Index

1.0 Introduction……………………………………………………………………………….3

2.0 What is
Histotechnology ?................................................... .................................................. ......4

2.1. History and evolution of Histotechnology……………………………………...4

2.2 Which samples are used?................................................ ..............................................5

2.3 Types of techniques used in this process and describe them…………………………7

2.4 Steps of histological techniques and describing

them………………………………………8

2.5 Methods by which samples are obtained…………………………………….10

3.0 Conclusion………………………………………………………………………………11

4.0 Bibliography………………………………………………………………………………...12

5.0 Annexes………………………………………………………………………………..13

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 Introduction

The object of study of histology is tissues (and the cells that compose them). In order
to study and understand them, you have two powerful tools that allow you to observe the
cellular and tissue microstructure: microscopy and the histological technique.

The histological technique is the series of ordered steps that allow tissue to be
prepared for observation through the microscope. The tissue is prepared for observation
according to the type of microscope that will be used.

The basic objectives of histotechnology are:

a. Produce sufficiently thin sections with the best possible morphological preservation,
so that they can be observed with the maximum resolution of the microscopes.

b. Preserve the biological and chemical characteristics of cells and tissues so that they
can be studied using specialized methods.

c. Allow most of the cellular and tissue structures to be studied in a single section.

d. Know the correlation between the morphology and function of cellular and tissue
structures.

In the case of bright field microscopy, the most common technique for preparing
samples is the ordinary histological or paraffin embedding technique. In this process, the
samples are infiltrated in paraffin, so that they have the appropriate consistency to obtain the
blocks with the samples or specimens. Once the blocks (inclusion) are obtained, they are cut
using equipment called a microtome and with which very thin cuts (micrometers thick) are
obtained, allowing the cellular and tissue structures to be observed. A step further that offers
the most detailed information corresponds to the moment of applying the stain, which gives
colors to the sample and thanks to this it is possible to identify various structures by
observing these preparations with the bright field microscope.

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 2.0 What is Histotechnology?

Histotechnology is the science that studies the technical foundations and the sequence
of manipulations necessary to carry out tissue analysis.

Histotechnologists work in laboratories where animal and plant tissues and organs are
observed with light microscopes (including human tissues).

Histotechnology is defined as the discipline that studies the scientific foundations and
the application of histological techniques, which ranges from obtaining the biological sample
to its transformation into a histological slide. The established procedures will allow the
preparation of the material to be observed under a microscope and analyze its topographic,
structural and dyeing characteristics, as appropriate.

2.1. History and evolution of Histotechnology

Data related to the study of human histology have been gathered since times before
Christ when the Greek Empedocles of Agrigento (495-430 BC. C.) philosopher and
politician, empirically described that the human body was made up of four elements; water,
air, earth and fire. Later Hippocrates of Cos (460 - 370 BC. C.) Ancient Greek doctor
considered the “father of medicine” postulated the theory of humors, which explained that the
body was composed of 4 humors (black humor, yellow humor, blood and bile) and that an
imbalance between them led to suffer from diseases so their treatments were aimed at
keeping them in balance. During all this time, science always had experimental behavior and
advances were developing slowly, until Andrés Vesalius (1514 – 1564), a Belgian anatomist,
began his studies in medicine and under the direction of Jacobus Sylvius and Jean Ferne he
reviewed the Galen's theories.

Vesalius, relying on his own observations, published a correction of Galen's Opera

omnia, and began writing his own anatomy text. Already in the microscopic stage In 1831
Robert Brown (1773-1857), Scottish physician, surgeon and botanist, published an article that
described an intracellular corpuscle which he called and assigned the term areola or nucleus
in eukaryotic cells, although Franz Bauer managed to describe it in 1804. briefly but did not
coin its demarcation. The postmicroscopic stage is characterized by the introduction of the
electron microscope into research. The electron microscope first created at the University of
Berlin, Germany in 1931 by the German physicist Erns Ruska (1906-1988) that used
electrons for image formation, allowing profiles to be achieved up to five thousand times
higher than those of best optical microscopes known up to that time. For his work in physics
and optical designs, including the electron microscope, he won a Nobel Prize in Physics in
1986. The evolution of this type of microscope meant an important advance for medicine

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(observation of parts of a cell, proteins, viruses, etc.) which led to the discovery of endless
microscopic structures. In 1949, the English cytologist and biochemist, Christian René de
Duve (1917-2013), discovered and investigated the physical functions of lysosomes and
peroxisomes, describing the process by which the action of lysosomes allows the introduction
of some substances inside. of the cell nucleus, this led him to win the Nobel Prize in
Physiology and Medicine in 1974. In 1973 George Emil Palade (1912 – 2008), a cellular
biologist born in Romania and naturalized in the United States, used the electron microscope
to continue studying the cell. He verified the presence of mitochondria, the Golgi apparatus,
and other cellular organelles, but he was also able to notice different structures, they were
microsomes formed by nucleic acids. The discovery of ribosomes is attributed to Palade and
for this reason he shared the Nobel Prize in Physiology or Medicine with Albert Claude and
Christian de Duve.

2.2 Which samples are used?

Fabrics can be obtained in different ways:

Biopsy :

Excisional biopsy - Incisional biopsy - Endoscopic biopsy - Colposcopic biopsy - Fine

needle aspiration puncture (FNAC) - Core needle puncture biopsy (Tru-cut)

Necropsy/autopsy (clinical or legal).

Fixation :

In this step, the tissue obtained is placed in a fixative substance, generally liquid, to
avoid post-mortem changes and to preserve the original shape of the tissue. One of the most
used fixatives is 10% formalin ( formaldehyde ). If we use the electron microscope later, we
will use it.

Washes:

The fabric must be washed to remove excess fixative (chemical). Excess fixative at
the time of infiltration, even in microtomy, could affect the histological sections , and
therefore should be washed with distilled water.

Dehydration is done using different alcohol solutions of increasing concentration.

Clearing:

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 In this step, the alcohol is replaced by a paraffin solvent. The most used is xylol (
xylene ) since as the sample is dehydrated, the xylol will enter the deepest part of the tissue.
The fabric also loses color and acquires a caramel tone.

Infiltration:

In this step the sample is placed in liquid paraffin, it is worth mentioning that
histological paraffin must be used. As said in the previous step, the tissue is completely filled
with xylol, now due to osmosis the xylol leaves and the paraffin enters.

Dehydration, clarification and infiltration can be carried out manually but today they are
carried out automatically in specific machines.

Inclusion:

Its purpose is to provide the tissue with a solid support that makes it possible to make
a very fine cut (3 to 5 microns), so it is of utmost importance that the medium used for
inclusion penetrates the interior of the tissue. Here paraffin blocks are formed within which
are the samples to be studied. There are also specialized machines for paraffin embedding of
tissues. After inclusion and subsequent drying, these must be cooled in a freezer for later
cutting. Can be carried out in Paraffin

Microtomy :

Very thin histological sections are made as required or customary in the laboratory
where the technique is performed. The cuts range from 0.5 microns to 8 or 10 microns. The
sections are placed in the flotation bath and 'fished' with a slide, marked with the date, the
type of tissue and the stain with which they will be processed. The cutting angle between the
microtome knife and the block must be between 10º and 15º. Once the cut is made, a distilled
water bath is given so that the paraffin stretches. A good histological section should have a
thickness of approximately 3-5 microns so that it can be easily penetrated by sunlight and
pass through the cell pores.

Staining:

The cells, by themselves, do not have coloration. Therefore, in order to observe tissue
morphology, they must be "stained." There are many types of stains to differentiate the
different structures or substances in tissues.

The most used or also called "routine" stain is hematoxylin and eosin (H&E). A dye
called hematoxylin is used that stains acidic substances or substances that contain them, such
as the nucleus that contains deoxyribonucleic acid ( DNA ). The yellowish eosin stains the
basic structures such as the cytoplasm and other eosinophilic organelles of the cell .

2.3 Types of techniques used in this process and describe them

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Obtaining the piece :

It consists of taking tissue samples from different organisms; A first condition is that
the tissue sample be taken from healthy, live and anesthetized individuals, this is achieved
with experimental animals; In the case of human tissues, samples are obtained from patients
undergoing surgery and biopsies, or from recently deceased corpses, taking care to section
only healthy organs and tissues. The separation of the organs should be done with scissors,
avoiding pressing and macerating the tissues; Once outside the body, the tissues are cut with
a fine scalpel or razor, without pressing; The pieces of tissue must measure 1 cm 3 thick but
must include all the elements of the organ.

The subsequent treatment of the tissue depends on the objective of the study; If you
wish to study living tissue, the sample is placed in culture media that allows the cells to be
kept alive during observation and subjected to vital staining, that is, using a dye that does not
cause damage to the cells such as blue. trypan; This dye derived from coal tar allows
macrophages to be identified in vivo because when mixed with water it forms a colloid,
which these cells phagocytose.

Fixation:

The study of morphology is more feasible in fixed cells that maintain their structure as
if they were alive; For this purpose, the tissues undergo the process of protein fixation or
insolubilization to suddenly stop vital cellular activity, stop the postmortem autolysis
processes, protect the cells from bacterial attack and putrefaction and prepare the tissues for
subsequent histological procedures. .

The methods used for fixation can be physical or chemical. The physical method
consists of rapid changes in temperature, with heat (not recommended because it produces
denaturation of proteins) and by immediate freezing by immersing the tissue in liquid
nitrogen; This produces hardening of the tissue leaving it ready to be cut (microtomy) in a
special device called a freezing microtome which may be mounted on a cryostat, a device
capable of maintaining the environment at a low temperature.

Chemical methods consist of exposing the tissue to chemical agents to cause the
formation of cross-linking bridges between adjacent tissue protein molecules. The most used
chemical agents in optical microscopy are simple or pure compounds such as 5%
formaldehyde or 10% formaldehyde or mixtures of compounds such as Bouin's liquid (picric
acid, formalin and glacial acetic acid) or Helly's liquid ( potassium dichromate, mercuric
chloride and formalin). In electron microscopy, the most commonly used fixative is
glutaraldehyde and osmium tetraoxide.

Two types of techniques are used for chemical fixation: the perfusion technique and
the immersion technique. In the first, the fixative is placed in a syringe and injected into the
animal's bloodstream so that it is distributed throughout the body; Once fixed we can dissect
it to obtain the organ of interest.

Dehydration:

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 The purpose of this process is to remove water from tissues by subjecting them to a
gradual concentration of aqueous solutions of a dehydrating agent, for example, ethyl alcohol
or acetone. It starts with concentrations of 50%, then 60, 70, 80, 90% to gradually reach
100% alcohol concentration. Immediately changing the tissue from water to a 100% alcohol
solution produces a rapid release of water which deforms the tissues, therefore it is necessary
to keep the tissue blocks for at least an hour in the different increasing concentrations of
alcohol. .

Clarification or diaphragm:

Since paraffin is not capable of mixing with alcohol, clarification is an intermediate

procedure that aims to eliminate alcohol from the tissues to saturate them with a substance
miscible with both alcohol and the paraffin solvent. The substance most frequently used is
xylene or xylol. The tissue sample is placed in a container with equal parts alcohol-xylol,
then in absolute xylol until the tissue becomes clear or transparent. Toluene, benzene or
chloroform can also be used as lightening agents.

2.4 Steps of histological techniques and describe them

Once the sample you want to study has been acquired, you must immediately proceed
to the next and crucial step: fixation.

Fixation: Fixation of a sample can be carried out by immersion or perfusion.

Immersion occurs when the sample is immersed in the fixative and it gradually penetrates
from the periphery to the center of the sample, while perfusion is promoted when the fixative
is injected into the body's bloodstream and, therefore, is distributed and fixes the tissues
according to the flow of circulation. In fixation, substances called fixatives are used; The
most commonly used fixative is formalin or 10 percent formalin.

In order for a sample to fix adequately, it must be soaked in the fixative in small
pieces (no larger than 1 cm 3 ); For each cubic centimeter of sample, 10 cm 3 of fixative must
be placed. The ideal time for formalin to penetrate the tissues is 24 to 48 hours. Once the
sample is fixed, you can proceed to the next step.

Dehydration: Once the sample is fixed, it is necessary to consider that the cells have
water inside, because it is an abundant tissue component. The samples are dehydrated with
alcohol in increasing concentrations. The water gradually leaves the tissues and, at the end of
this step, the water has been replaced with alcohol.

Clarification or difanization : Alcohol must be replaced with a lightening agent

(xylene, toluene or benzene), because paraffin is not miscible in alcohol, but it is miscible in
organic solvents such as lightening agents. In this step, the sample also acquires a certain
transparency.

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 Infiltration and inclusion : They are two simultaneous steps in which liquid paraffin is
used. Infiltration occurs when paraffin penetrates the interior of cells and tissue structures;
Embedding refers to the moment in which the tissue is embedded in paraffin, to form a block.

Cut or microtomy : Once the paraffin has solidified and has the right consistency,
then the tissue is ready to be cut. The tissue slices that must be obtained so that the sample
allows the passage of light from the microscope's optical system must have an average
thickness of 5-10 micrometers. The cuts are obtained with the microtome, which is the
instrument that allows us to obtain these very thin slices.

Deparaffinization : The sections must be deparaffinized, since the dyes are not
miscible in the paraffin and it would be impossible to dye them, so it is necessary to carry out
the reverse process: lightening agents are used to replace the paraffin. Such agents also confer
transparency to the cut.

Rehydration : Now in the cut the spaces are occupied by lightening agents, which
must be replaced with alcohol and at the end with water. In this process, the cut is subjected
to rehydration by immersion in alcohol in decreasing concentration.

Staining : The cuts, being so thin and having gone through the lightening agent, are
completely transparent. In order to differentiate structures, they are stained to give them
contrast. Sections can be stained with monochromic, dichromic or trichromic stains. This
section will be expanded later.

Mounting : Once the cut has been dyed, it must be protected to preserve it. Synthetic
resin (transparent) and a coverslip are placed on each section.

2.5 Methods by which samples are obtained.

Strictly speaking, this step is not considered within the histological technique; however,
any sample to be processed must first be obtained. In this section it is worth mentioning some
important concepts related to obtaining samples:

 Biopsy. The sample is obtained from a living individual.

 Necropsy. The sample is obtained from a corpse.

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 Incisional biopsy. A section of the lesion is obtained.

 Excisional biopsy. The entire lesion is removed.

 Types of biopsies. Depending on the type of tissue that needs to be obtained, it is the
appropriate type of biopsy. Consider some examples:

o Fine needle puncture and aspiration (FNAC). Liquid tissues such as blood are
obtained by this method.

o Core needle puncture and aspiration (PAAG). Red bone marrow, being a more
viscous tissue than blood, is obtained with a larger gauge needle.

o Exfoliative cytology. The cells that can be detached from the epithelia (such as
those from the endocervix and exocervix), from the oral cavity or from a
lesion, are obtained from a scraping or brushing.

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 3.0 Conclusion

Histotechnology is the science that studies the technical foundations and the sequence
of manipulations necessary to carry out the analysis of the tissues of living beings. The
objective of this is to know and develop staining, coloring and reaction, histochemistry and
immunochemistry methodologies, which allow the study and analysis of normal and
pathological tissues.

Histotechnology can be classified according to the type of sample or according to the

type of study. Depending on the type of study, they can be vital, supravital and postvital. A
histological sample is any tissue sample obtained from a healthy individual or animal with the
purpose of investigating its normal structure and composition.

Anatomopathological samples, on the other hand, correspond to those samples from

sick individuals, with the purpose of obtaining a diagnosis or investigating the origin of the
disease. These can come from biopsies, necropsies, cytological smears, experimental
pathology. An example of them is the biopsy, which is a sample of experimental tissue,
obtained from a living individual for anatomopathological study. Necropsy is, on the
contrary, studying dead tissues. In this way, there are samples for different histological
studies.

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 4.0 Bibliography

1. https://pacal.org/n/Datos/documentos/Pricipios%20histotecnologia%20aplicada.pdf

2. https://accessmedicina.mhmedical.com/content.aspx?
sectionid=150299454&bookid=1995&Resultclick=2

3. https://accessmedicina.mhmedical.com/content.aspx?
bookid=1502&sectionid=94733160
4. https://accessmedicina.mhmedical.com/content.aspx?
bookid=1995&sectionid=150299454#1138470829

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 5.0 Annexes

Brain and brainstem section .

- A. Piece of tissue in the fixator. B. Isolation of parts in

capsules.

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In A, a section of the trachea wall is observed, in which different tissues are identified:
connective tissue (▼), mucosal adenomers (♦), serous adenomers (→) and hyaline
cartilage (*). Smooth muscle is seen in B. Both sections are stained with hematoxylin-
eosin (HE). Photographs courtesy of Armando Zepeda and Francisco Pasos.

A PAS-stained liver section is shown in A. Within the hepatocytes,

magenta glycogen inclusions are observed. In B, a section of the
sublingual gland stained with Mayer's mucicarmine is observed, in
which it is possible to see the pink mucosal adenomers.

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