An image analysis study of vastus lateralis muscle fibers in malignant hyperthermia susceptible patients.

We studied the correlation between the in vitro contracture test (IVCT) performed in malignant hyperthermia (MH) and the muscle fiber type composition in 29 human vastus lateralis (VL) biopsy samples (from 12 women and 17 men) using a semiautomated image analyzer. Relative number, lesser diameter, global area, and spatial distribution of the muscle fibers were measured. In these and in 26 additional VL muscle biopsy samples of patients with other myopathies, we compared our morphometric data with the observations made by the pathologist. Among the MH group, type 1 fibers were larger in both malignant hyperthermia susceptible (MHS) men and women reaching statistical significance only in the latter. The relative number and global area were unchanged. In MHS patients relative number and global area of type 2A fibers were smaller. No changes in the parameters of type 2B fibers were found. In a minority of sections (14%) clustering was observed. Sex-related alterations in type 2 fiber characteristics were found between MHS patients. However, our findings do not clearly point towards a syndrome-induced alteration of size, number, global area, or distribution of type 1, 2A, and 2B muscle fibers in VL of MH patients. By morphometric analysis, we found several additional biopsy samples that met the interpretation of "abnormal" size and number of muscle fibers in human malignant hyperthermia than were reported by the pathologist.

Automated cytometry of fibre size and spatial distribution in the superficial masseter muscle of the rat at three ages.

An image cytometry program was applied to sections of the superficial masseter muscle of female and male 21-, 42- and 105-day-old rats. Lesser diameter and spatial distribution of more than 6000 muscle fibres were automatically measured in digital images from muscle sections stained for myofibrillar ATPase activity. In this muscle, only type 2A, 2B and 2C fibres were observed. At the three ages and in both sexes, 2A fibres were found to have the largest diameter and were the most frequent (> 54%). In the 21-day-old animals, females presented larger diameters than did males; in contrast, in the 105-day-old animals, the three fibre types were larger in males than in females. At all ages and in both sexes, type 2A occupied 32 to 80% more cross-sectional area than type 2B. Most images (98%) showed a random spatial distribution of their fibre types. All fibre types grew in diameter with age. The coefficient of variation of the diameter was age-independent and remained under 23%. The finding of an age-independent variable could have a practical application: an increase of the coefficient of variation (> 25%) can be considered as pathological, even without a perfect age-matched control.

Automated cytometry of muscle fibre sections: size and spatial distribution of the four fibre types in young and adult rats.

An automated cytometry program was applied to the extensor digitorum longus muscle of young and adult rats. Lesser diameter and spatial distribution of about 4000 fibres were measured in digital images from ATPase-stained muscle sections. All fibre types grow thicker with age, but the coefficient of variation of the diameter is age-independent. At both ages, 2B fibres have the largest mean diameter and are most frequent. 2A fibres in young rats present a diameter smaller or equal to type 1 but then show a faster increase in size; their relative number increases from 20 to 28%. Consequently type 2A displays the most important change with age. The spatial distribution of fibres is mathematically expressed; most images show a random distribution of type 1 and type 2 fibres. Taking into account the variation of fibre size of each type, the number of fibres which should be measured in order to reach a specified precision was calculated.

Automated analysis of muscle fibre images.

A technique for studying the distribution and size of different fibre types in muscles is proposed for automated analysis of individual fibres in optical density images from ATPase-stained muscle sections. After delineation, fibres may be classified into different histological types (1, 2A, 2B and 2C) using the measurement of their mean optical density (mOD). The densitometric measurements were obtained from three serial histological slides stained under different conditions. The delineation procedure is performed on one of the images: the resulting mask is fitted to the other images using a linear coordinate transform. Along with densitometric measurements, the lesser diameter of the fibres is computed. Both in processing and in analysis, extensive use was made of mathematical morphology tools. All software was implemented on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board.

Fully automated measurements by light microscopy of tissue sections using a cellular array computer.

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.