Measuring Fat and Vacuolar Space with Digital Pathology Frank Voelker, DVM, DACVP Principle Pathologist Trevor Johnson, MS Director, Image Analysis.

Measuring Fat and Vacuolar Space with Digital Pathology

Frank Voelker, DVM, DACVPPrinciple Pathologist

Trevor Johnson, MS Director, Image Analysis

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Originally An Algorithm for Measuring Fat

White Fat

Brown Fat

Lipid vacuoles in a variety of tissues (eg. Fatty liver or adrenal cortex)

Tissues composed of fibers or tubules (eg. skeletal muscle)

Tissues with hollow spaces or glandular lumena (eg. lung or thyroid gland)

Cerebral edema, demyelinization

Vacuoles in various cell types (eg. Lymphocytes)

What was actually needed: An algorithm for measuring circumscribed clear spaces in tissue

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Algorithm Requirements

Percent area of vacuoles or circumscribed spaces.

Total number of spaces

Mean area/diameter/perimeter of spaces

Histograms of areas, diameters or perimeters

Data Output:

Shape and Area Threshold ControlsHue Sensitivity AdjustmentMembrane Estimation or Completion

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Vacuole Algorithm Settings

Input and Output Windows

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How the Image is Analyzed

Define the tissue intensity

Determine the maximum and minimum vacuole areas

Determine how close each vacuole is allowed to resemble a circle and how tightly they are packed

Determine if Vacuole Perimeter Estimation (VPM) is needed

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Measuring White Fat Cells

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Measuring White Fat

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Measuring White Fat – Removing Small and Elongated Vacuoles

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Measuring White Fat – Removing Small Vacuoles

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Morphometric Considerations

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Remove Tissue (Just View Vacuole Spaces in Green)

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Remove Tissue (Parameter Too Low Including Tissue)

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Include Small Vacuoles

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Exclude Large Vacuoles

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Vacuole Analysis Fatty Liver

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Fatty Liver

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Measuring Brown Fat Vacuoles

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Measuring Brown Fat Vacuoles

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Measuring Brown Fat with Nuclear Counting

173 Nuclei

Area/No. Nuclei = Cell Size

No. Nuclei/No. Fat Droplets = No. Droplets/Cell

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Measuring Pulmonary Alveoli

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Measuring Pulmonary Alveoli

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Adrenal Gland

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Vacuole Analysis Adrenal

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Measuring Adrenal Vacuoles with Nuclear Counts

187 Nuclei

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Measuring Adrenal Vacuoles

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Measuring Bone Marrow Vacuoles

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Measuring Bone Marrow Vacuoles

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Measuring Skeletal Muscle Fibers

Type 1- Red Muscle. Slow twitch postural type muscle fiber. Aerobic oxidation.

Type 2- White Muscle Fast twitch fibers. ATPase +. Anaerobic oxidation

DenervationStatin-induced rhabdomyolysisNoninflammatory myopathiesCongential myopathiesSports physiology

Muscle fiber alterations are important in:

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Measuring Skeletal Muscle Fibers

In H&E sections, skeletal muscle fibers are poorly demarcated

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Measuring Skeletal Muscle Fibers

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Muscle Fiber Histogram

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Summary

An algorithm designed to measure vacuoles or circumscribed clear spaces in tissue was described in this presentation. The algorithm has proved useful in measuring clear spaces in a variety of tissues including white and brown fat, skeletal muscle fibers, pulmonary alveoli, fatty liver and adrenal cortex. Data outputs included percent as well as histograms and mean areas, diameters or perimeters of vacuoles or clear areas within tissue.