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Laboratory 1: Introduction to Pathology; Cell Injury,

Cell Adaptation and Cell Death

Part A: Examination of Specimens and Application to Differential Diagnosis

Key Concepts:
Systematic Examination of Gross Specimens
Systematic Examination of Microscopic Specimens
Approach to Differential Diagnosis

Explanatory Comments:

The purpose of subsequent laboratories is for you to begin to understand how to use your
knowledge of basic pathogenic mechanisms to understand the clinical and pathologic
manifestations of disease in patients. The laboratory exercises are centered around
hypothetical clinical cases matched with gross and histologic specimens that display
pathologic processes that could cause the signs and symptoms of disease described in the
case summary.

For each case, evaluate the provided information and specimens carefully, and answer the
questions. You may use any sources of information to answer these questions, including
texts, lecture notes, and consultation with fellow students and lab instructors. The
questions are designed to direct your thoughts about the cases rather than to test detailed
knowledge. Although the questions will usually address rather general issues, as you are
reaching your conclusions, you should think about and discuss the underlying basic
pathogenic events. The questions are an aid to learning the material, thus completion of
the questions does not indicate that you have learned all that is appropriate from the
laboratory material. A major learning objective of the laboratories is also to expand your
medical vocabulary. It will be useful to have a medical dictionary available.

Guide to Gross and Microscopic Examination of Specimens

The following guide to gross and microscopic examination of pathologic specimens was
adapted from material by Dr. R. D. Cardiff, Dept of Pathology, UC-Davis with additional
materials added by Dr. S. V. Smith, Dept of Pathology and Laboratory Medicine, UNC-
Chapel Hill. This systematic approach is recommended when studying all specimens.

I. Systematic Approach to Examination of Gross Pathology Specimens

The aim of this document is to present students with a framework for a logical,
systematic approach to analysis of gross specimens. The logical steps to analysis are as
follows:
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A. Identify the organ or tissue

This is easy in most instances, especially if you are presented with fresh, unfixed tissues
having all of their brilliant colors and characteristic textures intact. However, usually you
will be confronted with formalin-fixed tissue that has altered color and firmer texture. Yet
these specimens contain much information that will illustrate the effects of pathologic
processes on the body.

To identify the organ or tissue, focus your attention on familiar features. Examine
external and internal surfaces carefully for clues to the tissue source. Examine the
architectural configuration. Note the shape if the entire organ is available. Even when
they contain pathologic lesions, organs typically reflect their normal surface contours and
shapes which facilitate their identification. Note the color of the organ or tissues.
Is there a capsule or serosa? Is the cut surface hollow (gut), spongy (lung), solid (liver,
kidney, spleen, etc)? Does it demonstrate substructures typical of different organs? Note
the blood vessels supplying and draining the tissues, which tend to be characteristic for
different organ types. Also note excretory ducts (if any) for evidence of particular tissue
or organ type. Identify the normal and abnormal portions of the organ.

B. Identify lesions

Discern the pathologic lesions, i.e. the abnormal areas that reflect disease involvement.
The lesions are not typical of normal structures. Lesions may be focal, involving only a
small part of the specimen, or diffuse, permeating most or the entire specimen. Focal
lesions may include areas of necrosis (infarcts), focal inflammatory exudates (abscesses
and granulomas), fibrosis (scarring) and neoplasms (benign or malignant). Diffuse lesions
may include disseminated necrosis, widespread acute inflammation, leukemic or
neoplastic infiltrates, and parenchymal hypertrophy or hyperplasia. Focal lesions are
usually sharply demarcated from normal tissues, whereas diffuse lesions are more
vaguely delineated. Diffuse lesions may be reflected grossly only by an increase in the
size of an organ or thickness of a tissue. Remember that lesions may be discerned by
differences in color, texture, consistency, and/or shape.

Attempt to integrate the lesion, its location, and its impingement on normal structures
with its probable perturbing effect on tissue or organ function from a purely mechanical
or "space-occupying" effect. (i.e., does the lesion obstruct or narrow blood vessels, ducts,
airways, etc?) Does the lesion obliterate functioning subunits, such as pulmonary alveoli
or secretory units of secretory organs, etc?

C. Identify Key Diagnostic Features of Lesions

1. For focal lesions:

a. follows distribution of bloody supply
b. is solid or forms cavity
c. cavity has thin or thick walls; internal surface shaggy or smooth
d. lesion is expansile, clearly demarcated
e. lesion infiltrates normal tissues almost insensibly
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2. For diffuse lesions:

a. the organ is enlarged (organomegaly), either because of hypertrophy,
vascular congestion, leukemic infiltrate, edema, or inflammation
b. cardinal signs of acute inflammation
c. fibrosis (chronic inflammation)
3. For lesions on mesothelial surfaces:
a. smooth, dense, tough adhesions
b. shaggy, easily disrupted adhesions

Obviously, these features involve multiple complex mechanisms, which will initially
require help from your laboratory instructors to identify. You will progressively be able
to identify and understand these visual and tactile differences as you begin to understand
the mechanisms and cellular components of particular lesions.

D. Apply diagnostic criteria

You gradually learn criteria for making diagnostic decisions from your insight into the
natural history of disease, and the features of the different stages of the disease. This
requires an integration of macroscopic findings, histologic features, laboratory findings,
and clinical signs and symptoms for successful diagnosis. Making a diagnosis is an
"intellectual sifting" process in which you gradually approach the correct diagnosis
by exclusion, rather than by making a "snap or flash" determination. First, make an
evaluation of the possible underlying disease process and then attempt to narrow your
decision. Evaluate the lesion and assign it to a major category of disease. Remember
“VINDICATE”.

Major Categories of Pathologic Processes

Vascular
Inflammatory
Neoplastic
Drug
Infectious
Congenital (inherited, maldevelopment)
Allergic (autoimmune, immune injury)
Trauma (physical injury)
Endocrine (metabolic, nutritional imbalance)

Make a list of the possibilities, and decide why the lesion confronting you does or does
not fit each of the possibilities. This list is termed a "differential diagnosis", and it is an
essential method for good clinicians to reach a diagnosis. For example, once you have
constructed your list, ask yourself: "If this process is neoplastic (or inflammatory, or
vascular injury, etc.), then it should have the following diagnostic criteria." Does it?

If diagnostic criteria do not seem to apply, do not guess at the diagnosis. Beginning
students (and sometimes trained physicians) make the mistake of guessing or jumping to
conclusions on the basis of insufficient evidence. If you rush to an improper conclusion,
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your patient will suffer and you will get stuck with malpractice judgements. If the
diagnosis is unclear, retrace your choices and develop new information to derive a
differential diagnosis.

E. Record gross impression

Write out your conclusion. Commit yourself to an opinion or brief list of diagnostic
possibilities. This is an impression, not an absolute diagnosis. You will make your
diagnosis more precise by adding other information gained from the study of your
patient, such as clinical signs and symptoms, microscopic evaluation of histologic and
cytologic lesions, and other laboratory data (biochemistry, microbiology, etc). Remember
that many lesions cannot be precisely diagnosed at the gross level. Be sure to use all of
the information you can develop, including the examination of gross specimens.

F. Confirm gross impression

Confirm gross impression with microscopic examination and/or clinical laboratory

assessment. Proceed with a systematic approach to microscopic examination.

II. Systematic Approach to Microscopic Examination of Glass Slides

Analysis of histologic slides involves a formal process much like that described for gross
specimens, except that the microscope is used to aid in visualizing the tissue and cells.

A. Histologic Stains

The majority of sections that you will examine in the laboratory will be stained with
hematoxylin and eosin. Hematoxylin stains cellular structures that contain a net excess of
acidic (negatively charged) chemical groups deeply blue to purple. Eosin stains cellular
structures that contain a net excess of basic chemical groups pink to red. Thus,
hematoxylin is termed a basophilic stain (i.e., reactive with groups that "love" base =
acids), whereas eosin is acidophilic (i.e., reactive with groups that "love" acid = bases).
Eosinophilic is sometimes used as a synonym for acidophilic.

B. Examine Slide with Unaided Eye

View the tissue section with your unaided eye by looking at it with transmitted light, e.g.
hold the slide up to the ceiling light and examine the tissue section. Most normal tissue
will appear more-or-less uniformly pink. Note any irregular areas of staining. Deep blue
areas usually indicate foci rich in cell nuclei, such as malignant neoplasms or focal
inflammation. Pale pink areas frequently indicate a site of confluent cell necrosis, such as
an infarct, or scarring. At the least, such geographic patterns will indicate important areas
to investigate more closely.
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C. Examine Sections at Low Magnification

Examine the tissue sections at low magnification. Use the 4X objective to scan the slide
for normal and abnormal areas. Identify the tissue or organ by discerning normal
landmarks. Knowledge of normal histology is essential. Review if you have forgotten.
Identify the lesion(s) of interest. Use the 10X objective lens to further visualize these
areas, note the key histopathologic features, and confirm your diagnosis. Search for
diagnostically critical (i.e. pathognomonic) features. Discuss with laboratory instructor
for clarification of these features.

D. Examine Sections at Intermediate Magnification

The 20X objective may be used for assessment of cellular detail. After identifying the
type of lesion (inflammatory, neoplastic, etc.), evaluate the cytologic details at
intermediate magnification as needed. Rarely will you need to use the 40X (high dry)
objective. You should virtually never use the 100X oil-immersion objective unless you
are trying to visualize bacteria or cellular inclusions. The following chart gives the colors
of cellular structures in histologic sections stained with hematoxylin and eosin.

Colors of Cellular Structures on Conventional H&E Stain

Acidophilic: Basophilic: Other:

(pink-red) (blue-black)
Nucleus +
Nucleolus + +
Cytosol +
Ribosomes +
Mitochondria +
Protein +
Alcoholic hyaline +
Russell body +
Fat Clear, spherical droplets
Fluid Clear, irregular swelling of lysosomes,
mitochondria, endoplasmic reticulum
Glycogen Clear, irregular cytosol or lysosomal
accumulation
Hemosiderin Refractile, yellow-brown iron pigment
Lipofuscin Yellow-brown aging pigment; seen
especially in heart and neurons
Melanin Brown-black pigment in normal skin and
malignant melanoma
Bilirubin Green-brown accumulation in hepatocytes
Anthracotic pigment Black carbon in lungs and lymph nodes
Crystals Refractile, polarizable
Calcium +
Bacteria + +
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III. Finalize Your Diagnosis

Compare gross impression and histologic diagnosis, if both gross and histologic
specimens are available on the same tissue. Integrate clinical laboratory data and clinical
information (signs and symptoms) into final diagnosis.

Part B: Cell Injury and Cell Death

Key Concepts:
Fatty charge
Hemosiderosis
Hypertrophy
Hyperplasia
Coagulative necrosis
Caseous necrosis
Fat necrosis
Metaplasia

Apply a systematic approach to differential diagnosis for these pathologic processes.

Case 1

A 28-year-old alcoholic died in a car wreck. At autopsy, the liver was noted to be
markedly enlarged.

Examine the gross liver specimen for this case, and compare it to the normal liver tissue.
Note that the liver is very pale. Also feel the texture of the surface.

Examine slide A-2. There is no normal liver present. Examine the pale hepatocytes,
which show the histologic basis for the gross changes.

Question 1) What is the major pathologic lesion?

a. enzymic fat necrosis

b. fatty change of hepatocytes
c. hydropic change in tubular epithelial cells
d. hepatitis with hepatocyte apoptosis

Question 2) What is the likely mechanism of hepatic cell injury?

a. bile stones obstructing pancreatic duct

b. infarction caused by arterial thrombo-emboli
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c. abnormal lipid metabolism resulting from alcohol toxicity

d. trauma from automobile accident

Question 3) The clear vacuolated hepatocytes were filled with what substance (in vivo)?

a. water
b. bile
c. DNA
d. lipid

Case 2

A teenager with a severe congenital anemia had long standing hemolysis, which required
many blood transfusions. The patient eventually developed clinical evidence for cardiac,
hepatic and pancreatic dysfunction; including hepatomegaly, abnormal liver function
tests, and abnormal glucose tolerance.

Examine the gross liver specimen, and the histologic sections on slide B-70, which are
sections of diseased liver and pancreas stained with H&E (on the right hand side of the
slide) and a stain which stains iron blue (on the left hand side). (Normal pancreas is
available for reference on slide B-64).

Compare the H&E stained tissue to the iron stained specimens. Note the blue
hemosiderin pigment visible in the iron stained tissues. The H&E stained sections
demonstrate the body's response to the excess iron: parenchymal cell necrosis,
lymphocyte and macrophage infiltrates, and increased fibrous connective tissue (scar).

Question 4) The hemosiderin pigment in liver and pancreas came from what likely
source?

a. rare meat
b. spinach
c. hemolysis of red blood cells
d. rusty iron pots and pans used in cooking

Question 5) The cell injury in liver and pancreas is caused by:

a. tissue hypoxia secondary to anemia

b. systemic iron overload, increased cellular uptake, and resulting oxidative stress
c. increased production of lipofuscin by injured macrophages
d. hemorrhage into the tissues
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Case 3

You have been caring for a patient with difficult to control hypertension for many years.
Over the past several years, on physical exam, you have noted a progressive increase in
the intensity of the ventricular impulse on the chest wall and a shift in its position further
to the left. Recently, the patient had some dizzy spells and was found to have recurrent
arrhythmias. The patient died suddenly at home.

Examine the gross heart specimens. An abnormal heart representative of this patient's
disease (as well as a normal heart are available for examination.

Also examine slide A-13, which has two sections, one from a diseased and one from a
relatively normal heart. Compare the two specimens on the slide for myocyte number,
size and cellular contents. This is most easily done by positioning the slide so that you
see both specimens in one view (if possible).

Determine which specimens are normal and abnormal. Determine the gross and
microscopic structural lesions (abnormalities) in the diseased specimens.

Question 6) The major abnormalities are:

a. gross left ventricular wall thickening with cardiomyocyte hypertrophy

b. gross left ventricular wall thickening with cardiomyocyte hyperplasia
c. gross atrial enlargement with lipofuscin pigment deposition
d. atrial atrophy with cardiomyocyte apoptosis

Question 7) The pathologic process is:

a. myocardial infarction
b. viral myocarditis
c. myocardial hypertrophy
d. atrial fibrillation

Question 8) Chronic hypertension causes:

a. cardiac atrophy due to poor perfusion

b. hepatic necrosis due to right-sided heart failure
c. liquefactive necrosis due to increased blood pressure
d. cardiac myocyte hypertrophy due to increased workload

Case 4

A 59-year-old patient with a long history of tobacco abuse developed severe substernal
chest pain, sweating and pallor. Clinical evaluation included laboratory testing for
intracellular myocardial enzymes, which are released into the blood after myocardial cell
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death. These tests confirmed the presence of myocardial necrosis (i.e., infarction). After
initial improvement, the patient developed progressive heart failure. Ultrasound analysis
revealed a thrombus attached to the endocardial surface of the injured myocardium. Prior
to death, the patient developed marked cyanosis in several digits.

Gross and microscopic (slide A-5) preparations of spleen tissue are available for
examination. When examining the slide, first hold it up to the light and note the dark
area of normal tissue, and the large pale area of tissue injury. Compare the injured tissue
to the normal.

Question 9) What pathologic lesion is present in the histologic specimen?

a. enzymic fat necrosis

b. caseous necrosis
c. fatty degeneration
d. coagulative necrosis

Question 10) The pale zone in the spleen represents:

a. focal areas filled with pus

b. segments of coagulative necrosis
c. germinal centers
d. scar tissue

Question 11) Which is the most likely sequence of events in the pathogenesis of the
splenic abnormality:

a. Hyperperfusion due to hypertension producing oxidative stress and cell death

b. Tobacco-specific carcinogens inducing splenic apoptosis
c. Embolization of mural thrombus leading to splenic arterial infarcts
d. Embolization of necrotic cardiac myocytes leading to splenic infarcts

Case 5

A 50-year-old male debilitated by a chronic illness developed worsening coughing, night

sweats, weakness, easy fatigability, anorexia and fever. Chest radiographs revealed
pulmonary infiltrates, nodules and cavities. Sputum cultures documented tuberculosis.

Examine the gross lung specimen and slide A-9. Find the caseous necrosis in both.
When examining the slide, first hold it up to the light and note the multiple purple foci,
which are TB granulomas containing caseous necrosis. Two abnormal specimens are
mounted on each slide. (Slide B-22 is normal lung, if needed for reference).

Question 12) Histologic examination of lung revealed:

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a. foci of granulomatous inflammation composed of activated macrophages, giant cells

and a necrotic core
b. alveolar spaces filled with fibrino-purulent exudate
c. diffuse coagulative necrosis of alveolar epithelial cells with intra-alveolar hemorrhage.
d. no abnormality

Question 13) Unlike coagulative necrosis, caseous necrosis is:

a. caused by ischemia
b. caused by thrombo-emboli
c. caused by mycobacteria
d. caused by antibodies reacting with bacterial cell walls

Case 6

A 46-year-old female developed excruciating epigastric pain. Physical examination

revealed a markedly tender abdomen and a vague left abdominal mass. Laboratory tests
demonstrated an elevated serum amylase. A diagnosis of acute pancreatitis was made.

Examine the gross specimen of abnormal pancreas (which has so much distortion of
architecture that it is difficult to assess), and compare it to the piece of relatively normal
pancreas. Examine slide A-8. (Compare to slide B-64 normal pancreas if needed). Look
for enzymic fat necrosis in the adipose tissue on the periphery of the pancreas. These
regions contain normal as well as injured adipocytes.

Question 14) Gross inspection of the diseased pancreas revealed:

a. cavitating lesions filled with pus

b. chalky white areas in the fat and dark areas of hemorrhage
c. segments of ischemic necrosis
d. pancreatic ductular hyperplasia

Question 15) Which enzymes released from pancreatic parenchyma will cause necrosis of
lipocytes?

a. deoxyribonucleases
b. collagenases
c. lipases
d. amylases
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Case 7

An 87-year-old man with complaints of urinary frequency, was found to have and
enlarged prostate on physical exam.

Examine the gross specimens, compare normal to diseased. Compare slides C-78
(normal prostate) to slide 79 (prostatic hyperplasia). Contrast the glands (number per
viewing field, size) and stroma (distribution, amount).

Question 16) Cellular hyperplasia can arise as an adaptive response to stresses. It can
also occur as a nonadaptive, pathological change, such as prostatic
hyperplasia. Which of the examples below are adaptive responses.

a. breast duct hyperplasia during pregnancy

b uterine smooth muscle hyperplasia during pregnancy
c. lymph node germinal center hyperplasia during infections.
d. a, b, c

Case 8

A 42-year-old smoker who has no pulmonary signs or symptoms.

There is no gross specimen. Examine slide A-32, a cross-section of a bronchus.

Examine the entire epithelium lining the lumen. Normal bronchial epithelium is
pseudostratified ciliated columnar. Find the region(s) of metaplastic stratified squamous
epithelium.

Question 17) Metaplasia of the bronchial epithelium in this patient is:

a. a reversible adaptation to harmful components in smoke

b. an irreversible injury caused by harmful components in smoke

Additional study questions:

Question 18) Activated oxygen species cause cellular injury by:

a. inhibiting mitochondrial electron transport and ATP production

b. damaging membranes, proteins, and DNA
c. stimulating glycolysis
d. inhibiting cellular caspases

Question 19) Hydropic swelling is an indicator of:

a. acute, reversible cell injury

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b. apoptosis
c. necrosis
d. symbiosis

Question 20) An example of metaplasia is:

a. fatty degeneration
b. ischemic necrosis
c. squamous differentiation in bronchial epithelium
d. accumulation of lipofuscin pigment

Question 21) The executioners of apoptosis are:

a. Epidermal growth factor

b. Serum glucagon
c. Secretory immunoglobulin
d. Cellular caspases

Question 22) Necrosis differs from apoptosis by:

a. inducing inflammation strongly

b. stimulating hyperplasia
c. inhibiting clonal expansion
d. presence of nuclear degradation