ROUTINE HISTOTECHNIQUE

PART I

SPECIMEN HANDLING AND IDENTIFICATION

• Each laboratory has its own way of specimen

identification.

> Giving the tissue a unique accession number

> May include the year and month the specimen was

received.

• If multiple specimens are received on the same patient

from the same operation/procedure --- the specimen

may be given the same number followed by a

numerical or alphabetical designation.

SPECIMEN HANDLING AND

IDENTIFICATION

Bar codes are frequently used by clinical

laboratories.

The specimen container label and the

accompanying request form should include:

a. Patient’s name

b. Age or birth date

c. A medical record number

Label should be firmly attached to the body of

the container --- not to the lid of the container.

SPECIMEN HANDLING AND

IDENTIFICATION • The request form should have a provisional diagnosis

and brief clinical details.

• Any discrepancies in specimen identification or labeling

should be resolved prior to processing.

• Incorrect identification of any specimen of any specimen

results in the wrong diagnoses and incorrect treatment.

GROSSING Major components in grossing a specimen:

1. Reliable and rapid transfer of the specimen from surgery to pathology

2. Accurate identification of the specimen

3. Description of additional specimens received from the same patient

4. Gross description of the specimen’s normal and abnormal features

GROSSING Major components in grossing a specimen:

5. Recording the sites from which blocks of tissue

are taken

6. Recording markers that help with the correct

orientation

7. Identifying special studies requested and/or

needed.

SPECIMEN FROM DERMATOLOGY

@CORE BIOPSIES Larger core biopsies (4mm) --- should be bisected eccentrically and

embedded with cut surfaces down.

Small core biopsies (2mm) --- should be embedded totally without

cutting it.

@SHAVE BIOPSIES OF SKIN Depending upon the size of the biopsy, it may be bisected, trisected,

or cut into sections.

Excision biopsy

SPECIMEN FROM DERMATOLOGY @SHAVE BIOPSIES OF SKIN Most specimens of skin or other epithelial surfaces should be cut ---

all aliquots are embedded on edge.

Care should be taken with any pigmented lesions of the skin.

@EXCISIONAL BIOPSY Method of choice for surgical removal of melanomas but may be

sometimes removed by shaving

Shave skin biopsy

Excision biopsy

SPECIMEN FROM DERMATOLOGY

@ EXCISIONAL BIOPSY Biopsies of skin are examined to ensure that the lesion has been

completely removed and the original clinician’s diagnosis was correct.

Can be oriented using sutures or dyes.

@ RE-EXCISION SPECIMENS Original site of a lesion may need to be re-excised if:

> The margins are invaded by tumor

> Too close to the tumor --- melanoma or basal cell carcinoma.

NON-SKIN SPECIMEN

• Excisional biopsies

• Operative specimens --- tumors, unidentifiable

inflammatory masses, tissues removed prior to

transplantation, traumatic, congenital

malformations, or cosmetic surgical specimens.

• All specimens must be examined carefully ---

may harbor unsuspected malignant tumors

NON-SKIN SPECIMEN

• Important determinants of neoplastic specimens:

> Overall size of the tumor

> Depth of invasion into or through the tissue walls

> Involvement of margins and lymph nodes

METHODS OF FRESH TISSUE EXAMINATION

1. TEASING or DISSOCIATION

• A process whereby a selected tissue specimen is

immersed in a watch glass containing isotonic salt

solution, carefully dissected or separated, and examined

under the microscope.

2. SQUASH PREPARATION

• Crushing

• A process whereby small pieces of tissue not more than 1

mm in diameter are placed in a microscopic slide and

forcibly compressed with another slide or with a cover

glass.

METHODS OF FRESH TISSUE EXAMINATION

3. SMEAR PREPARATION • The process of examining

sections or sediments, whereby

cellular materials are spread

lightly over a slide by a wire loop

or applicator, or by making an

apposition smear with another

slide.

• Useful in cytological examinations

--- particularly for cancer

diagnosis

METHODS OF FRESH TISSUE EXAMINATION

a. STREAKING – with an applicator stick or platinum

loop, the material is rapidly and gently applied in a

direct or zigzag line throughout the slide, attempting to

obtain a relatively uniform distribution of secretion.

b. SPREADING – a selected portion of the material is

transferred to a clean slide and gently spread into a

moderately thick film by teasing the mucous strands

apart with an applicator stick

METHODS OF FRESH TISSUE EXAMINATION

b. SPREADING

• Little more tedious but with an advantage of

maintaining cellular interrelationships of the materials

to be examined.

• Recommended for smears preparations of fresh

sputum and bronchial aspirates and for thick mucoid

secretions.

METHODS OF FRESH TISSUE EXAMINATION

c. PULL-APART – done by placing a drop of secretion or

sediment upon one slide and facing it to another clean

slide.

• The material disperses evenly over the surface of two

slides.

• Slight movement of the two slides in opposite

directions may be necessary to initiate the flow of

materials.

METHODS OF FRESH TISSUE EXAMINATION

c. PULL-APART

• The two slides are then pulled apart with a single

uninterrupted motion, and the specimen placed under

microscope for immediate examination, or applied with

vital stains.

• Useful for preparing smears of thick secretions such as

serous fluid, concentrated sputum, enzymatic lavage

samples from GIT, and blood smear

METHODS OF FRESH TISSUE EXAMINATION

d. TOUCH PREPARATION – Impression smear

• A special method of smear preparation whereby the

surface of a freshly cut piece of tissue is brought into

contact and pressed on to the surface of a clean glass

slide, allowing the cells to be transferred directly to the

slide for examination.

• Advantage: Cells may be examined without destroying

their actual intercellular relationship

METHODS OF FRESH TISSUE EXAMINATION

4. FROZEN SECTION – is normally utilized when a rapid

diagnosis of the tissue in question is required.

• Especially recommended when lipids and nervous tissue

elements are to be demonstrated

METHODS OF FRESH TISSUE EXAMINATION

4. FROZEN SECTION • Applications:

a. Rapid pathologic diagnosis during surgery

b. Diagnostic and research enzyme histochemistry

c. Diagnostic and research demonstration of soluble

substances such as lipids and carbohydrates

d. Immunofluorescent and immunohistochemical staining

e. Some specialized silver stains --- particularly in

neuropathology

METHODS OF FRESH TISSUE EXAMINATION

4. FROZEN SECTION • LIQUID NITROGEN – generally used in

histochemistry and during operative procedures

> Most rapid of the commonly available freezing agents

> DISADVANTAGE:

* Soft tissue is liable to crack due to rapid expansion of the ice within the tissue --- producing ice crystals or freeze artifacts

METHODS OF FRESH TISSUE EXAMINATION

4. FROZEN SECTION • PROBLEM: Use of liquid nitrogen can cause a vapor

phase to form around the tissue --- acting as an

insulator that causes uneven cooling of tissue

(particularly in muscle biopsies) and making diagnostic

interpretation difficult.

> Can be overcome by freezing the tissue in

Isopentane, OCT, or Freon 2.2 --- has a high thermal

conductivity.

HISTOTECHNOLOGY

• Is the art and science performed by the

histotechnologist to produce a tissue section of good

quality that will enable the pathologist to diagnose the

presence or absence of disease.

• FIXATION – first and most critical step in

histotechnology

> Fixing or preserving fresh tissue for examination.

> The quality of the section on the slide is only as

good as the quality of the fixed tissue specimen.

FIXATION

• FIXATION – the process by

which the constituents of the

cells and tissues are fixed in a

physical, and partly in a

chemical state, so that they will

withstand subsequent treatment

with various reagents with a

minimum of loss, significant

distortion, or decomposition.

FIXATION

• Inadequate or poor fixation will result in a poorly processed tissue and will make it difficult for the pathologist to render a proper diagnosis.

• With fixation, the shape, structure, intercellular relationship and chemical constituents of tissues are preserved.

• Fixation prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body.

FIXATION

• To Preserve the tissue --- by stopping all cellular activities so that cells can be viewed under the microscope as if they are still in their original living state.

> Do not leave the tissue specimen in air for prolonged period of time --- dry out and will result in distortion of its morphologic appearance

> Leaving the tissue in water ---cause the cell to swell while a strong salt --- cause the cell to shrink.

FIXATION

1. ADDITIVE FIXATION

• The chemical constituent of the fixative is taken in

and becomes part of the tissue by forming cross-

links or molecular complexes and giving stability

to protein.

E.g. Formalin Osmium tetroxide

Mercury

FIXATION

2. NON-ADDITIVE FIXATION

• The fixing agent is not incorporated into the

tissue, but alters the tissue composition and

stabilizes the tissue by removing the bound water

attached to H-bonds.

E.g. Alcoholic fixatives

FIXATION

1. Hydrogen Ion Concentration

2. 2. Temperature

FIXATION

3. Thickness of section

4. Osmolality

FIXATION

5. Concentration

• Low concentrations of glutaraldehyde have

been found to be an ideal concentration for

immuno-electron microscopy.

6. Duration of fixation

• Buffered formalin – 2 to 6 hours during the day

the specimen is obtained

FIXATION

6. Duration of fixation

• Prolonged fixation:

* May cause shrinkage and hardening of tissue

* May severely inhibit enzyme activity and immunological reactions

• For electron microscopy, tissues should be fixed for 3 hours and then placed in holding buffer.

FIXATION

@ SPEED: Specimen should be placed in fixative as

soon as it is removed from the body.

* Done to prevent autolysis and putrefaction

@ PENETRATION: Time of fixation varies with

different types of tissue

* Formalin diffuses into the tissue at the rate of

1mm per hour.

FIXATION

@ VOLUME: The amount of fixative used has been 10

– 25 times the volume of tissue to be fixed

* Maximum effectiveness of fixation: 20x the

tissue volume

@DURATION OF FIXATION: Some tissues take longer

to fix than others, depending on their structure.

* Fixation time can be cut down by using heat,

vacuum, agitation or microwave

FIXATION

• Tissue selected for sectioning should be thin

enough to allow penetration by fixative within a

reasonable amount of time.

• To maintain an adequate fixation time of 4 – 6

hours, the recommended size of the tissue is 2

square cm, and no more than 4 mm thick.

• Most tissue can be cut and trimmed without prior

fixation, EXCEPT for the brain.

FIXATIVES

• Effects of Fixatives in General:

a. Harden soft and friable tissues and make the handling

and cutting of sections easier.

* Usually accelerated by the action of alcohol during

dehydration process.

b. Make cells resistant to damage and distortion caused

by hypotonic and hypertonic solutions used during

tissue processing

c. Inhibit bacterial decomposition

FIXATIVES • Effects of Fixatives in General:

d. Increase the optical differentiation of cells and

tissue components thereby rendering them

more readily visible during examination.

e. Act as mordants or accentuators to promote

and hasten staining or inhibit certain dyes in

favor of another.

f. Reduce the risk of infections during handling

and actual processing.

FIXATIVES

• CHARACTERISTICS OF A GOOD FIXATIVE:

1. Must be cheap, stable and safe to handle.

2. Must kill the cell quickly thereby producing

minimum distortion of cell constituents

3. Must inhibit bacterial decomposition and

autolysis

4. Must produce minimum shrinkage of tissue.

5. Must permit rapid and even penetration of

tissues.

FIXATIVES • CHARACTERISTICS OF A GOOD FIXATIVE: 6. Must harden tissues thereby making the cutting of sections

easier.

7. Must be isotonic --- causing minimal physical and chemical

alteration of the cells and their constituents.

8. Must make cellular components insoluble to hypotonic

solutions and render them insensitive to subsequent

processing.

9. Must permit the subsequent application of many staining

procedures to facilitate easier and more profitable

examinations.

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