6 Histopathology

Objectives
In this chapter we will study
• some of the factors leading to cellular injury and necrosis;
• the effects of cell damage or cell death on tissues and organs; and
• how histotechnologists and histopathologists study diseased tissues.

Cellular Injury and Tissue Damage in three ways: lipid peroxidation (destruction of unsaturated fatty
Cells are vulnerable to injury by such chemical and physical fac- acids), protein fragmentation, and DNA alterations. Of the three,
tors as oxygen deprivation (hypoxia), free radicals, toxic chemi- lipid peroxidation is probably the most destructive. When the free
cals, nutritional imbalances, temperature extremes, acids and radical (usually OH∙) reacts with the double bond of an unsatu-
bases, trauma, and radiation. Cells can often respond to such dis- rated fatty acid, the reaction generates peroxide. The peroxide then
turbances by making homeostatic adjustments in their metabolism, initiates a series of reactions that damage the plasma membrane
but severe disturbances may cause structural changes that can be and the membranes of the cell’s organelles, killing the cell. In pro-
observed microscopically. The study of such structural changes tein fragmentation, peroxide reacts with specific amino acid side
and the diagnosis of disease from histological evidence—that is, chains and irreversibly disrupts the secondary and tertiary struc-
the clinical application of histology—is called histopathology. tures of the protein, leading to fragmentation. When peroxides in-
Irreversible damage to cell structure and function leads to cell teract with many parts of the DNA molecule, DNA alterations are
and tissue necrosis—pathological death, as compared to apoptosis, induced that destroy the cell’s genetic functions.
the “programmed death” of cells that were destined to die in the
Chemicals
service of normal bodily development and function. Some of the
causes of cellular injury are described here. Chemical injury occurs when a toxic substance damages the vari-
ous membranes of the cell. This increases membrane permeability
Hypoxia and causes the cell and its organelles, especially the mitochondria
Hypoxia, or oxygen deficiency, is the most common cause of cell and endoplasmic reticulum, to swell with water. ATP synthesis and
death. It can result from ischemia (deficient blood flow to a tis- protein synthesis thus break down, gravely compromising the cell’s
sue), respiratory disorders such as emphysema that interfere with homeostasis. If unchecked, damage to the lysosomes also occurs,
oxygen exchange with the blood, or various kinds of poisoning and the cell undergoes autolysis (self-digestion).
that interfere with the blood’s ability to transport oxygen (carbon
Nutritional Imbalances
monoxide poisoning) or the tissues’ ability to use the oxygen they
receive (cyanide poisoning). Hypoxia forces cells to shift to anaer- Normal cellular function depends on the availability of adequate
obic fermentation, which may be harmful in two ways: (1) It may nutrients such as amino acids, glucose, lipids, minerals, and vi-
produce too little ATP to meet the cell’s needs, causing the cell to tamins. Either excessive or insufficient nutrient levels can harm
die from a shortage of usable energy, and (2) hypoxia produces cells. Usually our diets and the actions of the digestive and car-
lactic acid, which lowers the pH of the cell, especially in ischemic diovascular systems ensure that cells are appropriately nourished.
tissues where blood flow is inadequate to remove the lactic acid Some specific cellular effects of nutritional deficiencies are dis-
from the tissue. cussed in chapter 26.

Free Radicals Temperature Extremes

Free radicals are atoms or molecules that have unpaired electrons Temperature extremes are among the physical factors that can
causing them to be highly reactive in an effort of stealing elec- cause cellular injury. Freezing or chilling cells induces hypother-
trons from other atoms or molecules that need these electrons for mic injury. As ice crystals form and melt within cells, they dam-
im­portant normal physiological processes. Because of this elec- age the plasma membrane and cause an inflow of sodium ions
tron theft, they are termed oxidants. However, our cells produce and water, thus disrupting the cell’s osmotic balance. Depending
antioxidants to alleviate or minimize these deleterious actions. on the rate of chilling, hypothermia can also cause vasoconstric-
Sometimes the oxidant amounts overwhelm the antioxidant tion and ischemia, or it can paralyze the blood vessels in a dilated
amounts, thus the ex­cess oxidants (free radicals) cause oxidative state, increasing flow and producing severe tissue swelling. In
stress. These free radicals can be byproducts produced though a frostbite, blood clotting in the damaged vessels leads to ischemia
cell’s normal biochemical reactions (especially aerobic respira- and gangrene. Hyperthermia (excessive body temperature, either
tion) or through certain environmental exposures, such as radia- because of sun exposure or fever) damages cellular metabolism
tion, sunlight, and certain chemicals. Free radicals can kill cells by speeding up some enzymatic reactions more than others. This

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causes metabolic pathways to get out of synchrony with each other In a hospital setting, histopathology laboratories receive tissues
and leads to derangement of the cell’s homeostasis. Extreme heat and organs from operating and delivery rooms. Histotechnologists
“cooks” the cell’s structural proteins and enzymes, thus causing and pathologists often must prepare and examine the tissue and re-
the immediate tissue death characteristic of burns. port their findings while the patient is still on the operating table
so that the surgeon can determine the appropriate course of action.
Some Signs and Effects of Cellular Injury Before a tissue can be examined microscopically, it must be
sliced into tissue sections thin enough to see through—typically
Injured, failing cells are often unable to maintain normal plasma
about 7 µm thick, which is less than the thickness of many cells.
membrane function, so they accumulate fluid. The waterlogged
This is done with an instrument called a microtome, similar in
cells have a light-staining, “vacuolated” appearance (hydropic
principle to a butcher’s meat slicer but capable of far greater preci-
degeneration). In addition, failing cells are often unable to metabo-
sion. The microtome advances the tissue by fine degrees and shaves
lize fatty acids, and thus they accumulate lipids in the cytoplasm
off a thin slice each time the tissue passes over the blade of an ultra-
(fatty change). Fatty change is commonly seen in the liver cells of
sharp knife. However, tissues fresh from the body cannot be cut on
alcoholics, where gross examination (nonmicroscopic inspection)
the microtome because they are too soft; it would be like trying to
shows a yellowish, greasy-looking fatty liver. This can progress
slice a fresh loaf of bread with a very dull knife. The blade would
to an accumulation of fibrous scar tissue, giving the liver a lumpy
squash the tissue before it cut it and cause so much distortion in the
surface, a state called cirrhosis. Intracellular lipid accumulation is
tissue’s structure that the specimen would be useless for diagnostic
also characteristic of Tay-Sachs and Gaucher diseases (see chapter
purposes. Normally, the tissue has to be embedded in a supportive
4 of this manual).
block of paraffin before it is sectioned. A problem with this is that
Other substances, such as calcium salts and urate, may also
tissue is mostly water, and water and paraffin do not mix. There-
infiltrate damaged cells. Glycogen accumulation is characteristic of
fore, some preparation is needed before the embedding process.
Pompe disease and type II glycogen-storage disease (see chapter 4).
The traditional steps of tissue preparation carried out by a
Pigment accumulation can result from causes as diverse as tattoo-
histotechnologist are:
ing, sun exposure, diets high in carotene, or bilirubin accumulation
in liver failure. • Fixation The tissue is cut into small pieces and immersed
Dystrophic calcification is the accumulation of calcium salts in formalin or another chemical fixative, which prevents decay
in dead or dying cells. This is seen in chronic tuberculosis, ad- and somewhat hardens the tissue. Good preservation typically
vanced atherosclerosis, and injured heart valves. Even normal cells requires at least overnight immersion in the fixative.
accumulate calcium if the concentration of Ca2+ in the blood is
abnormally high. This is known as metastatic calcification, and it
• Dehydration The specimen is immersed in a series of etha-
nol baths of increasing concentration, ending with two baths
can result from vitamin D excess, Addison disease, or bone tumors of 100% ethanol, to remove all the water.
that decalcify the bones and raise the blood Ca2+ level.
Uric acid (or urate, the ionized form) is produced by the • Clearing The tissue is treated with a clearant such as xylol,
clove oil, or wintergreen oil to remove the ethanol. The clear-
breakdown of purines, a component of ATP and nucleic acids.
ant mixes with paraffin.
Tissues begin to accumulate crystals of sodium urate if the serum
urate concentration rises too high (above 4–5 mg/dL, depending on • Embedding The tissue is immersed in melted paraffin,
sex). This triggers an inflammatory condition called gout, which which infiltrates the tissue and replaces the clearant. The paraf-
may include extremely painful joint inflammation (gouty arthritis) fin is then allowed to cool slowly and harden into a solid block.
and the appearance of small, white nodules (tophi) under the skin.
Gout can result from abnormal purine metabolism or from insuffi-
• Sectioning The paraffin block is mounted on the microtome,
and a series of very thin slices are cut from it.
cient urate excretion by the kidneys. About half of all attacks of gouty
arthritis occur in the great toe, and the other half are distributed • Mounting The paraffin-embedded slices are mounted on
microscope slides with an adhesive such as albumin. The
mainly among the heel, ankle, instep, knee, elbow, and wrist. Gout paraffin is then dissolved out, leaving only a ghostly white or
is at least partially hereditary, and about 95% of its victims are men. gray tissue section on the slide.
Methods in Histopathology • Rehydration In preparation for staining with water-based
A histopathologist is a physician (M.D.) specialized in recog- dyes, the paraffin is dissolved away with a solvent, and the
nizing pathological changes in the microscopic appearance of slide is then immersed in a series of progressively lower con-
tissues and cells and making diagnoses based on this appearance. centrations of ethanol to rehydrate it.
A histopathologist is assisted by a staff that may include a • Staining The slide is immersed in one or more dye solu-
pathologist’s assistant, histotechnologists, and histotechnicians. tions that stain different components of the tissue different
A histotechnologist is a person who specializes in preparing colors, bringing out the contrast necessary for visual examina-
histological specimens for microscopic examination by the pa- tion. The most commonly used stains are a pair called hema-
thologist. A histotechnologist generally must have a baccalaureate toxylin and eosin (H&E). Hematoxylin stains cell nuclei blue
degree (preferably in science) and a year of on-the-job training. to violet, and eosin stains the cytoplasm pink. Different stains
Histotechnicians, who assist histotechnologists, typically have a are used for specialized purposes such as staining blood, fat,
2-year diploma or associate degree. or collagen.

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 • Clearing The stained tissue is treated with a clearing agent temperatures of −20°C to −50°C, in a chamber called a cryostat.
again to render it transparent, similar to the way a drop of oil Since freezing hardens the tissue, it takes the place of paraffin
on a piece of paper lets light shine through. embedding—and if paraffin is not going to be used, dehydration,
clearing, and rehydration (the most time-consuming steps) are
• Coverslipping If a specimen is to be kept permanently, a
not necessary. The frozen tissue once sectioned with a freezing
glass coverslip may be applied to the slide with an adhesive.
This is not necessary for routine diagnostic work. microtome is termed a frozen section. The frozen section is then
thawed and stained.
Specimen preparation can require several hours to days, be- Once the tissue specimens are prepared, they are examined
cause the fixation, clearing, dehydration, infiltration, and rehydra- by a pathologist. In some cases, when specialized methods of di-
tion steps must be carried out slowly and carefully to ensure good, agnosis are needed (such as an assay for estrogen receptors as in
useful preparations and to minimize distortion of the tissue. How- the case study that follows), tissue specimens may be sent out to a
ever, if a patient is on the operating table and a surgical decision regional laboratory for preparation and diagnosis. This is routine
must be made, there isn’t time for this classic technique. The pro- in the grading of Pap smears, for example.
cedure can then be accelerated by freezing the tissue, typically at

Case Study 6    Breast Tumors—A Day in the Histopathology Lab

Frances is a board-certified histotechnologist who works at a large Dr. Griffin shows Frances the slides of this biopsy. Mixed
urban hospital. Her job consists mainly of receiving tissues and among the normal fibrous tissue and adipocytes of the breast are
organs from surgeons and preparing them so they can be examined several irregularly shaped clusters of cells and dense fibrous tis-
by the pathologist, Dr. Griffin, for diagnostic purposes. sue. The cells resemble epithelium, but look immature and are not
One day, Frances receives specimens from two patients—Ms. arranged in epithelial sheets. They are not surrounded by a fibrous
Bennett and Ms. Malcolm—who have presented with lumps in capsule, but form tonguelike projections that extend into the sur-
their breasts. The oncologist has excised the lumps and sent them rounding fat and loose fibrous tissue of the breast. Speaking into a
to the histology lab. He needs to know whether the lumps are be- voice recorder, Dr. Griffin scans the slide and describes the cells
nign or malignant, and if malignant, whether they are local or in- as pleomorphic, hyperchromatic, and anaplastic (see “Selected
vasive so that he can decide whether to biopsy the axillary lymph Clinical Terms” for definitions). Many of the cells that Dr. Griffin
nodes for possible metastatic cancer. The patients remain in the points out exhibit mitotic figures. A small area of necrotic
operating room while the specimens are biopsied, so the frozen- tissue appears in the center of the lesion. Dr. Griffin, looking a
section method is used to speed up the diagnostic process. little downcast, says “Well, this one looks malignant—an invasive
Ms. Bennett, the first patient, is a single, 28-year-old gradu- ductal carcinoma, I’d say.” She adds that they will send the speci-
ate student. She had not been in the practice of doing breast self-­ men off to a lab to have a mitotic index and immunoperoxidase
examination (BSE), but was inspired by a newspaper article to start. assay performed.
To her dismay, while doing a BSE in the shower recently, she felt Some breast cancers are estrogen-sensitive, and the immuno-
a bean-sized lump in the lower outer quadrant of her right breast. peroxidase assay is a way of determining whether the malignant
She has visited her physician in deep fear that she has breast cancer. cells have estrogen receptors. To perform this test, immunoperox-
Dr. Griffin invites Frances to look at the specimen through a idase-coupled monoclonal antibodies that are specific for estrogen
dual-head microscope, pointing to a circular, well-circumscribed receptors in the cell nuclei are added to the slide. The slides are
mass surrounded by a layer of dense, irregular, fibrous connective then washed with a peroxide reagent. If any antibody is bound to
tissue. Around it are the adipose and areolar tissue typical of nor- nuclear receptors, it reacts with the reagent to produce a brown
mal breast tissue. The mass is composed of loose fibrous tissue and precipitate. Dr. Griffin shows Frances some older slides so that
cells that resemble those in the mammary ducts. On the basis of this she can see what a positive assay looks like. Most of the tissue is
examination, Dr. Griffin tells Frances that she is diagnosing this as gray in color, but the cell nuclei are brown. “That means this was
a benign tumor called a fibroadenoma, common in young women, an estrogen-sensitive tumor,” Dr. Griffin explains. “This is actually
and she phones Ms. Bennett’s oncologist with the information. good news. It gives Ms. Malcolm a better prognosis, because her
Ms. Malcolm, the other patient, is a 48-year-old attorney and cancer may respond to tamoxifen therapy.”
mother of four who has always been in the habit of doing BSEs.
Based on this case study and other information in this chapter,
She had a baseline mammogram when she was 36 and has been
answer the following questions.
getting mammograms every 2 years throughout her 40s, partly
because both her maternal grandmother and her older sister have 1. Review the characteristics of benign and malignant tumors.
had breast cancer. Additionally, a blood test has revealed she was What findings suggests that Ms. Bennett’s tumor is benign?
positive for the BRCA 1 gene mutation. Ms. Malcolm has never 2. Why might Ms. Malcolm’s doctor recommend biopsy of the
detected a lump in her own breasts, and yet when she had her last axillary lymph nodes?
biennial mammogram, a dense mass about 12 mm in diameter 3. Why would the mitotic index be relevant to a diagnosis of
appeared in the upper left quadrant of one breast. breast cancer?

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 4. Tamoxifen blocks estrogen receptors so that estrogen cannot 8. Mechanical stress or trauma often triggers the symptoms of
bind to them. How could tamoxifen help in the treatment of gouty arthritis in susceptible people. In view of this, explain
certain forms of breast cancer? why gouty arthritis affects the great toe more commonly than
5. Considering that X-rays are known to induce mutations and other joints.
the mutations can cause cancer why are women in certain 9. Patients with gout are often advised to drink 3 liters of
age groups advised to have mammograms? water daily. Why do you think this would help relieve their
6. Explain why prolonged anaerobic fermentation could cause the symptoms?
enzymes of a cell to become increasingly dysfunctional and 10. If you were using conventional paraffin-based histotechnique,
eventually lead to a shutdown of the cell’s metabolic pathways. but staining the tissue with an alcohol-based stain instead of
7. What similarity might you expect in the mechanisms of cell a water-based stain, what step in the preparation of the slide
injury seen in hypothermic injury and dystrophic calcification? could you omit?
Explain why both disorders may have similar effects on a cell.

Activity
Using the microscope observe a tissue slide and blood slide. Re- Look up the definitions of anaplasia, dysplasia, and metapla-
search why many tissue slides use hematoxylin and eosin staining sia. How are these terms applied to tumors?
and blood slides use Romanowsky stains.
Research the physiological significance of heat shock proteins
and stress proteins in cell injury.

Selected Clinical Terms

fibroadenoma A benign neoplasm, common in the breast, com- microtome An instrument that cuts tissue specimens into
posed of fibrous connective tissue, proliferating fibroblasts, extremely thin slices suitable for staining and microscopic
and anaplastic cells derived from the ductal epithelium of the examination.
mammary gland. mitotic figures Darkly staining aggregates of condensed
histotechnologist A specialist in the preparation of tissue chromosomes seen in stained cells, indicating that mitosis
specimens for microscopic examination. was underway when the cell was fixed.
hyperchromatic Staining more intensely than normal with mitotic index A count of the percentage of cells in a given
histologic stains. area of tissue that exhibit mitotic figures. An abnormally
invasive ductal carcinoma An advanced form of breast cancer high mitotic index indicates neoplasia (the development
in which malignant cells of the mammary ducts have broken of a tumor).
through the basement membrane of the duct epithelium and pleomorphic Variable in size and shape.
invaded the connective tissue stroma of the breast. This has
the worst prognosis of any form of breast cancer.

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