Unit 2.1. Fixation

Fixation

• first, most important & most crucial step

• killing, penetration, hardening of tissues

• alteration of tissues by stabilizing protein

• stopping all cell activities so that cells can be viewed under the microscope

stabilization of proteins

most important reaction in maintaining morphology of tissues

preserve the morphological and chemical integrity of the cells in as life like manner as possible

Primary aim of fixation

To harden and protect tissue from the trauma of further handling, so it is easier to cut and process for microscopy.

Secondary aim of fixation

Autolysis

______________ results from tissue digestion by intracellular enzymes releases when organelle membrane ruptures.

postmortem decomposition

Putrefaction

• bacterial decomposition or fungal colonization

• brought abt by microorganisms

1. To preserve the tissue

2. To prevent breakdown of cellular elements

3. To coagulate or precipitate protoplasmic substances

3 objectives of fixation

1. Physical Fixation

2. Chemical Fixation

2 MAJOR FIXATION METHODS

Physical FIxation

2 MAJOR FIXATION METHODS

• heating, microwaving, and cryo-preservation (freeze drying)

Heat Fixation

3 TYPES OF PHYSICAL FIXATION:

• simplest from

• each component is less soluble in water after fixation

• used for microbiology laboratory for bacterial smears

Microwave Fixation

3 TYPES OF PHYSICAL FIXATION:

• speeds fixation and can reduce times for fixation of some gross specimens and histological sections for more than 12 hrs to less than 20 mins

• not recommended for histopathology

• neurochemical substance in the brain (acetylcholine)

commercial glyoxal-based fixative which do not form vapors when

heated at 55°C

3 TYPES OF PHYSICAL FIXATION:

• efficient methods of microwave fixation

Cryo-preservation

• quenching ; -160 to -180°C

• desiccation

• sublimation ; -30 to -40°C

3 TYPES OF PHYSICAL FIXATION:

• “Freeze drying”

• special way of preserving tissues by rapid freezing (___________) of fresh tissue at _______

• and subsequently removed by ice water (___________) by a physical process of transferring the still frozen tissue block in a vacuum at a higger temperature of _________ (____________)

Freeze substitution

• Rossman’s formula

• absolute alcohol

3 TYPES OF PHYSICAL FIXATION:

• similar to freeze drying

• instead of fixing in an expensive vacuum drying apparatus, frozen tissue is fixed in a _____________ and dehydrated in a _______________

2 mm thick

thickness of fresh tissue for cryopreservation

Chemical Fixation

• immersion fixation

• perfusion fixation

2 MAJOR FIXATION METHODS

• immersing the spx in the fixative solution = “________________”

• for small animals or large organs (lung), by perfusing or injecting the vascular system with fixative = “________________”

paraformaldehyde and osmium tetroxide

For some specialized histochemical procedures, fixatives have occasionally been applied in the vapor form. For example, ____________________________ can be used to vapor-fix freeze-dried tissues.

1. Additive Fixation

2. Non-Additive Fixation

2 BASIC MECHANISMS IN FIXATION:

Additive fixation

2 BASIC MECHANISMS IN FIXATION:

• whereby 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 the protein.

formalin

osmium tetroxide

mercury

Additive fixatives example

Non-additive Fixation

2 BASIC MECHANISMS IN FIXATION:

• whereby 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 of certain groups within the protein molecule

• Upon removal of water, new cross-links are established within and among the protein molecules that stabilize the intercellular components, prevent autolysis and make them unsuitable for bacterial decomposition

alcoholic, cutting acetone

Non-Additive fixatives example

1. Speed

2. Rate of Penetration

3. pH

4. Temperature

5. Thickness of Section

6. Osmolality

7. Concentration

8. Amount of Fixative/Volume

9. Duration of Fixation

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

Speed (<1 hr)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• immediately fix tissues after removal

1 mm/hr

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• rate of penetration for formalin

neutral (6-8 pH)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• pH

room temperature (40°C)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Temperature for routine tissue processing

0-4°C

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Temperature for EM and histochemistry

formalin heated at 60°C

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Temperature for urgent biopsies/rapid fixation

40-42°C

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Temperature for automatic tissue processor

formalin heated at 100°C

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Temperature for tissues with tuberculosis

2 cm² and no more than 4 mm thick

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• recommended tissue size for routine processing

1-2 mm²

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• recommended tissue size for EM

2-4 mm

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• recommended tissue size for LM

2 cm²

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• recommended tissue size for lung spx

slightly hypertonic (400-450 mOsm)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• osmolality recommendation by the book

isotonic solution (340 mOsm)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• osmolality recommendation in practice

normal phosphate buffered saline (PBS)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• If the cells are fixed in a hypotonic solution, the cells may swell and burst.

• For that reason, we recommend using a ___________________________ based fixative.

10% formalin

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Concentration of formalin

• EM = 3%

• Immunohistochemistry = 0.25%

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• concentration of glutraldehyde in EM and ImmunoHCM

20:1 (20 x volume of tissue)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• amount of fixative for routine tissue processing

5-10:1

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• amount of fixative for osmium tetroxide (quite expensve)

20:1

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• amount of fixative for EM

not less than 50:1

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• amount of fixative for museum preparation

4-6 hrs

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• adequate fixation time

• fixed ASAP

• if not:

  • place in mortuary ref (4°C)

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• autopsy materials

• taped w cotton soaked in fixative or

• cut open before fixing

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• hollow organs (stomach, intestine)

• cover w several layers of gauze

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• air-filled lungs (may float)

• suspended whole in 10% NBF for 2-3 weeks

• undergo intravascular perfusion

• wash the blood with Ringer’s lactate

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Human brain

• undergo Lendrum’s method

  • 4% phenol immersion for 1-3 days

  • grossing/sectioning tissues before fixation

9 FACTORS INVOLVED IN FIXATION/PRACTICAL CONSIDERATIONS:

• Hard tissues (cervix, uterine, fibroid, etc)

1. Harden Tissues

2. Make cells resistant to damage

3. Increase optical differentiation to cells, acts as a mordant

3 General Effects of Fixatives

1. Need for immediate examination (urgency)

2. Type of spx

3. Tissue structure

4. Staining technique

4 Factors to Consider when choosing the right fixative

Zenker’s fluid

best fixative for liver/spleen

Picric acid fixative (Brasil’s fluid)

best fixative for glycogen structures

H&E staining

most compatible staining technique with 10% NBF

alcohol & cutting acetone

GENERAL RULE: All fixatives that are available are “additive fixative” except _________________

washing-out

• process of removing excess fixative from tissue after fixation to improve staining and to remove artifacts

Tap water

WASHING-OUT:

• removes excess formalin

• removes excess osmic acid

• remove excess chromates (Kelly’s, Zenker’s, and Flemming’s solutions)

• commonly used in practice

50-70% alchohol

WASHING-OUT:

• removes excess picric acid (Bouin’s solution)

alcoholic iodine

WASHING-OUT:

• removes excess merccuric chloride

Secondary Fixation/Post-mordanting

• process of placing an already fixed tissue in a 2nd fixative in order to

  • facilitate and improve the demonstration of a particular substance

  • make special staining technique possible

  • Ensure complete and further hardening and preservation of tissue

• not mandatory

Post-Chromatization

• a form of secondary fixation which uses a 2.5-3% potassium dichromate for 24 hrs to act as a mordant for better staining effects and to aid in cytologic preservation of tissues