EXAMINATION OF FRESH TISSUE

Question 1

is the microscopic study of the normal tissues of the body while histopathology is the microscopic study of tissues affected by disease.

Answer 1

Histology

Question 2

The procedures adopted for the preparation of material for such studies are known as

Answer 2

histologic or histopathologic techniques.

Question 3

The tissues are usually obtained during [?]. They range from very large specimens or whole organs to tiny fragments of tissue.

Answer 3

surgery, biopsy, or autopsy.

Question 4

The following surgical procedures are usually performed to obtain the specific-types of tissue that are submitted to a histology laboratory for processing:

Answer 4

Fine needle aspiration

core needle biopsy

incisional biopsy

excisional biopsy

Punch biopsy

Shave biopsy

Curettings

Question 5

is the simplest, least invasive test and uses the smallest needle to simply remove cells from the area of abnormality.

Answer 5

Fine needle aspiration

Question 6

This is not always adequate to obtain a diagnosis, depending on the area to be biopsied.

Answer 6

Fine needle aspiration

Question 7

removes not only cells, but also a small amount of the surrounding tissue.

Answer 7

core needle biopsy

Question 8

This provides additional information to assist in the examination of the lesion.

Answer 8

core needle biopsy

Question 9

takes out even more surrounding tissue. It takes out some of the abnormality, but not all.

Answer 9

incisional biopsy

Question 10

The doctor will slice into the lesion and remove only a portion of it.

Answer 10

incisional biopsy

Question 11

If the lesion is found to be cancerous, further surgery may be needed to remove or excise the entire lesion.

Answer 11

incisional biopsy

Question 12

generally removes the entire area in question.

Answer 12

excisional biopsy

Question 13

is considered the primary technique for obtaining diagnostic full-thickness skin specimens.

Answer 13

Punch biopsy

Question 14

It requires basic general surgical and suture-tying skills and is easy to learn.

Answer 14

Punch biopsy

Question 15

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.

Answer 15

Punch biopsy

Question 16

• where small fragments of tissue are “shaved” from a surface (usually skin).

Answer 16

Shave biopsy

Question 17

• where tissue is scooped or spooned to remove tissue or growths from body cavity such as endometrium or cervical canal.

Answer 17

Curettings

Question 18

Specimens are usually received in [?] but sometimes they arrive fresh and must be immediately fixed.

Answer 18

fixative (preservative)

Question 19

Tissue specimens received in the surgical pathology laboratory should have a request form that lists the [?] along with a description of the site of origin.

Answer 19

patient information and clinical history

Question 20

The specimens are accessioned by giving them a [?] that will identify each specimen for each patient. It is important that specimens are properly identified to minimize the risk of [?].

Answer 20

number

mislabeling

Question 21

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.

Answer 21

autolysis

Question 22

retarded by cold and accelerated at room temperature.

Answer 22

autolysis

Question 23

Autolysis is more severe in tissues that are rich in [?] and less rapid in [?].

Answer 23

enzymes (e.g. liver, brain, and kidney)

elastic and collagen tissues

Question 24

Methods of tissue examination may vary according to the [?] to be studied, and depends on the [?] to be evaluated.

Answer 24

structural and chemical components of the cells

nature and amount of the tissue

Question 25

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 [?] to demonstrate specific structures.

Answer 25

stained

Question 26

The glass slides are then mounted with [?] for permanent keeping.

Answer 26

coverslips

Question 27

Examination may be done on [?], depending on necessity.

Answer 27

fresh or preserved tissues

Question 28

have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as motion, mitosis, and phagocytosis to be observed.

Answer 28

Fresh tissues

Question 29

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.

Answer 29

Fresh tissues

Question 30

Methods of Fresh Tissue Examination

Answer 30

1. Teasing or Dissociation
2. Squash Preparation (Crushing)
3. Smear Preparation
4. Touch Preparation (Impression Smear)

Question 31

3. Smear Preparation

Answer 31

a. Streaking

b. Spreading

c. Pull-Apart

Question 32

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.

Answer 32

Teasing or Dissociation

Question 33

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 avoid forming bubbles.

Answer 33

Teasing or Dissociation

Question 34

It is either stained with a supravital dye or examined unstained by Phase Contrast or Bright Field microscopy.

Answer 34

Teasing or Dissociation

Question 35

It has the advantage of permitting the cells to be examined in the living state.

Answer 35

Teasing or Dissociation

Question 36

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.

Answer 36

Teasing or Dissociation

Question 37

The application of certain stains such as methylene blue can be also of great value.

Answer 37

Teasing or Dissociation

Question 38

The preparations, however, are not permanent.

Answer 38

Teasing or Dissociation

Question 39

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.

Answer 39

Squash Preparation (Crushing)

Question 40

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.

Answer 40

Squash Preparation (Crushing)

Question 41

The method of preparing the smear differs depending on the nature of the material to be examined.

Answer 41

Smear Preparation

Question 42

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.

Answer 42

Smear Preparation

Question 43

Alternatively, an apposition smear can be made using a second slide to obtain a relatively uniform distribution of secretion.

Answer 43

Smear Preparation

Question 44

Too thin or too thick smears have to be avoided, since they make the tissues less suitable for examination.

Answer 44

Smear Preparation

Question 45

Smears may be examined either as fresh preparations similar to that described for teased preparations, or by using a supravital staining technique.

Answer 45

Smear Preparation

Question 46

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.

Answer 46

Smear Preparation

Question 47

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.

Answer 47

Smear Preparation

Question 48

This technique is especially useful in cytological examinations, particularly for cancer diagnosis.

Answer 48

Smear Preparation

Question 49

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.

Answer 49

Streaking

Question 50

Too thin or too thick smears have to be avoided, since they make the tissues unsuitable for examination.

Answer 50

Streaking

Question 51

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.

Answer 51

Spreading

Question 52

It is a little more tedious than streaking, but has the advantage of maintaining cellular interrelationships of the material to be examined.

Answer 52

Spreading

Question 53

It is especially recommended for smear preparations of fresh sputum and bronchial aspirates, and also for thick mucoid secretions.

Answer 53

Spreading

Question 54

This is done by placing a drop of secretion or sediment upon one slide and facing it to another clean slide.

Answer 54

Pull-Apart

Question 55

The material disperses evenly over the surface of the two slides.

Answer 55

Pull-Apart

Question 56

Slight movement of the two slides in opposite directions may be necessary to initiate the flow of materials.

Answer 56

Pull-Apart

Question 57

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.

Answer 57

Pull-Apart

Question 58

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.

Answer 58

Touch Preparation (Impression Smear)

Question 59

It has an added advantage in that the cells may be examined without destroying their intercellular relationship.

Answer 59

Touch Preparation (Impression Smear)

Question 60

At times during the performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process.

Answer 60

Frozen Section

Question 61

The surgeon may want to know if the margins of his resection are free from tumor before closing.

Answer 61

Frozen Section

Question 62

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.

Answer 62

Frozen Section

Question 63

Immediate diagnosis is accomplished through the use of a [?], especially in intra-operative pathology to help the surgeon in choosing his next plan of action.

Answer 63

Frozen Section

Question 64

It is especially recommended when lipids and nervous tissue elements are to be demonstrated.

Answer 64

Frozen Section

Question 65

Frozen sections are usually done on muscle and nerve biopsies as well as on surgically removed tumors.

Answer 65

Frozen Section

Question 66

A fresh tissue is frozen on a microtome with C02, or on a [?], a cold
chamber kept at an atmospheric temperature of [?].

Answer 66

cryostat

-10° to -20° C

Question 67

The thin [?] 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.

Answer 67

Frozen Section

Question 68

For histochemistry, [?] 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.

Answer 68

cryostat sections

Question 69

The advantage is rapid processing time with less equipment requirement, and less need for ventilation in the laboratory.

Answer 69

Frozen Section

Question 70

The disadvantage is the relatively poor quality of the final slide.

Answer 70

Frozen Section

Question 71

Frozen sections, both fixed and unfixed, have many applications in histotechnology, and are commonly used for:

Answer 71

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

Question 72

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. The more commonly used methods of freezing include:

Answer 72

1. Liquid nitrogen
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
4. Aerosol sprays

Question 73

is generally used in histochemistry and during intra- operative procedures

Answer 73

Liquid nitrogen

Question 74

most rapid of the commonly available freezing agents.

Answer 74

Liquid nitrogen

Question 75

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.

Answer 75

Liquid nitrogen

Question 76

It also overcools urgent biopsy blocks, causing damage to both block and blade if sectioning is done at -70°C or below.

Answer 76

Liquid nitrogen

Question 77

The tissue snap-frozen in [?] must therefore be allowed to equilibrate to cryostat chamber temperature before sectioning is attempted.

Answer 77

Liquid nitrogen

Question 78

The majority of non-fatty unfixed tissues are sectioned well at temperatures between

Answer 78

-10oC and -25°C.

Question 79

One problem with the use of liquid nitrogen is that it causes a vapor phase to form around the tissue, acting as an insulator that causes uneven cooling of tissue, particularly of muscle biopsies, and making diagnostic interpretation difficult. This problem can be overcome by freezing the tissue in [?] that has a high thermal conductivity.

Answer 79

Isopentane, OCT, or Freon 2.2

Question 80

is liquid at room temperature.

Answer 80

Isopentane

Question 81

is usually suspended in a flask of liquid nitrogen until half-liquid and half-solid stage is reached.

Answer 81

Pyrex glass beaker containing isopentane

Question 82

The beaker is removed from the liquid nitrogen when small crystals start forming on the side of the beaker (approximately [?]), and the tissue to be frozen (affixed on a [?]) is dropped into the cooled liquid isopentane. This is an excellent method for freezing muscle tissue.

Answer 82

-170°C

cork disc, aluminum foil or cryostat chuck

Question 83

Tissue blocks can also be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a

Answer 83

C02 cylinder.

Question 84

The use of [?] has become increasingly popular in recent years, and is adequate for freezing small pieces of tissue except muscle.

Answer 84

aerosol sprays

Question 85

Quick- freezing spray cans of [?] have a distinct advantage of rapidly freezing blocks of any type of tissue.

Answer 85

fluorinated hydrocarbons (e.g., Cryokwik)

Question 86

Fresh, completely unfixed tissues, or tissues that have been briefly treated with formalin may not require embedding anymore; instead they may be frozen and cut in a [?].

Answer 86

freezing microtome or cryostat

Question 87

Two methods of preparing frozen sections may be resorted to:

Answer 87

1. Cold Knife procedure
2. Cryostat procedure (Cold Microtome)

Question 88

Tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a C02 cylinder, or by using a specially made piece of equipment known as cryostat.

Answer 88

Cold Knife Procedure

Question 89

Almost any microtome can be utilized for the purpose, provided means are made available for freezing and maintaining the specimen and the knife at low temperatures, usually by utilizing the carbon dioxide technique.

Answer 89

Cold Knife Procedure

Question 90

A piece of filter paper soaked in gum syrup is placed on the microtome stage, and short bursts of C02 are applied, freezing the filter paper to the stage.

Answer 90

Cold Knife Procedure

Question 91

The selected block of tissue, approximately 3-5 mm. thick, is then oriented on the stage, applied with a few drops of gum syrup and frozen solid with several intermittent bursts of CO2 , each for 1-2 seconds duration, at intervals of around 4 seconds. It should be frozen just to the point where it will be firm enough to section.

Answer 91

Cold Knife Procedure

Question 92

The tissue is then lifted up to the knife manually and trimmed until the surface is flat.

Answer 92

Cold Knife Procedure

Question 93

The surface is then warmed with the finger until the hard frozen tissue starts to thaw and becomes visible to the naked eye.

Answer 93

Cold Knife Procedure

Question 94

This is the DewLine, the point at which sections may then be cut at 10 μm thickness.

Answer 94

Cold Knife Procedure

Question 95

Sections do not form ribbons but rather stick to the knife blade and should, therefore, be removed with a camel hair brush or finger moistened with water.

Answer 95

Cold Knife Procedure

Question 96

They are then transferred to a dish of distilled water to separate, and picked up individually for mounting and staining.

Answer 96

Cold Knife Procedure

Question 97

The water dish is usually placed on a dark or black background, in order to see the sections which are usually colorless or very light in color.

Answer 97

Cold Knife Procedure

Question 98

Tissues that have been frozen too hard will usually chip into fragments when cut.

Answer 98

Cold Knife Procedure

Question 99

The surface of the block may then be softened by warming slightly with the ball of the finger or thumb.

Answer 99

Cold Knife Procedure

Question 100

Tissues that have not been sufficiently frozen will cut thick and crumble, and the block may come away from the stage.

Answer 100

Cold Knife Procedure

Question 101

More bursts of C02 gas should then be given to refreeze the block. Whether used in a cold environment or not, a different temperature between the tissue and the knife is usually employed, the latter being colder.

Answer 101

Cold Knife Procedure

Question 102

Using a cold knife in a controlled cold environment, optimum condition for sectioning shall be provided for by the following temperatures:

Answer 102

Knife -40° to - 60°C
Tissue -5° to - 10°C
Environment 0° to - 10°C

Question 103

The success of this procedure depends upon ambient temperature and humidity.

Answer 103

Cold Knife Procedure

Question 104

It is very hard, if not impossible, to cut sections in a hot or humid environment.

Answer 104

Cold Knife Procedure

Question 105

Ease of cutting and quality of sections are always improved if done in a cold room.

Answer 105

Cold Knife Procedure

Question 106

Sections thinner than 6 μ generally cannot be obtained even from tissues that section well, and with ideal conditions for sectioning.

Answer 106

Cold Knife Procedure

Question 107

an apparatus used in fresh tissue microtomy.

Answer 107

Cryostat

Question 108

consists of an insulated microtome housed in an electrically driven refrigerated chamber and maintained at temperatures near -20°C, where microtome, knife, specimen and atmosphere are kept at the same temperature.

Answer 108

Cryostat

Question 109

The optimum working temperature of cryostat is

Answer 109

-18 to -20°C.

Question 110

Majority of the sections can be cut in isothermic conditions, where the temperature for sectioning can be accurately established and controlled.

Answer 110

Cryostat Procedure

Question 111

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.

Answer 111

Cryostat Procedure

Question 112

Fresh frozen tissue requires that the tissue be maintained in the frozen solid state during cutting of section, thereby supporting and protecting the tissue from damage and distortion by the knife during the process of cutting.

Answer 112

Cryostat Procedure

Question 113

The tissue must be sufficiently cold to prevent compression and displacement of cell and tissue structures as the knife passes thru it.

Answer 113

Cryostat Procedure

Question 114

The microtome knife needs to be chilled and maintained at low
temperature to prevent complete melting of the tissue, thereby forming a sticky, distorted mass along the knife edge.

Answer 114

Cryostat Procedure

Question 115

When the tissue is too cold, on the other hand, resistance to cutting is increased, so that the tissue becomes brittle and is broken down into fragments upon cutting.

Answer 115

Cryostat Procedure

Question 116

should be left on at all times even when not in use, since it will require several hours to reach operating temperature from a room temperature start.

Answer 116

Cryostat

Question 117

It takes at least one hour for a knife to come down to operating temperature, so that a spare knife should always be kept inside the cryostat cabinet.

Answer 117

Cryostat Procedure

Question 118

To ensure that the sections will cut smoothly and freely onto the knife surface, the knife as well as the undersurface and edge of the anti-roll plate must be kept scrupulously clean and dry.

Answer 118

Cryostat Procedure

Question 119

Soft tissue paper, either dry or moistened with absolute alcohol, may be used to clean the knife and anti-roll plate.

Answer 119

Cryostat Procedure

Question 120

should be defrosted during the weekend, including cleaning and oiling of microtome with special low- temperature oil.

Answer 120

Cryostat

Question 121

The success of fresh tissue sectioning depends to a large extent on the temperature, both of the tissue and of the knife.

Answer 121

Cryostat Procedure

Question 122

Certain tissues such as fat or mucin, and hard or dense structures in a soft matrix require much lower temperatures to impart a suitable consistency for cutting.

Answer 122

Cryostat Procedure

Question 123

These are sectioned on the cryostat by lowering the tissue or knife temperature or both, either by placing the block holder in a bath of alcohol or acetone containing dry ice, or by exposing the tissue to carbon dioxide.

Answer 123

Cryostat Procedure

Question 124

[?] are generally used as mounting media for tissue blocks that need to be sectioned on a cryostat. The O.C.T. (Optimal Cutting Temperature) compound, Lab-Tek Products, Division of Miles Laboratories is especially recommended.

Answer 124

Synthetic water-soluble glycols and resins

Question 125

It is marketed in convenient 8 oz. plastic dispensers in three temperature ranges, depending on the tissue being cut:
[?] for brain, lymph nodes, liver, spleen, uterine curetting, soft cellular tumors;
[?] for non-fatty breast tissue, ovary, prostate, tongue, and GI tract;
[?] for fatty breast and omental tissue.

Answer 125

-5 to -15°C

-15 to -25°C

-35°C

Question 126

The cryostat is usually set at -18 to -20°C.

Answer 126

Mounting of Tissue Block

Question 127

Preferably, the tissue block should be 2-4 mm. thick in order to minimize the risk of the knife hitting the metal tissue block holder.

Answer 127

Mounting of Tissue Block

Question 128

Small fragments of tissue, such as curettings or brain biopsies, are placed on a thick base of O.C.T. compound.

Answer 128

Mounting of Tissue Block

Question 129

The blocks are then surrounded and covered with an additional matrix of O.C.T. compound, and frozen by liquid nitrogen.

Answer 129

Mounting of Tissue Block

Question 130

The frozen tissue is mounted on the microtome.

Answer 130

Mounting of Tissue Block

Question 131

Both the microtome knife and the tissue block are left in the cryostat for 15 minutes at -20°C, to ensure that they are cooled to the correct temperature.

Answer 131

Mounting of Tissue Block

Question 132

Sections between 5-10 μm are then cut slowly and steadily, removed from the knife with a camel hair brush, attached directly to slides of cover-glasses at room temperature, air​ dried, and fixed (optional).

Answer 132

Mounting of Tissue Block

Question 133

To mount cryostat sections after cutting, one edge of the glass slide is lowered gently until it is about 1/2 to 1 mm. from the knife face.

Answer 133

Mounting of Tissue Block

Question 134

The section will automatically transfer from the cold knife to the relatively warm slide.

Answer 134

Mounting of Tissue Block

Question 135

The slide should never be pressed down on the section, because this will cause a frost mark to remain where the section rested on the knife. If this happens, the frost mark should be wiped away with soft tissue paper.

Answer 135

Mounting of Tissue Block

Question 136

Overall, cryostat sections provide the simplest, quickest and least labor​- intensive method for producing frozen sections, and are routinely used for intraoperative and rapid diagnosis of surgical specimen.

Answer 136

Mounting of Tissue Block

Question 137

It should be noted that cryostats cut only individual sections, and do not form ribbons, as in paraffin blocks.

Answer 137

Mounting of Tissue Block

Question 138

Cryostat sections of fresh, unfixed tissue usually attach easily to the slide, even without adhesives, and will preserve enzymes and other substances that may be studied by histochemical techniques.

Answer 138

Freezing Previously Fixed Tissue

Question 139

The cryostat is also recommended for any technique requiring cold sectioning of fixed material, e.g., for fats and lipids, and for some special methods for the nervous system.

Answer 139

Freezing Previously Fixed Tissue

Question 140

Sections of formalin-fixed tissue, however, may not adhere to the slide, and will fall off or be detached during staining.

Answer 140

Freezing Previously Fixed Tissue

Question 141

Clean slides should be coated with albumin or chrome-glycerin jelly so that the fixed tissue will attach to the slide.

Answer 141

Freezing Previously Fixed Tissue

Question 142

Another way, albeit cumbersome, is to immerse the tissue block in boiling 10% buffered formalin for 1 to 2 minutes before freezing and sectioning for rapid surgical diagnosis.

Answer 142

Freezing Previously Fixed Tissue

Question 143

Special fixatives such as 10% formol calcium at 4°C may be used in histochemistry and for lipid demonstration.

Answer 143

Freezing Previously Fixed Tissue

Question 144

Tissues that have been fixed or stored in alcohol should be washed in water for 12-24 hours before sectioning, since alcohol inhibits freezing.

Answer 144

Freezing Previously Fixed Tissue

Question 145

Muscle and nerve biopsies are divided into separate portions that will allow for formalin fixation and paraffin embedding, unfixed snap-frozen for cryostat sections, fixation and resin embedding for electron microscopy (EM) and, in some rare cases, for biochemical immunoblotting studies.

Answer 145

Examination of nerve and muscle

Question 146

Multiple fixation processes are required because multiple techniques are to be used.

Answer 146

Examination of nerve and muscle

Question 147

The portion of a specimen intended for frozen section should be transported on top of wet ice, on saline-dampened gauze, and rapidly frozen within two hours.

Answer 147

Examination of nerve and muscle

Question 148

Upon receipt in the histology lab, specimen is oriented in O.C.T. (Optimal Cutting Temperature) compound and snap-frozen in liquid nitrogen/ isopentane for optimal results.

Answer 148

Examination of nerve and muscle

Question 149

Orientation, size, and expedient flash freezing are critical to obtaining undamaged sections of unfixed muscle fibers.

Answer 149

Examination of nerve and muscle

Question 150

Do not allow the tissue to freeze slowly or to soak up excess saline, as these will cause artifacts that can be seen microscopically and can interfere with diagnostic interpretation.

Answer 150

Examination of nerve and muscle

Question 151

A portion of the tissue is oriented on a piece of cardboard, fixed with 10% buffered formalin and processed for staining with routine Hematoxylin and Eosin (H&E staining).

Answer 151

Examination of nerve and muscle

Question 152

The biopsy portion for electron microscopy is fixed in a buffered solution of glutaraldehyde and postfixed in osmium tetroxide, usually by a specialist in electron microscopy.

Answer 152

Examination of nerve and muscle

Question 153

In some muscular degenerative disorders, biochemical techniques may also be required.

Answer 153

Examination of nerve and muscle

Question 154

For histochemical evaluation involving [?], the tissue needs to be chemically active, and the important chemical constituents should not have been removed, altered or displaced.

Answer 154

enzyme studies

Question 155

is deemed to be the most ideal and preferred means of preserving tissues in order to avoid complete or partial loss of enzymes consequent to chemical fixation.

Answer 155

frozen section

Question 156

Difficulties, however, arise in obtaining thin and serial sections of [?]; since cut sections of tissue tend to disintegrate and cannot be easily handled without prior fixation. These disadvantages will have to be considered in determining the necessity and advisability of such sections.

Answer 156

uniform thickness

Question 157

In addition to fresh frozen tissue sectioning, there are methods that may be resorted to, if chemical fixation of tissue blocks is to be avoided, namely:

Answer 157

1. Freeze-drying
2. Freeze substitution

Question 158

Like fresh frozen sections, these special techniques have the common principle of rapidly preserving the tissue block by [?]. The aim is to produce instant cessation of cellular activity thereby preventing chemical alteration of tissue and displacement of cellular tissue components.

Answer 158

freezing (quenching)

Question 159

must be rapid, accomplished within seconds to prevent the formation of ice crystal artefacts in tissue blocks and produce optimum tissue preservation.

Answer 159

Freezing

Question 160

The freezing agent commonly employed is [?], and the tissue is sectioned into thin slices using a [?] machine under very low temperature.

Answer 160

liquid nitrogen

cryostat

Question 161

The use of [?] and most recently of [?], which can be cooled to very low temperature in order to retain the fluidity of the freezing agents, have contributed much in giving higher conductivity to this liquefied gas.

Answer 161

isopentane, pentane and propane

dichloro-difluoromethane

Question 162

is a special way of preserving tissues by rapid freezing (quenching) of fresh tissue at -160°C and subsequently removing ice water molecules (dessication) by transferring the still frozen tissue block into a vacuum chamber at a higher temperature, e.g. -40°C (sublimation) without the use of any chemical fixative.

Answer 162

Freeze-drying

Question 163

This technique is generally not used in routine surgical laboratories, and is restricted to specialized or research laboratories.

Answer 163

Freeze-Drying

Question 164

A tissue around 2 mm. thick is plunged into isopentane or propane​- isopentane mixture which has been chilled to -160° to -180°C with liquid nitrogen.

Answer 164

Freeze-Drying

Question 165

This will effectively solidify the tissue in 2-3 seconds, thus preventing the formation of large ice crystals, autolysis and putrefaction.

Answer 165

Freeze-Drying

Question 166

The frozen tissue is then transferred into a high vacuum drying chamber maintained at a temperature of -30° to -40°C depending upon the size of the tissue.

Answer 166

Freeze-Drying

Question 167

Water is sublimated and dehydrated from the tissue, thereby completing the dessication process within 24-48 hours.

Answer 167

Freeze-Drying

Question 168

Once drying is completed, the tissue is removed, fixed and embedded, either in molten paraffin wax, water soluble waxes or celloidin.

Answer 168

Freeze-Drying

Question 169

Infiltration and impregnation are usually performed in a vacuum embedding oven.

Answer 169

Freeze-Drying

Question 170

The tissue is then sectioned in the usual routine manner and specific staining is applied, depending upon individual necessity.

Answer 170

Freeze-Drying

Question 171

This technique is generally time-consuming and expensive.

Answer 171

Freeze-Drying

Question 172

is by far the most time consuming part of the process, as certain tissues contain 70- 80% water by weight that has to be removed without damage to the tissue.

Answer 172

Drying

Question 173

Furthermore, freeze-dried materials are generally more difficult to section than ordinary paraffin blocks.

Answer 173

Freeze-Drying

Question 174

The tissue is brittle and inadequately supported due to the relatively short period for wax impregnation; hence, it is not advisable as a routine procedure.

Answer 174

Freeze-Drying

Question 175

The tissues are usually flattened directly into an albuminous glass slide with the aid of the finger.

Answer 175

Freeze-Drying

Question 176

Water must be avoided and warm alcohol, acetone, mercury are preferred.

Answer 176

Freeze-Drying

Question 177

Has many outstanding good features.

Answer 177

Freeze-Drying

Question 178

It produces minimum tissue shrinkage, and allows tissues to be processed in a fresh state.

Answer 178

Freeze-Drying

Question 179

It causes minimal chemical change on the cells, most especially on the protein components, and less displacement of tissue and cellular constituents.

Answer 179

Freeze-Drying

Question 180

This method avoids the chemical alteration of cellular components, the denaturation of proteins, destruction of enzymes, and loss of tissue constituents that usually occur in the usual histological processing.

Answer 180

Freeze-Drying

Question 181

This method is particularly important as far as enzyme studies are concerned.

Answer 181

Freeze-Drying

Question 182

In addition to demonstrating hydrolytic enzymes, mucous substances, glycogen and proteins, freeze-drying may be used for special studies, including:

Answer 182

1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and polypeptide
   hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent substances
5. Formaldehyde-induced fluorescence of biogenic amines (to
   demonstrate 5-hydroxytryptamine, adrenaline, and other
   catecholamines)
6. Scanning electron microscopy

Question 183

is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration.

Answer 183

Freeze-Substitution

Question 184

It is similar to freeze-drying in preparing and preserving tissue blocks for subsequent sectioning because both involve the rapid freezing of tissues and the subsequent infiltration and embedding of the frozen tissue block in paraffin or celloidin.

Answer 184

Freeze-Substitution

Question 185

The only variation is that the frozen tissue, instead of being subjected to dehydration in an expensive vacuum drying apparatus, is fixed in Rossman’s formula or in 1% Acetone and dehydrated in absolute alcohol.

Answer 185

Freeze-Substitution

Question 186

Infiltration and embedding is then carried out in the same way as in paraffin section.

Answer 186

Freeze-Substitution

Question 187

is based on rapid freezing of tissues followed by solution (“substitution”) of ice at temperatures well below 0°C. A 1 mm to 3 mm specimen is thrown into 3:1 propane-isopentane that is super cooled by liquid nitrogen to -175°C (with precautions).

Answer 187

Freeze-Substitution

Question 188

Cryostat sections are cut 8-10 μm, and transferred to water-free acetone (substituting fluid), and cooled to -70oC for 12 hours to 1 week in order to dissolve ice slowly without distorting tissue structure.

Answer 188

Freeze-Substitution

Question 189

The sections are floated onto coverslips or slides and allowed to dry for subsequent histochemical staining.

Answer 189

Freeze-Substitution

Question 190

This technique is relatively more economical and less time-consuming than freeze-drying.

Answer 190

Freeze-Substitution

Question 191

For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1% solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, or picric acid in ethanol.

Answer 191

Freeze-Substitution