Basic Principles

of Histology 1
INTRODUCTION
Histology (microanatomy) is the study of the human body at a tis-
sue or sometimes at a cellular level. As disease processes occur at
the molecular/cellular levels, manifestations of the disease processes
are readily and economically observed at the tissue level using a
microscope. To examine tissues under a microscope, several steps
to acquire, fix, and stain the samples are necessary. In each of the
preparatory steps, a variety of artifacts may be introduced to the tis-
sue samples. A variety of staining agents and methods are available
as are types of microscopes to help observe necessary cellular and
histologic features.

BASIC PRINCIPLES

TECHNIQUES IN HISTOLOGY
Methods Purpose
Tissue preparation
1. Tissue acquisition: Biopsy, surgical 1. Sampling tissue to examine
resection microscopically
2. Fixation: Placing tissue samples in a 2. Stopping tissue degradation, killing
fixative microorganisms
3. Processing: Series of chemical and 3. Removing water from tissue, infiltrat-
heat treatment ing the tissue with hardening agent
4. Embedding: Placing tissue into a hard- 4. Placing the tissue into rigid mold
ening agent (paraffin) in a tissue block
5. Sectioning 5. Slicing the tissue into thin sections
(7–12 μm)
6. Staining 6. Staining otherwise transparent tissues
with different types of dyes or chemi-
cals to observe cellular details

(continued)

1
2 LIPPINCOTT’S POCKET HISTOLOGY

TECHNIQUES IN HISTOLOGY (continued)

Methods Purpose
Staining methods
1. Hematoxylin and eosin 1. Staining basic or acidic
1
(H&E): Most common structures of the
staining method using a tissue
two dyes
a. Hematoxylin: Basic, a. Purple to blue dye:
positively charged b Attracted to acidic,
dye negatively charged
cellular structures
such as DNA and
RNA in nuclei and
on ribosomes in
cytoplasm
b. Eosin: Acidic, nega- b. Pink to red dye:
tively charged dye Attracted to basic,
positively charged
cellular structures
(many proteins) in
cytoplasm
2. Histochemistry: 2. Chemical reaction
Staining chemicals between the staining
bind or react with agent and tissue struc-
certain cellular tures generates color.
c
structures
c. Masson trichrome: c. Identifies connec-
Stains collagen and tive tissue content,
mucus blue, cyto- organization, and
plasm pink makeup
d. Periodic acid– d. Identifies areas of
Schiff (PAS): d high polysaccharide
Polysaccharide such concentration such
as glycogen turns as basement mem-
dark red color. brane and goblet
cells
3. Immunohisto- 3. Identifying cells or tis-
chemistry: Applies 3 sues that expresse the
specific antibody protein of interest
targeted at an anti-
gen of interest and
secondary antibody
tagged with chemical
agent that generates
brown color
 CHAPTER 1 • BASIC PRINCIPLES OF HISTOLOGY 3

Methods Purpose
Staining methods
4. Immunofluorescence: 4 4. Identifying cells or
Similar to immu- tissues that express
nohistochemistry the protein of interest,
in application of may be able to tag
specific antibody, but more than one specific
the secondary anti- protein with different-
body is tagged with colored fluorescence
fluorescent agent, can
tag more than one
specific protein with
different color

Additional Concepts
• Eosinophilia (acidophilia): Tendency for cell or tissue structures
to stain well with eosin, the acidic dye. Most cytoplasmic proteins
are eosinophilic (acidophilic); they stain particularly well with
eosin.
• Basophilia: Tendency for cell or tissue structures to stain well
with hematoxylin, the basic dye. Nuclei, nucleoli, and cytoplasmic
ribosomes are basophilic structures; they stain particularly well
with hematoxylin.
• Other naturally occurring pigments in cells
• Melanin: Black-brown pigments in certain types of cells such as
keratinocytes of the skin
• Lipofuscin: Yellow-brown pigment particles that accumulate
in certain types of cells such as cardiomyocytes, neurons, and
hepatocytes. Thought to be the residues of lysosomes
• Artifacts: Any artificial structures, defects, or observations that
were introduced during preparatory steps and are not naturally
present in vivo. Common artifacts observed in histologic tissue
slides include dust particles, separation or folding of tissue slice,
exaggeration of spaces between cells and tissues, and empty space
effect in previously lipid-filled areas.
4 LIPPINCOTT’S POCKET HISTOLOGY

CYTOLOGY
Structure Function Location
Nucleus
Oval to spheri- Storage of DNA Central to peri-
cal, basophilic 1 and regulation central in most
structure within of gene expres- cells
most cells sion
2
1. Nuclear 1. Forming a 1. Surrounding
envelope: Two tightly con- DNA content
phospholipid trolled bar-
b
bilayers rier between
the nucleus
and cyto-
c plasm
a. Nuclear a a. Regulating a. Through-
pore: transport out
Opening across nuclear
in nuclear 1 nuclear envelope
envelope envelope
2. Nucleolus: b 2. Ribosomal 2. Within
Small, round, RNA (rRNA) nucleus of
basophilic c assembly translation-
structure ally active
cells
3. Chromatin: 3. Organization 3. Within
DNA in orga- 1 of DNA nucleus
nized spool
form
b. Euchro- b. Areas b. Transcrip-
matin: a more tionally
Unspooled accessible active cells
chromatin, by tran- have more
relatively scription euchroma-
pale stain- proteins tin than
ing areas of hetero-
nucleus chromatin
c. Heterochro- c. Areas less c. Transcrip-
matin: accessible tionally
Tightly by tran- inactive
spooled scription cells have
chromatin, proteins more het-
darker stain- erochro-
ing areas of matin than
nucleus euchroma-
tin
 CHAPTER 1 • BASIC PRINCIPLES OF HISTOLOGY 5

Structure Function Location

Other major organelles
1. Golgi: Stack 1. Posttransla- 1. Perinuclear
of membrane- b tional in most
bound sacs 1 modification, cells; well
sorting, developed
packaging in secretory,
proteins translation-
a ally active
cells
a. Cis-face: a. Receiving a. Closer to
Flattened newly nucleus
sacs formed
proteins
b. Trans-face: b. Sending b. Farther
Curved sacs out from
modified nucleus
proteins to
appropri-
ate loca-
tions in the
cell
2. Mitochondria: 2. Large 2. Numerous
2
Spherical to amount of in cells that
elongated oval c adenosine generate and
structure with triphosphate expand much
two mem- (ATP) genera- energy
branes tion
c. Outer c. Forming c. Outer
d
membrane: an outer layer of
Smooth boundary, mitochon-
outer layer containing dria
ATP trans-
porters
d. Inner mem- d. Containing d. Inner layer
brane with machiner- of mito-
cristae, ies for chondria
complex aerobic
infoldings respiration
and large
amount of
ATP gen-
eration
(continued)
6 LIPPINCOTT’S POCKET HISTOLOGY

CYTOLOGY (continued)
Structure Function Location
Other major organelles
3. Rough 3. Protein syn- 3. Abundant in
3
endoplasmic thesis translation-
reticulum ally active,
(rER): Series secretory
of membrane- cells
bound tubules
and sacs with
ribosomes on
the outside
4. Smooth 4. Producing 4. Abundant in
endoplasmic membrane cells involved
reticulum materials, in lipid
(sER): Series lipid metabo- metabolism
of membrane- lism
bound tubules
without ribo- 4
somes
Cytoskeleton
Collection of fila- a Providing struc- Throughout cell
mentous fibers in
1 tural support, cytoplasm
various orienta- mechanism
tions in a cell for cellular
NH2 movements,
scaffolding
b and anchoring
for organelles;
COOH participating
in intracellular
trafficking
1. Actin fila- 1. Locomotion 1. Abundant
ments: Thin fil- of cells, cellu- in muscles
aments 6–8 nm lar processes; within
in diameter; forming contractile
lengths vary structural machinery,
a. Actin core of core of
monomer microvilli microvilli
subunits
2. Intermediate 2. Supporting, 2. Throughout
filaments: providing cytoplasm in
Rope-like 2 general most cells
filaments structural
8–10 nm in scaffolding to
diameter a cell
 CHAPTER 1 • BASIC PRINCIPLES OF HISTOLOGY 7

Structure Function Location

Cytoskeleton
Many different
types are pres-
ent but are
expressed in a
tissue-specific
manner
b. Eight tet-
ramers of
filamentous
monomer
protein
3. Microtubules: 3. Intracellular 3. Throughout
Hollow tubular transporta- cytoplasm
protein fibers tion, gen-
20–25 nm in eration of cell
diameter com- motility
posed of tubu-
lin proteins
a. Centriole:
3 24 nm a–b. Controll- a–b. Close
Cylinder of ing micro- to nucleus
short nine tubule
microtubule formation
triplets
b. Centro-
some: Two
centrioles at
right angle
to each a
other
c. Axoneme: c. Movement c. Core of
Cylinder of of cilia, cilia and
nine micro- flagella flagella
tubule dou- b
blets with
two single
c
microtu-
bules in the
center
8 LIPPINCOTT’S POCKET HISTOLOGY

Additional Concepts
• Tissue-specific intermediate filaments: There are several dif-
ferent types of intermediate filaments and they are expressed in
a tissue-specific manner (i.e., keratin intermediate filaments are
only expressed in epithelial-derived cells and vimentin intermedi-
ate filaments are only expressed in mesenchymal-derived cells).
Such specificity is useful when identifying the tissue origin of
metastatic or dedifferentiated tumors.
• Cytologic features indicating cellular activity: Large nucleus;
general euchromasia; distinct, large nucleolus (sometimes more
than one); well-developed Golgi; and basophilic cytoplasm indi-
cating abundant RNA associated with ribosomes all hint at rich
transcriptional and translational activity of the cell. On the other
hand, small and mostly heterochromatic nucleus, indistinct
nucleolus, and scant cytoplasm indicate cellular inactivity.

MICROSCOPY
Type Function
1. Standard microscopy utilizing natural
1
light to observe tissues stained with
H&E, other histochemistry and immu-
nohistochemistry
Light
a. Phase contrast microscopy: Utilizes
slight refractory differences
between cellular parts to observe
unstained tissues and live cells
2. Used to observe fluorescently dyed
2
tissues (immunofluorescence), utiliz-
Fluores- ing UV rays or lasers to excite the
cence fluorescence-tagged epitopes

3. Capable of focusing on a single plane

3
within a tissue, reducing the noise
created by other layers within the
tissue
Confocal
 CHAPTER 1 • BASIC PRINCIPLES OF HISTOLOGY 9

Type Function
4. Utilizes electrons rather than photons
a
to observe cellular structures at much
higher resolution
a. Scanning electron microscopy
allows observation of surface fea-
tures
b. Transmission electron microscopy
allows observation of cellular struc- b
Electron tures in 2-dimension