Morphometric Features

MyoPath extracts 37 unique morphometric features per sample from routine H&E-stained skeletal muscle sections. These features are organized into five biological categories and distilled into seven clinically interpretable pathology indicators.

Reference

Zhong H*, Gao M*, Ma S, Zhang W, Chen N, Jiao K, Zhu B, Song J, Yan C, Yue D, Xi J, Zhu W, Zhao C#, Luo S#. MyoPath: A Deep Learning Pipeline for Objective Morphometric Assessment of Skeletal Muscle Biopsies. Manuscript in preparation.

Seven Pathology Indicators

These seven features correspond to five pathological axes routinely assessed during muscle biopsy evaluation:

Pathological Axis	Indicator	Clinical Significance
Nuclear positioning	NCI	Centronuclear myopathy, DM1
Fiber size dysregulation	Fiber CV	Dystrophic & neurogenic processes
Fiber morphology distortion	Shape regularity	Fiber splitting, angular atrophy
Tissue replacement	Fat infiltration, Fibrosis	Late-stage dystrophy, denervation
Cellular reaction	Nuclear/muscle ratio, Inflammatory infiltration	Regeneration, inflammation

1. Nuclear Centralization Index (NCI)

Primary biomarker. Quantifies the average radial position of nuclei within muscle fibers.

$${\rho }_k=min(\frac{d_k^{\text{boundary}}}{r_i^{\text{eq}}},1.0),\text{NCI}=\frac{1}{N_{\text{analyzed}}}\sum _{k=1}^{N_{\text{analyzed}}}{\rho }_k$$

where $d_k^{\text{boundary}}$ is the distance from the nucleus centroid to the nearest fiber boundary, and $r_i^{\text{eq}}=\sqrt{a_i/\pi }$ is the equivalent circle radius.

NCI Range	Interpretation
< 0.03	Normal (subsarcolemmal nuclei)
0.03 -- 0.10	Mild centralization
0.10 -- 0.20	Moderate centralization
> 0.20	Severe centralization

Clinical evidence:

• Discriminated myopathy from controls: $p=1.3\times {10}^{-5}$, rank-biserial $r=0.69$
• DM1 showed the highest NCI (median 0.121), consistent with centronuclear pathology
• Correlated with CTG repeat count in DM1: Spearman $\rho =0.46$, $p=0.042$
• Significant dose-response trend across the GTEx myopathy spectrum (Jonckheere-Terpstra $p<{10}^{-4}$)

Nuclei are further classified into three radial zones:

Zone	Criterion	Metric
Peripheral	${\rho }_k\le 0.3$	peripheral_ratio (normal ~ 1.0)
Intermediate	$0.3<{\rho }_k<0.7$	—
Central	${\rho }_k\ge 0.7$	central_ratio (abnormal if > 0.05)

2. Fiber Size Variability Coefficient (Fiber CV)

Primary biomarker. The coefficient of variation of myofiber cross-sectional areas.

$$\text{Fiber CV}=\frac{{\sigma }_a}{\overline{a}}$$

Fiber CV Range	Interpretation
< 0.25	Normal
0.25 -- 0.40	Mild variability
0.40 -- 0.60	Moderate variability
> 0.60	Severe variability

Clinical evidence:

• Discriminated myopathy from controls: $p=2.9\times {10}^{-4}$, $r=0.58$
• Inversely correlated with grip strength in DM1: $\rho =-0.61$, $p=0.031$
• Increased with mutation severity in LGMD: 2x Missense (0.44) → LoF + Missense (0.49) → 2x LoF (0.65)
• Dimensionless and comparable across samples with different fiber calibers

3. Fiber Shape Regularity

Mean circularity (shape factor) of fiber cross-sections.

$${\text{Shape Factor}}_i=\frac{4\pi a_i}{p_i^2},\text{Mean Shape Factor}=\frac{1}{N_f}\sum _{i=1}^{N_f}{\text{Shape Factor}}_i$$

• 1.0 = perfect circle
• < 0.6 = irregular fiber morphology (splitting, angular atrophy, chronic remodeling)
• Normal range > 0.7

4. Fat Infiltration (%)

Percentage of the ROI occupied by adipose tissue.

$$\mathtt{\text{fat\_infiltration\_pct}}=\frac{A_{\text{fat}}}{A_{\text{ROI}}}\times 100\mathrm{\%}$$

• Normal range: < 5%
• A late-stage marker of dystrophic or denervation injury
• ROI-dependent: values vary with the location selected for analysis

WARNING

Fat and fibrotic replacement are nonspecific, late-stage changes that also occur with aging, disuse, and obesity. They are inherently ROI-dependent, making them less robust than NCI and fiber CV.

5. Fibrosis (%)

Connective tissue as a percentage of ROI area, computed by Boolean subtraction.

$$A_{\text{connective}}=A_{\text{ROI}}-A_{\text{muscle}}-A_{\text{fat}}$$$$\mathtt{\text{fibrosis\_pct}}=\frac{A_{\text{connective}}}{A_{\text{ROI}}}\times 100\mathrm{\%}$$

• Normal range: < 10%
• Reflects endomysial and perimysial fibrotic proliferation
• ROI-dependent

6. Nuclear/Muscle Ratio

Ratio of nuclei located within myofibers to total fiber count.

$$\mathtt{\text{nuclear\_muscle\_ratio}}=\frac{N_{\text{nuc}}^{\text{muscle}}}{N_f}$$

• Normal range: 1 -- 3
• Elevated values reflect nuclear proliferation, regeneration, or increased satellite cell activity
• Significant in HuashanMuscle cohort ($p=3.4\times {10}^{-5}$) but not in GTEx wasting spectrum

7. Inflammatory Infiltration

Nuclear density in connective tissue regions.

$$\mathtt{\text{density}}=\frac{N_{\text{nuc}}^{\text{conn}}}{A_{\text{connective}}/{10}^6}({\text{nuclei/mm}}^2)$$

• Normal range: < 2,000 nuclei/mm²
• High values suggest inflammatory cell infiltration or active fibroblast proliferation

---

Underlying Descriptive Features

The 37 features include the 7 pathology indicators above plus 30 underlying descriptive features.

Tissue Composition (10 features)

Feature	Unit	Description
roi_area_um2	µm²	Total area of the analyzed region of interest
muscle_fibers_count	count	Number of individual myofibers detected by Cellpose-SAM
muscle_area_um2	µm²	Sum of cross-sectional areas of all detected myofibers
muscle_area_pct	%	Myofiber area as fraction of ROI. Decreases with wasting, fat replacement, or fibrosis
fat_regions_count	count	Number of discrete adipose regions identified
fat_area_um2	µm²	Total adipose tissue area, excluding overlap with muscle annotations
connective_area_um2	µm²	Connective tissue area (ROI minus muscle minus fat)
connective_area_pct	%	Connective tissue as fraction of ROI
nucleus_area_um2	µm²	Total area occupied by all detected nuclei
nucleus_area_pct	%	Nuclear area as fraction of ROI. Elevated with increased cellularity

Fiber Size (7 features)

Feature	Unit	Description
fiber_mean_area_um2	µm²	Average cross-sectional area. Decreased in atrophy, increased in hypertrophy
fiber_median_um2	µm²	Median fiber area. Less sensitive to outliers
fiber_std_um2	µm²	Standard deviation of fiber areas
fiber_min_um2	µm²	Smallest detected fiber area
fiber_max_um2	µm²	Largest detected fiber area
fiber_q1_um2	µm²	25th percentile. Sensitive to grouped atrophy
fiber_q3_um2	µm²	75th percentile

Fiber Shape (3 features)

Feature	Unit	Description
shape_factor_std	dimensionless	Standard deviation of circularity across fibers
aspect_ratio_mean	dimensionless	Mean bounding-box elongation (1.0 = circular, > 2.0 = elongated)
aspect_ratio_std	dimensionless	Standard deviation of aspect ratio

Nuclear Distribution (7 features)

Feature	Unit	Description
nuclei_total_count	count	Total nuclei detected within the ROI
nuclei_in_muscle	count	Nuclei whose centroids fall within myofiber polygons
nuclei_in_connective	count	Nuclei in connective tissue regions
nuclei_unassigned	count	Nuclei not assigned to muscle or connective tissue
nuclei_per_fiber_mean	count/fiber	Average nuclei per myofiber (healthy: 1--3)
nuclei_per_fiber_std	count/fiber	Standard deviation of nuclei per fiber
nuclei_per_fiber_max	count	Maximum nuclei in any single fiber

Nuclear Localization (3 features)

Feature	Unit	Description
peripheral_ratio	0--1	Fraction of nuclei in the outer 30% of fiber radius (healthy ~ 1.0)
central_ratio	0--1	Fraction of nuclei in the inner 30% of fiber radius (abnormal if > 0.05)
multinucleated_fiber_count	count	Fibers containing more than one nucleus in cross-section

---

MyoPath Score

The MyoPath Score is a logistic regression composite of all seven pathology indicators:

$$\text{MyoPath Score}=\frac{1}{1+e^{-z}}$$$$z=-9.17+27.11\times \text{NCI}+3.96\times \text{Fiber CV}+8.22\times \text{Shape}+0.017\times \text{Fat\%}+0.043\times \text{Fibrosis\%}+0.123\times \text{NMR}+0.000001\times \text{Inflammation}$$

Performance:

• Training (GTEx): AUC = 0.788 (LOO-CV = 0.735)
• External validation (HuashanMuscle): AUC = 0.873 (without retraining)
• NCI and Fiber CV carry the largest standardized coefficients ($\beta =0.57$ and $0.53$)

Segmentation Performance

Tissue Layer	Method	Dice	IoU
Myofiber	Cellpose-SAM	0.92 ± 0.03	0.85 ± 0.06
Fat	Pixel classifier	0.95 ± 0.02	0.91 ± 0.03
Nucleus	Watershed	0.87 ± 0.04	0.78 ± 0.06
Connective tissue	Boolean subtract	0.88 ± 0.04	0.78 ± 0.06

Intraclass correlation coefficients exceed 0.88 for all seven pathology indicators.