Name

NameofofLearner:
Learner: ___________________________
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Section: ___________________________
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School: ___________________________
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Content
Content

MEDINA COLLEGE
Bulatok, Pagadian City

M Tel. No. (062) 2153721

O MLSP II
D
U The Complete Textbook of
Phlebotomy
L BSMT-1
E First Year – Second Semester
LEARNING MODULES
1
Name of Learner: ___________________________
Program: ___________________________
Year level & Section: _____________________

Prepared by:
Dhaiana Lyn C. Flores, RMT.
Instructor
 MEDINA COLLEGE INC
Pagadian City, Zamboanga del SurPrepared by:
Dhaiana Lyn C. Flores, RMT
Instructor
MEDINA COLLEGE INC. VISION and MISSION
Vision
An educational community of men and women
dedicated to the formation of the whole person with
professional competence and commitment to promote
local, regional and national development.
Mission
As an institution of higher learning, the
College commits itself to offer a well-rounded
program of liberal education and to provide varied
opportunities for students to grow professionally,
spiritually, socially and culturally. In the pursuit of this
mission, Medina College is guided by the following
commitments:
 As a CHRISTIAN COMMUNITY it
welcomes faculty, staff and students with
various religious beliefs and respects the
religious freedom of every member while
providing opportunities for them to grow in
their faith life.
 As an ACADEMIC COMMUNITY, it offers
quality and responsive education to
prepare its graduates for national and
global competitiveness.
As a FILIPINO COMMUNITY, it promotes the
formation of the student’s awareness of their civic
and social responsibilities and development of the
students’ pride for the nation’s rich cultural heritage.
This instructional material was collaboratively
developed and reviewed by educators from Medina
College – Pagadian City.
 Introductory Message
Hello learners. This Principles of Medical Laboratory
Science 2 (MLSP II) module, serve as a comprehensive
resource material on the various information’s your needed in
the field of phlebotomy. This module is carefully made to cater
all the necessary knowledge and information’s for you my
students to practice phlebotomy. This module enables you
students to be competent in phlebotomy at the end of the
semester. Competency assessments of students are included in
this module so you will know each step that is required in the
field of phlebotomy.
Learners, please read each page of this module carefully
and intensively, this will serve you well. Happy reading and
learning. Let’s learn by doing.
GENERAL DESCRIPTION
COURSE NO. : MLSP II
COURSE TITLE : PHLEBOTOMY
CREDIT : 3 Units
PRE-REQUISITE : MLSP I
CO-REQUISITE : NONE
COURSE DESCRIPTION:
This module; The Complete Textbook of
Phlebotomy emphasizes the relationship between
sample procurement and the laboratory. Student will
learn why samples are collected from patients.
Students will learn how important their phlebotomist
jobs are because most patients care decisions are
based on laboratory results.
This module will prepare students to the world
of phlebotomy and enables them to pass any of the
phlebotomy certification exams. The National
Accrediting Agency for Clinical Laboratory Sciences
(NAACLS) Phlebotomist Competencies was used to
ensure that all the information needed was included.
Course Intended Learning Outcomes
(CILO):
By the end of this course, students
should be able to:
 Acquire the basic concepts of phlebotomy
 Explains and understand the important of
phlebotomy.
 Discuss the principles and procedures of
phlebotomy and all the available competencies
written in this module.
 Develop the necessary skills required in the
preparation of career as a medical technologist
and as a phlebotomist in the future.
 Apply all the competencies in daily activities
set.
 Discuss the procedures and principles of
venipuncture
 Acquired basic concepts and skills of
venipuncture by evacuated tube method.
 Pass all the competencies required in this
course.
  History in phlebotomy
 Phlebotomy’s role in health care
 Areas of the hospital and health care settings
 Laboratories in the twenty-first century
 Laboratory staff
 The patient care partnership
 Professional attitude
 Professional grooming
 Advance directives
 Standards used in the laboratory
 Quality assurance in phlebotomy
 Review questions

Lesson 2: Safety in Phlebotomy

 Infection control
 Reducing exposure risk
 Procedure 1 Medical Asepsis Handwashing
 Procedure 2 Removing Contaminated Gloves
 Procedure 3 Donning and Removing a Mask, Gown,
Gloves, and Shoes Covers.
 Isolation techniques
 Occupational Safety and Health Administration
Standards

Course Outline Lesson 3: Basic Human Anatomy

 Basic Histology
 Type of Samples Obtained in Laboratory
 Types of Histological Preparation
 Responsibility of A Technician
PRELIM

MODULE I
Chapter 1. Introduction to Phlebotomy MIDTERM
 MODULE 2 Lesson 7: The Challenge of Phlebotomy.
.The Criteria of a Good Decalcifying Agents Area.
Chapter 4: Anatomy and Physiology of the Circulatory
 Formic Acid Sodium Citrate Method Procedure
System
 Decalcification of Bone Marrow Biopsy
 Basic Histopathology
 Use of Ion Exchange Resins
 Design of Histopathology Lab
 Chelating Agents
 Component Histopathology Lab
 Electrolytic Method
Lesson 5: Phlebotomy Equipment  Determination of End Point of Decalcification
 Types of Hazards Include the Following  Tissue Processing
 Factors Contributing to Laboratory Accidents  Dehydrating Agents
 Safety Precautions  Clearing
 Light Microscopy
Lesson 8. Caring for the Pediatric Patient.
Lesson 6: Phlebotomy Technique
 Properties of Paraffin Wax
 Aims and Effects of Fixation
 Points to Be Remembered During Use of Paraffin
 Amount of Fixative
Wax
 Properties of Fixatives
 Time of Impregnation
 Preparation of The Specimen for Fixation
 Microanatomical Fixatives
 Cytological Fixatives
 Cytoplasmic Fixatives
 Histochemical Fixatives FINAL
 Specimen Preparation
 Specimen Reception MODULE 4
 Gross Examination of Tissues
Chapter 9. Sample Considerations and Special
Procedures.
Trouble Shooting for Poor Sections

Lesson 10: Sample Preparation and Handling.

PREFINAL Theories of Staining
MODULE 3  Special Stains
  Classification
 Stains for The Detection of Connective Tissue M
 Connective Tissue O Lesson
 Cells D HISTOLOGICAL TECHN

1
 Fibers U HISTORICAL OV
L
Lesson 11: Customer Service.
E
Classification of Carbohydrates
 Mucins
1
 Techniques for The Demonstration of Carbohydrates

Lesson 12: Legal and ethical issues.

Intended Learning Outcomes

Lesson 13: Competency
Techniques of Microorganisms in Tissue
 Bacteria
 Fungi
 Viruses
 Parasites / Protozoa At the end of the lesson, the students should b
able to:
Lesson 14: CYTOLOGICAL TECHNIQUES

Lesson 15: TYPE OF SAMPLES OBTAINED IN 1. Discuss the origin of histopathology an

LABORATORY its technique;
Lesson 16: SAMPLING TECHNIQUES IN CYTOLOGY
Lesson 17: CYTOLOGICAL STAINING TECHNIQUES 2. Identify important persons behind th
advancement of histopathologica
techniques.

3. Name some of the important method use

 in histopathology. Lesson
Proper
Essay: Write it on a separate
sheet of paper.

Explain the interrelationship of cell, cytology,

histology, and histopathology.

PRE- TEST

Definition of terms: Write it on a separate sheet

of paper.

1. Histopathology BACKGROUND OF HISTOPHATOLOGY

2. Histologists
3. Trichrome Stains Under the microscope, there are many
4. Gram stain causes to examine cells and tissues. Medical,
5. Hematoxylin histological and biological research depends on
6. Microtome the normal structure knowledge and function of
7. Histological staining the cells, tissues, organs, and the systematic
architecture they constitute. The cells and other
constituents of tissue are ornamented in an
identifiable model in ordinary status (Bancroft &
Layton, 2013).
Changes in structure at a stage of
microscopic anatomy caused by a broad
spectrum of chemical and physical impacts are
overturned by alteration in an architecture at the
level of microscopical anatomy, and many
illnesses are distinguished by idea chemical and
structural defects that vary from the state of
normality (Anderson, 2011).
The main concept of cytopathology and
histopathology, a significant specialization of
modern medicine, is to identify these
modifications and interconnect them with certain
illnesses. Also, in biology, hematology, zoology,
microbiology, and botany, microscopy is a
significant part (Titford, 2009). Histology was
defined as the study of biological material's
microscopic anatomy as well as the paths of
structurally and functionally correlating individual
constituents. As it stands at the intersection
between physiology, biochemistry, and molecular
biology on one hand, and disease procedures
and their impacts on the other hand, it is the core
of medical and biological science. (Musumeci,
2014).
Many of diseases occurring at the level of
tissue. For example, cancer is predominating the
result of problematic overgrowth of tissue, and
some infections triggering tissue to necrotize.
For futuristic medical careerists, histology
purveys important insight into the development of
pathology (Wang et al.2019; Black, 2012).
Beginning with the growth of easy light
microscopes and methods to prepare thin slices
of histological and biological material, the
histology research began to create them suitable
for examination. (Lyiola & Avwioro, 2011)
Pioneer histologists learned a surprising
quantity about the form and structure of
biological material despite their simple equipment
and mildly inadequate prepared material. Such
studies resulted Virchow to suggest his cellular
theory of living organism architecture which
demonstrated the cell to be the most biological
material's functional unit (Alturkistani et al.2016).
Each cell was regarded as an individualistic
unit surrounded by a wall called the plasma
membrane and included all the equipment for its
function (Bancroft & Layton, 2013).
A vocabulary of histological techniques was
created in those early years, based on light
microscopic cell assessment and taken alone by
a finite understanding of cell biology and
physiology (Musumeci, 2014).
Modern research methods have
revolutionized our knowledge of cells. Cell
cloning in culture, protein sequencing, molecular
genetics, and electron microscopy methods have
also provided unpredictable insight into the
functioning of cells and tissue (Titford, 2013).
Histological staining is a chain of technical
procedures including tissue preparing using
histological stains to support microscopic
research (Wang et al.2019; Anderson, 2011;
Alturkistani et al.2016).
There are several phases in the histological
staining process involving: fixing, handling,
staining, sectioning and embedding (Titford,
2009). Substantial differences, biological and
molecular assays and immunological methods
have achieved substantial distinctions in the
methods used for histological staining and have
greatly facilitated tissue and organ studies
(Shostak, 2013).
Alturkistani and his companions (2016)
have demonstrated that the history of histology
shows that histological staining techniques had
altered considerably through chemical,
immunological techniques and molecular biology
assays, together mentioned to as histochemistry.
For the preparation of tissues for microscopic
research, early histologists used easily
accessible chemicals; these laboratory chemicals
are alcohol, mercury oxide for cellular tissue
hardening and potassium dichromate. Carmine,
silver nitrate, Giemsa, Trichrome Stains, Gram
stain, and Hematoxylin were the staining
techniques used (Alturkistani et al.2016).
A SHORT HISTORY OF HISTOPATHOLOGY
TECHNIQUE
The specialty of histopathology technique
dates back to 1838, when Johannes Miiller
published his book, On the Nature and Structure
Characteristics of Cancer, the first book on
histopathology. The first compound microscope
had been constructed earlier in 1591 but suffered
from severe optical problems. In 1673 Anton van
Leeuwenhoek started the development of simple
microscopes with single lenses but that gave
improved magnification and resolution.
The first microtome suitable for sectioning
animal tissues was constructed in 1848, with the
popular Cambridge Rocker (1885), Minot (1886),
and sledge microtomes (1910) manufactured
later. Paraffin wax for infiltration and support
during sectioning was introduced during the
mid1800s. Different laboratory chemicals were
investigated for use as fixatives. Formalin, widely
used today, was first used in 1893.Automated
tissue processors replaced hand processing
starting in 1945, and cryostats were first
manufactured in 1951. Enzyme histochemistry,
electron microscopy, and polarizing microscopy
all have become diagnostic tools during the last
50 years.

The widespread use of
immunohistochemistry began in the 1980s has
revolutionized cancer diagnosis and is still under
development. This article briefly reviews the
development of histopathology techniques in the
United States and United Kingdom from historical
times to present. (The J Histotechnol 29:99,
2006) Submitted December 21, 2005; accepted
with revisions March 28, 2006
Histological history indicates that the
histological techniques of staining, molecular
biology assays, chemical methods, and
immunological methods that jointly referred to the
word histochemistry have changed significantly.
In order to prepare histological sections of
tissues for microscopic studies, early
technologists used abundant chemicals; to
solidify tissue samples, these chemicals contain
potassium dichromate alcohol and mercury
chloride. Staining methods included silver nitrate,
Trichrome stains, Giemsa, carmine, Gram Stain,
and the routine for Hematoxylin-Eosin.
The results of literature review have shown
that the histotechnology and histopathology
depicted by staining processes have been
gradually improved. Requirements for accurate,
skilled, and less complexity staining processes
were battening. Numerous staining procedures
remain in use until the present time, as well as
other numerous staining procedures changed by
new immunohistological staining, non-cultural,
molecular and other sophisticated methods of
staining. Several staining techniques have been
avoided due to the medically proven toxicity of
the necessary chemicals. In order to improve
their efficiency, modified histological and other
strains had been altered and associated with
each other.
HISTORY OF HISTOLOGICAL TECHNIQUES
OF STAINING IN MEDICINE AND
BIOLOGICAL ASPECTS
A new field of disease diagnosis is
considered as the history of staining methods
referring to the application of histological
techniques (Rodrigues et al.2009). A seventeen
scientist Leeuwenhoek, who was an important
instrument in histology using substances such as
indigo, saffron and madder to stain parts of
materials then to study them its need to use
primitive microscopes, cited historical staining
methods by early histopathologists and surgeons
(Titford, 2009).
These early research groups used the
microscopic anatomy to delineate a connection
between cell variations as well as to distinguish a
standard structure of plant cells from that of the
animal (Bancroft & Layton, 2013). Consequently,
new methods have been developed to
strengthen in detail the cell structure study by
using several labor chemicals to reservation the understanding has been used for further studies
tissue in its natural shape before staining (Titford into new-histological tissue study methods
& Bowman, 2012). (Titford, 2009).

According to (Alturkistani et al.2016): in Many medicine centers lease pathologists,

1858, Joseph Von Gerlach was considered the
pioneer of microscopic staining when he
successfully used ammonia carmine to stain
histological sections of cerebellum and their
cells (Costa, et al. 2010). For microscopic
examinations, the pioneer histologist use thee
easily abundant chemicals in order to make
tissues ready for examination; potassium
dichromate, alcohol and mercuric chloride to
harden cellular tissue components are
represented these chemicals (Iyiola &
Avwioro 2011).
doctors, and surgeons in the early 19th century
to manage surgical problems (Titford & Bowman,
2012). It is this pathologist yield that breaks the
latest intraoperative staining methods for frozen
histological segments by customizing a unique
histopathology staining method. The paraffin
infiltration method was developed during this
moment (Shostak, 2013). According to Godwin
(2011), because of this achievement, the
nineteenth century researched malignant and
non-malignant tumors (Godwin, 2011).

These staining and fixative agents have

been effective, have been established and are
still accessible as staining methods in the
laboratory (Black, 2012). One of those stains still Summary
in use is the trichrome, which is used in kidney
and liver biopsies, and the silver nitrate used in of Learning
nerve tissue staining (Musumeci, 2014). Shostak
(2013) showed that the significant development
of histological stains was formalized in 1856 in
Germany by the growth of microscope
technology and an invention of aniline dye which
 Histological
produced the assortment techniques
of new were created in those early years, based on
histological
stains (Shostak, 2013).lightAt microscopic cell assessment
once, researches as and taken alone by a finite
well as understandingunderstanding
on tissues ofandcell biology
human and physiology
body anatomy have increased,
 Research groups and
used this
the microscopic anatomy to delineate a
connection between cell variations as well as to distinguish a standard
structure of plant cells from that of the animal.
 I. IDENTIFICATION

Name at least 4 staining methods and give each

characteristic, its function, and the specific tissues intended
to stain.

Individual Activity: Write this activity in

a separate paper.
Real-life
1. How can applicatio To validate your answers, feel free
histopathological to contact your course facilitator
techniques help n through any of the following:
medical practitioner and  Facebook: Dhaiane
people in our current Flores
situation today?  Contact No:
09561270937
 Email address: floresdhaianalyn@gmail.com

POST
TEST References:
 Additional references:
https://www.researchgate.net/publication/338456523_Histological
_Techniques_A_brief_Historical_Overview

M
O Lesson 5. Understand how
PAST, PRESENT AND FUTURE: OVERVIEW ON sections can be
D
U
L 2 HISTOLOGY AND HISTOPATHOLOGY photographed,
presented
reported.
and

E
1

Intended Learning Outcomes

At the end of the lesson,

the students should be
able to:

1. Define all the

important terms used in

PRE- TEST
histopathology.
2. Outline key features of a number of
pathological processes
3. Relate the histological appearance of
affected tissues to the underlying
pathology
4. Recognized the histological appearance
of a number of pathological tissues
Essay: Write it on a separate sheet of paper.

1. Histopathology
2. Histologists
3. Trichrome Stains Lesson
4. Gram stain
5. Hematoxylin Proper
6. Microtome
7. Histological staining

Essay: Write it on a separate sheet of paper. HISTOLOGY AND HISTOPATHOLOGY

Explain the interrelationship of cell, cytology,
histology, and histopathology
Histopathology and histology are often
times defined and described with one another.
The term of histology cannot be separate from
that of histopathology because the normal
histology understanding is fundamental for
interpretation of histo-pathological analysis
(Titford, 2006; Titford & Bowman2012).
To detect weather the cells or tissue are
healthy or unhealthy, it is to prepare
histological samples of a specimen and
examine them first. In addition, histo-pathologists
specialists must be able to educate histologists
about ordinary tissue morphology differences
and abnormalities, enhancing histo-pathological
interpretations (Coleman, 2006b).
We might then notice that there are
interconnections between histology and
histopathology. Histology was mentioned in its
own right in the 19th century as an
academic branch, and the first decades of the
20th century were a very productive interval
for new histopathology and histology staining
methods (Musumeci, 2014).
Indeed, histologists were awarded the
1906 Nobel Prize in Physiology and Medicine:
Camillo Golgi and Santiago Ramony Cajal.
Depending on different interpretations of the
same histological parts, they had
inconsistency interpretations of the brain
neuron structure. For his right theory, Cajal was
valued and Golgi for the staining method he
invented (Musumeci, 2014). There are many
different histological stains to be recognized
depending on the type of tissue .Several of the
stains were used more widely, while others are
used only to show very particular biological
tissue kinds. Prussian blue, which was launched
in 1774, is considered one of the oldest stains.
The response of Perl, identified in 1867, is
still commonly used for the identification of
intracellular iron and utilizes only Prussian blue
to locate hemosiderin in tissues (Musumeci,
2014).
The most popular histological stain used
for light microscopy is hematoxylin and eosin.
Hematoxylin colored the nuclei with dark purple
cells and eosin stains the cytoplasm of light pink
cells. H&E is, as opposed to a temporary stain,
a permanently histological stain. The (H & E)
techniques were first introduced in 1875–1878,
with consequently adjustments (1906)
(Coleman, 2006a).
Through the use of methylene dyes, Louis B.
Wilson was the first one to create a
histogical procedure for staining fresh-frozen
tissue from surgery (1906). Feulgen stain (1924)
is a coloring method used in histology to show
material of chromosomes or DNA of cell samples
and was the foundation for several subsequent
cell biological applications. In 1926, French
introduced Oil Red (ORO) and highlights the
existence of adipose tissue in new, frozen tissue,
a fat-soluble color, classified as one of the
Sudanese stains used since the late 18th
century. McManus (1946) made the Periodic
Acid-Schiff (PAS) method, which remains one of
the most common histopathology diagnostic
techniques of staining, canceled Best's carmine
(1906) to elucidate polysaccharides and is
commonly used in muscle and liver illness
(Musumeci, 2014).
Histology
Histology and Histopathology are often
discussed and described together. In fact, the
concept of histopathology cannot be separated
from that of histology since understanding of
normal histology is essential for histo-
pathological interpretation. It is indeed obvious
and necessary to prepare histology slides of a
sample or specimen and examine them first in
order to find out if the cells or tissue are healthy
or diseased. Moreover, expert histo-pathologists
should also be able to inform histologists about
normal variations of tissue morphology,
improving in this way histo-pathological
interpretations. We could state then, that
histology and histopathology are inter-
th
dependent. In the 19  century, histology was an
eminent academic discipline in its own right and
the first half of the 20th century was a very
productive period for new staining techniques in
histology and histopathology. Indeed the 1906
Nobel Prize in Physiology or Medicine was
awarded to histologists Camillo Golgi and
Santiago Ramon y Cajal. They had conflicting
interpretations of the neural structure of the brain
based on differing interpretations of the same
images. Cajal was appreciated for his correct
theory and Golgi for the staining technique that
he invented. Many of the centenary staining
techniques in cell biology and histopathology are
still used and continue to provide valuable
diagnostic information. Histology is the study of
the microscopic details and structures of
biological cells and tissues, using light,
fluorescence or electron microscopes, examining
a thin slice (called a "section") of tissues, that
have been previously prepared using appropriate
processes called "histological techniques". The
first microscope had been constructed in 1591
but had several optical problems. In 1673 Anton
van Leeuwenhoek developed a simple
microscope with a single lense but with improved
magnification and resolution. The first microtome
suitable for sectioning animal tissues was
constructed in 1848. During the 19th century
paraffin wax was introduced for infiltration and
support during sectioning. Over the years
different laboratory substances were investigated
for use as fixatives. Formalin was first used in
1893 and today is widely employed. In order to
better underline different biological structures,
histological stains are often used to modify or
enhance the colors of certain types of these
biological structures differently from the others
that may be located next to them or be in contact
with them. There are many different histology
stains selected according to the type of tissue to
be observed. Some stains are more widely used,
instead others are only used to study very
specific types of biological tissues. One of the
oldest stains was Prussian blue, introduced in
1774. Furthemore, Perl's reaction, discovered in
1867, is still widely used to localize intracellular
iron and uses just Prussian blue for the
histochemical localization of hemosiderin in
tissues. Hematoxylin and eosin (H&E stain) is the
most commonly used histology stain for light
microscopy. Hematoxylin stains the nuclei within
cells blue and eosin stains the cytoplasm of cells
pink. H&E is a permanent histology stain, as
opposed to a temporary stain. The hematoxylin
and eosin staining techniques were first
described in 1875–1878, with later modifications.
Hematology took advantage from the introduction
in the 1890s of Romanovsky-type staining for
blood smears, including Giemsa's and May-
Grunwald staining which are still fundamental in
clinical practice. Louis B. Wilson was the first to
develop a method using methylene dyes to stain
fresh-frozen tissue of surgical specimens (1906).
The periodic acid-Schiff (PAS) technique of
McManus (1946) is still one of the most common
diagnostic staining methods in histopathology, it
superseded Best's carmine (1906) to stain
polysaccharides and is widely used in liver and
muscle disease. Oil Red (ORO), introduced by
French in 1926 highlights the presence of fat or
lipids in fresh, frozen tissue sections. ORO is a
fat soluble diazo dye, and is classified as one of
the Sudan dyes used since the late 1800s.
Feulgen stain (1924) is a staining technique used
in histology to identify chromosomal material or
DNA in cell specimens and has been the basis of
many subsequent applications in cell biology.
The Ziehl–Nielsen stain for Mycobacterium
tuberculosis (1883) and Gram's stain for bacteria
(1884) are examples of diagnostic bacteriological
and pathological techniques, which are still used
over a century since their discovery. Paul Ehrlich,
who received the Nobel Prize in 1908 for his
discovery and his work on "magic bullets",
developed the use of dyes to combat disease i.e.
methylene blue (1891) to fight malaria and trypan
red (1904) against the trypanosomes. Trypan
blue remains in widespread as a vital stain,
particularly in highlighting cataracts during eye
surgery. These staining methods are generally
inexpensive, reliable, fast, produce permanent
preparations that are easy to interpret and
archive, and deliver information for diagnoses
that cannot be achieved by other means. For
these reasons they remain useful and
irreplaceable tools in the histology discipline.
Histopathology
Histopathology is the diagnosis and study
of diseases of the tissues, and involves
examining tissues and/or cells under a
microscope. Histopathologists are responsible for
making tissue diagnoses and helping clinicians
manage a patient’s care.
Histopathology regards biological tissues
and cells with their microscopic changes or
abnormalities that can be the causes or the result
of diseases. The main application of
histopathology in clinical medicine, is in the
examination of a biopsy (i.e., a surgical sample
or specimen taken from patients possibly for
diagnosis and screening of various tumors) by a
specialist physician called a pathologist. The
pathologist may be more accurately referred to
as a histopathologist. However, for some
histopathologists the microscopic examination of
diseased tissues may be only a relatively minor
part of their overall professional responsibilities.
A pathologist or histopathologist studies
specimens of cells and tissue samples removed
from the patients, processed using special
histological techniques (histological slides and
sections prepared and stained in order to make
the sample ready for observation with a
microscope). Either a light microscope or an
electron microscope may be used to examine
histology slides. The first book on the
specialization of histopathology techniques,
entitled, "On the Nature and Structural
Characteristics of Cancer" was written in 1838 by
Johannes Müller. Enzyme histochemistry,
electron microscopy, and polarizing microscopy
have all become diagnostic tools during the last
50 years. Actually, the use of electron
microscopy is rare, except for specific contexts or
disciplines such as renal pathology. The use of
immunohistochemistry, still under development
with new antibodies and biomarkers, began in
the 1980s and has revolutionized cancer
diagnosis. The histology laboratory experienced
several changes in the mid 20th century when
cryostats, enclosed tissue processors, plastic
cassettes, and disposable knives were
introduced. The emergence of
immunohistochemistry in the 1980s was a
revolution among the histopathology methods
and made obsolete the necessity for the use of
electron microscopy. New techniques include
flow cytometry, fluorescence in situ hybridization
(FISH), DNA and genetics studies, proteomics,
telepathology, and digital imaging. The field of
proteomics will be widened by the discovery of
new biomarkers and new immunohistochemical
tests. These new tests must be accepted by the
medical community, and therefore histology
laboratories in various hospitals will have to work
together to standardize their protocols.
Telepathology will be implemented for
consultation and diagnosis. However, technicians
will have to be sure that a slide prepared in one
laboratory when sent as a scan to another
laboratory for diagnosis conforms to certain
criteria. Thus, specimen collection, fixation, and
processing will need to be consistent between
laboratories. In less-developed countries these
telepathology techniques and equipment will be
of benefit to patients.
We can be proud that basic histological,
histochemical and immunohistochemical
methods have a very long and productive history
and continue to give us useful information. We
have an enormous debt to the pioneers who
discovered stains for coloring tissues and
combating disease together with subsequent
techniques. These old methodologies still
continue to play an important role in the
histopathology laboratory and remain at the
forefront of research in these disciplines with an
enormous long-term impact in cell biology and
molecular biology. Histology is the tool for
accessing a specific knowledge of the
microscopic organization of the organs,
microscopic anatomy, which is essential to
understand the histopathology for a possible
diagnosis. Although today in the research field
these disciplines may seem displaced by the in
vitro study of cell biology, molecular biology,
genetics studies and proteomics, in my opinion,
remain a key to help and sustain, with the in
vivo study of tissue and organs, an efficient
clinical practice. In confirmation, it is known that
histopathology is an essential tool in diagnosis
and the experience of the histopathologist is
prime and irreplaceable for the correct
interpretation of the data obtained. Although
immunohistochemical analyses and molecular
biology are useful for diagnostic purposes, light
microscopy remains preeminent in cytological
and histological diagnosis on a daily basis.
Hematoxylin and eosin stain are still the gold
standard for diagnosis, also in malignancy
diagnosis that is based on the interpretation of
cytological and architectural features, while
immunohistochemistry and molecular biology are
ancillary tools which can provide useful
information in confirming histologically-based
diagnosis.
THE IMPPORTANCE OF HISTOPATHOLOGY
IN DIAGNOSIS, TREATMENT AND
PREVENTION OF DISEASES.
Histopathologists are doctors who work
closely with other clinical specialties. They can
reach a diagnosis by examining a small piece of
tissue from the skin, liver, kidney or other organ.
This is called a biopsy.
They examine the tissue carefully under a
microscope, looking for changes in cells that
might explain what is causing a patient’s illness.
Around 20 million histopathology slides are
examined in the UK each year
CANCER DIAGNOSIS
Histopathologists provide a diagnostic
service for cancer; they handle the cells and
tissues removed from suspicious ‘lumps and
bumps’, identify the nature of the abnormality
and, if malignant, provide information to the
clinician about the type of cancer, its grade and,
for some cancers, its responsiveness to certain
treatments.
With the help of sophisticated imaging
techniques, biopsy tissue can now be obtained
from previously inaccessible sites such as the
pancreas or retroperitoneum (behind
the peritoneum, the membrane lining the
abdominal cavity). Tissue is then processed,
usually overnight, before being examined under
a microscope.  In certain limited circumstances
using special techniques, the specimen can be
examined immediately. With rapidly  changing
developments in molecular pathology,
pathologists are leading the way with new
techniques such as fluorescence in-situ
hybridization.
(FISH) and polymerase chain reaction (PCR), to
map the genetic material in tissues or tumours,
which are essential in the management of many
cancers.
THE ROLE OF THE HISTOPATHOLOGIST
Many histopathologists specialise in
specific organs such as the liver or skin,
dissecting (‘cutting up’ or ‘trimming’) tissues for
viewing under the microscope on a daily basis.
For large specimens, such as samples of bowel
or breast following surgery, these are dissected
to select the most appropriate areas to examine
under microscope. Histopathologists write
reports on specimens, consult literature (past
and current research findings), and many also
have teaching and research responsibilities.
They will also attend multi-disciplinary meetings
so their findings can be discussed with other
clinicians. Treatments are then planned in detail
and tailored to each individual patient.
Histopathologists also work directly with
patients, for example, they may carry out
procedures such as fine needle aspiration in
head and neck or breast clinics. They
increasingly have key responsibilities for cancer
screening, at the moment for breast, bowel and
cervical cancer, with other programmes expected
in the near future.
Histopathologists also examine cells in smears,
aspirates or bodily fluids (cytopathology), for
example in urine or cervical smears. Other
subspecialties include forensic pathology,
neuropathology and paediatric pathology.
PATHOLOGICAL PROCESSES -
INFLAMMATION AND INFECTION
Histological examination of tissues can
help diagnose disease, because each condition
produces a characteristic set of changes in the
tissue structure. There are such a wide variety of
diseases that histology alone usually cannot
produce a diagnosis, although in some cases the
histological appearance is definitive. For
example, a pathologist might see signs of a viral
infection in the brain, because of tissue damage
and inflammation, but would be unable to tell
what virus is responsible; to identify the virus
might require immunohistochemistry (IHC) for the
viral protein or more likely, the diagnosis would
be confirmed by the symptoms or serology.
Conversely, the appearance of 'owl-eye' cells in
the brain is diagnostic of a particular type of
measles infection (Figure 1). Normally
histopathology reports only form one part of the
disease picture that the clinician is assembling.

Figure 1 Owl-eye bodies in infected neurons
of a child with subacute sclerosing
panencephalitis (SSPE) are characteristic of
this type of viral infection, which is produced
by a variant of the measles virus.
Although diseases are very diverse, the
responses made by the body are more limited
and fall into specific categories. For example,
inflammation, may be seen in response to an
infection or as a result of physical damage or as
part of an autoimmune disease, where the
immune system attacks components of the body.
The following sections outline some of the more
common pathological processes and relate them
to examples which can be seen in the virtual
microscope.
INFECTION
Infection can affect any tissue of the body,
producing cell damage and inflammatory
reactions. Viruses are generally too small to be
seen in the light microscope, but their presence
can often be inferred by the changes they
produce in tissue, even if their identity requires
confirmation by immunohistochemistry, serology
or molecular biology.
Bacteria can be seen in the light
microscope using high magnification objective
lenses; however, the numbers of bacteria that
are present in a tissue can be highly variable
even in one disease. A classic example of this
variability is leprosy, where there may be very
large numbers of bacteria in the skin
(lepromatous leprosy), or very few (tuberculoid
leprosy). Distinguishing the type of bacteria in a
thin section of a lesion generally requires
specialized histological stains, although the
morphology of the bacteria may also be
informative (Figure 2). As with viral infection, the
histological findings are an adjunct to serology
and microbiology in producing a diagnosis.
 Figure 3 A blood smear from a patient with a
malarial infection. Several of the erythrocytes
are infected, and the appearance is typical of
the schizont phase of infection. Scale bar =
50µm.

Figure 2 Gas gangrene in muscle - the

ACUTE INFLAMMATION
micrograph shows a colony of bacteria,
stained with haematoxylin. The bacteria have
Inflammation is a common response to
the characteristic shape and growth pattern
tissue injury or infection. Acute
of Clostridia. Scale bar = 20µm.
inflammation develops quickly and resolves
within days, whereas chronic inflammation can
last for months or years, usually because of the
Identification of parasites is often difficult by
persistence of the initiating factor. The
serological methods; however, the appearance of
histological appearance of acute inflammation is
parasite-infected cells (e.g. malaria) or the
quite different from chronic inflammation and the
parasites themselves is absolutely characteristic
distinctive features can point to the initiating
of the particular infection (Figure 3).
agent. For example, an infection of the skin
Consequently, diagnosis of parasitic infections
with Staphylococcus aureus usually produces an
relies substantially on the initial histological or
acute inflammatory response, whereas infection
haematological findings.
with Mycobacterium leprae (leprosy) typically
produces persistent infection and chronic
inflammation.

There are three main components of

inflammation (Figure 4):
1. An increase in the blood supply to the
affected area, caused by dilation of
arterioles supplying the area.
2. An increase in the permeability of
capillaries, which allows larger serum
molecules such as antibodies to enter the
tissue.
3. Migration of leukocytes from the blood into
the tissues - the cells cross the endothelial
cells, which line the venules, and then
move out into the tissue. This process is
mediated by signalling molecules called
chemokines, which are bound to the
endothelial surface.
Figure 4 The three main features of
inflammation are controlled at different
places in the vasculature.
The diagram shows a longitudinal section
through an arteriole, capillary and venule.
Smooth muscle in the arteriole’s controls blood
flow into the site of inflammation. Exudation of
serum molecules occurs in capillaries as
endothelial cells retract in response to
inflammatory mediators. This allows antibodies
and molecules of the complement system to
enter the site of inflammation. Migration of
leukocytes takes place in venules, partly
because the shear force is lowest in venules and
partly because signalling molecules are present
on the endothelium of the venules which attract
leukocytes at this point.
All of these processes bring the defence
systems of the body to the affected area. The
blood contains a number of proteins that stop
bleeding, help clear infection and induce repair or
regeneration of the tissues. It also contains
different types of leukocyte (white blood
cells), each of which has evolved to deal
with different types of infection. One of the
key histological differences between acute
and chronic inflammation is seen in the
sets of leukocytes that are present in the
tissues. In acute inflammation
polymorphonuclear neutrophils usually
predominate, whereas macrophages and
lymphocytes predominate in chronic
inflammation. Eosinophils are often prevalent in
sites of helminth infections. Hence the
characteristics of inflammation are determined
both by the tissue in which it occurs and by the
initiating agent and its persistence.
CHRONIC INFLAMMATION

Chronic inflammation is seen in diseases

where there is persistent infection, usually
because the pathogen can resist the body's
immune defences. If the infection is cleared,
chronic inflammation resolves, but residual
damage may still be evident in the tissues.
Chronic inflammation also occurs in many
autoimmune diseases; in autoimmunity the target
of the immune response is one of the body's own Table 1 Autoimmune diseases
proteins or cellular components, and
Disease Organ Target Hist
consequently the stimulus for inflammation
antigens
cannot be cleared, although the condition may
improve if the normal controls that prevent Hashimoto's Thyroid Thyroglobuli Destru
autoimmune reactions are restored. thyroiditis n
Thyroid
peroxisomes
AUTOIMMUNITY
Goodpasture's Kidney, Basement Dama
The immune system normally recognises syndrome lung membranes a
and tolerates all of the body's own tissues.
However, in some conditions the immune system Myasthenia Skeletal Acetyl Deg
reacts against 'self', resulting in autoimmune gravis muscle choline
disease. The targets may be individual receptor at n
molecules found in a specific tissue, or antigens
present in many tissues or in the extracellular Pemphigus Skin, Desmosome Se
matrix. Table 1 gives some examples of mucosa proteins in
autoimmune diseases and the target antigens. keratinocytes
An example of a tissue-specific autoimmune
disease is Hashimoto's thyroiditis, in which Diabetes - Islets of Pancreatic Select
lymphocytes recognise thyroglobulin and a type I Langerhans beta cells cell
thyroid peroxisome antigen (Figure 5). Insulin, GAD
 the thyroid follicles are destroyed by an
(enzyme)
immune reaction against components of the
Rheumatoid Joint IgG thyroid, cartilage
Erosion of articular including thyroid peroxisomes and
arthritis antibodies, thyroglobulin.
by fibrous, inflammatory Scale bar = 50µm.
cartilage
components. The histological appearance of
autoimmune disease depends on the nature of
Systemic Kidney, DNA and the immune response and the target organ.
lupus skin, CNS intracellular However, a characteristic of many organ-specific
erythematosus antigens diseases is that autoantibodies bind to the
antigen within the tissue and recruit inflammatory
cells. In this case, direct immunofluorescence
microscopy can be used to identify the presence
of antibodies, which goes a long way towards
providing a diagnosis of the disease (Figure 6). It
is also possible to detect autoantibodies in the
blood of patients, using the same technique; the
patient's serum is first incubated with normal
tissue to allow any autoantibodies to bind, and
these are then detected, by direct
immunofluorescence or immunohistochemistry.
Examination of the stained sections can
determine not just whether there are
autoantibodies in the serum, but also indicate
what the target antigen might be, depending on
where the autoantibodies are located in the cells.

Figure 5 Hashimoto's thyroiditis is an

example of an autoimmune disease, in which

Figure 6 Autoantibodies to Islets of
Langerhans in the pancreas in type-1
diabetes may be demonstrated by
immunofluorescence (Courtesy of Dr B.
Dean)
HYPERSENSITIVITY
Hypersensitivity is defined as an immune
response, where the reaction is out of proportion
to the damage caused by the antigen or
pathogen and does more harm than good.
Autoimmune diseases are by their very nature a
type of hypersensitivity reaction; however, there
are many instances where the immune reaction
against an antigen or a pathogen is out of
proportion to the damage that it causes.
A simple example is hay fever or asthma
induced by pollen, where
the pollen itself is clearly
harmless, but the
inflammatory reactions,
especially in the lung, can
be life-threatening. In
some infectious diseases,
such as M. tuberculosis, a
significant component of
the pathology is the
collateral damage caused
to lung tissue by the
ongoing immune reaction
against the bacteria. Obviously, the bacterial
infection is itself potentially damaging, but the
severity of the disease in different individuals is
at least partly due to the variability in their
immune responses.
Diseases such as multiple sclerosis are
even more complex. In this case, it is suspected
that there is an autoimmune reaction, although
the target antigen is unclear, and there is clearly
a hypersensitive response taking place in the
brain. The fact that this immune response is
particularly damaging is partly related to the
nature of the CNS, which is delicate and normally
shielded from immune and inflammatory complexes from the circulation in organs where
reactions. filtration occurs, particularly the kidney.
Hypersensitivity reactions can be classified
into four main types depending on the type of
immune response that causes them. Although
the causes of hypersensitivity are beyond the
scope of this course, the histological appearance
of the different types of hypersensitivity reactions
is often distinctive and can aid in diagnosis.
Referring to the examples given above, hay fever
and allergic asthma are examples of type-1
hypersensitivity reactions, which develop rapidly
following exposure to antigen.

They are characterized by neutrophils and

eosinophils in mucosal and submucosal tissues Figure 7 Autoantibodies in pemphigus bind to
of the respiratory tract; basophils are also components of the desmosome, a cellular
common in the bronchial wall in asthma. In structure that connects cells to their
contrast, tuberculosis is an example of a type-4 neighbours. The antibodies disrupt inter-
hypersensitivity reaction, which develops slowly, cellular adhesion causing blistering of the
in association with chronic inflammation, and is skin. The section is stained with a fluorescent
characterized by macrophages and T- antibody which detects bound auto-
lymphocytes. The other types of hypersensitive antibodies (IgA) of the patient. (Courtesy of
reaction are due to antibodies in tissues. For Dr R. Mirakian and Mr P. Collins).
example, the
autoantibodies seen SCARRING AND
in pemphigus (Figure FIBROSIS
7) are an example of
a type-2 reaction, Scarring and
whereas type-3 fibrosis are seen
reactions are caused when the cells of a
by the deposition of tissue are damaged
antigen-antibody or killed and
regeneration of the normal tissue architecture
cannot take place. For example, in cirrhosis of
the liver, the normal hepatocytes are damaged
and do not regenerate effectively. The tissue is
repaired and replaced by cells such as
fibroblasts, which lay down extracellular matrix
components including collagen, which can be
seen by appropriate histological stains.
The cells which carry out the repair vary
from one tissue to another. For example,
following damage to the CNS, a group of glial
cells called astrocytes replace damaged
neurons, forming a glial scar. Obviously, this scar
tissue cannot carry out the normal function of
nervous tissue, but it also can actively prevent
the tissue from regenerating - neurons do not
regrow their axons through glial scars. Similarly
scar tissue in the skin will usually lack
characteristic features of normal skin, such as
hair follicles and sweat glands.
Fibrosis also occurs in some infections,
particularly if the infectious agent cannot be
cleared, fibroblasts lay down areas of
extracellular matrix, which walls off the infection.
For example, schistosomiasis (a worm infection)
in the liver often results in areas of fibrosis
surrounding the individual parasites.
Fibrosis and scarring are end-stages of a
pathological process in which the body is unable
to regenerate normal tissue and does the best it
can by patching up the remaining tissue to limit
further damage.
WOUND HEALING, ANGIOGENESIS AND
TISSUE REGENERATION
In many cases cells can divide and
regenerate the tissue, restoring it to virtually
normal. For example, the basal cells of the skin
epidermis can divide to cover a scratch or a
graze, provided that it does not extend over too
great an area. Epidermal cells from hair follicles
can contribute to the regeneration, provided that
the damage has not gone too deep. In this case
there is a balance between regeneration from the
epidermis and repair from the dermal layers, the
outcome of which will determine whether a scar
is formed or not. The process of normal tissue
regeneration can be favoured by closing wounds
with stitches, or skin grafts. Conversely, if the
damage persists or the area of damage is large,
fibrosis and scarring prevail.
The ability to regenerate varies greatly
between cell types. For example, neuronal cells
have a very limited capacity to regenerate
(regrow) their axons if they have been severed,
and virtually no capacity to replace themselves
by cell division. By contrast, hepatocytes have
enormous potential for division, which can be
seen following removal of a portion of the liver,
following surgery; the remaining cells can divide
to fully restore the liver to its original size.
In tissue such as skeletal muscle,
regeneration is characterized by an increase in
the thickness of myofibers (hypertrophy), but
without significant increase in their number. The
same effect is seen with adipocytes, which
increase or decrease in size (i.e. the volume of
the lipid-filled vesicle) in response to fasting or
over-eating rather than by changes in cell
number. In such tissues, the histological
appearance can give an indication of tissue
damage that has taken place a long time
previously.
angiogenic cytokines, including VEGF
(vascular endothelial cell growth factor). The
cytokines and locally released enzymes
cause the breakdown of the vessel walls of
arterioles and venules, and sprouting of cells,
including pericytes in the vessel wall.
Endothelial cells proliferate and migrate out
of the vessel into the tissue. They reorganize
to form capillaries which interconnect
(anastemosis) and link to venules, thereby
forming a new capillary netwo
PATHOLOGICAL PROCESSES - NEOPLASIA

Angiogenesis is the process by which new HYPERPLASIA, DYSPLASIA AND

blood vessels grow into tissues, forming NEOPLASIA
capillaries.
Cell division is normally a highly regulated
process. The numbers of
cells in any tissue is
usually fairly constant,
although some tissues
can respond to
physiological demand by
an increase in cell
number.

Other types of cell

may increase in numbers
in response to appropriate
stimuli. For example, in a
guitar player, the basal
Figure 8 Ischemic regions of tissue release
cells of the epidermis in the fingertips can
proliferate to produce hard pads of keratin
(calluses) caused by repeated contact with the
strings. Cell proliferation and the consequent
increase in cell numbers seen in these two
examples is called hyperplasia. It is a normal
physiological response to demand placed on a
tissue. The numbers of each cell type are
controlled specifically. For example, the numbers
of erythrocytes in the blood is controlled by a
hormone, erythropoietin; an increase in
erythrocyte numbers does not produce any
concomitant increase in leukocyte numbers,
since leukocyte subsets are each subject to their
own controls on cell number.
If cell division becomes poorly regulated,
cells may lose some of their morphological
characteristics and/or functions. The tissue
becomes disordered in appearance, often with
an increase in the numbers of immature cells,
and greater variability between cells. This
appearance is called dysplasia. It should be
emphasised that dysplasia does not necessarily
show that the cells have become cancerous;
however, it does suggest underlying changes in
the cells, which may predispose to cancer. In this
sense dysplasia may be a stage on the way to
cancer development. For example, when
histologists screen cervical smears, they are
particularly looking for changes in the normal
morphology of the cells which indicate pre-
cancerous changes.
Neoplasia is the term used to describe the
development of tumours or cancerous tissue.
The development of a tumour requires a series of
changes in the biology of the cell, with
progressive loss of the controls that limit cell
division. Even a cell which is undergoing
uncontrolled proliferation will not necessarily be
malignant. Malignancy typically arises when the
dividing cells invade the normal tissue and move
away from their site of origin. Because of the
great variety of different tumours, it is impossible
to generalise. Nevertheless, it is very important
for a pathologist to be able to distinguish
between a benign tumour and a malignant
cancer, since the treatment required will usually
be radically different. Consequently, pathologists
often grade tumours according to how
malignant/invasive they are. Histologists can get
some impression of the rate of cell division within
a tissue according to the number of mitotic
figures - the number of cells with the nucleus
showing the characteristic pattern of separating
chromosomes, seen as the cell divides (Figure
9). Invasion of tumour cells within the tissue can
be estimated by observing where the cells are in
relation to their normal position and in relation to
other cells in that tissue, and this forms an
important element in the pathological report on a
tumour.
 cell; nevertheless,
metastasis accounts for
90% of cancer-related
deaths, so identification
of metastatic tumours is
important both for
prognosis and
treatment. Pathologists
recognise metastatic
tumours, because the
affected organ contains
clumps of cells which
Figure 9 A mitotic figure in a carcinoma of the are completely uncharacteristic. In some cases
breast (arrowed) indicates cell division. The the primary tumour-type can be recognised
number of mitoses, together with other because it has retained some distinctive
factors, are used to grade the tumour. characteristics of the original cell-type. However,
as noted above, the original identify of tumour
cells is not always self-evident and this is
METASTASIS particularly true of metastatic tumours. Hence, it
may be possible to observe a metastatic tumour
Tumours can also move away from their in a tissue, but be unable to identify the primary
original tissue by invading blood vessels or cell-type and hence the original site of the
lymphatic ducts and being carried to distant sites. tumour, at least by H&E staining. In this case
This process is called metastasis. Tumour cells additional staining, particularly
that are carried through lymphatics will usually immunohistochemistry is valuable to identify the
metastasize to local lymph nodes - this is the original cell type, because it can provide an
reason that surgeons may remove lymph nodes important guide for patient-scanning, further
as well as the original tumour to treat a cancer. surgery, radiotherapy and drug treatment.
Tumours that metastasize via the blood must first
invade a blood vessel at the initial tumour site,
and then exit the blood vessels in a different
organ to establish a new tumour site. Such an
event is relatively rare for any individual tumour PATHOLOGICAL PROCESSES - CELL DEATH
APOPTOSIS AND NECROSIS Although the loss of individual cells is
histologically undramatic, the cumulative loss of
When cells die, they do so in two main cells in such degenerative conditions can cause
ways: by apoptosis or necrosis (Figure 10). major loss of function in the affected tissue.
Apoptosis is programmed cell death; the cell dies Moreover, cell loss may be accompanied by the
as part of its normal programme of development, accumulation of products of tissue breakdown,
or it may be lacking in growth factors, or it may which are histologically evident.
be instructed to die by cells of the immune
system, because it has become infected. Even
pre-cancerous cells may be propelled into
apoptosis, by the normal cellular controls that
check the development of tumours.
In all cases, apoptosis is a highly ordered
process. If it occurs as part of a developmental
process, it does not induce inflammation - the
dead cells are quietly removed by phagocytes
within the tissue. Hence, it is often very difficult to
identify apoptotic cells within tissues, since they
are usually individual cells, with small condensed
nuclei and little cytoplasm. Cell death in
degenerative conditions (e.g. Alzheimer's
disease) appears to occur by apoptosis.
Figure 10 Schematic diagram comparing the
events that occur in cells undergoing
death by necrosis (left) or apoptosis
(right). The first events in necrosis are
irregular condensation of chromatin,
swelling of the mitochondria and
breakdown of membranes and
ribosomes. The cell is eventually
disrupted, releasing its contents and
inducing an inflammatory reaction. In
contrast, a cell undergoing apoptosis
shows condensation of the nucleus
into fragments and shrinkage of the cell. The
nucleus and cytoplasm break up into
fragments called apoptotic bodies, which are
phagocytosed by mononuclear phagocytes.
In contrast necrosis is wholesale
unregulated cell death caused by lack of
nutrients or infection. For example, the failure of
the blood supply to an organ due to thrombosis
(see below) will cause massive cell death due to
lack of oxygen (ischaemia). A large area of cell
death caused by ischaemia is called
an infarction. Another example of cell necrosis
is seen in severe viral infections with cytopathic
viruses (e.g. polio). Necrosis is an uncontrolled
process and the dying cells release their
contents. Areas of necrosis are characterised by
infiltration with inflammatory cells; macrophages
and neutrophils enter the area over a number of
days and weeks in order to clear the dead cells
and associated cellular debris. Such large areas
of cell loss and inflammation are frequently easily
seen in pathological specimens, even without
microscopic examination (Figure 11).
Figure 11 An area of necrosis in the
lung caused by a thromboembolism is visible
on the left of this section. The area on the
right includes surviving lung tissue, which is
thickened and has areas of fibrosis. Scale bar
= 2mm.
THROMBOSIS AND EMBOLISM
Blood clots may form in vessels for a
variety of reasons. A blood clot is called a
thrombus, and the process by which it forms
is thrombosis. Embolism occurs when
something is carried through the circulation from
one site to another. When a thrombus breaks
away and is carried through the circulation, it is
referred to as a thromboembolism. Other
examples of emboli are tumour cells or air-
embolism, where air is accidentally introduced
into the circulation by a physician.
Thromboembolism can block the downstream
blood vessels; emboli formed in veins pass
through the heart to block arteries in the other
side of the circulation, while thrombi formed in
arteries can block vessels in the organ where
they form. Exactly which vessels may become
blocked also depends on the size of the
embolism, and the site determines what damage
may follow.
DEGENERATIVE DISEASES AND STORAGE
DISORDERS
Cell loss occurs in many tissues with age;
the effects are particularly notable in tissues that
have a limited capacity for regeneration, such as
nerves in the central nervous system, the retina
of the eye and the sensory cells of the inner ear.
Histologically, it is more difficult to identify
something that is not there than a change in the
structure of the tissues. In diseases such as
Alzheimer's disease there is progressive loss of
neurons and shrinkage of the brain, which may
be more evident in the gross pathology, although
counting the relative numbers of cells within an
area can also give some histological indication of
the cell loss. For example, the relative numbers
of neurons relative to glial cells falls in areas
affected by Alzheimer's disease. More evident
are characteristic accumulations of proteins.
Degenerating neurons leave tangles of fibres
(neurofibrillary tangles) produced be
degenerating components of the cytoskeleton. In
addition there are extracellular accumulations of
'amyloid' within the brain. It is debated whether
these deposits are the cause or consequence of
the disease, or both.
production of amyloid is a primary event, and in
others it is secondary to infection or a tumour.
The actual protein type varies, depending on the
cause of condition. In some cases it affects
individual organs, such as the brain in
Alzheimer's disease, but in the so-called
'systemic amyloidoses' many organs may be
affected, including the lung, kidney, heart and
spleen.
There are a number of hereditary
conditions in which the person lacks enzymes
that break down particular macromolecules; they
are collectively called storage diseases because
the components that cannot be degraded within
lysosomes accumulate and form insoluble
deposits. This is particularly noticeable in the
brain, where they are often associated with
neurodegenerative diseases (Figure 12).

Amyloid is an extracellular insoluble

deposit of protein, and amyloidosis refers to the
diseases in which amyloid occurs. In some cases

Figure 12 Protein aggregates in brain cells
associated with human degenerative diseases.
Arrows highlight (a) extracellular plaques in prion
disease, (b) extracellular plaques (blue) and
neurofibrillary tangles (yellow) in Alzheimer's
disease, (c) nuclear aggregates in Huntington's
disease, (d) cytosolic aggregates known as
'Lewy bodies' in Parkinson's disease, (e) nuclear
aggregates in amyotrophic lateral sclerosis and
(f) accumulations of sphingolipids in the
distended neurons of a patient with Niemann-
Pick disease.
PHOTOGRAPHY AND REPORTING
IMAGE ACQUISITION
Digital photography has superseded the
use of film for obtaining images of histological
sections and many microscopes have a digital
camera attached. An image obtained from a slide
generally only includes a tiny proportion of the
section, however microscope systems are
now available that can scan entire slides,
providing very large images. Such images
can be transmitted electronically, so that a
pathologist can 'view' a section from a
distant location. Such systems are being
increasingly used, although they are still
very much the exception to the standard
practice where the pathologist observes
and records their observations at their own
hospital.
Images are not usually obtained for routine
work. Since the sections are stored for many
years it is always possible to return to them later.
However, for presentations, images are
essential, and there is some skill in selecting
suitable areas of the section to illustrate a point.
Journals require a minimum of 300dpi for
histological images, usually in jpg or tiff formats.
If you are preparing images for publication, it is
essential to generate images of acceptable
quality and format, by checking the requirements
on the journal website beforehand.
MAGNIFICATION AND SCALE BARS
When you see micrographs in older text-
books, a magnification is usually stated in the
legend (e.g. x100). Strictly, this should mean that
the magnification of the illustration in the book is
100-fold larger than the original item. However,
there is occasionally some ambiguity. For
example, the statement can mean that the
picture was taken using a microscope set with a
100x magnification (10x objective, 10x eyepiece).
Since the light path to the camera is not the
same as the light path to the eye (which passes
through the eyepieces), these magnifications are
not meaningful. Moreover publishers may
increase or decrease the size of a micrograph to
fit the available space. The stated magnification
in the legend should then be corrected, but often
it is not.

For these Summary of

reasons, the use
of scale-bars
Learning
has replaced a statement of magnification. A
scale bar, corresponding to a convenient unit of
length, is added to the image taken by the
camera, and is then an integral part of that
image. If the image is Histopathology and histology
increased or reduced in are often times defined and
size thereafter, the described
scale barwith one another.
changes in The term of histology cannot be
separate
proportion, so that it is always from tothat
possible seeofthehistopathology because the normal
histology
correct size of the cells or tissue. understanding is fundamental for interpretation of
histo-pathological analysis
 Histopathology is the diagnosis and study of diseases of the
tissues, and involves examining tissues and/or cells under a
microscope. Histopathologists are responsible for making
tissue diagnoses and helping clinicians manage a patient’s
care.
 Histopathologists are doctors who work closely with other
clinical specialties. They can reach a diagnosis by examining
a small piece of tissue from the skin, liver, kidney or other
organ. This is called a biopsy
 Histological examination of tissues can help diagnose
 POST
TEST II. IDENTIFICATION

_______1. What stain could you

use to identify M. tuberculosis in a section of lung?
2. What stain could you use to identify N.
gonhorrea in a urethral smear?
3. How does collagen appear in H&E staining?
What stains show collagen
Real-life more effectively?
4. Under what circumstances would you expect
application new vessels to grow into
tissues?
5. What process occurs as mountaineers
acclimatise to high altitude?
Why?
Individual Activity: Follow the instructions 6. If a thrombus is formed in the veins of the leg,
where is it likely to end up?
below. 7. If thrombi form on the tricuspid valves of the
2. In a regular size drawing book (short heart (leading to the
aorta), where might the emboli end up?
bond paper size) draw figure 1-12. 8. What stain can be used to identify amyloid
deposits in tissues?
9. can last for months or years, usually because
of the persistence of the
initiating factor. 
10. Can affect any tissue of the body, producing
cell damage and
inflammatory reactions. 
 DEAR STUDENTS, PLEASE WRITE YOUR
To validate your answers, feel free to contact your course
ANSWER IN EVERY ACTIVITY ON A SEPARATE SHORT
facilitator through any of the following: BOND PAPER. AND PROVIDE A SHORT BROWN
 Facebook: Dhaiane Flores ENVELOPE FOR YOR ACTIVITIES TO BE SUBMITTED
 Contact No: 09561270937 BEFORE PRELIM EXAM.
 Email address: floresdhaianalyn@gmail.com
PLEASE BE GUIDED.
THANK YOU.

References:
Additional references:
https://www.researchgate.net/publication/338456523_Histological
_Techniques_A_brief_Historical_Overview

https://www.open.edu/openlearn/ocw/mod/oucontent/view.php?
printable=1&id=2312#:~:text=Histological%20examination%20of
%20tissues%20can,the%20histological%20appearance%20is
%20definitive.