بسم الله الرحمن الرحيم ( سَنُرِيهِمْ آيَاتِنَا فِي الْآفَاقِ وَفِي أَنفُسِهِمْ حَتَّى يَتَبَيَّنَ لَهُمْ أَنَّهُ الْحَقُّ أَوَلَمْ يَكْفِ بِرَبِّكَ أَنَّهُ عَلَى كُلِّ شَيْءٍ شَهِيد ( ٌ فصلت : 53 صدق الله العظيم

HISTO-PATHOLOGY Introduction & Techniques Dr. Abdel Monem H. Lubbad. A.Professor Of Pathology (M.D.) Consultant of Pathology Islamic University -Gaza

Epidemiology Epidemiology Etiology - Causes Etiology - Causes Pathogenesis - Evolution Pathogenesis - Evolution Morphology - Structural Changes Morphology - Structural Changes Clinical Significance – Functional Changes Clinical Significance – Functional Changes Management Management Complications Complications Prevention Prevention Study of Disease: (Pathology)

Etiology - Study of Causes Etiology - Study of Causes Pathogenesis - Step by step evolution Pathogenesis - Step by step evolution Morphology - Structural Changes Morphology - Structural Changes Clinical Significance* Clinical Significance* Four aspects of Pathology: Important.!

Branches of clinical Pathology: Histopathology Histopathology Cytopathology Cytopathology Haematology Haematology Forensic Pathology Forensic Pathology Immunology Immunology Chemical Pathology Chemical Pathology Genetics Genetics Toxicology Toxicology Microbiology Microbiology Anatomic Pathology

Learning Pathology: General Pathology General Pathology Common changes in tissues. Common changes in tissues. Systemic Pathology Systemic Pathology Specific changes in organs. Specific changes in organs.

Techniques in Pathology: Gross Pathology: Gross Pathology: Light Microscopy: Light Microscopy: Histopathology, Cytology, Autopsy Histopathology, Cytology, Autopsy Histochemistry, Biochemical Histochemistry, Biochemical Immunohistochemistry Immunohistochemistry Electron Microscopy Electron Microscopy Cell Cultures, Medical Microbiology Cell Cultures, Medical Microbiology Molecular Pathology Molecular Pathology

Histo-Pathology Dr. Abdel Monem H. Lubbad A.Professor of Pathology (M.D.) Consultant of Pathology Islamic University –Gaza

HISTOTECHNOLOGY “The technique of processing the tissues submitted for histopathological study until the preparation of the stained section on a glass microscopic slide ready for study is known as Histotechnology ” “The technique of processing the tissues submitted for histopathological study until the preparation of the stained section on a glass microscopic slide ready for study is known as Histotechnology ” And persons specializing in this technique are known as Histotechnologists. And persons specializing in this technique are known as Histotechnologists.

Surgical Specimen Clinical Details Clinical Details Adequate specimen Adequate specimen Proper Fixative Proper Fixative 10% buffered Formalin 10% buffered Formalin

Gross Examination: Description: Specimen weight & measurement (approx) Specimen weight & measurement (approx) Consistency Consistency Photo * Photo * Cut section Cut section

Taking Samples: Edge of lesions. Wall of cysts. Wall of cysts. Include normal areas. Include normal areas. Avoid necrotic area. Avoid necrotic area. Whole specimen if small. Whole specimen if small. Direction, mark Direction, mark

Inking the Margins* To mark surgical margin. To mark surgical margin. Spread of lesion Spread of lesion Malignancy Malignancy Adequacy of removal Adequacy of removal Different colors to identify margins Different colors to identify margins

Fixation: Specimen bits are placed in porous cassettes Specimen bits are placed in porous cassettes Not more than 5mm thick Not more than 5mm thick In 10% formalin In 10% formalin 1mm/hour fixation 1mm/hour fixation ~ 6 hour ~ 6 hour

Fixation: After fixation is Replacing aqueous formalin with alcohol in gradual sequence (70, 95, 100%) to make way for paraffin. After fixation is Replacing aqueous formalin with alcohol in gradual sequence (70, 95, 100%) to make way for paraffin.

Clearing: Removal of alcohol with “xylene” that will be miscible with the embedding medium (paraffin) Removal of alcohol with “xylene” that will be miscible with the embedding medium (paraffin) Impregnating with paraffin. Impregnating with paraffin.

Embedding: Paraffin block with embedded tissue Paraffin block with embedded tissue consistency to cut consistency to cut Paraffin blocks taken for sectioning Paraffin blocks taken for sectioning

Tissue Processing: Preservative Preservative Provides stability Provides stability Protects from infection Protects from infection Prevents autolysis Prevents autolysis Permits sectioning and staining Permits sectioning and staining

Sectioning: Microtome Microtome 3-10 microns 3-10 microns Ribbon of sections Ribbon of sections taken on hot water bath taken on hot water bath

Picking up sections: Floating sections onto slides Floating sections onto slides Common “float” artefact Common “float” artefact

Microscope slide preparation: Taking the section onto slide Taking the section onto slide Flat, no air bubbles, no stretch or breaks. Flat, no air bubbles, no stretch or breaks.

Automated Staining: Routine stain H&E Routine stain H&E Hematoxylin (basic) Hematoxylin (basic) Eosin (acidic) Eosin (acidic) Nucleus is acidic and cytoplasm is relatively basic Nucleus is acidic and cytoplasm is relatively basic Special stains Special stains

Coverslipping: Clearing - xylene Clearing - xylene Thin glass coverslips to protect the section Thin glass coverslips to protect the section Using mounting media (Eg. DPX, Resins, Canada balsam etc.) Using mounting media (Eg. DPX, Resins, Canada balsam etc.)

Reporting: Additional sections Additional sections Deeper / Thinner sect. Deeper / Thinner sect. Special Stains/tech. Special Stains/tech. Reference.. Reference.. Discussions with Clin. Discussions with Clin. Diagnosis Diagnosis Report Typing Report Typing Despatch. Despatch. (>3-5 days) (>3-5 days)

Cytopathology: Cytopathology is study of cells in diagnosis of disease. Cytopathology is study of cells in diagnosis of disease. Exfoliative & Non-Exfoliative - cytology. Exfoliative & Non-Exfoliative - cytology. Exfoliative: Cell samples are collected from normally shedding tissues like epithelium. Spatula or brush to enhances collection. Exfoliative: Cell samples are collected from normally shedding tissues like epithelium. Spatula or brush to enhances collection. Non-Exfoliative: Cells samples collected by needles with suction pressure. (FNAC) Non-Exfoliative: Cells samples collected by needles with suction pressure. (FNAC)

PAP* Smear Normal:

PAP Smear - Abnormal:

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Special Techniques:

Light Microscopy  Kohler Illumination  Condenser  Objectives  2 to 4x - Low power  100x lens – Oil Imm.  Eye piece of 10x and objective of 40x = 400 times magnification.

Normal Stomach

Normal Skin

Normal Skeletal Muscle

Normal Kidney

Summary 1. Grossing 2. Fixation 3. Processing 4. Embedding 5. Sectioning 6. Staining 7. Mounting

Some Special Techniques

Frozen Sections: Freezing acts as embedding agent by forming minute ice crystals within cells. Freezing acts as embedding agent by forming minute ice crystals within cells. More rapid (5min), More rapid (5min), Liquid nitrogen. Liquid nitrogen. Freezing Microtome

Immunohistochemistry Antigen antibody reaction Antigen antibody reaction Ab Tagged with marker Ab Tagged with marker Simple Dye Enzyme (peroxidase) Fluorescent Dye Radioactive Dye Marker Sec. Antibody Pri. Antibody Tissue Antigen

Melanoma +ve for HMB-45

B cell Lymphoma – CD20

Breast Cancer Estrogen Receptor Antigen Tamoxifen Sensitive

Polarized Microscopy  Under Polarized light, Some materials have the property of "birefringence" which is the ability to pass light in a particular plane.  Eg. Crystals, fat, fibers. Amyloid etc.

Cardiac Amyloidosis

Urine Oval Fat Bodies

Fluorescent Microscopy  Property of materials that causes them to absorb light at a shorter (UV) wavelength, and to emit light at a higher (visible) wavelength  Auto-Fluorescence  Immuno-Fluorescence

ANA – Diffuse Pattern

ANA – Nucleolar Pattern

Electron Microscopy  Electron beam instead of light.  Magnified images are typically from 1000X to 50,000X. (Light microscope is x).  Gluteraldehyde fixative.  Glass knives.  Specimen is mounted on a metal grid.

Membranous GN

Minimal Change GN

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