Aughey E., Frye F.L. Comparative veterinary histology with clinical correlates

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1.4 Four cassettes

containing paraffin-

embedded tissue ready for

sectioning by a microtome.

1.3 A small piece of paper listing special staining

requests is embedded on the opposite side of the

cassette. In this instance, haematoxylin and eosin

(H & E) and periodic acid–Schiff (PAS) stains will be

applied to separate duplicate tissue sections.

Introduction

1.2b1.2a

1.2 (a) Once the tissue has dehydrated, (b) a histology laboratory technician

embeds it in a melted paraffin wax<plastic polymer compound.

1.3

1.4

1.5 Using the finely honed microtome blade, the

histology technician cuts a thin ribbon of paraffin-

embedded tissue.

1.5

1.6 The tissue section is transferred to a water bath and

is floated onto the glass microscope slide with a fine

camel’s hair brush. Note the matt black finish of the

water bath, which facilitates visualizing the nearly

transparent tissue section.

1.6

dry, and the section adheres to the glass slide (1.7).

Removal of the paraffin wax by a suitable solvent,

such as xylene, and rehydration allows the tissue to

be examined unstained; this has no advantage over

the direct examination of living cells. It is necessary

to stain the component cells and tissues selectively

and make a permanent preparation for examination

with the light microscope; a selection of these tech-

niques is described later (see Staining technique).

Decalcification is necessary for tissue with ossi-

fied or calcified components before paraffin embed-

ding, otherwise the hardness of the tissue will result

in difficulty in cutting the sections, causing artefacts.

Specimens are fixed in formalin or other chemical

fixatives, and then transferred to the decalcifying

solution to allow removal of the mineral salts. Most

of these decalcifying agents contain acids such as

formic, malic, glacial acetic, hydrochloric or nitric.

Freezing

A cryostat, a microtome confined to a freezing

chamber, is required to cut frozen sections (1.8).

These may be from fixed or from unfixed tissue.

The advantage of this method is that the time

between taking the sample and examining it under

the microscope is much reduced. A biopsy may be

taken and examined while the patient is still in the

operating room. Fat-containing cells retain the lipid

content and the tissue is often more life-like in

appearance than non-frozen sections. The disad-

vantages of this method are tissue distortion, caused

by the freezing and thawing, and thicker sections.

Once the sections are cut and mounted on glass

slides, conventional staining techniques are used.

Consequence of freezing unfixed tissues

When unfixed tissues are frozen and then thawed

before being chemically fixed, their delicate cell

membranes may become distorted or ruptured, or

both, by the forces induced by the expansion and

contraction of the intracellular fluid as it freezes

and thaws. Therefore, if tissues are to examined

histologically, unfixed specimens must not be

frozen. Examples of tissues that were frozen before

histological fixation and processing are illustrated

in 1.9.

Comparative Veterinary Histology with Clinical Correlates

1.7 Slides bearing thin tissue sections are warmed on a

thermal table, which causes evaporation and enhances

the adhesion of the sections onto the glass surfaces, and

smooths out irregularities, which is preparatory to xylene

clearing.

1.7

1.8 Frozen tissue sections are created with the use of a

cryostat, which is a conventional microtome enclosed

within a freezing temperature chamber. Whereas tissue

enzymes and some other cellular constituents cannot be

detected in paraffin-embedded sections, specially stained

frozen tissue sections reveal them. The advantages of

frozen tissue sections are that they require less time than

paraffin-embedded sections to process, and stained tissue

specimens obtained during surgery can be examined and

interpreted while the patient is still in the operating

room. Fat-containing cells retain their lipid contents

because they are not dissolved by xylene.

1.8

The rehydrated sections of tissue are now

immersed in a solution of one or more stains; any

excess stain is removed during this process. The

slides are dried again, cleared in xylene, and per-

1.9 Histological sections of

(a) myocardium, (b) kidney and

C. constrictor) that were frozen

before being fixed in 10% neutral

buffered formalin solution. Note the

disruption and distortion of the

histological architecture, and the loss

of all but the erythrocytic nuclei.

Such destruction can be prevented if

the tissues are fixed before freezing.

H & E. a, ×62.5; b, ×62.5; c, ×62.5.

1.9a

1.9b

1.9c

manently mounted beneath a glass or plastic cover-

slip using a mounting medium that is xylene mis-

cible. Sections that require special stains are

stained and given individual coverslips, as shown

Introduction

in 1.10. Sections requiring standard haematoxylin

and eosin (H & E) stain are stained and cover-

slipped by automated machines that process the

tissues and then dispense an appropriate volume

of mounting medium, apply a coverslip and com-

press the finished mounted slide to remove any

trapped bubbles of air (1.11). The completed

stained microsections are placed onto the surface of

a warming table beneath a fume hood, where the

xylene in the mounting medium evaporates. This

final step fixes the coverslip firmly to the tissue and

glass slide, forming a permanent ‘sandwich’ that can

be handled without dislodging any portion of the

stained section.

Staining techniques

Numerous different dyes in various combinations

are formulated into stains that are used to impart

specific and reproducible colouration. Many of these

dyes possess positive and negative electrical charges

and are attracted or repulsed by electrostatic charges

that are characteristic of certain tissue constituents.

In order for some dyes to combine with tissue com-

ponents, a metallic salt, termed a mordant, is

required. The combination of a dye with an appro-

priate mordant forms a ‘lake’ and carries a positive

electrostatic charge. Dye-mordant combinations

with positive charges are cationic and are termed

1.10 Slides that have

received special staining are

coverslipped manually.

Exposure of laboratory

personnel to potentially

toxic xylene vapours is

reduced by conducting the

coverslipping operations

beneath a vacuum fume

hood.

1.10

1.11 When large volumes

of slides with standard H & E

stain must be coverslipped,

an automatic coverslipping

machine is employed.

1.11

Comparative Veterinary Histology with Clinical Correlates

‘basic’ stains. These cationic basic lakes combine

electrochemically with negatively charged tissue con-

stituents, such as nuclear chromatin, other nucleo-

proteins, and phosphate groups. Some dyes are

inherently basic without requiring the addition of a

mordant; they carry their own positive electrostatic

charge. Basic fuchsin, toluidine blue and methylene

blue are examples of naturally basic stains. Con-

versely, anionic or ‘acidic’ dyes carry a negative or

anionic charge, and are called ‘acidic’ because they

are attracted to and combine with tissue constituents

that possess a negative electrostatic charge. Eosin

is an example of an acidic stain. Differential stain-

ing is possible because some tissues may be acidic,

basic or amphoteric. Thus, the pH of the extracel-

lular fluid causes their electrostatic charge to vary

and, as a result, their acceptance of acidic and basic

stains varies.

Many special dye combinations, some requiring

rare metallic salts, are used to stain certain tissue types

and constituents, micro-organisms, metabolic by-

products and so on. Many formularies containing

recipe-like staining formulae are available and new

staining techniques are continually being developed.

Examination of living cells with the light micro-

scope yields very little information. Therefore, thin

sections of tissue, after they have been excised and

processed, are stained with special dyes to enable

detailed observations to be made on their struc-

ture. The most widely used staining technique is H

& E. Haematoxylin stains a deep purple colour and

acts as a basic stain (basophilic). Eosin is pink to

red in colour and acts as an acid stain (acidophilic

or eosinophilic). Haematoxylin reacts with deoxyri-

bonucleic acid and ribonucleic acid, and eosin reacts

with cytoplasmic proteins and a variety of extra-

cellular structures. Thus, nuclei and rough endo-

plasmic reticulum stain blue to purple and

cytoplasm stains pink to red depending upon the

concentration of the basic and acid components of

the cell (1.12).

Specialized staining methods are used to illustrate

particular features. Osmic acid reacts with fat to give

a grey–black colour (1.13), periodic acid Schiff (PAS)

1.12 Digital pad (dog). The nuclei are

stained deep blue (arrowed). (1) The

cytoplasm and fibres are stained varying

shades of pink with eosin. (2) Fat cells are

unstained, the fat is lost during processing.

H & E. ×160.

1.12

1.13 Longitudinal section (LS nerve (dog).

The myelin sheath surrounding the nerve

fibre reacts with osmic acid and stains black;

the supporting connective tissue is

unstained. Osmic acid. ×250.

1.13

Introduction

and alcian blue reveal glycosaminoglycans (1.14 and

1.15), silver impregnation displays reticular fibres

and some aspects of nervous tissue (1.16 and 1.17),

and Masson’s trichrome differentiates between con-

nective tissue and muscle (1.18 and 1.19). Some cir-

cumstances require the combination of two or more

staining methods to yield the maximum informa-

tion. There are many other methods available, but

only the commonly used ones are mentioned here.

1.17 Stellate cells in the cerebellum (cat). This method is

used specifically to illustrate the cytoplasmic processes of

the neurons of the central nervous system (arrowed).

Cajal’s uranium silver. ×250.

1.17

1.14 Duodenum (dog). The mucus-secreting goblet cells

react with PAS (arrowed). Haematoxylin/PAS. ×125.

1.14

1.15 Cervix (sheep). The epithelial cells lining the cervix

react with either alcian blue or PAS, illustrating chemical

differences in the types of mucus secreted. Alcian

blue/PAS. ×200.

1.15

1.16 Adrenal (horse). The reticular fibres form a

fine network in (1) the capsule and (2) as a delicate

supporting framework for the adrenal secretory

cells. The method of Gordon and Sweet for reticular

fibres. ×125.

1.16

Examination of living material with the electron

microscope has necessitated the development of

new techniques in preparation procedures to illus-

trate the arrangement of organelles, membranes and

cell contents (1.20). It has been further refined to

provide a three-dimensional picture without dis-

tortion (1.21). All of these techniques are now

strandard tools in histology and have advanced our

understanding.

Comparative Veterinary Histology with Clinical Correlates

Introduction

1.19 Kidney (dog). In this trichrome stain the connective

tissue is stained a blue/green. Gomori’s trichrome. ×125.

1.19

1.18 Tongue (dog). The muscle is stained red and the

connective tissue is stained green. Masson’s trichrome. ×50.

1.18

1.20 Transmission electron micrograph

of a fibroblast (sheep). (1) Nucleus,

(2) nuclear membrane, (3) cisternae of

rough endoplasmic reticulum (RER),

(4) plasmalemma, (5) mitochondria,

(6) fat droplet and (7) collagen fibrils.

×8000.

1.20

1.21 Scanning electron micrograph

of kidney (dog). (1) Renal tubule,

(2) interstitial connective tissue, (3) free

erythrocyte – a biconcave disc with the

typical indentation. ×675.

1.21

Microscopy

The examination and study of normal cells and tis-

sues by microscopy is called histology or microscopic

anatomy. The study of abnormal cells and tissues is

histopathology. An understanding of the normal is

essential for the recognition of the abnormal.

Investigative microscopes range from the simple light

microscope to the sophisticated high-resolution elec-

tron microscope. In between lie a wide variety of spe-

cialized microscopes to meet special needs, such as

phase contrast, polarizing and fluorescence micro-

scopes, and the scanning electron microscope.

Units of histological measurement

A micrometre (+m) is equal to a millionth part of

a metre and is the unit of measurement of the light

microscope; a red blood cell is approximately 8 µm

in diameter.

A nanometre (nm) is equal to a billionth part of

a metre. The thickness of the basal lamina of an

epithelial cell is 70 nm, which can be resolved using

the electron microscope.

Light microscopy

The light microscope is the instrument most com-

monly used for the visualization of cells and tissues.

With it magnifications of up to 2000 times are pos-

sible. The limit to the size of the structure that can

be distinguished with the light microscope is limited

by the physical nature of light. The wavelength of

visible light ranges from 0.4 to 0.7 µm. Therefore,

even with the best optical system available the res-

olution, or resolving power, of the light microscope

is limited to 0.2 µm, and anything smaller than that

will not be clearly distinguished.

In order to achieve the best results a few basic

preliminary checks must be made.

• Ensure that the glass slide is clean, free from dust

and smears.

• Ensure that the microscope condenser, objec-

tives and ocular lenses are clean < take great

care to clean the microscope with soft lens tis-

sues.

• Set the microscope up for critical illumination for

each objective by:

(1) closing the iris diaphragm (the substage con-

denser diaphragm),

(2) adjusting the condenser until the circular area

of illumination has a sharp edge, and

(3) making sure the condenser is centred by using

the adjusting screws.

Always begin with the lowest objective and increase

the magnification slowly.

Transmission electron microscopy

This microscope uses an electron beam instead of

a light source and allows resolution of structures

as small as 1 nm. Small pieces of tissue (cubes not

more than 1 mm on a side) are fixed rapidly (to

avoid artefacts induced by tissue degradation) in

cold glutaraldehyde-based fixative, dehydrated and

embedded in epoxy resin. Sections are cut at

0.03–0.05 µm on an ultramicrotome using a glass

or a diamond knife, mounted on copper grids and

stained with heavy metal solutions such as lead sul-

phate and uranium nitrate. The vapours of fixa-

tives used for electron microscopy processing are

volatile and hazardous to the eyes and mucous

membranes, so an exhaust hood or adequate ven-

tilation is essential.

Scanning electron microscopy

Solid pieces of tissue fixed in a glutaraldehyde fix-

ative, are dried, coated with gold and placed in the

microscope. The electron beam scans the specimen

and a three-dimensional representation of the sur-

face is obtained.

Artefacts induced by

histological processing

The preparation of tissue sections involves a number

of stages during fixing, dehydrating, paraffin embed-

ding, sectioning, deparaffinizing, rehydrating, stain-

ing and coverslipping. Each of these processes

necessitates the manipulation of tissue specimens and

laboratory reagents, thus providing opportunities for

errors to be made. Just one flawed laboratory tech-

nique can spoil the final result. Some of the common

artefacts are illustrated in 1.22<1.26.

Comparative Veterinary Histology with Clinical Correlates

Introduction

1.22 Ovary (sheep). A knife mark, caused by a nick in the

microtome’s cutting edge, leaves a straight line across the

section (arrowed). Masson’s trichrome. ×25.

1.22

1.23 Uterus (cat). Shrinkage of the adhesive medium

used to mount the coverslip to the slide captures air and

causes bubbles. H & E. ×65.5.

1.23

1.25 Cloacal bursa (bird). Raised areas, overlapping folds

and cracked and separated tissue are present because it is

often difficult to flatten the tissue completely, particularly

in very thin sections. H & E. ×125.

1.25

1.24 Spleen (bird). When crystals accumulate in the stain

solutions or are not removed during standard processing,

stain deposits precipitate onto the surfaces of the tissue

section. H & E. ×25.

1.24

1.26 Spleen (dog). Compression of the paraffin-

embedded tissue causes parallel ‘chatter’ marks.

H & E. ×25.

1.26

Comparative Veterinary Histology with Clinical Correlates

Clinical correlates

In order to appreciate the often subtle alterations

that accompany disease or other physical abnor-

malities, it is useful to compare the characteris-

tic changes by which histopathological diagnoses

are made and classified. To that end, clinical cor-

relates sections are inserted throughout this text.

It is important to note that in many instances the

tissues comprising an organ of one species are

similar or even identical to those found in a dis-

parate species.

Generally, there are fewer substantive differ-

ences within a phylogenetic group of animals

than between different groups of animals. For

instance, the livers of sheep, cattle, horses,

swine, dogs and cats are relatively quite similar;

the liver tissues of many fish resemble the

hepatic tissue found in amphibians; and the

hepatic tissues of many reptiles resemble those

observed in birds. Because of these characteris-

tic similarities and differences, we have selected

examples of tissues that are particularly instruc-

tive in order to avoid showing repetitively the

same tissues for every animal, irrespective of its

phylogeny. However, examples from a wide

variety of species are included for purposes of

comparison.

These correlates are placed where they most

readily illustrate specific, clinically significant

medical conditions. Recognizing normal tissue

facilitates interpreting the often subtle alterations

in abnormal tissues. Where appropriate, the

physiological attributes or significance, or both,

of a particular organ or structure are discussed

briefly so that their importance to the survival of

the animal becomes apparent.