Fixation of tissues and organs for educational and scientific purposes

1. Fixation of tissues and organs for educational
and scientific purposes

2. 1. Goal of fixation
• • The primary goal of fixation to preserve tissues for scientific and educational
purposes. The tissue is being fixed by fixation is more in more natural, or "life-
like" condition
• On this slide, left we see the image of the kidney stained during the pathological
treatment of the patient, and the right picture of the kidney after fixation is
done for the purpose of education. It is macroscopically visible that there has
not been a significant change in the volume and tissue morphology, the only
change in color resulting from chemical reactions occurring during the fixation
process itself
• Also, apart from the possibility of fixation of certain organs, the fixation is used
for whole bodies, that is, for the fixation of cadavers (donated for scientific
purposes) used for educational purposes for students. By fixation, the donated
body can be preserved for a longer period of time, thus enabling our students
to have the highest quality education possible. The pictures show parts of a
cadaver who went through fixation with specially isolated sections and
structures for student education
• Tissue fixing agents act by stopping enzymatic degradation - autolysis - caused
by cellular enzymes and tratophage - caused by enzymes of bacteria
• The fixation mechanism (the most common) is the process of irreversible cross-
linking of the amino group of an amino acid with the nitrogen of the adjacent
amino acid using the -CH2- group, METHYLENE BRIDGE.
• The consequence of cross-linking the amino acids is the hardness of the tissue
to be fixed

3. 1. Fixing agents
•
There are four main groups of fixatives: ALDEHYDE (formaldehyde,
glutaraldehyde), OXIDIFIED AGENTS (OXYGETHYDEXIDE, POTASSIUM
PERGANANE), ALCOHOLIC FIBERS (methyl alcohol, ethyl alcohol) and FIXED
BASE METALS (mercury chloride, picric acid)
•
Aldehydes are the most widely used fixatives in anatomic studies, based on
formaldehyde
•
Fixation is always a physical and chemical process, as besides chemical
reactions, it involves the process of diffusion or perfusion of tissue fixation.
Diffusion means suspending the fixative preparation, whereby it slowly and
gradually penetrates into the tissue, while perfusion involves the injection of
the fixative directly into the tissue, and is usually used when the preparation is
voluminous and the diffusion is hampered. It is also important to note that
already fixed tissue can act as a physical barrier for further fixation or
penetration of further fixation into the tissue
•
FORMALDEHID is a low molecular weight compound, in standard conditions it
is in a gaseous aggregate state and is very well dissolved in water.
•
FORMALIN, consisting of 37 - 40% formaldehyde and 60 - 63% water. In
formalin, most of the formaldehyde molecules exist in the form of water-
soluble shorter polymers, and only a small part is made of polymers that are
very poor or water-soluble (paraformaldehyde)
•
Ideal fixatives contain as many monomeric formaldehyde as possible because
they can only successfully react with proteins. The dilution of water-based
fixation causes depolymerization of formaldehyde within a few days. If, instead
of water, a buffer solution with physiological pH values is used instead of water,
the depolymerization is almost instantaneous because in slightly alkaline
waste, the hydroxide ions catalyze the hydrolysis of the polymer
•
In formalin, the formaldehyde molecules are hydrated and methane diol
(methylene glycol) is formed. Molecules of methylene glycol if they are long
then spontaneously form polymers (polyoxymethylene glycol). In neutral to
slightly alkaline conditions (approximately physiological pH) depolymerization

4. of polyoxymethylene glycol takes place, and the free methylene glycol molecule
is then dehydrated to carbonyl formaldehyde. And the hydrated and
dehydrated forms possess the fixing properties. During the fixation process,
formaldehyde penetrates into the tissue in the form of methylene glycol and
only afterwards, due to the shift in the balance of the reaction, the methylene
glycol is converted to formaldehyde
•
The described reactions take place only in the neutral to slightly alkaline
medium, so it is important to ensure that the tissue that we physically meets
meets this criterion. One way is to thoroughly remove blood from the
preparation or cadaver as the presence of deoxygenated blood makes the free
formaldehyde molecules ineffective. This is because acidic conditions of
primary amine (NH2) react with present H + forming NH3 and can not react
with formaldehyde. • Formalin is preferably added to 10% methanol as
methanol interferes with polymerization and thus decelerates the formation of
paraformaldehyde. Moreover, commercially known paraformaldehyde almost
always contains 10% methanol. Therefore, such paraformaldehyde tissue is
fixed in two phases: first using methanol (alcohol fixation), resulting in
dehydration of the tissue surface, i.e. a certain level of tissue scavenging and
hardening; in the second phase, after sufficient diffusion of formaldehyde in
tissue, denaturation fission begins by cross-linking the amino acids in proteins
•
• If it stays in the air for a longer period of time, formalin is oxidized, causing
formic acid to form. Formalin contaminated formic acid has poorer fixing
properties
•
Formaldehyde is a carcinogenic (alkylating agent). It raises skin irritation,
•
conjunctiva, respiratory mucous membranes and severe headaches
•
Ideal fixation should have certain properties:
•
Do not harden tissue, organ or body
•
Provides perfect coloring of the tissue for analysis
•
Not to be toxic to humans and animals
•
Whether the price is acceptable

5. •
Ideal fixative DOES NOT exists
2. . Fixation procedure
• Exclusively use of formaldehyde does not produce good results. To
optimize the fixation of cadaver and individual organs, formaldehyde
should be combined with other agents. It is important to keep in mind
that certain protocols described cannot be universally used for the fixation
of cadavers and organs, but there are specialized protocols only for
cadavers, or only for isolated organs. Most formaldehyde fixants
significantly change the natural color of the tissue in a greyish-yellowish
shade. Several protocols have been developed to achieve satisfactory
preservation of the natural color of the tissue
• Below are examples of protocols that are used to preserve individual
isolated organs, that is, to fix the whole cadaver
• Kaiserling's protocol is used for the preservation of certain isolated
organs for the purpose of teaching. The advantage of this protocol is the
ability to preserve approximately the natural color of the tissue
• Preparation of solutions:
• Kaiserling I: 85 g potassium acetate, 45 g of potassium nitrate, 4800 mL
of formalin (3-4%)
• Kaiserling II: ethyl alcohol, 80-95%
• Kaiserling III: 200 g of potassium acetate, 300 mL glycerine, 900 mL of
tap water
• Protocol:
• Fix the body in the Kaiserling solution for one to five days, depending on the
size of the organ, or the thickness of the wall. In the case of a larger organ (for
example, a liver), it is recommended to inject a fixative directly into the

6. parenchyma. Fixation in Kaiserling and causes tissue decolorization. Then
transfer the organ to the Kaiserling II solution for several hours - in it, the
acidic hematin will cross into alkaline hematine, which approximates the color
of hemoglobin and gives the body a near natural color. The preparation
should be left in Kaiserling II until a satisfactory dye is achieved. If the
preparation is left in this solution for longer, there will be irreversible tissue
decoloration. The organ is stored in a Kaiserling III solution
• The standardized formula for the fixation of cadavers by Pretorius is a
customized formula according to Woodburn and Lawrence (1952), and serves
to prepare the body for dissection for teaching purposes. The formula has
proved empirically very successful because using this protocol does not
increase the fungus on the cadaver. The advantages of this protocol are low
cost and satisfactory durability of cadavers. The main disadvantage is the
smectic dyeing of the tissue
• Preparation of fixation:
- 96% ethanol
- Formalin (3-4%) 500 mL
- Plumbing water 750 ml
- 80% liquid phenol 3,5 L
• Protocol:
- The prepared fixative with the pump is injected into the radial artery at a
pressure of 1 to 1.5 kPa, until the full frame is completely filled. It takes about
25 L fixation for a 60 kg cadaver
• Maintenance:
- The welding can very quickly fail if it is improperly maintained and stored.
Proper storage involves closing a cadaver covered with a cloth dipped in a
humectant in a plastic bag and storing it in a cold room. Maintenance involves
regularly spraying a humectant while working on a cadaver.
• Preparation of humectant:
- Glycerin - 250 mL
- 80% liquid phenol - 250 mL
- Tap water - up to 1 L

7. Reference
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Aug; 33(8):845-53.
• Grizzle WE, Fredenburgh JL, Myers RB. Fixation of tissues. In: Bancroft JD, Gamble M, editors.
Theory and Practice of Histological Techniques. 6th ed. Philadelphia, USA: Elsevier; 2008. pp.
56–63.
• Kiernan JA. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are
and what they do. Microscopy Today. 2000;1:8–12.
• International Agency for Research on Cancer. IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, Volume 88 (2006): Formaldehyde, 2-Butoxyethanol and 1-
tertButoxypropan-2-ol.
• Brenner, E. (2014). Human body preservation–old and new techniques. Journal of anatomy,
224(3), 316-344.
• Pulvertaft, RJV. (1950). Museum techniques: a review. Journal of clinical pathology, 3(1), 1.
• Waters, BL. (2009). Museum techniques. Handbook of Autopsy Practice, 151-156.
• Pretorius, WF. (1997). Formula for embalming of cadavers for student dissection and the
modification thereof for plastination. J mt Soc Plastination, 9(2), 28-30.
• Thavarajah R, Mudimbaimannar VK, Elizabeth J, Rao UK, and Ranganathan K. Chemical and
physical basics of routine formaldehyde fixation. J Oral Maxillofac Pathol. 2012 Sep-Dec, 16(3):
400–405.