CHAPTER 3: EXAMINATION OF FRESH TISSUE
Histology is the microscopic study of the normal tissues of the body while histopathology is the
microscopic study of tissues affected by disease. The procedures adopted for the preparation of
material for such studies are known as histologic or histopathologic techniques. The tissues are
usually obtained during surgery, biopsy, or autopsy. They range from very large specimens or
whole organs to tiny fragments of tissue. The following surgical procedures are usually
performed to obtain the specific-types of tissue that are submitted to a histology laboratory for
processing:
1. Fine needle aspiration is the simplest, least invasive test and uses the smallest needle
to simply remove cells from the area of abnormality. This is not always adequate to
obtain a diagnosis, depending on the area to be biopsied.
2. A core needle biopsy removes not only cells, but also a small amount of the
surrounding tissue. This provides additional information to assist in the examination of
the lesion.
3. An incisional biopsy takes out even more surrounding tissue. It takes out some of the
abnormality, but not all. The doctor will slice into the lesion and remove only a portion of
it. If the lesion is found to be cancerous, further surgery may be needed to remove or
excise the entire lesion.
4. An excisional biopsy generally removes the entire area in question.
5. Punch biopsy is considered the primary technique for obtaining diagnostic full-thickness
skin specimens. It requires basic general surgical and suture-tying skills and is easy to
learn. The technique involves the use of a circular blade that is rotated down through the
epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4- mm cylindrical
core of tissue sample.
6. Shave biopsy - where small fragments of tissue are “shaved” from a surface (usually
skin).
7. Curettings - where tissue is scooped or spooned to remove tissue or growths from body
cavity such as endometrium or cervical canal.
Specimens are usually received in fixative (preservative) but sometimes they arrive fresh and
must be immediately fixed. Tissue specimens received in the surgical pathology laboratory
should have a request form that lists the patient specimens are accessioned by giving them a
number that will identify each specimen for each patient. It is important that specimens are
properly identified to minimize the risk of mislabeling.
Once tissues are removed from the body, their proteins and cells are digested and broken down
by their own enzymes, independent of a bacterial action. This process is known as autolysis,
which is retarded by cold and accelerated at room temperature. It is more severe in tissues that
are rich in enzymes (e.g. liver, brain, and kidney) and less rapid in elastic and collagen tissues.
Methods of tissue examination may vary according to the structural and chemical components
of the cells to be studied, and depends on the nature and amount of the tissue to be evaluated.
,Fresh tissues
- are usually examined when there is an immediate need for evaluation. On the other
hand, a better and more effective means of studying tissues, whether normal or
abnormal, is by examination of adequately preserved sections and smears that are
stained to demonstrate specific structures. The glass slides are then mounted with
coverslips for permanent keeping.
Examination may be done on fresh or preserved tissues, depending on necessity. Fresh tissues
have the advantage of being examined in the living state, thereby allowing protoplasmic
activities such as motion, mitosis, and phagocytosis to be observed. Its use is limited, however,
because of the fact that tissues examined in the fresh state are not permanent, and therefore,
are liable to develop the changes that have usually been observed after death.
Methods of Fresh Tissue Examination
1. Teasing or Dissociation
● is a process whereby a selected tissue specimen is immersed in isotonic salt solution
such as normal saline or Ringer’s solution in a petri dish or watch glass, carefully
dissected with a needle and separated by direct or zigzag spread using an applicator
stick.
● Selected pieces of the tissue are transferred carefully to a microscope slide and
mounted as a wet preparation underneath a cover glass, care being taken to examine
unstained by Phase Contrast or Bright Field microscopy.
● It has the advantage of permitting the cells to be examined in the living state. The use of
the phase contrast microscope greatly increases the structural detail of the cells
examined in the living state, allowing movement and mitotic division to be observed. The
application of certain stains such as methylene blue can be also of great value. The
preparations, however, are not permanent.
2. Squash Preparation (Crushing)
● is a process whereby small pieces of tissue (not more than one mm. in diameter) are
placed in a microscopic slide and forcibly compressed with another slide or with a cover
glass.
● If necessary, a supravital stain may be placed at the junction of the slide and the cover
glass, and allowed to be absorbed by the tissue through capillary attraction.
3. Smear Preparation
● The method of preparing the smear differs depending on the nature of the material to be
examined. As a general rule, smears are made either by spreading the selected portion
of the specimen over the surface of the slide with a platinum loop. Alternatively, an
apposition smear can be made using a second slide to obtain a relatively uniform
distribution of secretion. Too thin or too thick smears have to be avoided, since they
make the tissues less suitable for examination. Smears may be examined either as fresh
preparations similar to that described for teased preparations, or by using a supravital
, staining technique. Smear preparations can be made permanent by fixing them while still
wet, staining them to demonstrate specific structures and inclusions, and mounting the
cleared specimen beneath a cover glass with a suitable mounting medium. This is useful
for preparing smears of thick secretions such as serous fluids, concentrated sputum,
enzymatic lavage samples from the gastrointestinal tract, and blood smears. This
technique is especially useful in cytological examinations, particularly for cancer
diagnosis.
○ Streaking -With an applicator stick or a 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. Too thin or too thick smears have to
be avoided, since they make the tissues unsuitable for examination.
○ 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. It is a little more tedious than streaking, but has the
advantage of maintaining cellular interrelationships of the material to be
examined. It is especially recommended for smear preparations of fresh sputum
and bronchial aspirates, and also for thick mucoid secretions.
○ Pull-Apart – This is 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 the two slides. Slight movement of the two slides in opposite directions
may be necessary to initiate the flow of materials. The two slides are then pulled
apart with a single uninterrupted motion, and the specimen is placed under the
microscope for immediate examination, or applied with vital stains.
○ Touch Preparation (Impression Smear) – This is 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 by Phase Contrast microscopy
or staining for light microscopic study. It has an added advantage in that the cells
may be examined without destroying their intercellular relationship.
4. Frozen Section
At times during the performance of surgical procedures, it is necessary to get a rapid diagnosis
of a pathologic process. The surgeon may want to know if the margins of his resection are free
from tumor before closing. An unexpected disease process may be found that requires
immediate diagnosis so the surgeon can decide what to do next, or it may be necessary to
determine if the appropriate tissue has been obtained for further workup of a disease process.
Immediate diagnosis is accomplished through the use of a frozen section, especially in intra-
operative pathology to help the surgeon in choosing his next plan of action. It is especially
recommended when lipids and nervous tissue elements are to be demonstrated. Frozen
sections are usually done on muscle and nerve biopsies as well as on surgically removed
tumors.
, A fresh tissue (very thin slices: 10-15u) is frozen on a microtome with C02 or on a cryostat, a
cold chamber kept at an atmospheric temperature of -10° to -20°C. The thin frozen sections
are mounted on a glass slide, fixed immediately and briefly in liquid fixative, and stained using
similar staining techniques as in traditional wax embedded sections.
For histochemistry, cryostat sections give much faster results than paraffin sections. However,
the morphological detail and resolution of frozen sections are usually inferior compared to the
quality of tissue that has been embedded in paraffin.
The advantage of the frozen section method is rapid processing time with less equipment
requirement, and less need for ventilation in the laboratory. The disadvantage is the relatively
poor quality of the final slide.
APPLICATIONS: Frozen sections, both fixed and unfixed, have many applications in
histotechnology, and are commonly used for:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances such as lipids and
carbohydrates
4. Immunofluorescent and immunohistochemical staining
5. Some specialized silver stains, particularly in neuropathology
The tissue for freezing should be fresh, and freezing should be done as quickly as possible.
Slow freezing can cause distortion of tissue due to ice crystal artifacts.
RAPID FREEZE: slow freezing causes distortion due to ice crystal artifacts
The more commonly used methods of freezing include: (FAST-FREEZE: opposite that of
slow freeze seen in crystals being formed). Agents used for rapid freese include:
1. Liquid nitrogen
● Most rapid and commonly available
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
● Freezing microtome
4. Aerosol sprays
● Not for muscle tissue
1. Liquid nitrogen is generally used in histochemistry and during intra-operative
procedures, and is the most rapid of the commonly available freezing agents. Its main
disadvantage is that soft tissue is liable to crack due to the rapid expansion of the ice
within the tissue, producing ice crystals or freeze artifacts. It also overcools urgent
biopsy blocks, causing damage to both block and blade if sectioning is done at -70°C or
below. The tissue snap-frozen in liquid nitrogen must therefore be allowed to equilibrate
to cryostat chamber temperature before sectioning is attempted. The majority of non-
fatty unfixed tissues are sectioned well at temperatures between -10°C and -25°C.
Histology is the microscopic study of the normal tissues of the body while histopathology is the
microscopic study of tissues affected by disease. The procedures adopted for the preparation of
material for such studies are known as histologic or histopathologic techniques. The tissues are
usually obtained during surgery, biopsy, or autopsy. They range from very large specimens or
whole organs to tiny fragments of tissue. The following surgical procedures are usually
performed to obtain the specific-types of tissue that are submitted to a histology laboratory for
processing:
1. Fine needle aspiration is the simplest, least invasive test and uses the smallest needle
to simply remove cells from the area of abnormality. This is not always adequate to
obtain a diagnosis, depending on the area to be biopsied.
2. A core needle biopsy removes not only cells, but also a small amount of the
surrounding tissue. This provides additional information to assist in the examination of
the lesion.
3. An incisional biopsy takes out even more surrounding tissue. It takes out some of the
abnormality, but not all. The doctor will slice into the lesion and remove only a portion of
it. If the lesion is found to be cancerous, further surgery may be needed to remove or
excise the entire lesion.
4. An excisional biopsy generally removes the entire area in question.
5. Punch biopsy is considered the primary technique for obtaining diagnostic full-thickness
skin specimens. It requires basic general surgical and suture-tying skills and is easy to
learn. The technique involves the use of a circular blade that is rotated down through the
epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4- mm cylindrical
core of tissue sample.
6. Shave biopsy - where small fragments of tissue are “shaved” from a surface (usually
skin).
7. Curettings - where tissue is scooped or spooned to remove tissue or growths from body
cavity such as endometrium or cervical canal.
Specimens are usually received in fixative (preservative) but sometimes they arrive fresh and
must be immediately fixed. Tissue specimens received in the surgical pathology laboratory
should have a request form that lists the patient specimens are accessioned by giving them a
number that will identify each specimen for each patient. It is important that specimens are
properly identified to minimize the risk of mislabeling.
Once tissues are removed from the body, their proteins and cells are digested and broken down
by their own enzymes, independent of a bacterial action. This process is known as autolysis,
which is retarded by cold and accelerated at room temperature. It is more severe in tissues that
are rich in enzymes (e.g. liver, brain, and kidney) and less rapid in elastic and collagen tissues.
Methods of tissue examination may vary according to the structural and chemical components
of the cells to be studied, and depends on the nature and amount of the tissue to be evaluated.
,Fresh tissues
- are usually examined when there is an immediate need for evaluation. On the other
hand, a better and more effective means of studying tissues, whether normal or
abnormal, is by examination of adequately preserved sections and smears that are
stained to demonstrate specific structures. The glass slides are then mounted with
coverslips for permanent keeping.
Examination may be done on fresh or preserved tissues, depending on necessity. Fresh tissues
have the advantage of being examined in the living state, thereby allowing protoplasmic
activities such as motion, mitosis, and phagocytosis to be observed. Its use is limited, however,
because of the fact that tissues examined in the fresh state are not permanent, and therefore,
are liable to develop the changes that have usually been observed after death.
Methods of Fresh Tissue Examination
1. Teasing or Dissociation
● is a process whereby a selected tissue specimen is immersed in isotonic salt solution
such as normal saline or Ringer’s solution in a petri dish or watch glass, carefully
dissected with a needle and separated by direct or zigzag spread using an applicator
stick.
● Selected pieces of the tissue are transferred carefully to a microscope slide and
mounted as a wet preparation underneath a cover glass, care being taken to examine
unstained by Phase Contrast or Bright Field microscopy.
● It has the advantage of permitting the cells to be examined in the living state. The use of
the phase contrast microscope greatly increases the structural detail of the cells
examined in the living state, allowing movement and mitotic division to be observed. The
application of certain stains such as methylene blue can be also of great value. The
preparations, however, are not permanent.
2. Squash Preparation (Crushing)
● is a process whereby small pieces of tissue (not more than one mm. in diameter) are
placed in a microscopic slide and forcibly compressed with another slide or with a cover
glass.
● If necessary, a supravital stain may be placed at the junction of the slide and the cover
glass, and allowed to be absorbed by the tissue through capillary attraction.
3. Smear Preparation
● The method of preparing the smear differs depending on the nature of the material to be
examined. As a general rule, smears are made either by spreading the selected portion
of the specimen over the surface of the slide with a platinum loop. Alternatively, an
apposition smear can be made using a second slide to obtain a relatively uniform
distribution of secretion. Too thin or too thick smears have to be avoided, since they
make the tissues less suitable for examination. Smears may be examined either as fresh
preparations similar to that described for teased preparations, or by using a supravital
, staining technique. Smear preparations can be made permanent by fixing them while still
wet, staining them to demonstrate specific structures and inclusions, and mounting the
cleared specimen beneath a cover glass with a suitable mounting medium. This is useful
for preparing smears of thick secretions such as serous fluids, concentrated sputum,
enzymatic lavage samples from the gastrointestinal tract, and blood smears. This
technique is especially useful in cytological examinations, particularly for cancer
diagnosis.
○ Streaking -With an applicator stick or a 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. Too thin or too thick smears have to
be avoided, since they make the tissues unsuitable for examination.
○ 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. It is a little more tedious than streaking, but has the
advantage of maintaining cellular interrelationships of the material to be
examined. It is especially recommended for smear preparations of fresh sputum
and bronchial aspirates, and also for thick mucoid secretions.
○ Pull-Apart – This is 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 the two slides. Slight movement of the two slides in opposite directions
may be necessary to initiate the flow of materials. The two slides are then pulled
apart with a single uninterrupted motion, and the specimen is placed under the
microscope for immediate examination, or applied with vital stains.
○ Touch Preparation (Impression Smear) – This is 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 by Phase Contrast microscopy
or staining for light microscopic study. It has an added advantage in that the cells
may be examined without destroying their intercellular relationship.
4. Frozen Section
At times during the performance of surgical procedures, it is necessary to get a rapid diagnosis
of a pathologic process. The surgeon may want to know if the margins of his resection are free
from tumor before closing. An unexpected disease process may be found that requires
immediate diagnosis so the surgeon can decide what to do next, or it may be necessary to
determine if the appropriate tissue has been obtained for further workup of a disease process.
Immediate diagnosis is accomplished through the use of a frozen section, especially in intra-
operative pathology to help the surgeon in choosing his next plan of action. It is especially
recommended when lipids and nervous tissue elements are to be demonstrated. Frozen
sections are usually done on muscle and nerve biopsies as well as on surgically removed
tumors.
, A fresh tissue (very thin slices: 10-15u) is frozen on a microtome with C02 or on a cryostat, a
cold chamber kept at an atmospheric temperature of -10° to -20°C. The thin frozen sections
are mounted on a glass slide, fixed immediately and briefly in liquid fixative, and stained using
similar staining techniques as in traditional wax embedded sections.
For histochemistry, cryostat sections give much faster results than paraffin sections. However,
the morphological detail and resolution of frozen sections are usually inferior compared to the
quality of tissue that has been embedded in paraffin.
The advantage of the frozen section method is rapid processing time with less equipment
requirement, and less need for ventilation in the laboratory. The disadvantage is the relatively
poor quality of the final slide.
APPLICATIONS: Frozen sections, both fixed and unfixed, have many applications in
histotechnology, and are commonly used for:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances such as lipids and
carbohydrates
4. Immunofluorescent and immunohistochemical staining
5. Some specialized silver stains, particularly in neuropathology
The tissue for freezing should be fresh, and freezing should be done as quickly as possible.
Slow freezing can cause distortion of tissue due to ice crystal artifacts.
RAPID FREEZE: slow freezing causes distortion due to ice crystal artifacts
The more commonly used methods of freezing include: (FAST-FREEZE: opposite that of
slow freeze seen in crystals being formed). Agents used for rapid freese include:
1. Liquid nitrogen
● Most rapid and commonly available
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
● Freezing microtome
4. Aerosol sprays
● Not for muscle tissue
1. Liquid nitrogen is generally used in histochemistry and during intra-operative
procedures, and is the most rapid of the commonly available freezing agents. Its main
disadvantage is that soft tissue is liable to crack due to the rapid expansion of the ice
within the tissue, producing ice crystals or freeze artifacts. It also overcools urgent
biopsy blocks, causing damage to both block and blade if sectioning is done at -70°C or
below. The tissue snap-frozen in liquid nitrogen must therefore be allowed to equilibrate
to cryostat chamber temperature before sectioning is attempted. The majority of non-
fatty unfixed tissues are sectioned well at temperatures between -10°C and -25°C.
