FRESH TISSUE EXAMINATION

Learning Objectives:

1. To be able to differentiate histotechnologyand histotechnologist.2. To be able to differentiate the advantages and disadvantages of fresh tissue examination.3. To be able to know the basis for method used in fresh tissue examination.4. To be able to name and differentiate the methods of fresh tissue examination.5. To be able to understand the fresh frozen method.

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Chapter 1:FRESH TISSUE EXAMINATION

Histotechnology - is the art and science performed bythe histotechnologist to produce a tissue section of goodquality that will enable the pathologist to diagnose the presenceof disease.

The tissue may be done fresh or preserved.

Advantage of fresh tissue examination: The specimenmay be in living state, therefore may observe protoplasmicactivities (motility, mitosis, phagocytosis, pinocytosis).

Disadvantage: not permanent, and therefore liable to changes

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Chapter 1:FRESH TISSUE EXAMINATION

The choice of tissue examination method would dependon the following conditions;

1. Cell Structure and chemical components

2. Amount and nature of tissue

3. Urgency of result

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Frozen tissue – 10 -15 u in thickness cut from frozen tissue on a microtome with CO2 or on cryostat.

A cryostat is cold chamber kept at an atmospheric temperature of -10 to -20 deg. C. The frozen

sections are then transferred to a slide, and examined under light microscopy.

Sequence of fresh frozen tissue examination by cryostat

a b c d e

The tissue should be fresh and processed quickly. Slow freezing can cause tissue distortion due to

ice artifacts. The more commonly used methods of freezing include;

1. Liquid nitrogen

2. Isopentane cooled by liquid nitrogen

3. Carbon dioxide gas

4. Aerosol sprays

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Chapter 2: Fixation and Fixatives

Learning objectives:

1. To understand the process of autolysis

2. To determine the contributing factors in autolysis.

3. To discuss why certain tissues are affected severely by autolysis.

4. To know the objectives of fixation and qualities to serve its objective.

5. To understand the factors involve in fixation.

6. To know the types of fixative.

7. To understand the properties of formaldehyde.

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Fixation process

• Fixation is usually the

first stage in a multistep

process to prepare a

sample of biological

material for microscopy

or other analysis.

• Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned.

• For example, immunohistochemistry utilizes antibodies which bind to a specific protein target.

• Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalin for around 24 hours is typically used.

Chapter 2:Fixation and Fixatives

Once tissues are removed from the body, they undergo a process of self-destruction or autolysis. Soon after tissue death, the intracellular enzymes break down the protein and then the cell eventually undergo liquefaction. – Autolysis.

Properties of Autolysis:

1. independent of a bacterial action

2. retarded by cold

3. accelerated at 30 degree C temperature, inhibited at 50 degrees Celsius

4. more severe in tissue which are rich in enzymes (e.g. liver, brain, and kidney) and less rapid in elastic and collagen tissues.

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Types of fixationThere are generally three types of fixation process:

1. Heat fixation:

After a smear has been allowed to

dry at room temperature, the slide is

gripped by tongs or a clothespin and

passed through the flame of a Bunsen

burner several times to heat-kill and

adhere the organism to the slide

2. Perfusion:Fixation via blood flow. The fixative is injected into the heart with the injection volume matching cardiac output.

The fixative spreads through the entire body, and the tissue doesn't die until it is fixed.

This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms)

3. Immersion:

The sample of tissue is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed.

The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account.

• Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine.

• Its effects are reversible by excess water and it avoids formalin pigmentation. Other benefits include: Long term storage and good tissue penetration.

• It is particularly good for immunohistochemistry techniques.

• Also the formaldehyde vapour can be used as a fixatives for cell smears.

.• HOPE Fixative

Hepes-glutamic acid buffer-mediated

organic solvent protection effect (HOPE)

gives formalin-like morphology,

excellent preservation of protein

antigens for immunohistochemistry

and enzyme histochemistry, good RNA

and DNA yields.

COMPOUND FIXATIVES

1. Formalaldehyde fixatives

• 10% formal saline

• Formalin 100 ml

• Sodium chloride 8.5 grams

• Tap water 900 ml

2. Buffered 10% formalin

Formalin 100 ml

Acid sodium phosphate monohydrate 4 g

Anhydrous disodium phosphate 6.5 g

Tap water 900 ml

ALCOHOLIC FIXATIVES

1. Carnoy’s fixatives

Absolute alcohol 60 ml

Chloroform 30 ml

Glacial acetic acid 10 ml

2. Picric acid fixatives

Bouin’s fluid

Rossman’s fluid

Gendre’s fluid

3. Mercuric chloride containing fixatives

Formal sublimate

Zenker’s solution

Susa fluid

Helly’s fluid