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HISTOPATHOLOGYTissue Handling and Examination

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Histopathology Technical Summary

Identification of tissue and clinical information (Surgeon)

Grossing (Pathologist) Tissue sampling (Pathologist) Fixation (Technician) Processing (Technician) Embedding (Technician) Sectioning (Technician) Section ‘floating out’ (Technician) Histochemical Staining (Technician) Slide Mounting (Technician) Light microscopy and Reporting (Pathologist) Tissue disposal or storage, slide and block

archiving and retrieval (Technician)

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The Surgical Specimen (Surgeon)

The surgeon removes the tissue specimen from the patient

This specimen may be a small ‘biopsy’, a tissue sample (such as this appendix) or a large ‘resection’ specimen.

There is no identification attached to the tissue but it is the duty of the surgeon to ensure that the specimen container and the clinical request form have the patient’s details written on them.

The container may be a small pot, a small bucket or a large bucket.

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The Surgical Specimen (Surgeon)

Clinical Details Adequate

specimen Proper Fixative 1:10 proportion

tissue:fixative in 10% buffered Formalin

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Booking in the Specimen (Technician)

The tissue sample is received in the Department of Histopathology by the Technician.

Booking or logging in of the case is done

Checking of the details on the specimen pot and clinical form

Checking that the tissue is adequately immersed in appropriate fixative.

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Gross Examination (Pathologist):

Grossing or ‘Cut Up’ is prepared by the Technician who also attends the Pathologist.

Small biopsies and, in some Labs, other tissue specimens may be grossed by experienced Technicians.

Whole specimens are described with weight & measurement.

The number of pieces taken is recorded.

Representative sections or all embed the tissue.

Plastic cassettes have been pre-labelled by the Technician prior to or during grossing.

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Gross Examination (Pathologist):

Partially fixed tissue portions, 0.5 cm thick, are selected by the Pathologist and placed in the plastic cassette (2.0 x 1.5 x 0.5 cm).

A metal or plastic lid is then placed on the cassette.

A Technician takes the cassette away to be ‘processed’.

At this stage the surgical pathology number identification is on the plastic cassette; the tissue itself has no separate identification.

Any left over tissue after grossing is returned to the container and removed for storage by the Technician.

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Taking Samples (Pathologist):

For large specimens and organs, sampling guidelines are given by the Royal College of Pathologists

Sample gross abnormality. Sample specimen margins. Include normal areas. Avoid necrotic areas. Whole specimen if small. Check that the surgical

number on the specimen pot and form matches the number on the plastic cassette.

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Loading Plastic Cassettes Containing Partially Fixed Tissue into a Modern Tissue Processor (Technician)

At this stage, tissue is partially fixed and within a plastic cassette

A variety of designs of automated tissue processors are used which can load dozens of cassettes.

This process will run on a programme, usually overnight.

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Fixation (Technician):

This older version of a tissue processor, with less specimen capacity, demonstrates the sequence of events.

After fixation is done Replacing aqueous

formalin with alcohol in gradual sequence (70, 95, 100%) to make way for paraffin.

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The Stages of Processing - Clearing (Technician):

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

This is done before impregnating with paraffin.

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Embedding (Technician):

Following the processing schedule, the processor is unloaded.

The fixed and processed tissue pieces are removed from the plastic cassette, oriented and placed in to a stainless steel ‘mould’

At this stage, the tissue samples are ‘oriented’ by the technician

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Embedding (Technician):

Embedding in paraffin wax involves pouring hot wax into the mould containing the oriented fixed tissue.

The paraffin wax tissue block contains the fixed, embedded tissue.

There is usually no separate identification of this tissue block.

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Embedding (Technician):

This tissue block is then transferred back to the plastic cassette.

Heat is used to partially melt the wax allowing the block to ‘stick’ to the plastic cassette.

Paraffin wax ensures consistency when cutting sections for microscopy.

Paraffin blocks are then taken for sectioning.

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The Tissue Block

Fixed and processed tissue pieces

Embedded in paraffin wax to form a block (1.5 x 2.0 x 0.5 cm)

The block is ‘stuck’ to a plastic cassette on which is written the patients surgical pathology number as identification.

This block will be stored temporarily on lab benches while awaiting sectioning.

Ultimately, this block will be stored in boxes or files in the lab and will be accessible for further sectioning

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The Purpose of Tissue Fixation and Processing:

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

Fixed, as opposed to fresh, tissue cells may lose antigens (proteins) - so for immunohistochemistry, ‘antigen retrieval’ methods need to be employed; nucleic acids (DNA/RNA) may also become denatured or lost.

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Sectioning (Technician): A rotary Microtome is

used to cut sections from the tissue in its wax block.

The microtome may be manual or electric.

3-10 micron, thin sections are cut

Ribbons of sections will be cut from each block

These tissue ribbons are then taken on to a hot water bath or ‘floated out’

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Picking up sections – ‘Floating Out’ (Technician):

‘Floating’ sections onto slides in a heated water bath

Common “float” artefact may occur at this stage

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Microscope slide preparation (Technician):

Taking the unstained section onto the glass slide

Flat, no air bubbles, no stretch or breaks.

The surgical pathology number, taken from the plastic cassette is marked on the glass slide.

Once a suitable number of sections are ready they are batched and dried in an oven for several hours.

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Histochemical Staining (Technician):

Routine stain haematoxylin and eosin (H&E)

Hematoxylin (pink) (basic)

Eosin (blue)(acidic) The cell nucleus is acidic

and the cytoplasm is relatively basic

Special stains may be requested at this stage

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Coverslipping (Technician):

Clearing - xylene Thin glass

coverslips are used to protect the tissue section on the glass slide. Using mounting media (Eg. DPX, Resins, Canada balsam etc.)

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Slide Labelling (Technician): The surgical pathology

number which identifies the patient and the sample is hand written by the Technician on to the top of the mounted glass slide.

The writing may be in pencil or marked in to the glass with a diamond pen.

A paper identification label is hand written or printed by the Technician and then stuck on the top of the slide.

These labelled slides are batched and then given to the Histopathologist for microscopy and reporting.

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The H&E Stained Tissue Section on a Glass Slide, Ready for Microscopic Analysis:

From the initial selection of the tissue pieces by the Histopathologist, placed in the labelled plastic cassette…..

To the 5 micron section of fixed tissue, stained with H&E prepared by the Technical staff…..

Now ready for microscopic analysis.

Special stains may be requested at this stage as more sections can be cut from the stored block.

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Reporting (Histopathologist): The Histopathologist

reviews the stained, fixed, thin tissue section on the glass slide under the microscope.

At this stage a diagnostic, descriptive Histopathology Report may be produced (hand-written on to the form or dictated).

The Report will be typed by a Secretary.

This report will be stored on a computer and there will be paper copies.

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The Role of the Histopathology Report Report Despatch is

usually between 48 hours to 5 days following booking in of the tissue sample in to the Histopathology Department

The Histopathology opinion puts the patient in to a diagnostic category that guides further clinical management.

The Histopathologist does not decide upon or carry out patient treatment.

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Further Work (Histopathologist/s):

Additional sections may be requested.

Deeper / Thinner sections may be requested.

Special Stains/techniques.

External referral. Discussions with

Clinicians.

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Tissue Disposal, Archiving and Retrieving Tissue Slides and Blocks

(Technician)

Once the Report has been issued, left-over tissue within containers and buckets will be disposed of.

Slides and blocks are stored at ambient temperature in ‘open’ boxes within or near the Histopathology Lab.

This allows for easy access to allow technical staff to cut extra sections for special techniques or to send out of the Department.

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Light Microscopy

Kohler Illumination Condenser Objectives (2x to 65x) 2 to 4x - Low power 100x lens – Oil

Immersion Final magnification =

eye piece of 10x and objective of 40x = 400 times magnification.

eg. Photomicrograph of H&E of Normal Skin (x 20 objective)

Keratin

Granular Cell layer

] Suprabasal Cell Layer

Basal Cell Layer

Papillary Dermis

Reticular Dermis

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Some Special Diagnostic Techniques

Frozen sections

Immunohistochemistry

Polarised light microscopy

Fluorescence microscopy

Electronmicroscopy

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Frozen Sections: Freezing acts as

embedding agent by forming minute ice crystals within cells.

More rapid (5min)

Liquid nitrogen.Freezing Microtome

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Immunohistochemistry Antigen/antibody

reaction Antibody tagged

with a visual marker Simple Dye Enzyme (peroxidase) Fluorescent Dye Radioactive Dye

Marker

Sec. Antibody

Pri. Antibody

Tissue Antigen

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eg. Melanoma +ve for HMB-45

(brown staining in cell cytoplasm)

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eg. B cell Lymphoma – CD20

(brown staining of cell membrane)

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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.

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eg. Cardiac Amyloidosis

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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

eg. ANA – Nucleolar Pattern

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Electron Microscopy

Electron beam instead of light. Magnified images are typically

from 1000X to 50,000X. (Light microscope is 10-1000x).

Gluteraldehyde fixative. Glass knives. Specimen is mounted on a metal

grid.

Eg. Kidney – Membranous Glomerulonephritis