Introduction to Histology

2
Maura Bríd Cotter and Massimo Loda

2.1 Introduction to Histology 2.2 Specimen Types

“Histology” is the examination of normal cells Specimens received for histological examination
and tissue and is performed with the aid of a include both cytology specimens and histopatho-
microscope. In contrast, “histopathology,” a logy specimens, examples of which are listed in
subdiscipline within pathology, refers to the Table 2.1. Cytology specimens are taken with
study of diseased tissues, and will be discussed the aim of examining tissue at a cellular level [1].
separately in Chap. 3. Histologists have the These specimens, therefore, include samples of
specialized skills necessary to process and stain free cells or tissue fragments. The most common
various tissue samples, while histopathologists sample type is a fine needle aspiration (FNA),
are physicians with the skills necessary to inter- where a very thin needle and a syringe are used
pret the histological slides. The routine specimen to acquire a small amount of cells or fluids from a
types received in a histology laboratory, and their lesion (e.g. thyroid cyst [2]). Bodily fluids, such
preparation using routine histological techniques, as urine [3, 4], or cerebrospinal fluid [5], etc., can
are first introduced below, followed by an also be processed. Another special sampling
introduction to the important components of a technique is when cells are gently scraped or
normal human cell and the histology of various brushed from an organ (e.g. cervical smear [6]).
normal tissue types. In contrast, histopathology specimens include
whole organs or small samples of larger tissues.
A needle core biopsy is the most common type of
sample where, in comparison to a FNA, a large
needle is used to remove a greater quantity of
tissue. Other sampling techniques available to
clinicians include excisional biopsies where an
entire lesion is surgically excised, or incisional
biopsies where part of a larger lesion is removed.

M.B. Cotter
M. Loda (&)

Department of Pathology, Dana-Farber Cancer
Institute and Brigham & Women’s Hospital, Harvard
Medical School, Boston, MA 02115, USA
e-mail: massimo_loda@dfci.harvard.edu

© Springer International Publishing Switzerland 2017 11

M. Loda et al. (eds.), Pathology and Epidemiology of Cancer,
DOI 10.1007/978-3-319-35153-7_2
12 M.B. Cotter and M. Loda

Table 2.1 Examples of Cytopathology Histopathology

cytopathological and
histopathological Fine needle aspiration (e.g. thyroid cyst) Biopsies (e.g. biopsy of a breast mass)
specimens Smears (e.g. cervical) Surgical specimens (e.g. prostatectomy)
Bodily fluids (e.g. urine) Autopsy specimens (e.g. kidney)

Table 2.2 highlights the various stages of tissue

2.3 Specimen Examination preparation and summarizes the purpose of each
and Sampling stage mentioned below.

Grossing of histopathology specimens [7]

involves careful examination by the pathologist 2.4.1 Fixation of Tissue
including a specimen description, weight, and
measurement of dimensions [8]. Photographs can Fixation involves submerging the sampled tissue
be taken and relevant surgical margins inked. in chemical substances (i.e. fixatives) in order to
Thorough dissection is performed in order to prevent tissue digestion by enzymes or bacteria
locate representative areas suitable for sampling. and to preserve as much as possible of its mor-
Biobanking [9] can also be completed at this phologic and chemical characteristics. Fixatives
stage, which involves taking small samples of promote cross-links between proteins and form a
fresh tissue and is used, for instance, to create gel that maintains the in vivo relations of tissue
cell lines, isolate stem cells, generate organoids components to each other [14]. There are a
or ex vivo organotypic cultures [10, 11] for number of reagents that can be used for fixation,
storage in a tissue biobank. Touch preparations each of which has differing penetration rates.
(or “touch preps”) can also be made using fresh Formaldehyde [15] is the most commonly used
tissue [12, 13], where the specimen is gently agent for histopathology and when dissolved in
touched against a clean glass slide, an imprint water, it is referred to as “formalin.” One part
made, and later examined. formalin is typically diluted with nine parts water
to produce a 10 % formalin solution, a concen-
tration that is optimal for tissue fixation [16].
2.4 Preparation of Histological This solution penetrates tissue at about 1 mm an
Slides hour [17], therefore, biopsies are generally sub-
mitted for processing the same day as received,
The sampled tissue is placed into a plastic cas- while larger specimens (e.g. a mastectomy) are
sette and undergoes a series of steps in order to not processed the same day as received as they
prepare the tissue for histological examination. require a longer fixation period.

Table 2.2 The stages of tissue preparation and the purpose of each stage
Stage Purpose
Sampling To choose the most representative areas of the specimen
Fixation To preserve tissue morphology and chemical composition
Dehydration To remove fixative and cell water and replace with dehydrating fluid
Clearing To remove dehydrating fluid and replace with clearing fluid
Embedding To impregnate with liquid paraffin and make the tissue resistant to sectioning
Sectioning To make tissue sections available for histological analysis
2 Introduction to Histology 13

2.4.2 Processing of Tissue selectively stain various tissue elements. The

constituents that react with basic dyes do so
The processing of tissue includes dehydration, because of acid in their composition (e.g.
clearing, and embedding steps. Dehydration nucleoproteins), while acidic dyes stain basic
involves the removal of fixative and water from tissue components (e.g. cytoplasmic proteins). Of
the tissue and their replacement with dehydrating all routine stains, the combination of hema-
agents by placement in increasing concentrations toxylin and eosin, or the “H&E stain” [19], is the
of ethanol. Next, the clearing step involves most commonly used and dates as far back as the
replacing the dehydrating fluid with a lipid sol- 1870s. This stain is considered the gold standard
vent (e.g. xylene). The tissue is subsequently in histology and in a typical tissue section, nuclei
removed from the cassette and placed in a molten are stained blue/purple, whereas the cytoplasm
wax-filled mold for embedding. At this point, and surrounding matrix have varying degrees of
orientation of the tissue within the mold is criti- pink staining [20]. Therefore, the H&E stain has
cal, as it will determine the plane through which the ability to reveal structural information with
the section will be cut. Incorrect placement of specific functional implications. Special stains
tissues may result in diagnostically important [21] use a slightly different technique to stain
areas being missed or damaged later [18]. Elon- particular structures (e.g. Masson’s trichrome for
gate tissues should be placed diagonally across muscle and collagen fibers [22]) or pathogens
the block (e.g. core biopsy), while tubular (e.g. Ziehl–Neelsen for acid-fast bacteria [23]).
structures (e.g. vas deferens) are embedded so as When routine or special staining cannot provide
to provide transverse sections showing all tissue all the diagnostic answers required, histopathol-
layers. Specimens that have an epithelial surface ogists can use advanced staining techniques
(e.g. skin surface) are embedded in such a way as including immunohistochemistry (IHC) or in situ
to provide sections in a plane at right angles to hybridization (ISH).
the surface.

2.5.1 Immunohistochemistry
2.4.3 Sectioning of Tissue
IHC is a multistep technique involving the
The waxed cassette is next placed in an instru- interaction of a target antigen (i.e. the protein of
ment with fine blades called a microtome. interest) with a specific antibody tagged with a
Rotation of the drive wheel moves the block visible label [24]. The aim is to detect the pres-
holder a controlled distance forwards, the blade’s ence of elevated levels or the absence of a par-
edge strikes the tissue block, and thin sections are ticular target antigen. Antibodies can be coupled
cut and affixed to a glass slide. Sections are with an enzyme, such as horseradish peroxidase
usually four microns thick so that a single layer (HRP), requiring the use of a light microscope
of cells can later be seen under the microscope. for visualization, or with fluorescent chemical
Following thorough drying of the tissue sections compounds requiring the use of a fluorescent
are ready for staining. microscope. Different labeling techniques are
available including the direct or indirect labeling
method. In a direct assay, a fluorophore-labeled
2.5 Staining of Tissue antibody, such as fluorescein isothiocyanate, can
react directly with the target antigen. This tag
As most tissues are colorless, methods of staining allows immediate visualization of the antigen. In
tissues have been developed to make them visi- contrast, in an indirect assay, an unlabeled pri-
ble, while also allowing distinctions to be made mary antibody is used. This binds to the target
between tissue components. This is done by antigen and an enzyme-labeled secondary anti-
using mixtures of acidic or basic dyes that body binds to the primary antibody. In general,
14 M.B. Cotter and M. Loda

Fig. 2.1 Illustration of

immunohistochemistry

primary antibodies are raised against the antigen probe), or RNA strand (or riboprobe) is used to
of interest and are unlabeled, while secondary localize a specific DNA or RNA sequence. The
antibodies are raised against IgG of the primary probe hybridizes to the target sequence with
antibody. Finally, a chromogenic substrate must elevated temperature (i.e. denaturation) and
be added for visualization (e.g. diaminoben- excess probe is washed away. If the probe is
zidine), which reacts to produce a brown pre- already fluorescent, it will detect the site of
cipitate in the presence of the HRP enzyme. hybridization directly. If the probe is chro-
A simplistic illustration of these two assays is mogenic, an additional step is needed to visualize
shown in Fig. 2.1. Recent advances in this area the probe [29]. A simplistic illustration of ISH is
include multiplexing of antigens [25, 26], shown in Fig. 2.2. Multiplexing using ISH can
whereby multiple stains can be performed on the also be performed [30], followed by spectral
same tissue section, followed by analysis using imaging for the detection and subsequent
digital imaging software. deconvolution of multiple signals.

2.5.2 In Situ Hybridization 2.6 The Frozen Section

While IHC involves the detection of marker The “frozen section” is an alternative tissue
proteins in tissue, ISH can detect target ribonu- preparation technique where, in contrast to rou-
cleic acid (RNA) or deoxyribonucleic acid tine processing, is a rapid histological examina-
(DNA) sequences [27]. Different detection sys- tion done on fresh tissue. The sample is quickly
tems are then used to visualize the presence of placed into cryoprotective embedding medium
the target sequence. Fluorescence ISH (FISH) and cut in a refrigerated microtome (i.e. cryostat).
uses fluorescent dyes and fluorescent microscopy The sections are then stained with H&E. This
while chromogenic ISH (CISH) uses chro- technique is used, for example, where the sur-
mogenic dyes and brightfield microscopy [28]. In geon needs a tumor margin to be examined to
this process, a complementary DNA strand (or ensure that it has been adequately removed, or to
2 Introduction to Histology 15

Fig. 2.2 Illustration of

in situ hybridization

confirm a diagnosis of cancer intraoperatively core biopsies are taken [35]. The cores are then
[31, 32]. The diagnosis should ideally be con- inserted into a separate paraffin block (i.e. the
veyed to the clinician within 20 min after receipt recipient block) [36] in a precisely spaced, array
of the tissue within the pathology laboratory, pattern. This process is repeated multiple times
termed “the turnaround time” [33]. Despite the where finally, one paraffin block is made up of
speed of the procedure, one major disadvantage hundreds of tissue core biopsies from many
of the frozen section technique is that “freezing donor blocks (Fig. 2.3). Simultaneous analysis of
artifacts” are frequently seen which can obscure molecular targets at the DNA, mRNA, and pro-
tissue morphology and cellular detail. Examples tein levels can then be performed under identical
of freezing artifacts seen, include nuclear ice conditions. Epidemiologists, histopathologists,
crystals, bubbles, vacuolated cytoplasm, nuclear and researchers can subsequently analyze data
chromatin changes, tissue cracking, etc. There- following quantitative digital image analysis
fore, this method is only used when an urgent [37]. Therefore, the development of TMA tech-
intraoperative diagnosis is needed. nology has allowed the efficient study hundreds
of different tissue samples concurrently [38] and
is an invaluable research tool.
2.7 Tissue Microarray
Construction
and Evaluation 2.8 Microscopes, Automated
Imaging, and Digital
The tissue microarray (TMA) construction pro- Software
cess [34] involves a region of interest first being
identified and marked on a histology slide. This Many different microscopes are available today,
same area is then marked on the corresponding each of which have varied applications and
paraffin tissue block (i.e. the donor block) and modifications that contribute to their usefulness
16 M.B. Cotter and M. Loda

Fig. 2.3 Tissue microarray construction

Table 2.3 Classification, type, light source, and function of various microscopes
Classification Microscope type Light Description
source
Optical Light microscope (or Visible This is the most commonly used microscope with strong
“compound”) light magnifying power, used for the study of cells,
chromosomes, and DNA
Dissecting Visible This contains lenses in different angles that provides 3D
microscope (or light viewing, used for forensics, fine repair, microsurgery
“stereoscope”)
Fluorescence UV light This is a special type of light microscope where, instead of
microscope light reflection and absorption it uses UV light to view cells
Digital microscope Visible This makes use of the optical lens and charge-coupled
light device (CCD) sensors to magnify objects and includes a
camera for high quality recording
Electron Transmission Electron This microscope is used for studying cells and
electron microscope beam microorganisms and can produce images as small as 1 nm
(TEM) in size
Scanning electron Electron This is less powerful than the TEM but can provide 3D
microscope (SEM) beam viewing of objects and is used for studying cells and small
particles of matter

(Table 2.3). The upright microscope is the most through the field diaphragm and condenser lens
common configuration and has binocular eye- beneath the stage, up through the histology slide
pieces, high power compound objective lenses, containing tissue, into the objective lens, and
and a precision sample stage [39]. In contrast, an finally up to the camera and/or eyepiece. A sim-
inverted microscope is essentially an upside- plistic illustration of this light path is shown in
down, upright microscope. As brightfield and Fig. 2.4. The fluorescence microscope is similar
fluorescence microscopy are the most frequent to the conventional brightfield microscope with
types performed in histology labs, only these will added features to enhance its capabilities [39].
be introduced in this chapter. While the conventional microscope uses visible
Brightfield microscopy is performed using a light (*400–700 nm), the fluorescence micro-
light microscope with a light source commonly scope uses much higher intensity light source
projecting from the back of the microscope. This that causes excitation of fluorophores (i.e. the
light travels upwards from beneath, projects excitation light). The light is absorbed by the
2 Introduction to Histology 17

Fig. 2.4 Light paths of

brightfield and fluorescence
microscopes

fluorophores, which causes them to emit a complex algorithms for quantitation of

longer, lower energy wavelength light. This immunostaining. Tissue can be automatically
fluorescent light (i.e. the emission light) can be segmented into gland or stromal targets and cells
separated with filters designed for that specific can also be segmented into nuclei and cytoplasm
wavelength. In the fluorescence microscope using various algorithms. This allows the trans-
(Fig. 2.4), the light initially travels to a filter lation of extent and intensity of immunostaining
cube. Inside the filter cube it passes through an into a continuous variable, more amenable to
excitation filter to a dichroic mirror (or beam- large-scale bioinformatics analyses.
splitter), which sends light down through the
objective lens and onto the histology slide con-
taining tissue. The emitted fluorescence from the 2.9 The Normal Human Cell and its
specimen then travels back up through the Components
objective lens, into the filter cube, through the
dichroic mirror and through an emission filter. The human cell is the basic structural and func-
From here it continues traveling upwards toward tional unit of the body and its main compart-
the camera and/or eyepiece and can be recorded ments are the nucleus and the cytoplasm, which
or viewed [39]. are completely separate entities that work toge-
Modern digital pathology combines the power ther to keep the cell functioning [41]. Histolog-
of the microscope with electronic detection and ically, the nucleus, nucleolus, and cytoplasm can
advanced computerized analysis and is now easily be distinguished on routine H&E. Anti-
progressively replacing previously subjective, bodies can also be used to selectively stain the
semiquantitative manual scoring with precise nucleus, cytoplasm, or cytoplasmic membrane
quantification of protein expression [40]. A sen- also, depending on the target of interest. While
sor is used to obtain an image, which is then the structures within the nucleus and cytoplasm
displayed on a computer monitor using charge- cannot be seen using routine microscopy, their
coupled device technology. Automated scanning functions are introduced briefly, as knowledge of
can also be performed using a robotic loader. this basic information is imperative for an
Associated imaging software packages for both understanding of the topics discussed in forth-
brightfield and fluorescence purposes provide coming chapters.
18 M.B. Cotter and M. Loda

2.9.1 The Cytoplasm and its 2.9.2 The Nucleus and Gene
Organelles Expression

The cytoplasm surrounds the cell nucleus and is The nucleus is the largest organelle found in
surrounded by a plasma membrane, which sepa- human cells and is surrounded by a nuclear
rates the interior of the cell from the outside envelope with nuclear pore complexes that allow
environment. This membrane is selectively per- material to move in and out [54]. It contains
meable and controls the movement of substances genetic information in the form of DNA. DNA is
in and out of the cell. The cytoplasm is made a complex molecule consisting of two antiparal-
largely of cytosol fluid containing multiple orga- lel strands of nucleotide bases, each with a
nelles (“little organs”) suspended within it that backbone of sugar (deoxyribose) molecules
carry out specific directions of the nucleus. These linked together by phosphate groups [55]. Each
organelles include mitochondria, endoplasmic sugar molecule is linked to a base, which is
reticula, the Golgi apparatus, vacuoles, and lyso- attached by hydrogen bonds to a base on the
somes (Fig. 2.5) among others. Mitochondria other strand in a complementary fashion so that
function to produce most of the cell’s energy in the adenine (A) bonds with thymine (T) and guanine
form of adenosine triphosphate (ATP) [42] and are (G) bonds with cytosine (C) [56]. DNA is orga-
also involved in processes such as cell signaling nized into highly compact, regular units called
[43], cellular differentiation [44], cell growth [45], chromosomes. Within the nucleus is a structure
cell cycle control [46], and cell death [47]. There called the nucleolus, which contains RNA,
are two types of endoplasmic reticula, the rough ribosomal proteins, and functions as the site of
endoplasmic reticulum (RER) and the smooth ribosome synthesis. RNA differs from DNA in
endoplasmic reticulum (SER) [48]. The SER is that RNA molecules are single-stranded, the
involved in lipid metabolism [49], carbohydrate backbone sugar is ribose, and it contains uracil
metabolism, and detoxification, while the RER (U) in place of thymine [57].
contains ribosomes on the surface where active Human genetic testing has made significant
protein synthesis occurs [50, 51]. The Golgi advances in the past decade [58]. The identifi-
apparatus concentrates and packages proteins cation of certain sequences in order to diagnose
from the RER inside the cytoplasm prior to being genetic diseases can be done by performing
transferred to their appropriate destinations [52]. sequencing on a blood sample, fresh tissue, or
Vacuoles are involved in the storage and intra- paraffin embedded tissue. DNA sequencing [59]
cellular digestion of molecules and lysosomes is the process of determining the nucleotide order
contain enzymes responsible for the breakdown of DNA fragment. As mRNA is generated by
of proteins, nucleic acid, carbohydrates, lipids, transcription from DNA, reverse transcription
cellular debris, and foreign organisms [53]. must be performed (using a reverse transcriptase
enzyme) for RNA sequencing [60]. Following
this, complementary DNA (cDNA) fragments are
generated, PCR amplification executed, a library
created, and sequencing performed comparing
results to a reference genome.

2.10 Basic Histology of Normal

Tissues

“Tissues” refer to groups of similar cells per-

forming similar functions (e.g. cardiac myocytes).
Fig. 2.5 The human cell and its components “Organs” refer to groups of tissues (e.g. the heart)
2 Introduction to Histology 19

and “organ systems” include groups of organs Cells of epithelial tissues are usually tightly
that function together (e.g. the cardiovascular packed together and form a continuous sheet or a
system). Tissue is composed of various cells solid aggregation of cells. They lack intracellular
together with a surrounding extracellular matrix spaces and are united by several types of junc-
(ECM). There are four fundamental tissue types tional specializations [64] (i.e. tight junctions
including epithelia, connective tissue, muscle and [65], desmosomes [66], hemidesmosomes [67]
nervous tissue, each of which will be introduced etc.). Therefore, epithelia have only one free
separately below. surface (i.e. apical surface), which is exposed at
the body surface or at the lumen of a duct, tube,
or vessel. The lower surface of an epithelium (i.e.
2.10.1 Epithelia basal surface) [68] rests on an underlying base-
ment membrane, which is a thin sheet of collagen
Epithelial cells are generally classified as “cov- and glycoproteins, which acts as both a scaffold
ering and lining epithelia” which are found lining and a selectively permeable membrane allowing
the cavities of the body and surfaces of structures, water and small molecules through [69].
or as “glandular (or secretory) epithelia” [41]. Two special categories of epithelium are
Glands refer to single cells or groups of cells that pseudostratified and transitional epithelium [41].
secrete protein, mucus, or lipid. This includes Pseudostratified columnar epithelium is so called,
“endocrine glands” which secrete into extracel- because of an apparent stratification, however, all
lular spaces (i.e. secrete internally) and “exocrine of the cells are attached to the basement mem-
glands” which secrete into ducts (i.e. secrete into brane (Fig. 2.6). Therefore, it is really simple
the external environment). Epithelial cells can epithelium, despite giving the impression of
also be classified based on the number of cell stratification. Transitional epithelium (or urothe-
layers present and the shape of the cells in the top lium) is stratified epithelium lining the walls of
layer (Fig. 2.6). Epithelial tissue can, therefore, the urinary tract. The term refers to the fact that it
be one cell thick (i.e. simple epithelium), or two may appear as stratified cuboidal to squamous in
or more cells thick (i.e. stratified epithelium) [61]. appearance depending on the extent of bladder
There are three basic cell shapes based on distention. Specific names are also given to
microscopic appearance, including squamous epithelium in certain locations. Endothelium is a
(flat and wide), columnar (tall), and cuboidal term given to simple epithelium lining blood
(cube shaped) cells. Consequently, by describing vessels (vascular endothelial cells) [70] or
the number of cell layers and the surface cell lymphatics (lymphatic endothelial cells) [71].
shape the different forms of epithelia can easily be Mesothelium is a name given simple squamous
classified. In some cases a third feature, namely epithelium lining the major body cavities [72], for
specialization of the cell surface, e.g., kera- example, the peritoneal epithelium lining the
tinization [62] or the presence of cilia [63], is abdominal organs [73].
included. Stratified squamous epithelium that is
exposed directly to the environment (e.g. the
skin), can show keratinization (i.e. a layer of dead 2.10.2 Connective Tissues
cells is present on the surface), while those that
are not directly exposed (e.g. the oral cavity) are Connective tissues form a scaffold that epithelial
only partially keratinized or nonkeratinized. The tissues lie on, and nerve and muscle tissues are
presence of cilia (i.e. hair-like motile processes) is embedded. They are classified as connective tis-
another specialization seen in simple columnar sue proper and specialized connective tissues,
epithelium present on the surface of these cells. including adipose tissue, cartilage, bone, and
This surface adaptation helps propel substances blood (Fig. 2.7). All connective tissues are char-
along, e.g., the airways contain cilia to propel acterized by various individual cells scattered
mucus. within an extracellular space filled with an ECM
20 M.B. Cotter and M. Loda

Fig. 2.6 Illustration of

epithelial cell types

[41]. Variations in the composition of the ECM another type of collagen (type III). They are
determine the properties of the connective tissue. usually not visible using routine H&E, but can be
In general, the ECM comprises ground substance demonstrated using special stains (e.g. reticulin)
and various fibers (i.e. collagen, reticular or [22]. Like reticular fibers, elastic fibers [74]
elastic fibers) woven into a network. Ground require special stains to be visualized also (e.g.
substance supports the connective tissue cells, elastin). Once stained, elastic fibers appear as
binds them together, and permits the diffusion of fine, dark, undulating fibers within the tissue.
nutrients and other dissolved substances between The principle cells in connective tissue proper
capillaries and cells. There are many known types include fibroblasts that secrete collagen fibers
of collagens, with type I being the most abundant. and ground substance [75], together with mac-
Histologically, collagen appears as irregular, rophages, mast cells, and adipocytes among
wavy fibers arranged singly or in small groups. others. Fibroblasts have elongated nuclei with a
Reticular fibers are very fine fibrils consisting of moderate amount of cytoplasm that tapers at the
2 Introduction to Histology 21

Fig. 2.7 Illustration of

connective tissue cell types

ends under the microscope. They are usually contrast, the principle cells in bone tissue are
found alone and contain an elliptical nucleus osteoblasts, osteocytes, and osteoclasts. The
showing finely stippled (i.e. “dot-like”) chro- osteoblast is involved in bone deposition at the
matin and one or two nucleoli. Macrophages are bone surface and secretes the matrix of bone (i.e.
large, round cells, with vesicular nuclei [76]. In osteoid), which becomes calcified following
some cases, a brown pigment is seen within deposition [78]. As they are trapped within the
them, which is the result of lysosomal action on ECM, they become osteocytes [79]. The osteo-
ingested red blood cells. Mast cells are small, cytes are located in lacunae and fine channels
ovoid cells with spherical, eccentric nuclei, and (canaliculi) containing osteocyte cell processes,
basophilic granules [77]. In adipocyte cells, the connecting lacunae to each other. The third cell
nucleus appears flattened with the cytoplasm type, the osteoclast, is associated with bone
forming a very narrow rim around a large central resorption [80] and does this by secreting
lipid droplet. During routine preparation of his- enzymes that acidify the matrix. These are large,
tological slides fat is dissolved, therefore, the multinucleated cells with a ruffled border histo-
adipocytes actually appear empty. When adipo- logically. In order to be able to visualize bone
cytes are seen in large numbers, the tissue is tissue, the specimen is usually placed in a
referred to as adipose tissue. decalcifying solution (e.g. formic acid) [81],
There are three types of cartilage tissues that which removes calcified material so that good
differ in the type of fibers they contain within the quality paraffin sections can be prepared that will
ECM. These include hyaline, fibrocartilage, and preserve the microscopic elements.
elastic cartilage, with hyaline being the most Blood is traditionally classified as a special-
abundant type. Histologically, hyaline cartilage ized form of connective tissue, even though it has
has a basophilic appearance on H&E. Chondro- a different function in comparison to other con-
cytes (cartilage cells) produce the matrix of car- nective tissue types. It has a highly fluid ground
tilage and are seen within lacunae (i.e. matrix substance (i.e. plasma), which comprises mainly
cavities), singly or in clusters of 2–8 cells. These water together with salts, proteins, nutrients,
groups are called isogenous groups and are hormones, and waste material. The cellular
derived by mitosis from a single chondrocyte. In component of blood is produced by the bone
22 M.B. Cotter and M. Loda

Table 2.4 Histologic description and function of hematopoietic cells

Cell type Histologic description Function of hematopoietic cell
Erythrocyte Flat or oval-shaped cell with no nucleus Responsible for the transportation of oxygen in
the body
Neutrophil Multilobated nucleus, often with 3–5 lobes Involved in the acute inflammatory response
Eosinophil Eosinophilic granular cytoplasm and a Associated with the allergic response and
bilobed nucleus parasitic infections
Basophil Basophilic granular cytoplasm and a bilobed Responsible for allergic response by releasing
nucleus histamine
Monocyte Large cell, with a large, indented Precursors for tissue macrophages, which
(“kidney-bean” shape) nucleus and abundant engulf and digest foreign microorganisms, dead
cytoplasm cells, or debris (i.e. phagocytosis)
Lymphocyte Small, round cell with a deeply staining Precursors of natural killer cells, B and T
spherical nucleus surrounded by a thin rim lymphocytes involved in the acute
of basophilic cytoplasm inflammatory response. B cells further
differentiate into plasma cells
Megakaryocyte Large cell, with a large pale multilobated Precursors of thrombocytes (or platelets)
nucleus and abundant cytoplasm formed by budding from megakaryocytes and
contribute to hemostasis

marrow in a process termed hematopoiesis [82]. 2.10.3 Muscles

These hematopoietic cells are derived from
multipotent hematopoietic stem cells. Following Muscles are responsible for maintaining posture,
division, the resulting daughter cells (myeloid or locomotion, and movement of the internal organs
lymphoid progenitor cells) can commit to alter- (e.g. contraction of the heart). Three kinds of
native differentiation pathways depending on muscle tissues are found in different organs of the
growth factors involved. Finally, blood cells are body, including skeletal muscle, cardiac muscle,
divided into three lineages including the ery- and smooth muscle (Fig. 2.8). Skeletal muscle
throid lineage (reticulocytes and erythrocytes), generally forms the muscles attached to bones
the lymphoid lineage (T cells, B cells [83] and and by contracting, these muscles move joints.
natural killer cells [84]) and the myeloid lineage Cardiac muscle (myocardium) forms the mass of
(granulocytes [85, 86], megakaryocytes [87], and the heart. Smooth muscle is a component of the
macrophages [88]). Table 2.4 lists the histologic walls of many hollow organs within the body,
description and function of various hematopoi- such as the digestive tract. By contracting,
etic cells, and their basic structures are also smooth muscle propels the contents along the
illustrated in Fig. 2.7. tube it surrounds (e.g. the intestine), or regulates

Fig. 2.8 Illustration of

three kinds of muscle tissue
cell types
2 Introduction to Histology 23

the amount of fluid flowing through it (e.g. the skull) and the spinal cord (within the vertebral
blood vessels). Skeletal and cardiac muscle is canal), while the PNS is composed of nerves (i.e.
referred to as “striated” muscle as they show light cranial nerves from the brain and spinal nerves
and dark bands when viewed under the micro- from the spinal cord) and ganglia (i.e. nerve cell
scope. Smooth muscle cells do not have visible clusters). Neurons (nerve cells) are specialized
striations, however, they do contain the same cells that respond to stimuli and conduct elec-
contractile proteins arranged in a different pat- trical impulses to and from all body organs. All
tern. While muscles are conventionally classified neurons have the same basic structure consisting
based on morphology (i.e. striated or smooth of the cell body (soma) containing the nucleus,
muscle), they can also be classified based on cytoplasm and organelles, and nerve processes
function (i.e. voluntary or involuntary muscle). that conduct the signals [89]. Nerve processes
Histologically, muscle tissue cells (myocytes) include the axon, which carries signals away
are elongated and spindle-shaped with little from the cell body, and dendrites, which carry
intervening extracellular material. Smooth mus- signals toward the cell body. There are three
cle and cardiac muscle myocytes contain one basic shapes to neurons, bipolar (i.e. consisting
nucleus, while skeletal muscle is multinucleate. of a single axon and single dendrite), pseu-
The unusual microstructure of myocytes has dounipolar (i.e. consisting of a single axon with a
lead to the use of specialized terminology, central and a peripheral branch), and multipolar
including the sarcolemma (plasma membrane), (i.e. consisting of a single axon and numerous
the sarcoplasm (cytoplasm), the sarcoplasmic dendrites), illustrated in Fig. 2.9. There are four
reticulum (endoplasmic reticulum), and sarco- types of supporting cells (or central neuroglia) in
somes (mitochondria). the CNS, including oligodendrocytes [90],
microglia [91], astrocytes [92], and ependymal
cells [93]. In contrast, the Schwann cell is the
2.10.4 Neural Tissues principle supporting cell in the PNS (or periph-
eral neuroglia) [94]. The oligodendrocyte or
The nervous system is divided anatomically into Schwann cell wraps around axons of neurons to
the central nervous system (CNS) and the form the myelin sheath that ensures rapid con-
peripheral nervous system (PNS). The CNS duction of nerve impulses [95]. This sheath is not
consists structurally of the brain (within the continuous and gaps between neighboring cells

Fig. 2.9 Illustration of

basic neuron types
24 M.B. Cotter and M. Loda

are called nodes of Ranvier [96]. Neurons are 9. Elger BS, Caplan AL. Consent and anonymization in
seen histologically as irregular or stellate in research involving biobanks: differing terms and
norms present serious barriers to an international
shape and are multipolar. They have a large cell framework. EMBO Rep. 2006;7(7):661–6.
body with a large, round, and pale (euchromatic) 10. Helgesson G, Helgesson G, Dillner J, Carlson J,
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