muscular marker selected from the group consisting of a muscular dystrophy-linked protein and a muscle fiber membrane biomarker. 
     
     
         3 . The method of  claim 2 , wherein the muscular dystrophy-linked protein is a protein product of a gene that when mutated, or otherwise disrupted, gives rise to at least one muscular dystrophy disorders. 
     
     
         4 . The method of  claim 2 , wherein the muscular dystrophy-linked protein is selected from the group consisting of dystrophin and utrophin. 
     
     
         5 . The method of  claim 2 , wherein the muscle fiber membrane biomarker is selected from the group consisting of spectrin and merosin. 
     
     
         6 . The method of  claim 1 , wherein the digital images are captured by a method selected from the group consisting of chromogenic image capture and fluorescence image capture. 
     
     
         7 . The method of  claim 1 , wherein the muscle fibers are identified using at least three phases of image processing, including: initial segmentation, closure of gaps for incomplete membranes, and removal of false positive membranes. 
     
     
         8 . The method of  claim 7 , wherein the first phase of image processing identifies fiber membranes using staining intensity differences selected from the group consisting of fiber membrane staining, background staining outside of fibers, and staining within the interior region of a fiber. 
     
     
         9 . The method of  claim 7 , wherein the second image processing phase closes remaining gaps in incomplete fibers using a method selected from the group consisting of fiber end continuation, gap maximum, and expansion processes. 
     
     
         10 . The method of  claim 7 , wherein the third image processing phase removes false positive membranes and membrane segments by a method selected from the group consisting of removal of isolated fiber membrane segments and fiber region borders, removal of fiber membranes outside of morphometric feature thresholds, removal of fiber membranes outside of staining feature thresholds, and removal of fiber membranes inside a larger fiber membrane object. 
     
     
         11 . The method of  claim 1 , wherein the morphometric and staining features relate to the presentation of muscular dystrophy-linked protein staining, and are selected from the group consisting of the total area of the tissue section, the total area of muscle fiber membranes, individual muscle fiber membranes in the tissue section in an object-based manner, muscle fiber membrane width, and muscle fiber membrane crest. 
     
     
         12 . The method of  claim 11 , wherein the membrane crest is a continuous ridge of high staining intensity values around the diameter of a muscle fiber membrane. 
     
     
         13 . The method of  claim 11 , wherein staining features extracted by the algorithm process pertain to the appearance of muscular dystrophy-linked protein staining and morphometric features pertain to the physical presentation and distribution of muscular dystrophy-linked protein staining. 
     
     
         14 . The method of  claim 1 , wherein the morphometric and staining features of muscular dystrophy-linked protein staining are combined using at least one of linear, non-linear, and logical operators to derive a summary score of disease status. 
     
     
         15 . The method of  claim 1 , wherein the assessment of disease severity is used to determine a patient's status, wherein the status is selected from the group consisting of diagnosis of muscular dystrophy, prognosis of the normal course of disease, monitor treatment efficacy for muscular dystrophy, and selection of muscular dystrophy patients as candidates for a specific therapeutic intervention. 
     
     
         16 . The method of  claim 1 , wherein annotations are placed around each identified muscle fiber object identified by the algorithm process. 
     
     
         17 . The method of  claim 16 , wherein the annotations can be a rectangular geometry or an outline fit to the shape of each fiber. 
     
     
         18 . The method of  claim 1 , further comprising calibrating the image analysis features. 
     
     
         19 . The method of  claim 19 , wherein the calibration is performed using at least one reference selected from the group consisting of a reference digital image and a reference tissue section.