MA-OSEP Histology, microscopy, anatomy and disease

Histology, microscopy, anatomy and disease

About this free course

This free course is an adapted extract from the Open University course .

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You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

There you’ll also be able to track your progress via your activity record, which you can use to demonstrate your learning.

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Contents

Week 1: Histology

- Introduction

- 1 What is histology?

- 1.1 A histology department in action

- 1.2 How histological slides are produced

- 1.3 Staining techniques

- 1.4 Immunohistochemistry

- 1.5 Cells and tissues

- 2 Using a light microscope

- 2.1 Basic functions of the virtual microscope

- 2.2 Normal blood smear

- 2.3 Malaria

- 2.4 Considerations for interpreting sections

- 3 Measurements on the microscope

- 3.1 Observing different types of leukocyte

- 3.2 An example cell count

- 3.3 Acute myeloid leukaemia

- 3.4 Identify an abnormality – part I

- 3.5 Identify an abnormality – part II

- 4 This week’s quiz

- 5 Summary of Week 1

Week 2: Tissues and cells

- Introduction

- 1 Identifying cell types within tissues

- 1.1 Recognising common cell types

- 1.2 Histology of the skin

- 1.3 Skin sections

- 1.4 Histology of the gut

- 1.5 Epithelium in the gut

- 1.6 How the virtual microscope images were produced

- 2 Identifying tissues

- 2.1 Introduction to different tissues

- 2.2 Structure of a lymph node

- 2.3 Immunohistochemistry

- 2.4 Immunohistochemistry technique

- 2.5 Using IHC to identify B cells and T cells

- 3 Putting it into practice

- 4 Summary of Week 2

Week 3: Tissue structure and function

- Introduction

- 1 Functions of tissues

- 1.1 Structure–function relationships

- 2 Tissue structure and functions

- 2.1 Secretion: functions of the breast

- 2.2 Movement: functions of muscle

- 2.3 Structure and strength: functions of bone and cartilage

- 2.4 Excretion: functions of the kidney

- 2.5 Histology of the kidney

- 2.6 Communication: functions of the nervous system

- 2.7 Normal tissues

- 3 This week’s quiz

- 4 Summary of Week 3

Week 4: Recognising disease

- Introduction

- 1 Infection and inflammation

- 1.1 Infection

- 1.2 Inflammation

- 1.3 Histopathology for the diagnosis of disease

- 1.4 Infection

- 1.5 Identify an infection

- 2 Cell death and degeneration

- 2.1 Cell death in the CNS

- 2.2 Histopathology of the nervous system

- 3 Tumours

- 3.1 Metastasis

- 3.2 Neoplasia and hyperplasia

- 3.3 Put it into practice

- 4 End-of-course quiz

- 5 End-of-course summary

- Tell us what you think

- Where next?

References

Acknowledgements

Week 1: Histology

Introduction

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Welcome to this Open University course on histology. This course is designed for students studying human biology at school
or university, medical laboratory scientists and anyone interested in biomedical science.

Histology is the study of tissues and their structure, and histopathology labs are found in most hospitals. In the course
you will see a histology department in action and discover how histological slides are produced.

Watch the following video in which David Male describes what you’ll be learning about over the next four weeks.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

David has been a Professor of Biology at the OU since 1999, and has a particular interest in the use of technology for teaching
science. You will have a chance to experience one such approach during the course, by using a virtual microscope to examine
a number of biomedical samples, just as scientists and medical professionals would explore such samples in a lab.

Before you start, The Open University would really appreciate a few minutes of your time to tell us about yourself and your
expectations of the course. Your input will help to further improve the online learning experience. If you’d like to help,
and if you haven't done so already, please fill in this optional survey.

1 What is histology?

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Histology is the study of tissues and their structure. The structure of each tissue is directly related to its function, so
histology is related to anatomy and physiology.

Similarly, histopathology is the study of tissues affected by disease. This is something that can be very useful in making
a diagnosis and in determining the severity and progress of a condition. Disease processes affect tissues in distinctive ways,
which depend on the type of tissue, the disease itself and how it has progressed.

Histopathology units are found in most hospitals and there are also independent private laboratories. The services provided
by these laboratories can be accessed by healthcare professionals, such as general practitioners (GPs).

Because of the great variety of tests that are available, and the high level of skill that is needed to carry them out and
interpret them, many laboratories specialise in particular tissues or types of diagnosis. For example, a neuropathology laboratory
will focus on understanding diseases that affect the nervous system.

Histology is also used extensively in biomedical research, to identify the causes and possible treatments for disease. This
type of research may take place in a hospital laboratory but it is more often carried out in universities, research institutes
and pharmaceutical companies.

The conventional view of a histopathologist is someone looking down a microscope. Most histological work does indeed involve
the preparation of tissues for microscopy, observation of sections and reporting of the findings. However, a pathologist can
often tell a great deal about a tissue without using a microscope.

For example, the brain of a person affected by multiple sclerosis has distinct lesions (areas of damage or injury) that are
a few millimetres across. These are called plaques, and can readily be seen in a tissue sample with the human eye (see the
darker areas highlighted with arrows in Figure 1 below).

Figure 1 A cross-section through a human brain, with arrows indicating the presence of lesions (Caroldoey, 2015).

View description - Figure 1 A cross-section through a human brain, with arrows indicating the presence ...

Such large specimens that can be examined macroscopically (by eye) are usually only available post-mortem (after death) or
following surgical removal of tissue. In contrast, biopsy specimens, which consist of just a needleful of cells or a flake
of tissue, can be extracted at any time, but can only be examined microscopically.

In this course we will first teach you how to use a basic light microscope and then show some sections of various human tissues,
presented via a virtual microscope, which mirrors the functions of a real microscope.

Once you are familiar with the normal appearance of different tissues, we will start to introduce sections from diseased tissues,
and relate their appearance to the normal physiology of the tissue and pathological changes that have occurred.

Note that the virtual microscope tool will work in all modern browsers on desktop computers, laptops and tablet devices. However,
we recommend completing the course on a desktop computer or laptop for better viewing of the sections and to enable integration
of microscopy with the course text, images and video in separate windows.

1.1 A histology department in action

The following video shows the work of a busy histology department at Milton Keynes Hospital, in central England.

In the video, Peter Mooney, the Laboratory Manager for the department, takes you through the different steps involved in the
production of sections, staining and initial analysis of slides for diagnostic histopathology. These steps will be described
in more detail later in the course.

Please note that this video is around 17 minutes long, so you may prefer to watch it in stages.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

1.2 How histological slides are produced

A number of distinct steps are involved in producing histological slides, ready for analysis. Some of these steps are described
below.

Fixation

As they die, cells release enzymes that start to break down components of the tissue. This is a process termed autolysis.

The degree of breakdown depends on the tissue and what has happened. For example, post-mortem tissue is not usually taken
until several hours or days after the person has died, and this would have undergone much more autolysis than a surgical specimen.
Also, different components of tissue vary in their sensitivity to enzymatic digestion. A histologist has to be aware of all
these processes and distinguish between changes that are due to a disease and those due to tissue autolysis.

However, it is important that the original structure of the tissue is preserved before it reaches the histology laboratory,
so that it can be fully analysed. To minimise tissue breakdown, samples are often placed in a solution of fixative, in a process
known as fixation.

Different fixation procedures are required depending on what method is needed for a later step in this process, namely staining.
(You will read about staining shortly.) Because of this, it is important to know what techniques will subsequently be applied
to a tissue sample when it is taken.

In addition to preventing autolysis, fixation may serve to retain the structure of the tissue and limit microbial growth that
could otherwise make analysis more difficult.

Embedding

Tissue that has been received in the laboratory then needs to be prepared for sectioning (the process of cutting it into very
thin sections so it can be viewed with a microscope). A variety of instruments are used to cut the sections and the protocol
depends on the application.

In most cases the tissue requires embedding in a medium, which allows thin sections to be cut cleanly. Most tissues for routine
histology are embedded in wax. This occurs when water is removed from the tissue and progressively replaced by wax, which
can be solidified later to make a tissue block.

The tissue is progressively dehydrated by immersing it in successively higher concentrations of alcohol before transfer to
the organic solvent (e.g. xylene) and finally embedding in wax. In a large histology laboratory, much of this tissue processing
is automated, to save time and to produce consistent results.

Sectioning

A number of devices are available for cutting tissue into sections for analysis:

- a microtome: cuts thin sections (1–50 μm) from fixed tissue

- a vibratome: uses a vibrating blade to cut thicker sections (100–200 μm) from fresh or fixed tissue

- a cryostat: cuts sections from deep-frozen blocks, usually of unfixed tissue.

Most sectioning in routine histopathology departments is done with a microtome producing sections of ~3 μm thickness, from
tissue that has been embedded in wax, as Figures 2 and 3 below show.

Figure 2 A histologist examines a tissue specimen in a wax block to establish its orientation.

View description - Figure 2 A histologist examines a tissue specimen in a wax block to establish its ...

Figure 3 The wax block containing the tissue is sectioned on a microtome.

View description - Figure 3 The wax block containing the tissue is sectioned on a microtome.

1.3 Staining techniques

Most cells and cellular elements are virtually transparent, so it is difficult to distinguish individual cells and cellular
structures when viewed with a microscope. However, this issue can be countered if staining agents are applied to the sample
while it is being made ready for analysis.

Histologists have developed a wide variety of different staining techniques to identify different elements within tissues
when they are looked at with a microscope. This methodology is referred to as histochemistry.

The most commonly used stain for medical diagnosis uses a combination of the dyes haematoxylin (which is blue) and eosin (which
is red). This approach is usually abbreviated to ‘H&E’ staining. An example of some tissue stained using H&E is shown in the
slide image below.

Figure 4 Section across the wall of the human aorta stained conventionally with haematoxylin and eosin.

View description - Figure 4 Section across the wall of the human aorta stained conventionally with haematoxylin ...

However, there are some structures that do not stain well with H&E, so different stains have been developed that allow the
identification of particular tissue elements (e.g. collagen). These alternative stains include Giemsa, Masson’s trichrome
and elastic Van Gieson (see the slide below, Figure 5), and they are described in more detail shortly.

Figure 5 Section across the wall of the human aorta stained with elastic Van Gieson, which identifies the elastic fibres (black)
in the media of the artery and the collagen (red) in the adventitia.

View description - Figure 5 Section across the wall of the human aorta stained with elastic Van Gieson, ...

A wide variety of other histochemical stains are also available, each of which can help identify particular structures. Some
are relatively simple to apply, merely requiring that the section is dipped in the stain for a set time. Others require a
number of sequential steps, and in some cases the results can be surprisingly variable or unpredictable. For example, the
silver staining technique, originally developed by Camillo Golgi and shown in Figure 6, is notably variable.

Figure 6 Neurons visualised with a modification of the Golgi silver staining method.

View description - Figure 6 Neurons visualised with a modification of the Golgi silver staining met ...

Some of the more commonly used staining techniques are outlined below.

- Giemsa stain consists of a mixture of methylene blue and eosin. It is mostly used on blood films, where it stains red blood
cells (erythrocytes) pink and the different types of white blood cells (leukocytes), allowing their identification according
to the size and shape of their nuclei. It also binds to some pathogens (e.g. spirochaetes and syphilis) and it is, therefore,
particularly useful if infection is suspected.

- Luxol fast blue/cresyl violet is used to identify myelin, a component of the nervous system which insulates the nerves and
stains blue, while other elements of the nervous system stain pink or violet.

- Masson’s trichrome stain uses three dyes to stain different structures. It is valuable for distinguishing elements of connective
tissue. Typically the cell cytoplasm, muscle and keratin are stained red, nuclei are stained black and collagen is stained
blue.

- Periodic acid schiff (pas) stains carbohydrates magenta, including components of the basal lamina, surface glycoproteins on
cells, and intracellular carbohydrates, such as glycogen in hepatocytes. Cells that secrete mucus are also strongly stained.

- Congo red is used to identify deposits of protein in tissue called amyloid.

- Toluidine blue is a particularly versatile dye that stains nuclei blue, and can be used to differentiate between different
types of granules (e.g. within mast cells).

- Van Gieson stain binds to collagen in the extracellular matrix, staining it pink. It is often combined with a stain for elastic
fibres (elastic Van Gieson) which stains black, allowing the two major elements of connective tissue to be differentiated.

1.4 Immunohistochemistry

In the last 30 years, staining methods using antibodies have been increasingly developed. When applied to staining for the
light microscope, these techniques are collectively referred to as immunocytochemistry (ICC) for the staining of cells or
immunohistochemistry (IHC) for the staining of tissues.

Antibodies are proteins synthesised by one of the cell types of the immune system, but their use for staining other cells
has revolutionised histology, as they allow the identification and localisation of individual molecules. Antibodies specifically
bind and detect individual proteins. They can therefore be selected to identify the presence or location of a single protein
that identifies a single cell type, or is diagnostically discriminating.

A molecule that binds to, and is recognised by, an antibody is called an antigen, and, in the context of histology, such antigens
are often referred to as markers, since they act as ways of recognising a particular cell.

Staining with antibodies is particularly valuable for distinguishing between different cell types in a tissue. For example,
different classes of lymphocyte appear virtually identical in size and shape, but they can be distinguished through staining
according to their surface markers. Similarly, in Figure 7 some nerve cells (neurons) are clearly visible among other cells
in the cortex of the brain as a result of this type of staining technique.

Figure 7 Neurons in the cortex of the brain are identified by immunohistochemistry using an antibody directed against neurofilaments.

View description - Figure 7 Neurons in the cortex of the brain are identified by immunohistochemistry ...

1.5 Cells and tissues

Cells have distinctive shapes and functions which depend on how they have developed and their interactions with other cell
types. It is difficult to be precise, but there are probably at least 200 different types of cell within the body.

Disease processes affect individual cells in many ways. They may cause them to:

- die

- change their shape

- divide

- move

- invade other tissues.

Any of these changes also affect the anatomy of the tissue.

Understanding the changes that are characteristic of a disease requires a detailed knowledge of the normal appearance of cells
and tissues, and the range of normality. Many tissues change considerably with age, so that a feature that is normal in an
adult would not be normal in a child. For example, the thymus gland gradually decreases in size with age, so a large thymus
in an old person could indicate some underlying pathology.

Primary and secondary changes

The histological changes seen in a tissue may be primary, a cause of the disease process, or secondary, a consequence.

For example, if blood pressure is high due to vascular disease, it may cause an increase in the volume of the heart muscle,
as the heart finds it more difficult to pump blood into the circulation. A further consequence may be a corresponding decrease
in the volume of the chambers of the heart so that less blood is pumped with each contraction.

Interpreting the changes seen histologically requires a sound understanding of the underlying disease processes.

Identifying cells and structures

As you have read, it is difficult to see many cellular structures using a light microscope, so dyes are often applied to samples
to stain the contents and make them visible. There are two main ways of staining cells.

- The traditional method involves the use of dyes that selectively bind to different structures within the cell.

- More recently immunohistochemistry techniques have emerged that use antibodies to stain individual molecules.

Most of the sections you will view in this course have been stained with haematoxylin and eosin (or H&E). The cell nucleus
containing DNA binds haematoxylin and is, therefore, blue, as can be seen in Figure 8 below. Inactive cells usually stain
dark blue, while more active cells are paler. Dead cells have shrunken compacted nuclei which may be fragmented.

Figure 8 A section of the adrenal gland (cortex), stained with haematoxylin and eosin. Cell nuclei are stained deep blue and
the cytoplasm is pink. The capsule, the outer envelope, of the organ (large arrow) is also pink. The erythrocytes (red blood
cells) seen within small blood vessels (small arrow) are red. Scale bar = 50 µm.

View description - Figure 8 A section of the adrenal gland (cortex), stained with haematoxylin and eosin. ...

The cytoplasm of a cell stained with H&E may range from pink to purple. More active cells have diffuse purple staining ribonucleic
acid (RNA) in the cytoplasm indicating that they are producing protein. Many cells also have granules which are acidophilic
(pink) or basophilic (purple) depending on whether they have taken up eosin or haematoxylin.

The cytoplasm may also contain non-staining vacuoles. For example, fat cells (adipocytes) have a large central vacuole containing
fat.

2 Using a light microscope

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

The remainder of this week focuses on the use of a light microscope for histological purposes. You will have plenty of opportunity
to put your learning into practice by using the virtual microscope tool to explore some real samples, and later on to take
measurements from them.

Histology departments use light microscopes with transmitted light. Microscopes come in many different types, each of which
has distinct functions and applications. However, in a routine histology department, virtually all of the work is done using
a light microscope with transmitted light. This means that the light passes through the section to the objective lens, as
shown in Figure 9.

Figure 9 Elements of a light microscope.

View description - Figure 9 Elements of a light microscope.

Here, light from the lamp is focused on the specimen by the substage condenser. The objective collects light from the specimen
and focuses it to form an image within the barrel of the microscope. The eyepiece allows this image to be viewed as if it
were a projection in the plane of the section.

Resolving power

It is important to understand some of the limitations of a light microscope.

First, the absolute limit of resolution of a light microscope is determined by the wavelength of light being used (λ) and
the properties of the optical components of the microscope in question (). The limit of resolution is defined as the smallest distance () at which it is possible to distinguish two separate items.

Optics tells us that the relationship between these properties is:

= λ /(2 x )

Using the best optical components the value of  is approximately equal to 1. Consequently, the resolving power of a microscope is approximately equal to  of the wavelength of light passing through the sample.

So for example, green light has a wavelength of 550 nm. This means that structures such as the nucleus (~5000 nm), mitochondria
(~1000 nm) and large vesicles (>200 nm) can be resolved by a light microscope. Smaller structures, however, cannot be resolved.

Another limitation of a light microscope is the degree of magnification. No matter how the optical system is configured, increasing
the overall magnification beyond 1000x cannot resolve further detail of the cell structure. For this reason, the highest objective
found on light microscopes is typically 100x. When used in association with a 10x eyepiece, it gives the maximum of 1000x
magnification.

Finally, be aware that the quality and thickness of the section affects what can be resolved. In general, thicker sections
allow lower resolution than thin sections. The sections produced for diagnostic pathology are generally about one cell thick,
which allows a clear view of the structure of a single cell.

2.1 Basic functions of the virtual microscope

The following video shows you the basic functions of the virtual microscope that you will use throughout this course. It demonstrates
the processes of stage movement, focusing, illumination and the use of different objectives (the parts of the microscope that
focus the light into an image at a given magnification).

Video content is not available in this format.

View transcript - Uncaptioned interactive content

In the next section you will have your first opportunity to use the virtual microscope for yourself.

2.2 Normal blood smear

As you work through this course you will be directed to the virtual microscope to examine lots of different types of samples.
You’ll start by looking at a normal blood smear.

Activity 1

Open the virtual microscope in a new browser window or tab. This is helpful so you can operate it while still having access to the instructions you are
reading now.

Load Slide 1 (‘Normal blood smear’), which is available from the Week 1 collection in the drop-down menu above the slides
and familiarise yourself with the basic controls, including the slide box, the focus and lighting controls, the stage movement
controls and the objectives.

When you first access the slides you will find that they are blurry. This mirrors the experience that you would have when
you initially looked at a slide under a light microscope, and can be easily remedied by changing the focus settings and the
light level.

If you are struggling to focus a given slide, you can click on the spanner icon and click the checkbox for ‘Load slides in
focus’, and this will clarify the image. We urge you to try focusing the image manually first though, if possible.

Once you have mastered the microscope’s basic controls, use the legend to identify different cell types in the blood from
Slide 1 (‘Normal blood smear’). The predominant cell type is the red blood cell (erythrocyte), which carries oxygen to the
tissues, and helps to remove carbon dioxide. Leukocytes, or white blood cells, are involved in immune defence against infection.

There are a variety of different types of leukocytes, all with distinct functions. You will learn more about these cells,
and how to identify them, later on in the week. You might like to keep the virtual microscope open as you work through the
week.

2.3 Malaria

Having explored the virtual microscope in the previous activity and looked at a normal blood smear, you will now use it to
start comparing different samples. Specifically, you will be identifying infected red cells in a blood sample.

Activity 2

First, open the virtual microscope in a new browser window or tab.

Spend a few minutes comparing the normal blood smear (Slide 1 in Week 1) with the sample marked ‘Malaria’ (Slide 2 in Week
1), in the virtual microscope. Identify similarities and differences between them.

Note down the differences you notice.

Provide your answer...

View discussion - Untitled part

Now take a brief look at the slide for ‘Sample 3’ (Slide 3). How is this blood smear different from ‘Normal blood smear’ (Slide
1) and ‘Malaria’ (Slide 2) samples that you have just looked at?

Make a note of any differences that you observe.

Provide your answer...

View discussion - Untitled part

2.4 Considerations for interpreting sections

Activity 2 introduced you to the idea of comparing the content of different slides, but there are a number of other considerations
to take into account when interpreting samples.

Orientation

Most histological sections are 2D slices, from a 3D piece of tissue. Exactly what will be seen on the microscope slide depends
on the plane of the section; that is, the position of the microtome cut, in relation to the anatomical structures in the tissue.

Clinicians attempt to cut tissues in a plane that allows the underlying structure of that tissue to be seen and interpreted
most easily. For example, sections of the intestine and skin are usually cut in transverse section (across the tube), so that
all layers of the tissue are visible. Similarly, skin sections are cut perpendicular to the surface of the skin.

Positioning the tissue before embedding and knowing its orientation are, therefore, critically important, so that the sectioning
is done in the correct plane. For biopsy specimens there is no latitude for deciding the plane of the section, as the direction
of the needle tract determines what tissue is available and how it is oriented.

Pathologists expect to view sections in their best orientation as this then gives them the best opportunity of identifying
abnormalities. Many anatomical structures are not readily visible in thin, 2D sections, simply because most of the structure
is not present. In this case, the 3D structure needs to be interpreted from what can be seen.

In order to give you some insight into this problem, spend a few minutes looking at the three structures in the figure below
and thinking about what the different sections would look like if they were taken along the planes indicated by lines shown
(i.e. A–A’, B–B’ and C–C’). You can try making simple diagrams of the sections that would result from sectioning along these
lines, if you wish.

Figure 10 Understanding the plane of a section for (a) a blood vessel, (b) a sample of kidney tissue and (c) a neuron. Note
that for the neuron, the thickness of the section is significant in relation to the thickness of the cell.

View description - Figure 10 Understanding the plane of a section for (a) a blood vessel, (b) a sample ...

This exercise should make you appreciate that it is not just the orientation of the section, but also the position (depth)
into the tissue or tissue block that affects the appearance of the structures. For example, the blood vessel in (a) can appear
as one or two structures, depending on the level of the section within the block.

For this reason, it may be necessary to section a block at different levels to obtain suitable slides. (The initial cut-up
of the tissue should have ensured that the area of interest is within the block so it should not be necessary to hunt through
the block to find it.)

Size of sample

Another problem in interpreting sections is that some structures are too big to be seen in a thin section, because much of
the cell and its overall architecture cannot be seen. The neuron (image (c)) in Figure 10 is a good example of this.

One solution is to cut thicker sections in order to capture more structures. However, as noted earlier, resolution is generally
better in thin sections because in thick sections some cells will overlie others so that distinguishing details of individual
cells or the tissue structure may be more difficult. Moreover staining thick sections requires much longer for the stains
or antibodies to permeate the tissue, which slows the process of analysing and interpreting the material.

3 Measurements on the microscope

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Sometimes it is important to measure the size of a cell, the distance between cells or the number of cells in a given area.
Most light microscopes have the option to add a graticule for determining distances or a grid into the light path, to allow
such measurements to take place.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

These elements are usually added by exchanging one of the eyepieces for another with a graticule built into it. Therefore,
the appearance of the graticule is fixed according to the eyepiece that is used; changing the objective does not change the
appearance of the graticule.

A consequence is that the graticule has to be calibrated for each objective. This is most readily done by viewing a slide
that has rulings etched into its surface, and observing how many units on the graticule correspond to, for example, 1 mm on
the slide. Such a slide is shown below.

Figure 11 The appearance of an etched slide on a microscope stage when viewed with an eyepiece that includes a graticule.

View description - Figure 11 The appearance of an etched slide on a microscope stage when viewed with ...

The video above shows you how to calibrate an objective using a graticule and etched slide on the virtual microscope, as well
as introducing a grid feature that you can apply. The grid can be used to measure the number of items within a particular
area, for example, the density of blood vessels or the numbers of cells within a defined area of tissue. These are skills
you will use in later parts of the course.

3.1 Observing different types of leukocyte

One of the most basic histological tests carried out is the differential white blood cell count in which the numbers of different
types of leukocyte are counted. This process can help clinicians to diagnose diseases.

There are five main types of leukocyte, each with different functions.

1. Neutrophils are normally the predominant leukocyte in blood. They are involved in fighting infection by internalising (phagocytosing)
foreign material, including many pathogens. Neutrophils may be increased in response to bacterial infection or inflammatory
disease, although they may decrease in severe infection, or in response to cytotoxic drugs used for cancer therapy.

2. Eosinophils increase in response to some parasite infections and in allergies.

3. Basophils increase in some long-standing inflammatory conditions or in some hypersensitivity reactions.

4. Lymphocytes may increase in bacterial or viral infections. Decreased lymphocyte numbers are seen, for example, in the later stages of
HIV infection.

5. Monocytes are scavengers that phagocytose pathogens and migrate into tissues to clear debris following infection, inflammation or cell
damage. They may be increased in inflammatory conditions.

The cells differ in appearance when viewed in section under a microscope, as shown in the images below.

Figure 12 The appearance of the five main types of leukocyte.

View description - Figure 12 The appearance of the five main types of leukocyte.

The normal range for each of these cell types is quite variable, and is expressed as number of cells per litre of blood (see
the table below). For example, if you counted 100 leukocytes, you would expect to find that 20–40 of them would be lymphocytes.

Table 1 Normal ranges for a differential blood leukocyte count

Leukocyte type

Cells per litre

Percentage

Neutrophils

2–7 x 10^9

40–80%

Eosinophils

0.02–0.5 x 10^9

1–6%

Basophils

0.02–0.1 x 10^9

<2%

Lymphocytes

1–3 x 10^9

20–40%

Monocytes

0.2–1.0 x 10^9

2–10%

Values outside the normal range often indicate some kind of disease. High levels of any of them can indicate overproduction
of that cell type, for instance, a leukaemia.

3.2 An example cell count

This video shows you how to use the virtual microscope to identify leukocytes in a blood smear and carry out a differential
cell count.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

You will carry out a cell count of your own in the next activity.

3.3 Acute myeloid leukaemia

The blood smear on Slide 4 comes from a person who has acute myeloid leukaemia. Myeloid cells are the immature form of leukocytes
and would not normally be found in the blood. Usually, they would develop in the bone marrow into neutrophils, basophils or
eosinophils. However, in leukaemia there is overproduction and large numbers of myeloid cells are seen in the blood.

Activity 3

Open the virtual microscope in a new window or tab. Find Slide 4 in the ‘Week 1’ category.

Carry out a differential leukocyte count on the blood smear on Slide 4 from the ‘Week 1’ category within the virtual microscope.
Aim to count at least 100 leukocytes.

3.4 Identify an abnormality – part I

In the next activity you will be using the virtual microscope again.

Activity 4

Open the virtual microscope in a new window or tab.

Carry out a differential leukocyte count on the blood smear shown on Slide 5 in the ‘Week 1’ category within the virtual microscope.

Consider these questions. You may find it helpful to refer to Figure 12 showing the appearance of the cells and Table 1 showing
the relative abundance of different types of leukocyte.

- Is the differential blood count within the normal range?

- Do the lymphocytes appear normal?

- What is the diagnosis of this condition?

Provide your answer...

View discussion - Untitled part

How did you get on? Did you manage to derive the same conclusions? Also, please don’t worry if you were not able to derive
all of these answers – it requires a certain amount of practice to count cells and draw conclusions from your observations.
The next activity provides another opportunity for you to develop your skills in this area.

3.5 Identify an abnormality – part II

In the next activity you will be using the virtual microscope again.

Activity 5

Open the virtual microscope in a new window or tab.

Spend a few minutes comparing Slide 6 in the ‘Week 1’ category within the virtual microscope with the normal blood smear (Slide
1). Can you identify anything unusual in this smear?

Note down your thoughts.

View discussion - Activity 5

4 This week’s quiz

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

The following questions will help you understand and practise this week’s material.

Complete the Week 1 quiz now.

Open the quiz in a new window or tab then come back here when you’re done.

5 Summary of Week 1

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Congratulations, you’ve now reached the end of Week 1.

You should have some insight into the role of histology in a hospital laboratory and in biomedical research. You should also
be familiar with the capabilities of a simple light microscope, and be able to operate all the features of the virtual microscope.

You have been introduced to some of the processes involved in preparing material for microscopy, including fixation, sectioning
and staining.

You have also been shown how to recognise different types of cells in blood and carry out a differential leukocyte count.
Abnormalities in the appearance of the cells, or in their numbers, in the blood, can aid the diagnosis of genetic conditions,
infection, inflammation and allergies. The differential cell count is a key guide in the diagnosis of different types of leukaemia.

In the next part of the course you will be introduced to a variety of different types of tissues, using the virtual microscope.
The aim is for you to be able to recognise the normal appearance of each of these tissues, and be able to identify the cell
types in them.

You can now go to Week 2.

Week 2: Tissues and cells

Introduction

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Welcome to Week 2.

In the following video, David Male describes the subjects you will be covering this week, as you build on your ability to
identify different types of cell.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

1 Identifying cell types within tissues

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

This is a short introduction to some of the types of cells that you will encounter when you start to examine tissues under
the microscope.

Cells in the body differentiate into many types as they develop, and within a single organ there may be many different types
of cell. For example, in the liver the major cell type is the hepatocyte, which carries out many metabolic functions, but
there are also cells associated with blood vessels, bile ducts and immune defence, as well as various structural cells.

Despite the diversity of cells there are a number of patterns of cell organisation which occur in many different tissues.
Some key examples are described in the text below.

Epithelial cells

Epithelial cells form tight cohesive sheets of cells which cover many of the body surfaces, such as the skin or the gut. They
also form secretory glands, such as the breast, tear ducts, sweat glands and salivary glands. The ducts leading from the secretory
cells in these tissues are lined with other types of epithelium.

A layer of epithelial cells may be just a single cell thick, or may be several cells deep. This difference affects their overall
shape, which may be cuboidal, columnar or stratified (flattened). You can see some of this variation in the diagram below.

Figure 1 Arrangement of epithelial cells lining a secretory gland, its duct and the epithelial surface.

View description - Figure 1 Arrangement of epithelial cells lining a secretory gland, its duct and the ...

Endothelial cells

Another type of cell that is found in layers is the endothelial cell. These form a single layer that lines blood or lymphatic
vessels.

Muscle cells

Muscle cells fall into three main types.

- Striated muscle fibres form the voluntary muscles of the body, i.e. the ones that you can choose to move.

- Smooth muscle is a contractile tissue found in many tissues which operates involuntarily. It is found in the gut and around
blood vessels and many glands and ducts.

- Cardiac muscle is a specialised muscle found only in the heart.

Nerve cells (neurons)

Neurons have long processes (axons) which extend away from the nerve body. They form both the:

- central nervous system (CNS), namely the brain, spinal cord and retina of the eye

- the peripheral nervous system, which describes any nervous tissue outside the CNS.

Nerves run through virtually all tissues of the body, and there are large networks of nerves in tissues such as the gut.

Adipose tissue (adipocytes)

This type of cell stores fat, and is found in many tissues, not just in the main fat storage areas of the body. Adipocytes
have a large central vacuole where lipids (fats) are stored.

Leukocytes (white blood cells)

As you read and observed in Week 1, leukocytes come in a number of different types. They are found in all tissues of the body
(not just the blood), where they carry out the function of immune defence.

Lymphoid tissues, such as lymph nodes, tonsils, adenoids and Peyer’s patches (a lymphoid tissue of the intestine), contain
highly organised groupings of leukocytes that activate and coordinate the body’s immune response to infection.

Extracellular matrix proteins

Bone, tendons and connective tissues are mostly formed of extracellular matrix proteins which are produced by specific cell
types within the tissue. For example, collagen is a major extracellular matrix protein, and it is formed in several different
types, depending on its function within the tissue.

1.1 Recognising common cell types

This video describes how to use the virtual microscope tool to recognise some of the different cell structures that you have
just read about.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

You will explore some of these cell types in more detail as you progress through the rest of the course. However, what is
important to recognise here is the fact that these distinct structures commonly recur in very different types of body tissue.

1.2 Histology of the skin

The skin is the first tissue we’ll examine in more detail from a histological perspective.

The outermost layer of the skin, the epidermis, is formed by epithelial cells (see part (a) of the diagram below). In this
case the epithelium is a multi-layered stratified structure, and its thickness depends on where on the body the skin is located.

Beneath the epidermis lies the dermis, which contains sweat glands, sebaceous glands that produce an oily secretion to lubricate
the skin, and hair follicles. A number of cell structures are found in this layer of the skin, as illustrated in the figure.
The nerves end in various sensory organs in the dermis, and they innervate the muscle which is attached to the hair.

Figure 2 Structure of the skin showing the various types of cells and tissues: (a) epithelial cells in the epidermis; (b)
connective tissue; (c) muscle; (d) epithelial cells lining the duct of a sweat gland; (e) adipose tissue below the dermis.

View description - Figure 2 Structure of the skin showing the various types of cells and tissues: (a) ...

Take a few moments to familiarise yourself with the overall arrangement of the structures in the skin.

1.3 Skin sections

Next you’ll examine structures in the skin in more detail via the virtual microscope.

Activity 1

Open the virtual microscope. (Remember, you may find it easier to open the virtual microscope in a new browser window or tab, to make it easier to refer
back to these instructions.)

Load Slide 1 (from the ‘Week 2’ collection) onto the stage. This sample is a section through the skin of a fingertip. Spend
a few moments looking at the slide, and making use of the legend provided.

You should have noticed that the epidermis is particularly thick and ridged towards the right-hand side of the sample. In
the skin the epithelial cells are called keratinocytes because they form keratin, the thick impervious layer of the skin surface.

You may have also observed in this sample that the skin of the fingertips does not have hairs, but it does have sweat (eccrine)
glands and a high density of sensory organs in the dermis. These sensory organs include structures such as the Pacinian corpuscles,
which specifically respond to touch and pressure, enabling us to feel the external environment.

Finally for this slide, spend a few moments trying to identify the connective tissue of the dermis and some blood vessels.

Now turn your attention to Slide 2 from the same collection. This is from pigmented skin.

Identify the cells in the epidermis which have high levels of the pigment melanin. Also, compare the thickness of the epidermis
on this section with that on Slide 1. You should see that the contrast between their respective thicknesses is significant.

1.4 Histology of the gut

The next type of tissue that we’ll examine more closely is that which forms the gut. Although the shape and dimensions of
the gut vary along its length, the organisation of the different cell types that make up the gut wall is essentially similar
throughout.

There are three main layers in the gut wall, as shown in Figure 1 below. Starting from the inside of the wall and working
outwards these layers are the:

- mucosa

- submucosa

- muscularis externa.

Each of these layers, in turn, contains several cell types. Note that the adventitia (the name for the outer covering of any
organ) does not form an integral part of the gut wall.

Figure 3 Cross-section of the gut showing the arrangement of the different tissue elements at the level of the ileum (small
intestine).

View description - Figure 3 Cross-section of the gut showing the arrangement of the different tissue ...

Let’s consider the composition of the three main layers of the gut wall in more detail.

Mucosa

In some areas such as the mouth, oesophagus and anus, where some physical protection is needed to prevent infection, the epithelium
is several layers thick, and is something like that of the skin.

However, in most parts of the gut, the properties of the epithelium are rather different from those of the skin. Here the
epithelium is only a single cell thick, with the apical membrane of these cells exposed to the lumen of the gut and the basal
membrane facing the submucosal cells. All epithelial cells have two faces – the basal membrane lies on the connective tissue
of the basal lamina and the apical membrane faces the surface of the tissue or duct.

These epithelial cells are specialised to perform digestive or absorptive roles. For example, the epithelial cells in the
small intestine have small hair-like projections called microvilli on their apical surface.

Finally, in the stomach and intestines the epithelium is frequently invaginated (i.e. folded inwards) to form glands which
consist of groups of secretory epithelial cells. Different types of gland are found in different regions of the gut.

Submucosa

The submucosa is a loose matrix of connective tissue in which blood and lymph vessels lie, and a large number of neuron cell
bodies are grouped together as ganglia.

The enteric neurons (i.e. those associated with the gut) extend to the mucosa, where they influence both the production of
digestive secretions by the epithelial cells and the state of dilation of the mucosal blood vessels.

Muscularis externa

The muscularis externa is a thick layer of smooth muscle. In almost all parts of the gut it consists of two separate muscle
layers that lie at right angles to each other.

- The outer layer is longitudinal, that is it runs lengthways along the gut

- The inner muscle layer fibres encircle the wall of the tube.

Successive contraction and relaxation of these muscle layers results in peristalsis, which are waves of movement that propel
the food through the gut.

Familiarise yourself with the overall anatomy of the gut before you move on to the next activity.

1.5 Epithelium in the gut

Look at some actual tissue samples for different parts of the gut.

Activity 2

Open the virtual microscope in a new window or tab.

Examine Slides 3–5 from the ‘Week 2’ category within the virtual microscope.

The slides depict sections taken from the stomach (fundus region), illeum and colon, respectively. Spend a few minutes looking
at the samples and note the different types of epithelium lining the tissue.

In each case, aim to identify the typical structures that denote the three main layers of gut tissue:

- the mucosa

- the submucosa

- the smooth muscle of the muscularis externa.

1.6 How the virtual microscope images were produced

In this video, David Male discusses The Open University’s virtual microscope project.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

As the collection of slides available has been drawn from a number of laboratories, it gives you the opportunity to explore
samples that you might not normally have access to.

2 Identifying tissues

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Learn to identify different types of tissue using evidence provided by the virtual microscope.

In this video, David Male discusses how to use the virtual microscope to examine a histological specimen.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

David emphasises how important it is to make an overall scan of the sections before focusing on specific areas and selecting
an objective that can identify the features of interest in those areas.

2.1 Introduction to different tissues

The rest of this week focuses on the task of identifying different types of tissue using evidence provided by the virtual
microscope. Specifically, this involves working through Activity 3, looking at Slides 6–11 from the ‘Week 2’ collection.

Activity 3

Open the virtual microscope in a new browser window or tab and find the ‘Week 2’ collection. Slides 6–11 have been chosen
to introduce a range of different tissues, so that you can recognise them again later on in the course. Use the instructions
below, and the associated legends to navigate around the sections.

Lung (Slides 6 and 7)

Compare the normal lung (Slide 6) and the ‘inflated’ lung (Slide 7). You should be able to see that the alveoli are much larger
on the inflated lung, which was injected to inflate it, before processing.

Both sections are from normal tissue. However, this pair of slides illustrates that the way in which a tissue is prepared
can affect its appearance.

Blood vessels (Slide 8)

Arteries and veins often run together as pairs, although they appear quite different. The vein has a much thinner wall and,
therefore, has collapsed and is less prominent than the artery in this slide.

The section also includes a number of smaller blood vessels. Arteries branch into arterioles and then into capillaries, the
smallest blood vessels. Capillaries drain into venules, which feed into the veins.

Liver (Slide 9)

The liver has a complex organisation with an ‘open’ circulation. The circulation is described in this way because the blood
within the organ is not confined within blood vessels.

Instead, the liver is supplied by the arterial blood (hepatic) artery and receives blood from the gut (hepatic portal vein).
Blood flows through the liver along sinusoids, rather than capillaries.

Pancreas (Slide 10)

The pancreas contains two distinct types of tissue.

- The exocrine pancreas produces digestive enzymes.

- The Islets of Langerhans produce a number of hormones, including insulin, which control aspects of metabolism.

Notice that the Islets can most easily be identified using the lowest magnification objective.

Thymus (Slide 11)

The thymus is a lymphoid organ, in which a type of leukocyte known as T lymphocytes (or ‘T cells’) develop. (You have already
encountered lymphocytes in the blood smears.) After their initial development in the thymus, T cells distribute to other lymphoid
organs, including lymph nodes, the spleen and Peyer’s patches. The tissues where lymphocytes initially develop, thymus and
bone marrow, are referred to as primary lymphoid organs, and the other organs, such as lymph nodes, are secondary lymphoid
organs. T cells are involved in the recognition of infected cells and they also control and coordinate many other elements
of immune responses.

T cell development occurs during the early years of life, and the thymus becomes gradually smaller with age. Notice the distinct
areas within the tissue. T cell development starts in the cortex (outer layer) and the cells mature as they progress to the
medulla (centre) of the organ.

2.2 Structure of a lymph node

Lymph nodes are encapsulated organs that are strategically placed along the lymphatic network. Here they can trap foreign
material (antigens), which are presented to the lymphocytes by antigen-presenting cells, to initiate an immune response.

The lymphocytes are densely packed in the lymph node, and the tissue is organised both to facilitate the interactions needed
to generate an immune response against the antigen and to promote rapid division of the responding lymphocytes.

Lymph nodes vary in size (from a few millimetres to 1–2 cm), and are distributed in different areas of the body. They are
linked in chains by lymphatic ducts, so that fluid flowing out of one lymph node via the efferent lymphatic vessel becomes
the inflow to the next in line, via the afferent lymphatics.

The fluid in question is called lymph, and is derived from the tissue that carries cells and foreign material to the lymph
nodes. Eventually, lymph returns to the bloodstream via one of the body’s two major lymphatic ducts.

Secondary lymphoid organs can be thought of as guard posts that are strategically placed to intercept any infectious agent
that enters an area of the body. So, for example, the lymph nodes in the axilla of the arm (the armpit) will intercept infections
which enter that part of the body.

Lymphocytes located in the local lymph nodes are responsible for the initial recognition of the infection and the development
of the immune response. Once the immune response has developed, the cells will migrate out from the lymph node to the blood
and, ultimately, cells will move to the site of infection to combat the pathogen there.

Figure 4 Structure of a lymph node.

View description - Figure 4 Structure of a lymph node.

Lymph nodes have a well defined structure with different sub-regions. Different types of leukocyte are localised within the
regions so that they can interact with each other appropriately to initiate and develop the immune response. Antigens and
cells enter the node through afferent lymphatics, and cells and fluid leave through the efferent lymphatic. Cells (lymphocytes)
can also enter the node from the blood by migrating across the specialised high endothelial venules. Within the node, cells
distribute themselves to distinct zones. B cells proliferate and develop within the follicles of the cortex, while T cells
are primarily located in the paracortex. The capsule, medullary cords and hilus are fixed structural elements of the tissue.

Activity 4

Open the virtual microscope in a new browser window or tab.

Look at Slide 12 in the ‘Week 2’ category within the virtual microscope. This slide is a section from a lymph node that you
learned about above.

Now spend a few moments exploring Slide 12, and try to identify some of the structures described. (Don’t forget to use the
slide’s legend to help guide your navigation of the sample.)

2.3 Immunohistochemistry

As you read in Week 1, the staining of cells and sections with antibodies has revolutionised histology as it allows the identification
and localisation of individual molecules.

A molecule that binds to, and is recognised by, an antibody, is called an antigen, and, in the context of histology, such
antigens are often referred to as markers, since they act as ways of recognising a particular cell. Many markers are designated
by a CD number. Indeed, more than 250 markers have been assigned CD designations.

As you read previously, immunohistochemistry (IHC) is particularly valuable for distinguishing different cell types in the
diagnosis of cancer. For example, different classes of lymphocyte appear virtually identical in size and shape (morphology),
but they can be distinguished according to their surface markers. All T lymphocytes express CD3, and the two major subpopulations
of helper T cells and cytotoxic T cells express CD4 and CD8, respectively. Therefore, a T cell lymphoma can be tracked in
different tissues using these markers.

Figure 5 Immunohistochemistry. Double immunolabelling of an Islet of Langerhans in the pancreas identifies insulin-producing
cells (blue) and glucagon-producing cells (brown).

View description - Figure 5 Immunohistochemistry. Double immunolabelling of an Islet of Langerhans in ...

Another use for IHC is in guiding treatment. For example, many breast cancers require oestrogen to divide, but a drug called
tamoxifen can bind to the oestrogen receptor on the cancerous cells, blocking this proliferative effect. However, this only
applies to some cancers, as others do not have an oestrogen receptor and are therefore not susceptible to tamoxifen therapy.

A researcher or clinician can use IHC to identify whether a breast cancer removed by surgery expresses the oestrogen receptor.
The clinician wants to treat the patient so as to prevent any secondary tumours from growing and with this information they
can decide whether or not tamoxifen therapy is appropriate.

2.4 Immunohistochemistry technique

A standard IHC protocol involves treatment of the section with an antibody that recognises the marker. This is referred to
as the primary antibody.

The primary antibody is then recognised by a secondary antibody that is linked to an enzyme, or several copies of the enzymes.
Such reagents are sometimes called conjugates, although this term can mean different things in other contexts.

Finally, the section is treated with a chromogen, a reagent that is acted upon by the enzyme, to deposit an insoluble coloured
compound onto the cell, where the original primary antibody had bound.

Figure 6 Principles of immunohistochemistry. (a) Unlabelled primary antibodies bind to the antigen on the section. (b) Labelled
secondary antibodies bind to the primary antibodies. The secondary antibodies have an enzyme attached. (c) Chromogen is converted
by the enzyme into an insoluble stain on the section.

View description - Figure 6 Principles of immunohistochemistry. (a) Unlabelled primary antibodies bind ...

2.5 Using IHC to identify B cells and T cells

T cells and B cells are the two major subtypes of lymphocyte. B cells may ultimately differentiate into antibody-producing
cells, while T cells perform many functions in immune defence.

Within lymph nodes B cells tend to distribute to the follicles, and T cells to areas of the paracortex. The distribution can
be readily seen by IHC staining of the cells with appropriate antibodies against their cell surface markers: CD3 for T cells
or CD20 for B cells.

Activity 6

Open the virtual microscope in a new window or tab.

Look at Slides 13 and 14 from the ‘Week 2’ category.

Spend a few moments looking at where the T cells and B cells are distributed. Then compare these lymph node sections with
the H&E stained section in Slide 12.

3 Putting it into practice

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

The final activity this week you will investigate three sample sections.

This is an opportunity for you to put into practice what you’ve learned this week about identifying types of tissue and will
increase your familiarity with the virtual microscope.

Activity 6

Open the virtual microscope in a new window or tab. Find Slides 15–17 from the ‘Week 2’ slide set.

For each slide, answer the questions and then click to reveal the answer.

Slide 15

- What is the tissue in Slide 15?

- Does the tissue in Slide 15 appear normal?

Provide your answer...

View answer - Untitled part

Slide 16

- What is the tissue in Slide 16?

- Does the tissue in Slide 16 appear normal?

Provide your answer...

View answer - Untitled part

Slide 17

- What is the tissue in Slide 17?

- Does the tissue in Slide 17 appear normal?

Provide your answer...

View answer - Untitled part

4 Summary of Week 2

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Having reached the end of the second week of the course you should now be able to identify a number of tissue types, including
skin, lung, gut, liver, pancreas and lymph node.

In the process you have learned about tissue organisation, and should be able to recognise some of the cell types found within
the organs mentioned above.

Finally, from a practical perspective, you have learned how to scan across a slide systematically, and you should now be starting
to look for abnormalities in tissue structure based on your understanding of what healthy tissue looks like.

Next week you will be examining some more tissues and relating their structure to their functions.

Please note that there is no quiz this week.

You can now go to Week 3.

Week 3: Tissue structure and function

Introduction

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Welcome to Week 3 of the course.

This week you will look at a selection of tissues that have different functions. You will continue your work with the virtual
microscope tool to explore how the structure and function of these tissues are related.

As David Male explains, the relationship between tissue function and structure is a key consideration for histologists and
other medical professionals, because understanding this association can support the diagnosis of disease.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

1 Functions of tissues

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

The structure of each tissue of the body is organised to carry out its own specific functions, and this is reflected in the
arrangement of the cells and its histological appearance.

The week focuses on five functions of tissues, namely:

- secretion

- movement

- strength

- excretion

- communication.

As you should already be aware, different tissues are capable of different functions. However, these functions may be interrelated.
For example, the cells of the endocrine system secrete hormones, which are involved in communication between different tissues.

Secretion is an example of a function that is particularly specialised. Many cell types release molecules into the extracellular
environment, but secretion is a specific function carried out by epithelial cells. Some examples are given in Table 1.

Table 1 Tissues and secretion

Tissue

Secretion

Thyroid

thyroid hormones

Breast

milk

Salivary gland

saliva

Tear ducts

tears

Exocrine pancreas

digestive enzymes

Islets of Langerhans

insulin, glucagon

Stomach epithelium

acid, intrinsic factor

Cells that carry out secretion generally store the secreted proteins either inside the cell, in secretory vesicles, or in
extracellular depots (e.g. the thyroid gland). The secreted material is released when the cell or tissue receives an appropriate
signal, as seen in the Figure 1 below.

Figure 1 Secretion of proteins produced in the Golgi apparatus. Secretion may either be constitutive (continuous) or the secreted
molecules may be stored in vesicles and then released when a signal is received.

View description - Figure 1 Secretion of proteins produced in the Golgi apparatus. Secretion may either ...

Tissues respond to a variety of stimuli. For example, the thyroid responds to thyroid-stimulating hormone (TSH) released by
the pituitary gland, which promotes the division of thyroid epithelial cells and the release of thyroid hormones.

It is important when viewing a tissue to consider how it may have adapted to physiological changes or signals from other cells.
For example, what factors can you think of that might cause the expansion (hypertrophy) of muscle cells in the heart (cardiac
myocytes)?

There are a number of factors you could identify to explain this observation, including: a programme of heavy exercise or
training; high blood pressure, requiring greater effort from the heart to pump blood; and heart valve regurgitation, causing
inefficient pumping of blood into the circulation. Understanding the likely cause of things that you observe in tissue sections
is key, as it allows decisions to be made as to what medical intervention (if any) is required.

1.1 Structure–function relationships

The cells within tissues are generally arranged to carry out the function of the organ most effectively. Consider the lung
as an example.

The lung’s primary function is gas exchange. Red blood cells carry oxygen from the lungs to the other tissues and return carbon
dioxide to the lungs. The physiology of the blood transport systems need not concern us here, but the relationship of the
cells in the lung is optimised for gas exchange as shown in the diagram.

Figure 2 Schematic diagram of an alveolus, in contact with pulmonary capillaries (Villee, 1989).

View description - Figure 2 Schematic diagram of an alveolus, in contact with pulmonary capillaries ...

Another tissue that you have already seen is the gut. (Recall that the appearance of the gut varies greatly along its length,
although the overall arrangement of the layers of cells is basically the same throughout.)

The main site for absorption of nutrients is in the intestine, which has a very large surface area, with numerous villi. The
villi have a rich blood supply and lymphatic drainage; therefore, this region of the gut is particularly well suited for their
function.

Figure 3 Schematic diagram of villi in the small intestine.

View description - Figure 3 Schematic diagram of villi in the small intestine.

The rest of this week guides you through studying the structure–function relationships of a range of tissues, as grouped by
the main functionalities we identified earlier. You will start by looking at secretion, before progressing on to movement,
strength, excretion and communication.

As you learn to recognise each tissue, try to relate the histological appearance to the 3D structure of the organ and its
underlying function.

2 Tissue structure and functions

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

In the next sections you'll learn about specialist tissues for secretion, movement, strength, excretion and communication
and examine them under the virtual microscope.

Secretion: Functions of the thyroid

The thyroid is a two-lobed endocrine (hormone-secreting) gland located in the neck, as can be seen in Figure 4 below.

Figure 4 Location of the thyroid gland (adapted from BTF, 2016).

View description - Figure 4 Location of the thyroid gland (adapted from BTF, 2016).

The gland produces two related hormones: thyroxine (T4) and tri-iodothyronine (T3), which are involved in the control of metabolic
rate. T4 contains four molecules of iodine and T3 contains three molecules of iodine. Deficiency of iodine leads to proliferation
of the cells in the thyroid, in response to the unmet demand for the hormones.

Histologically, normal thyroid tissue consists of many follicles lined with a single layer of epithelial cells called thyrocytes.
These cells synthesise a protein (thyroglobulin), which acts as a precursor and storage site for the thyroid hormones. The
thyroglobulin is secreted and stored inside the thyroid follicles.

When stimulated by thyroid-stimulating hormone (TSH) from the pituitary, thyroglobulin is internalised (phagocytosed) by the
thyrocytes and broken down to release the hormones, which are then secreted into the blood.

Thyroid histology is altered in conditions such as Graves’ disease (as a result of an overactive thyroid) and in Hashimoto’s
thyroiditis, an autoimmune disease, where the body’s own immune system attacks and destroys the thyroid epithelium.

Activity 1

Having read a little about the structure and function of the thyroid you will now examine a sample of tissue using the virtual
microscope.

Open the virtual microscope in a new window or tab. Find Slide 1 in the ‘Week 3’ category. Slide 1 shows a thyroid lobule containing about 50 follicles
lined with thyroid epithelium, which produce the hormones thyroxine (T4) and tri-iodothyronine (T3).

Spend a few moments familiarising yourself with the structure of this sample, as guided by the slide’s interactive legend.

2.1 Secretion: functions of the breast

The breast is another example of a secretory tissue.

Structurally, the breast consists of a number of lobes connected by lactiferous (milk-carrying) ducts to the nipple. Each
lobe is a compound gland branching into lobules and alveoli lined with epithelial cells, which are supported in a loose matrix
of connective tissue. The connective tissue includes accumulations of adipocytes (fat cells).

During pregnancy the duct system and alveoli expand considerably as the epithelial cells lining the alveoli are responsible
for milk secretion. At this time the connective tissue is correspondingly reduced. The alveoli are surrounded by contractile
cells (myocytes), which put pressure onto the alveoli to promote milk secretion.

After weaning, when breast milk is no longer required, the alveoli regress, leaving just the duct system in place.

Figure 5 A diagram showing breast anatomy and histology.

View description - Figure 5 A diagram showing breast anatomy and histology.

Activity 2

Open the virtual microscope in a new window or tab. Find Slides 2 and 3 in the ‘Week 3’ category.

Spend a few minutes comparing the histology of a normal breast (Slide 2) and a lactating breast (Slide 3). Notice the expansion
of the alveoli and the secretory epithelium in the lactating breast. These sections demonstrate how a tissue can vary greatly
in its histological appearance as a result of normal physiological changes.

2.2 Movement: functions of muscle

Focus now shifts to the function of movement, and the muscle tissue that facilitates it.

All muscles are contractile and excitable and they contain the proteins myosin and actin, which are responsible for contraction.
However, the functions of the muscle types and the arrangement of cells within them vary. As you read earlier in the course,
from a histological perspective there are three different types of muscle:

- striated (or skeletal) muscle

- smooth muscle

- cardiac muscle.

Skeletal muscles are the voluntary muscles, attached to bones via tendons, which allow voluntary movement of the body. Muscle
tissue is made from a collection of highly specialised cells, known as muscle fibres. These are formed by the fusion of individual
muscle cells (myocytes).

The muscle tissue is surrounded by supporting and protecting bands of fibrous connective tissue called fascia. The plentiful
nerve and blood supplies which an active muscle requires are located within fascia.

Figure 6 The internal structure of a skeletal muscle fibre (Gottfried, 1994).

View description - Figure 6 The internal structure of a skeletal muscle fibre (Gottfried, 1994).

Within a myofibril, actin and myosin are interleaved, producing the characteristic striated (striped) appearance, which can
be seen under the microscope and is shown schematically in the figure below. When a muscle contracts the actin and myosin
slide across each other, causing the striations to bunch up.

Energy is required to relax the muscle (surprisingly the default condition is contraction). When a muscle runs out of energy
it contracts, as occurs in cramp and rigor mortis.

Figure 7 Arrangement of actin and myosin in a myofibre produce striations. The upper diagram shows the relaxed state and the
lower diagram the contracted state.

View description - Figure 7 Arrangement of actin and myosin in a myofibre produce striations. The upper ...

In contrast to skeletal muscle, both cardiac and smooth muscle are involuntary muscles, meaning that we cannot voluntarily
control their contraction. In cardiac muscle the myocytes form a network, with crosslinks and intercalated discs between the
cells. They have striations comparable to those in skeletal muscle.

Figure 8 Arrangement of myocytes in cardiac muscle.

View description - Figure 8 Arrangement of myocytes in cardiac muscle.

Smooth muscle is found in the wall of blood vessels and many hollow organs, including the uterus, the bladder and along all
parts of the gastrointestinal tract.

Activity 3

Open the virtual microscope in a new window or tab. Find Slides 4–6 in the ‘Week 3’ category.

See if you can identify the characteristics of the three different types of muscle: striated muscle (Slide 4), cardiac muscle
(Slide 5) and smooth muscle (Slide 6).

Note that smooth muscle is illustrated in Slide 6 in the wall of an artery. However, it can also be seen in different areas
of the gut (see Slides 3–5 in the ‘Week 2’ category).

2.3 Structure and strength: functions of bone and cartilage

Tissues such as bone and cartilage give our bodies structure and strength. The examples used are bone and cartilage.

There are many different types of cartilage, including articular cartilage, which covers the joints at the end of the bones.
However, the example provided in the slide set that you will examine shortly is from the trachea (windpipe). This structure
has bands of hyaline cartilage, which strengthen the trachea, to resist collapse or closure as the head moves.

Figure 9 (a) Diagram and (b) photograph of a partially sectioned long bone (femur), showing its internal structure. Erythrocytes
and leukocytes are produced from stem cells located primarily in the red marrow. Yellow marrow produces factors (cytokines)
that promote the development of the blood cells (Tortora and Grabowski, 1993).

View description - Figure 9 (a) Diagram and (b) photograph of a partially sectioned long bone (femur), ...

Notice in these strong tissues how the cells form only a small proportion of the total tissue mass. They are responsible for
forming, remodelling and repairing the tissue, but the strength is provided by the extracellular matrix (and mineralisation),
including proteins such as collagen and elastin in tendons.

Mineralisation produces highly ordered structures around the cells, and is arranged to resist the stresses placed on the tissue.
The hardest tissues of the body – bones and teeth – are mineralised with calcium hydroxyl apatite.

Activity 4

Open the virtual microscope in a new window or tab. Find Slides 7–8 in the ‘Week 3’ category.

First, take a look at the section of bone shown in Slide 7. Identify the cells, osteoblasts (which produce the bone) and osteoclasts
(which remodel it), and familiarise yourself with their appearance and structure.

Next, in the section of trachea shown in Slide 8, identify the chondrocytes that form the connective tissues, and in each
case relate the position of the cells to the extracellular structures.

2.4 Excretion: functions of the kidney

The kidneys are excretory organs that perform three main functions to produce urine, filtration, reabsorption and secretion.
They are situated at the back of the peritoneal cavity.

A cross-sectional slice through the kidney shows an outer layer (renal cortex), a middle layer (renal medulla), and an inner
area (renal pelvis), where the ureter widens to join the kidney. These three structures are shown in Figure 10.

Figure 10 Diagram of the kidney structure with an enlarged diagram of a nephron. The ureter carries urine from the kidney
to the bladder.

View description - Figure 10 Diagram of the kidney structure with an enlarged diagram of a nephron. ...

Within each kidney there are approximately one million structures called nephrons, each of which acts as an independent filter
and urine-processing unit. A nephron consists of a renal corpuscle, which lies in the cortex, and a long tube which collects
and processes the filtered fluid, called a renal tubule.

At the renal corpuscle, a network of very small-diameter blood capillaries, known as the glomerulus, comes into close contact
with the closed end of the tubule, which is composed of a single layer of epithelial cells – the Bowman’s capsule.

In this specialised region, fluid is filtered out of the blood capillaries, across the epithelial cells and into the lumen
of the tubule. The filtrate then passes along the tubule, which is convoluted (in some cases looping down into the medulla),
before finally joining with the renal pelvis, where the urine is emptied into the ureters.

It is during its passage along the tubule that the contents of the filtrate are processed, and urine is formed. Most of the
filtered water, glucose, amino acids, sodium and other ions are reabsorbed by the epithelial cells of the tubules. Waste substances
are either not reabsorbed at all, or only partially reabsorbed.

Some molecules and ions are also secreted into the tubule by the epithelial cells and, together with waste products which
remain in the filtrate, are excreted in the urine.

Cells of the kidney thus perform three main functions to produce urine:

- filtration occurs in the glomerulus/Bowman’s capsule

- reabsorption and secretion occur in the tubules.

The following diagram illustrates the blood supply to different parts of the nephron. High pressure within the glomerulus
promotes the initial filtration of the blood plasma, and the network of vessels associated with the loop of Henle allows reabsorption
of ions and water.

Figure 11 The blood supply to different parts of the nephron.

View description - Figure 11 The blood supply to different parts of the nephron.

2.5 Histology of the kidney

As the anatomy and histology of the kidney is quite complex, this video gives you a short tour of the section, before you
look at it yourself in the virtual microscope.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

Activity 5

Open the virtual microscope in a new window or tab. Find Slide 9 in the ‘Week 3’ category.

Now it’s your turn to identify the main areas that exist within the kidney sample shown in Slide 9. Try to locate each of
the following:

- the glomerulus

- Bowman’s capsule

- convoluted tubules

- the loop of Henle

- collecting tubules.

2.6 Communication: functions of the nervous system

The function of the nervous system, and the cells that comprise it, is to communicate signals that originate both within the
body and in the external environment.

As you read earlier in the course, the nervous system is divided into two main parts.

- the central nervous system (CNS) includes the brain, spinal cord and retina of the eye

- the peripheral nervous system (PNS) is the network of nerves outside the CNS that runs through virtually all other tissues.

Both parts are illustrated in Figure 12.

Figure 12 a) The human nervous system showing the CNS and PNS. (b) A diagram of a small peripheral nerve showing the mixture
of nerve fibres travelling from the CNS (efferent neurons) which control muscle movement, and nerves travelling to the CNS
(afferent neurons) which convey sensory information.

View description - Figure 12 a) The human nervous system showing the CNS and PNS. (b) A diagram of a ...

There are a greater abundance of nerve cells in some parts of the body than others. For example, there is a dense plexus of
nerves associated with the gut, which is know as the enteric nervous system. This network of cells controls peristalsis –
the coordinated contraction of the muscles along the gut wall that moves matter through the digestive system.

The roles and structures of neurons

Around half of the cells that comprise the nervous system are called neurons.

Neurons typically consist of a cell body, which contains the nucleus of the cell. Dendrites extend from the cell body, and
this is where information (signals) are received. Each cell also has a single axon, which relays signals onwards to other
neurons or to other cells such as muscle. A number of possible arrangements of neural structures are depicted below in Figure
13.

Figure 13 Different types of neuron. (a) With the cell body at the end of the axon. (b) With the cell body to the side of
the axon. (c) With no processes. (d) With an insulating sheath of myelin around the axon, which speeds nerve conduction. (e)
A small nerve network showing inputs (neurons 2, 3 and 4) to neuron 1, and its output to neuron 5.

View description - Figure 13 Different types of neuron. (a) With the cell body at the end of the axon. ...

The roles and structures of other cell types

The other cells that comprise the nervous system perform supporting functions for the neurons, and are collectively known
as glia. Examples of glia are astrocytes, oligodendrocytes, Schwann cells and microglia, and these are described in more detail
below.

Astrocytes control many metabolic functions within the nervous system and they signal between neurons and the vascular system
to control blood flow.

Many nerves have a sheath of insulating material (myelin) around their axons, which allows faster conduction of the signal
(see part (d) of the previous figure). Such nerves are said to be myelinated, to distinguish them from unmyelinated nerves.
In the CNS the myelin sheath is produced by oligodendrocytes, and in the PNS it is produced by Schwann cells.

About 10% of the cells in the CNS are microglia; they are the representative of the immune system within the nervous system,
and they respond to infection, damage and inflammation.

The anatomy and connectivity of neurons in the brain is complex and cannot be discerned in regular histological sections.
Moreover, the area that is seen in a single section is quite limited. Nevertheless, it is important to be able to recognise
the appearance of CNS tissue and some peripheral nerves.

Activity 6

Open the virtual microscope in a new window or tab. Find Slides 10–13 in the ‘Week 3’ category.

The slide collection includes two examples of a peripheral nerve (Slides 10 and 11). Study these sections and identify the
axons and the elements of the accompanying blood vessel.

There are also sections from two different areas of the brain: the cerebral cortex (Slide 12) and the cerebellum (Slide 13).
Notice the completely different arrangement of the neurons in these two areas.

2.7 Normal tissues

You have nearly reached the end of this week, and this is a good opportunity to try to apply some of the things you’ve learned
about observation and the structure–function relationship that exists in tissues.

Activity 7

Open the virtual microscope in a new window or tab. Use the slides from the course so far to consider the following questions and make notes.

1. What differences can you see between the two sections of breast tissue in Slides 2 and 3 from the ‘Week 3’ collection?
How do these differences relate to the function of the tissue?

Provide your answer...

2. What differences are there between the types of skin shown in Slides 1 and 2 from the ‘Week 2’ collection? How might this
relate to the functions of different areas of the skin?

Provide your answer...

3. What other types of skin are seen on the body? If you look at an area of skin on your forearm, does it all appear uniform?
Would you consider a mole (nevus) to be a normal area of skin?

Provide your answer...

3 This week’s quiz

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Now test your ability to recognise tissues, and specific features within them.

The questions that follow require you to observe Slides 14, 15 and 16 in the virtual microscope. We recommend that you open the tool in a new browser window or tab, so you can easily alternate between the slides and the
questions.

Complete the Week 3 quiz now.

Open the quiz in a new window or tab then come back here when you’re done.

4 Summary of Week 3

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

By this stage in the course you should be able to identify some additional tissue types, including thyroid, breast, different
types of muscle, kidney, bone, some types of cartilage and some areas of the CNS.

In this week specifically we have highlighted the relationship between the structure of a tissue and its function. We have
also alerted you to the possibility that many tissues vary in their appearance, depending on normal physiological changes.
A section that appears ‘atypical’ may not appear as such because of a pathological change.

In Week 4 you will be examining some tissues which do have pathological changes, and you will gain some insight into how histology
is used for the diagnosis of disease.

You can now go to Week 4.

Week 4: Recognising disease

Introduction

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Welcome to the final week of the course.

Infection can affect any tissue of the body, producing cell damage and inflammatory reactions.

As David Male explains in the video below, the focus of this week is on studying pathological sections, namely those that
show histological evidence of disease.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

The sections that you will look at have been organised around three main types of pathology:

- infection and inflammation

- degeneration and cell death

- tumours (specifically hyperplasia, dysplasia and neoplasia).

You will learn more about these topics as you progress through the week.

In the end-of-course quiz you’ll look at a number of different pathological sections to identify the abnormalities, to try
to deduce the underlying causes and, finally, to venture a diagnosis of what the disease might be.

1 Infection and inflammation

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

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
they would be unable to tell what virus is responsible for these changes. To identify the virus might require immunohistochemistry
(IHC) for a specific viral protein or, more likely, the diagnosis would be confirmed by other symptoms and/or examination
of a blood sample (serology).

In some cases the histological appearance is characteristic of a specific disease. For example, the appearance of ‘owl-eye’
cells in the brain, as shown in the micrograph below, is diagnostic of a particular type of measles infection. However, normally
histopathology reports only form one part of the picture of a disease that the clinician is trying to assemble.

Figure 1 Owl-eye bodies in infected neurons of a patient with subacute sclerosing panencephalitis (SSPE).

View description - Figure 1 Owl-eye bodies in infected neurons of a patient with subacute sclerosing ...

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, as a result of physical damage or as part of an autoimmune
disease, where the immune system attacks components of the body.

You’ll start looking at pathology in more detail by looking at two common causes of infection.

1.1 Infection

Infection can affect any tissue of the body, producing cell damage and inflammatory reactions. There are many microbiological
agents that cause infection, but two of the most common are viruses and bacteria.

Viruses

Viruses are generally too small to be seen with the light microscope. However, their presence can often be inferred by the
changes they produce in tissue, even if their identity requires confirmation by IHC, serology or molecular biology.

Bacteria

Bacteria can be seen with the light microscope using high magnification objective lenses, but 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), depending
on the precise nature of the infection.

Distinguishing the type of bacteria in a thin section of a lesion generally requires specialised histological stains, although
the morphology (shape) of the bacteria may also be informative, as can be seen in the micrograph below.

Figure 2 Gas gangrene in a muscle. The micrograph shows a colony of bacteria, stained with haematoxylin. The bacteria have
the characteristic shape and growth pattern of clostridia. Scale bar = 40 μm.

View description - Figure 2 Gas gangrene in a muscle. The micrograph shows a colony of bacteria, stained ...

As with viral infection, the histological findings are an adjunct to serology and microbiology in producing a diagnosis, rather
than a stand-alone explanation.

Activity 1

Based on what you learned earlier in the course, can you identify what type of infection can be detected in the blood smear?

Figure 3 (Scale bar = 20 μm)

View description - Figure 3 (Scale bar = 20 μm)

View answer - Activity 1

1.2 Inflammation

Inflammation is a common response to tissue injury or infection, and occurs when the body’s defence systems are brought to
the site of the damage.

There are three main components of inflammation as illustrated in the diagram below (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. The migration of leukocytes from the blood into the tissues: the leukocytes cross the endothelial cells, which line the venules,
and then move out into the tissue.

Figure 4 The three main features of inflammation.

View description - Figure 4 The three main features of inflammation.

The blood that is brought to the site of the injury 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 have evolved to deal with different types of infection.

Inflammation can be separated into two main types.

- Acute inflammation develops quickly and resolves within days.

- Chronic inflammation can last for months or years, usually because of the persistence of the initiating factor.

More details about both types of inflammatory response, and how they can be recognised in tissue samples, is provided below.

Acute inflammation

The histological appearance of acute inflammation is quite different from that of chronic inflammation and the distinctive
features can point to the initiating agent. For example, an infection of the skin with Staphylococcus aureus usually produces an acute inflammatory response, whereas infection with Mycobacterium leprae (leprosy) typically produces persistent infection and chronic inflammation.

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 (parasitic worms). 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. The immune system normally recognises and tolerates all of the
body’s own tissues. However, in some conditions the immune system reacts against a tissue resulting in autoimmune disease.

In autoimmunity the target of the immune response is one of the body’s own proteins or cellular components, and consequently
the stimulus for inflammation cannot be cleared, although the condition may improve if the normal controls that prevent autoimmune
reactions are restored. The targets may be individual molecules found in a specific tissue, or antigens present in many tissues
or in the extracellular matrix.

Table 1 gives some examples of such diseases and the target antigens.

Table 1 Autoimmune diseases affecting specific tissues

Disease

Organ(s)

Target antigens

Histological appearance

Hashimoto’s thyroiditis

Thyroid

Thyroglobulin, Thyroid peroxisomes

Destruction of thyroid follicles with severe inflammation

Goodpasture’s syndrome

Kidney
Lung

Basement membranes

Damage to kidney glomerulus and/or lung alveoli

Myasthenia gravis

Skeletal
muscle

Acetyl choline receptor

Degeneration of the motor endplate at nerve/muscle junction

Pemphigus

Skin
Mucosa

Desmosome proteins in keratinocytes

Separation of layers of epithelium

Type 1 diabetes

Islets of Langerhans

Pancreatic beta cells, insulin, GAD (enzyme)

Selective damage and loss of cells of pancreatic islets with inflammation

Multiple sclerosis

Brain
Spinal cord

Components of myelin

Focal areas of inflammation and loss of myelin in the central nervous system

Inflammation is associated with many types of tissue damage, although the appearance will vary depending on the tissue and
the initiating factor. The slide collection that you will examine shortly includes some examples of inflammation as a consequence
of infection.

1.3 Histopathology for the diagnosis of disease

In this video, David provides some guidance for the systematic examination of slides, based on an understanding of what the
normal tissue looks like.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

When viewing a section, it is important to note and report any changes objectively. Interpretation of what has caused the
changes takes considerable experience. Even if you have a good idea of the underlying cause, observations and interpretation
should be clearly distinguished.

1.4 Infection

This activity will show you examples of diseases that affect tissues with which you are familiar. In each case the normal
tissue section is included (Slides 2 and 4), so that you can compare normal and diseased tissue more easily. You should use
the legends to navigate around the sections.

Activity 2

Open the virtual microscope in a new window or tab. Find Slides 1–4 in the ‘Week 4’ category.

Look at Slides 1 and 2. They are sections of lung. Slide 1 is from a patient with miliary tuberculosis, which resulted from
an infection with the bacterium mycobacterium tuberculosis. Notice how the bacteria have produced cell death (necrosis) in
the centre of the tubercle, and the distinctive appearance of the chronic inflammatory reaction surrounding it.

Look at Slides 3 and 4. They are sections of kidney. Slide 3 is from a patient who had chronic inflammation caused by a bacterial
infection (Staphylococcus aureus). The bacteria are not visible with the H&E stain, but notice the infiltration of the kidney cortex by large numbers of leukocytes
(macrophages, neutrophils and lymphocytes).

1.5 Identify an infection

In this activity you'll identify the tissue and try to identify any abnormality.

Activity 3

Open the virtual microscope in a new window or tab. Find Slide 5 in the ‘Week 4’ category.

Look at Slide 5 and identify the tissue. Try to identify any abnormality in it. You may find it helpful to look back and compare
this section with normal tissue. for example Slide 1 in the ‘Week 1’ collection.

When you have noted the appearance of the tissue, think about the pathological changes that can be seen and what is causing
them.

Provide your answer...

Answer these questions about your findings:

1. What tissue was on Slide 5?

Lung

Liver

Cerebral cortex

Thyroid

Lymph node

2. What abnormality did you identify on Slide 5?

the liver cells (hepatocytes) contain many pale staining vesicles

there is evidence of haemorrhage (bleeding) into the tissue

the capsule of the tissue is ruptured

there are cysts containing a multicellular organism

View answer - Untitled part

3. What pathological processes did you see on Slide 5? Select all that apply.

acute inflammation with many neutrophils

chronic inflammation with macrophages

cell death (necrosis)

fibrosis – the deposition of fibrous connective tissue

View answer - Untitled part

A scientific approach to these slides involves:

- observation and description of the tissue and any changes

- interpretation of the findings and possible inferences.

These two stages correspond to the ‘Results’ and ‘Discussion’ sections in scientific papers. The observations and description
should be clear, accurate and unequivocal. The interpretation may or may not be correct. A histopathologist will give their
best opinion, perhaps with a differential diagnosis (probability) for the underlying cause of any changes in the tissue.

Remember that the finding ‘no unusual features’ is just as important in reaching an accurate diagnosis of disease, as the
identification of any histopathological change.

2 Cell death and degeneration

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Cell loss occurs in many tissues with age but the effects are particularly notable in tissues that have a limited capacity
for regeneration, such as nerves in the CNS, the retina of the eye and the sensory cells of the inner ear.

When cells die they do so in two main ways, by apoptosis or necrosis. This is illustrated in Figure 5.

Figure 5 Schematic diagram comparing (a) necrosis and (b) apoptosis. The first events in necrosis are irregular condensation
of the nucleus, 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.

View description - Figure 5 Schematic diagram comparing (a) necrosis and (b) apoptosis. The first events ...

Apoptosis

Apoptosis is programmed cell death; the cell dies as part of its normal programme of development, it may be lacking in growth
factors or it may 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. Therefore, 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. Although the loss of individual
cells is histologically undramatic, the cumulative loss of cells in such degenerative conditions can cause major loss of function
in the affected tissue. Moreover, cell loss may be accompanied by the accumulation of products of tissue breakdown, which
are histologically evident.

Necrosis

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 (ischaemia) due to thrombosis will cause massive cell death due to
lack of oxygen. 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 and 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, as can be seen in the micrograph below.

Figure 6 An area of necrosis in the lung caused by blockage of the blood supply 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 = 2 mm.

View description - Figure 6 An area of necrosis in the lung caused by blockage of the blood supply is ...

2.1 Cell death in the CNS

Cell death and degeneration within the nervous system is referred to as neurodegeneration. These processes occur in a number
of genetic conditions as well as conditions associated with aging including Alzheimer’s disease and Parkinson’s disease.

Cell loss occurs in many tissues with age but the effects are particularly notable in tissues that have a limited capacity
for regeneration, such as nerves in the CNS, 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. More evident are characteristic accumulations of proteins. Degenerating neurons leave tangles
of fibres (neurofibrillary tangles) produced by degenerating components of the cytoskeleton. In addition, there are extracellular
accumulations of amyloid within the brain.

Amyloid

Amyloid is an extracellular insoluble deposit of protein, and amyloidosis refers to the diseases in which amyloid occurs.
In some cases 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 the 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 lungs, 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;
examples of this are shown in the micrographs below.

Figure 7 Protein aggregates in brain cells associated with neurodegenerative disease. 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) Lewy bodies in Parkinson’s disease, (e) nuclear aggregates in amyotrophic lateral
sclerosis.

View description - Figure 7 Protein aggregates in brain cells associated with neurodegenerative disease. ...

2.2 Histopathology of the nervous system

In the activity below you will examine sections from the nervous system in conditions in which neurons die. These are Alzheimer’s
disease, dementia with symptoms of Parkinson’s disease, frontotemporal lobe dementia and cerebrovascular disease.

Activity 4

Open the virtual microscope in a new window or tab. Find Slides 6–14 in the ‘Week 4’ category.

Start with Slide 6. Slide 6 is from a normal cortex for comparison. The outer layer of the brain, the cortex, contains large
numbers of neuronal cell bodies and appears as ‘grey matter’. Beneath the cortex are areas with a high proportion of axons
and myelin-producing oligodendrocytes, which appear as ‘white matter’.

Next look at Slides 7 and 8. They are from the cortex of a person with Alzheimer’s disease. Slide 7 shows the loss of neurons
from the outer layers of the cerebral cortex. Slide 8 is stained to show accumulation of the protein β-amyloid; the accumulation
of the protein in discrete plaques is very evident.

Next look at Slides 9 and 10. They are from the midbrain of a person with dementia with Lewy bodies (DLB) and symptoms of
Parkinson’s disease. In this condition neurons from particular regions of the brain, controlling movement, are selectively
lost. In addition, intracellular accumulations of protein, termed Lewy bodies, are detected by an H&E stain (Slide 9). The
Lewy bodies can be identified by immunohistochemical staining with antibodies that recognise the protein α-synuclein (Slide
10).

Next look at Slides 11 and 12. They show frontotemporal lobe dementia (FTLD). In this condition there is severe neuronal loss
from the cortex (Slide 11) and neurons often have protein inclusions. However, unlike Alzheimer’s disease, amyloid plaques
and tangles are absent. Slide 12 is a section from the spinal cord, which shows motor neuron loss.

Finally, look at Slides 13 and 14. They are from a patient who had cerebrovascular disease. Slide 13 shows areas of cell death
caused by lack of an adequate blood supply (infarcts). In this case the condition is associated with thickening of the walls
of the blood vessels, narrowing the vessel lumen and deposition of β-amyloid, a condition termed amyloid angiopathy (Slide
14).

Slides 7–14 were kindly loaned for imaging by Dr Andrew King and Dr Safa Al-Saraj from King’s College Hospital, London.

3 Tumours

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

Cell division is normally a highly regulated process. The number of cells in any tissue is usually fairly constant, although
some tissues can respond to physiological demand by an increase in cell number. Tumours disrupt this regulation.

Hyperplasia

What process occurs as mountaineers acclimatise to high altitude? The number of erythrocytes in their blood increases. But
why does this happen? The fall in the level of oxygen in the air at altitude means that the capacity of the blood to carry
oxygen increases, to compensate. There is a progressive increase in the number of erythrocytes over a period of weeks as the
bone marrow responds by increasing production.

Other types of cell may increase in number in response to appropriate stimuli. For example, in a guitar player, the basal
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.

Dysplasia

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 number 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 them 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

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 that 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 showing the characteristic pattern of separating chromosomes, seen as the cell divides. 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.

Figure 8 A mitotic figure in a carcinoma of the breast (arrowed) indicates cell division.

View description - Figure 8 A mitotic figure in a carcinoma of the breast (arrowed) indicates cell ...

3.1 Metastasis

Tumours can also move away from their original tissue by invading blood vessels or lymphatic ducts and being carried to distant
sites. This process is called metastasis.

Tumour cells that are carried through lymphatics will usually metastasise to local lymph nodes; this is the reason that surgeons
may remove lymph nodes as well as the original tumour, to treat a cancer.

Tumours that metastasise via the bloodstream 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
cell; nevertheless, metastasis accounts for 90 per cent 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 that are completely uncharacteristic.
In some cases, the primary tumour type can be recognised because it has retained some distinctive characteristics of the original
cell type. However, the original identity of tumour cells is not always self-evident and this is particularly true of metastatic
tumours.

Therefore, it may be possible to observe a metastatic tumour in a tissue, but be impossible to identify the primary cell type
and the original site of the tumour, at least by H&E staining. In this case additional staining, particularly immunohistochemistry,
is valuable to identify the original cell type, because it can provide an important guide for patient scanning, further surgery,
radiotherapy and drug treatment.

3.2 Neoplasia and hyperplasia

For this activity, refer to the ‘Week 4’ category within the virtual microscope, Slides 15–18.

Activity 5

Open the virtual microscope in a new window or tab. Find Slides 15–17 in the Week 4 category.

Start with Slide 15. It shows a primary liver carcinoma, a cancer which has arisen from a liver cell. Such primary tumours
are relatively uncommon in the liver. Notice the irregular cell morphology, the lack of normal liver anatomy and the invasion
of the normal tissue by the cancer. (For comparison look back at the normal liver on Slide 9 in Week 2.)

Next look at Slide 16 which is also from a liver. In this instance the tissue contains a secondary tumour, an adenocarcinoma,
which has arisen from epithelial cells. This tumour is a metastasis from the primary site, which has been carried to the liver
through the blood. However, it is not possible to tell from the H&E staining where the original tumour was located.

Look at Slide 17. It shows uncontrolled cell growth (hyperplasia) in the skin caused by a viral infection (Molluscum contagiosum). In some cases viral infection can lead to neoplasia; however, viral infections are generally controlled by the immune system.
(Look at Slide 18 to compare this with normal skin.)

3.3 Put it into practice

In the next activity, you are invited to investigate, identify and comment on two slides drawing on what you’ve learned.

When reporting on sections it is important to clearly distinguish observations and interpretations. You might want to make
a short written report on these two sections, before responding to the questions. Then compare your observations and conclusions
with the answers.

Activity 6

Slide 19

Open the virtual microscope in a new window or tab. Find Slides 19 and 20 in the ‘Week 4’ category.

Look at Slide 19. Identify:

- the tissue shown in Slide 19

- whether the section is normal or abnormal.

Provide your answer...

View discussion - Slide 19

Now try to identify what kind of pathological reaction can be seen in the tissue on this slide. What might cause this type
of change?

Provide your answer...

View answer - Untitled part

Slide 20

Look at Slide 20. Identify:

- the tissue shown in Slide 20

- whether the section is normal or abnormal.

Provide your answer...

View discussion - Slide 20

Compare Slide 20 with Slide 1 from Week 3. Make notes about:

- what type of reaction you can see in the tissue on this slide

- what might have caused this type of change, which has resulted in enlargement of the thyroid.

Provide your answer...

View answer - Untitled part

4 End-of-course quiz

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

The final quiz of this course pulls in material from each of the weeks and presents you with some problems, where histology
can help in understanding biology or in the diagnosis of disease.

Some of the questions are straightforward and directly related to material presented in the course. Others are intended to
make you think about the implications of what you have learned. Do not worry if you cannot immediately see or deduce the correct
answer, histology is usually only one part of the picture when it comes to making a diagnosis of disease.

Complete the end-of-course quiz now.

Open the quiz in a new window or tab then come back here when you’re done.

5 End-of-course summary

You can experience this free course as it was originally designed on OpenLearn, the home of free learning from The Open University
– http://www.open.edu/openlearn/science-maths-technology/histology-microscopy-anatomy-and-disease/content-section-overview

You have now reached the end of the course and, in this final week, we have introduced you to the concept of pathological
changes in tissues, including infection, inflammation, cell death and degeneration, neoplasia and hyperplasia.

These biological processes occur individually and sometimes concurrently in normal tissues and in different diseases. Understanding
the normal appearance and function of a tissue allows for the identification of potential abnormalities, which inform the
diagnosis of disease.

This course has focused on several important tissues of the body and provided an overview of their structure and function.
It has also introduced you to light microscopy and histology within the context of specific diseases.

In the video below David gives a summary of the course and suggests where to take your study of histology and histopathology
next.

Video content is not available in this format.

View transcript - Uncaptioned interactive content

David mentions The Open Science Laboratory where you can explore a collection of 320 annotated histological and histopathological slides.

Tell us what you think

Now you’ve completed the course we would again appreciate a few minutes of your time to tell us a bit about your experience
of studying it and what you plan to do next. We will use this information to provide better online experiences for our learners
and to share our findings with others. If you’d like to help, please fill in this optional survey

Where next?

If you’ve enjoyed this course you can find more free resources and courses on OpenLearn.

Making the decision to study can be a big step and The Open University has over 40 years of experience supporting its students
through their chosen learning paths. You can find out more about studying with us by visiting our online prospectus.

You may be particularly interested in our courses and qualifications in Science.

References

Holman, D. W., Klein, R. S. and Ransohoff, R. M. (2011) ‘The Blood-Brain Barrier, Chemokines and Multiple Sclerosis’, Biochimica et Biophysica Acta, vol. 1812, no. 2, pp. 220–30 [Online]. Available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3005102/ (Accessed 19 April
2018).

British Thyroid Foundation (BTF) (2016) Your Thyroid Gland [Online]. Available at http://btf-thyroid.org/information/your-thyroid-gland (Accessed 19 April 2018).

Gottfried, S. (1994) Human Biology, St Louis and London, Mosby.

Tortora, G. J. and Grabowski, S. R. (1993) Principles of Anatomy and Physiology, 7th edn, New York, HarperCollins.

Villee, C. A. (1989) Biology, 2nd edn, Philadelphia, Saunders College Publishing.

Acknowledgements

This free course was written by David Male.

Except for third party materials and otherwise stated (see terms and conditions), this content is made available under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Licence.

The material acknowledged below is Proprietary and used under licence (not subject to Creative Commons Licence). Grateful
acknowledgement is made to the following sources for permission to reproduce material in this free course:

Week 1

Images

Course image: © Likoper / Shutterstock.com

Figure 6: with kind permission of Dr Dimitri P. Agamanolis, M.D.

Figure 7: courtesy of Dr Eva Del Valle Suarez.

Figure 10: © David Male.

Figure 12: © Nicolas Grandjean, Dr Graham Beards/These files are licensed under the Creative Commons Attribution-Share Alike Licence
(http://creativecommons.org/licenses/by-sa/3.0/), Bobjgalindo/This file is licensed under the Creative Commons Attribution-ShareAlike
4.0 International Licence (https://creativecommons.org/licenses/by-sa/4.0/), CS99

Tables

Table 1: Image assets: Nicolas Grandjean, Dr Graham Beards/These files are licensed under the Creative Commons Attribution-Share Alike
Licence (http://creativecommons.org/licenses/by-sa/3.0/), Bobjgalindo/This file is licensed upder the Creative Commons Attribution-ShareAlike
4.0 International Licence (https://creativecommons.org/licenses/by-sa/4.0/), CS99

Audio-visual

Section 1.1: © The Open University https://creativecommons.org/licenses/by-nc-nd/4.0/

We are especially grateful to Peter Mooney for his tour around the histopathology department at Milton Keynes Hospital.

Week 2

Images

Figure 4: © Roitt, I. et al. (1998) ‘Major lymphoid organs and tissues’, Immunology, 5th edn, Mosby Internation Ltd.

Figure 5: courtesy of Susan Van Noorden.

Audio-visual

Section 1.6: © The Open University and its licensors https://creativecommons.org/licenses/by-nc-nd/4.0/

Week 3

Images

Figure 2: © adapted from Villee, C.A. (1989) Biology, 2nd edn, Philadelphia, Saunders College.

Figure 3: images © The Open University and its licensor Dr R. Dourmashkin/Science Photo Library).

Figure 4: © adapted from British Thyroid Foundation (BTF) (2016) Your Thyroid Gland http://btf-thyroid.org/information/your-thyroid-gland.

Figure 5: adapted from © 2012–2016 McMaster Pathophysiology Review (MPR).

Figure 6: from Gottfried, S. (1994) Human Biology, St Louis and London, Mosby.

Figure 9: from Tortora, G. J. and Grabowski, S. R. (1993) Principles of Anatomy and Physiology, 7th edn, HarperCollins Publishers Ltd.

Week 4

Images

Figure 1: © Brostoff, J. et al. (1991) Clinical Immunology, Gower Medical.

Figure 2, 3 and 6: © David Male

Figure 7: © Image copyright holders a–e: Claudion, S. (2003) ‘Unfolding the role of protein misfolding in neurodegenerative diseases’,
Nature Reviews Neuroscience, vol. 4, 2003 © Nature Publishing Group

Figure 8: © Woo, E. K. et al. (2005) ‘Myoepithelial carcinoma of the breast: a case report with imaging and pathological findings’,
British Journal of Radiology, vol. 78, May 2005. The British Institute of Radiology

Interactive

For providing slides to examine in the Virtual Microscope, special thanks goes to:

Cambridge University, Department of Pathology

University of Leicester

King’s College Hospital, London

University College Hospital, London

Milton Keynes Hospital, Department of Histopathology

All other organisations who have donated to the OpenScience Laboratory.

Every effort has been made to contact copyright owners. If any have been inadvertently overlooked, the publishers will be
pleased to make the necessary arrangements at the first opportunity.

Don't miss out

If reading this text has inspired you to learn more, you may be interested in joining the millions of people who discover
our free learning resources and qualifications by visiting The Open University – www.open.edu/openlearn/free-courses.

Activity 6

Untitled part

Answer

This tissue is from a normal lung, and it was seen earlier this week. Notice the open structure of the alveoli where gas exchange
takes place, which is a characteristic of a normal lung.

This section was included to see how well you can recognise a tissue from its histological appearance. Remember that even
in a histopathology laboratory it is just as important to correctly identify normal tissues as it is to identify diseased
tissue.

Back

Activity 6

Untitled part

Answer

This section of skin includes an area of wound healing. The area of a skin wound contains so-called granulation tissue (8615,
861), consisting of newly-formed capillaries (8615, 1257), leukocytes (8939, 1000) and proliferating fibroblasts (10466, 826).
The collagen (11114, 1293) in the dermis is still rather disorganised.

This section was included to see whether you could spot these changes in the skin, even if you could not work out what had
caused them. Now that you know what these changes are, navigate to the coordinates above in the Virtual Microscope to see
them for yourself.

Back

Activity 6

Untitled part

Answer

This section has come from a person with cirrhosis of the liver. Cirrhosis of the liver is the result of chronic liver damage,
which is caused by, for example, alcohol abuse.

The section shows broad bands of fibrosis (7450, 3650), which are areas of connective tissue with extracellular matrix proteins
and fibroblasts. These broad bands of fibrosis are separating the lobules (5385, 3650), in which the hepatocytes show fatty
change (5550, 3360).

Nodules (8226, 4081) of regenerating liver cells may be present, but the fibrosis disrupts the normal blood flow through the
liver from the portal vein to the hepatic vein.

Back

Activity 1

Answer

This is a blood smear from a patient with a malarial infection. Several of the erythrocytes are infected. Malarial infection
of blood was shown in the ‘Week 1’ slide set in the virtual microscope.

Back

Activity 3

Untitled part

Answer

Correct:

there are cysts containing a multicellular organism

Wrong:

the liver cells (hepatocytes) contain many pale staining vesicles

there is evidence of haemorrhage (bleeding) into the tissue

the capsule of the tissue is ruptured

This section of liver is from a person infected with a parasitic worm (schistosomiasis). An immature fluke can be seen at
[2948, 2145].

Back

Activity 3

Untitled part

Answer

Correct:

chronic inflammation with macrophages

fibrosis – the deposition of fibrous connective tissue

Wrong:

acute inflammation with many neutrophils

cell death (necrosis)

Around the parasites are many leukocytes [2706, 2387] including lymphocytes, macrophages and eosinophils. The parasites have
been walled off by fibrous tissue [2759, 2211].

Back

Activity 6

Untitled part

Answer

The section of lung shows areas of low grade inflammation (8480, 2904) with some thickening of the walls of the alveoli (7285,
2903).

This biopsy came from a patient with cytomegalovirus infection. Some macrophages (6637, 1215) in the alveoli have dark-staining
intra-nuclear inclusions (6475, 802). Although the virus itself cannot be seen with the light microscope, its appearance on
infected cells is characteristic. Navigate to these coordinates to make sure that you can identify these features.

You would probably not be able to identify the type of infection from the histology alone. However, you might have worked
out that an infectious agent is one likely cause of inflammation in the lung.

Back

Activity 6

Untitled part

Answer

The tissue shows hyperplasia of the thyroid, which is typically produced by a lack of iodine in the diet and/or abnormally
high production of thyroid-stimulating hormone (TSH) by the pituitary.

The thyroid epithelial cells are dense and form clumps within the acini (2421, 3842), but the overall anatomy appears normal,
so there is no evidence that the cells are neoplastic. Moreover, hyperplasia has occurred throughout the gland.

Neoplasia generally arises in one area of a tissue (due to gene mutation in one cell or group of cells), and there is usually
some relatively normal tissue still present, which may have been included in the histological section. Notice also that the
acini in Slide 20 have scalloped edges in some places, caused by the active removal of stored colloid to release the thyroid
hormones.

All of these observations indicate hyperplasia, rather than neoplasia. This section illustrates how understanding the normal
function of a tissue can shed light on the histological changes that have occurred.

Back

Activity 2

Untitled part

Discussion

One difference that you may have noticed is that of the colours of cells in the samples. Red blood cells do not normally contain
a nucleus or nucleic acids so they do not stain blue. However, infected cells containing the malaria parasite (Plasmodium) are stained blue. The appearance of an infected cell depends on how far the parasite has progressed through its developmental
stages.

Back

Activity 2

Untitled part

Discussion

You may have observed that there are similarities between the ‘Malaria’ slide and the slide for ‘Sample 3’

The reason for this is that ‘Sample 3’ is another example of malaria in a blood smear. However, in this case, more red cells
have been infected than in the ‘Malaria’ slide, and there are numerous parasites within each cell.

From this evidence you could deduce that ‘Sample 3’ represents blood taken at a later stage of infection than that of the
‘Malaria’ slide. At this later stage the infected cells are called ‘schizonts’, and you can see an example of them at position
(4718, 2868) in Slide 3.

Back

Activity 4

Untitled part

Discussion

The following conclusions can be drawn from analysis of Slide 5:

- The differential blood count is not within the normal range. There are very large numbers of lymphocytes in the smear.

- Additionally, the lymphocytes do not appear normal. The cells and their nuclei have an irregular shape.

- The diagnosis of this condition is a chronic lymphocytic leukaemia: the blood contains very high numbers of abnormal lymphocytes.

Back

Activity 5

Discussion

You may have noticed that some of the blood cells shown in Slide 6 look distinctly different from those found in the normal
blood smear (Slide 1).

Specifically, many of the red blood cells in Slide 6 are elongated or have a sickle shape, compared to those in the normal
blood smear. This is an example of sickle cell disease.

Sickle cell disease occurs as a result of a genetic variation in haemoglobin, which causes it to become less soluble. In people
who have two genes for the condition (homozygous), the haemoglobin distorts the erythrocytes, producing the characteristic
sickle cells, which are less able to flow through the blood capillaries.

Back

Activity 6

Slide 19

Discussion

The tissue shown in Slide 19 is from a lung and it is not normal. You can compare this section with Slide 6 from Week 2, which
is from a normal lung. In particular, the area near the upper right of the section (8000, 4000), does not look like the normal
tissue. Navigate to these coordinates in the Virtual Microscope to see them for yourself.

Back

Activity 6

Slide 20

Discussion

The tissue shown in Slide 20 is from a thyroid and it is not normal. You can compare this section with Slide 1 from Week 3,
which comes from a normal thyroid. Slide 20 comes from a patient with a goitre, i.e. an enlarged thyroid. The changes are
seen throughout the section.

Back

Figure 1 A cross-section through a human brain, with arrows indicating the presence of lesions (Caroldoey, 2015).

Description
This is an image of a cross-section of a brain.

Back

Figure 2 A histologist examines a tissue specimen in a wax block to establish its orientation.

Description
This is a photograph of a woman in a lab coat examining a tissue specimen.

Back

Figure 3 The wax block containing the tissue is sectioned on a microtome.

Description
This is a photograph of a woman in a lab boat using a machine.

Back

Figure 4 Section across the wall of the human aorta stained conventionally with haematoxylin and eosin.

Description
This is a section across the wall of the human aorta.

Back

Figure 5 Section across the wall of the human aorta stained with elastic Van Gieson, which identifies the elastic fibres (black)
in the media of the artery and the collagen (red) in the adventitia.

Description
This is a section across the wall of the human aorta.

Back

Figure 6 Neurons visualised with a modification of the Golgi silver staining method.

Description
This is an image of neurons visualised with a modification of the Golgi silver staining method.

Back

Figure 7 Neurons in the cortex of the brain are identified by immunohistochemistry using an antibody directed against neurofilaments.

Description
This is an image of neurons in the cortex of the brain.

Back

Figure 8 A section of the adrenal gland (cortex), stained with haematoxylin and eosin. Cell nuclei are stained deep blue and
the cytoplasm is pink. The capsule, the outer envelope, of the organ (large arrow) is also pink. The erythrocytes (red blood
cells) seen within small blood vessels (small arrow) are red. Scale bar = 50 µm.

Description
This is an image of a section of the adrenal gland.

Back

Figure 9 Elements of a light microscope.

Description
This is a drawing of a light microscope. From left to right the labelled parts are: lamp, field condenser, iris diaphragm,
substage condener, specimen, objective, image plane, eyepiece, eye.

Back

Figure 10 Understanding the plane of a section for (a) a blood vessel, (b) a sample of kidney tissue and (c) a neuron. Note
that for the neuron, the thickness of the section is significant in relation to the thickness of the cell.

Description
This figure is made up of three separate drawings. The first (a) is bifurcating blood vessel, the second (b) is loop of Henie
(kidney) and the third (c) is a neuron.

Back

Figure 11 The appearance of an etched slide on a microscope stage when viewed with an eyepiece that includes a graticule.

Description
This drawing shows a view down a microscope.

Back

Figure 12 The appearance of the five main types of leukocyte.

Description
This figure is made up of five different images: the first (1) is Neutrophils, the second (2) is Eosinophils, the third (3)
is Basophils, the fourth (4) is Lymphocytes and the fifth (5) is Monocytes.

Back

Figure 1 Arrangement of epithelial cells lining a secretory gland, its duct and the epithelial surface.

Description
This is a drawing of the arrangement of epithelial cells lining a secretory gland, its duct and the epithelial surface.

Back

Figure 2 Structure of the skin showing the various types of cells and tissues: (a) epithelial cells in the epidermis; (b)
connective tissue; (c) muscle; (d) epithelial cells lining the duct of a sweat gland; (e) adipose tissue below the dermis.

Description
This is an illustration of the structure of the skin.

Back

Figure 3 Cross-section of the gut showing the arrangement of the different tissue elements at the level of the ileum (small
intestine).

Description
This is a drawing of a cross-section of the gut.

Back

Figure 4 Structure of a lymph node.

Description
This is a cross-section showing the structure of a lympth node.

Back

Figure 5 Immunohistochemistry. Double immunolabelling of an Islet of Langerhans in the pancreas identifies insulin-producing
cells (blue) and glucagon-producing cells (brown).

Description
This image shows double immunolabelling of an Islet of Langerhans in the pancreas.

Back

Figure 6 Principles of immunohistochemistry. (a) Unlabelled primary antibodies bind to the antigen on the section. (b) Labelled
secondary antibodies bind to the primary antibodies. The secondary antibodies have an enzyme attached. (c) Chromogen is converted
by the enzyme into an insoluble stain on the section.

Description
This image shows the principles of immunohistochemistry.

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Figure 1 Secretion of proteins produced in the Golgi apparatus. Secretion may either be constitutive (continuous) or the secreted
molecules may be stored in vesicles and then released when a signal is received.

Description
This image shows the secretion of proteins produced in the Golgi apparatus.

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Figure 2 Schematic diagram of an alveolus, in contact with pulmonary capillaries (Villee, 1989).

Description
This is a schematic diagram of an alvelous.

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Figure 3 Schematic diagram of villi in the small intestine.

Description
This is a schematic diagram of villi in the small intestine.

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Figure 4 Location of the thyroid gland (adapted from BTF, 2016).

Description
This is an illustration showing the location of the thyroid gland.

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Figure 5 A diagram showing breast anatomy and histology.

Description
This is an image showing breast anatomy and histology. It contains the following text: The breast is composed of glandular
and stromal tissue. Glandular tissue includes the ducts and lobules. Stroma comprises area between lobes. Each lobe drains
into a major lactiferous duct that dilates into a lactiferous sinus beneath the areola opening onto the nipple. Each terminal
duct lobular unit is composed of branched tubuloalveolar glands organised into lobes. Inner layer of myoepithelial cells provide
structural support to the lobules and assist in milk ejection during lactation. Outer layer of lobular luminal epithelial
cells produce milk during lactation.

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Figure 6 The internal structure of a skeletal muscle fibre (Gottfried, 1994).

Description
This shows the internal structure of a skeletal muscle fibre.

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Figure 7 Arrangement of actin and myosin in a myofibre produce striations. The upper diagram shows the relaxed state and the
lower diagram the contracted state.

Description
This shows the arrangement of actin and myosin in a myofibre produce striations.

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Figure 8 Arrangement of myocytes in cardiac muscle.

Description
This shows the arrangement of myocytes in cardiac muscle.

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Figure 9 (a) Diagram and (b) photograph of a partially sectioned long bone (femur), showing its internal structure. Erythrocytes
and leukocytes are produced from stem cells located primarily in the red marrow. Yellow marrow produces factors (cytokines)
that promote the development of the blood cells (Tortora and Grabowski, 1993).

Description
This shows a diagram and photograph of a partially sectioned long bone (femur), showing its internal structure.

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Figure 10 Diagram of the kidney structure with an enlarged diagram of a nephron. The ureter carries urine from the kidney
to the bladder.

Description
This is a diagram of the kidney structure with an enlarged diagram of a nephron.

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Figure 11 The blood supply to different parts of the nephron.

Description
This shows the blood supply to different parts of the nephron.

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Figure 12 a) The human nervous system showing the CNS and PNS. (b) A diagram of a small peripheral nerve showing the mixture
of nerve fibres travelling from the CNS (efferent neurons) which control muscle movement, and nerves travelling to the CNS
(afferent neurons) which convey sensory information.

Description
This is an outline of a human showing the nervous system.

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Figure 13 Different types of neuron. (a) With the cell body at the end of the axon. (b) With the cell body to the side of
the axon. (c) With no processes. (d) With an insulating sheath of myelin around the axon, which speeds nerve conduction. (e)
A small nerve network showing inputs (neurons 2, 3 and 4) to neuron 1, and its output to neuron 5.

Description
This is made up of two images: one showing the cell body at the end of the axon, and the other showing the cell body to the
side of the axon.

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Figure 1 Owl-eye bodies in infected neurons of a patient with subacute sclerosing panencephalitis (SSPE).

Description
This is an image showing owl-eye bodies in infected neurons.

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Figure 2 Gas gangrene in a muscle. The micrograph shows a colony of bacteria, stained with haematoxylin. The bacteria have
the characteristic shape and growth pattern of clostridia. Scale bar = 40 μm.

Description
This shows gas gangrene in a muscle.

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Figure 3 (Scale bar = 20 μm)

Description
This shows a blood smear.

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Figure 4 The three main features of inflammation.

Description
This shows the three main features of inflammation: arteriole, capillary and venule.

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Figure 5 Schematic diagram comparing (a) necrosis and (b) apoptosis. The first events in necrosis are irregular condensation
of the nucleus, 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.

Description
This is a schematic diagram comparing necrosis and apoptosis.

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Figure 6 An area of necrosis in the lung caused by blockage of the blood supply 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 = 2 mm.

Description
This is an area of necrosis in the lung caused by blockage of the blood supply

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Figure 7 Protein aggregates in brain cells associated with neurodegenerative disease. 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) Lewy bodies in Parkinson’s disease, (e) nuclear aggregates in amyotrophic lateral
sclerosis.

Description
This shows protein in brain cells associated with neurodegenerative disease.

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Figure 8 A mitotic figure in a carcinoma of the breast (arrowed) indicates cell division.

Description
This shows a mitotic figure in a carcinoma of the breast.

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Uncaptioned interactive content

Transcript

DAVID MALE

Hello, I'm David Male and welcome to this course on histology and histopathology, the study of tissues and diseases underneath
the microscope. In the first part of the course, we are going to introduce you to microscopy and teach you how to use a microscope.
A lot of what we will do is taught through a virtual microscope which emulates many of the functions of a real microscope.

We have a very large collection of tissue sections, which we've obtained from different universities and hospital departments,
showing both normal structures and disease structures. We'll be showing you what happens in histopathology department in a
hospital, as well. Then we'll be showing you how to calibrate a microscope and obtain images from it.

Later on, we'll show you how to examine blood smears and to do a differential leukocyte count. Leukocytes are white blood
cells and they're involved in protection and combating infection in the body.

By the end of the week, you should be confident in using a microscope, knowing how the controls work, and be able to recognise
the different types of cell in a blood smear, and whether they are normal or abnormal.

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Transcript

DAVID MALE

Today we're at the histopathology department at Milton Keynes hospital. And I want to introduce you to Peter Mooney, who's
the laboratory manager here. And this is a busy histopathology department. And Peter's going to introduce us to the kind of
work that goes on here.

PETER MOONEY

Yes. Welcome, David. Welcome to a relatively busy histology department. We do about 400 to 500 specimens a week. We get specimens
from GP surgeries. We got them from outpatient clinics, and of course, from inpatients and theatres.

That's the main source. We do get the occasional autopsy specimen. But that's few and far between. The majority are from clinics
within the hospital. It could be dermatology, endoscopy.

DAVID MALE

OK. And from the time that you receive a specimen, how long does it take til it actually hits the desk of a pathologist?

PETER MOONEY

Right. Yeah. This is a problem we have with clinicians. They take a blood sample, press a button, out comes a result. With
histology, it's quite different.

It depends very much on the size of the specimen. If it's a very small biopsy, it arrives midday, we could have a result maybe
tomorrow. If it's a much larger specimen, like a breast or colon specimen, it can take one or two days to get a result. So
it's very much dependent on the type of specimen and the size of the specimen.

DAVID MALE

So can you take us through the process as it goes here?

PETER MOONEY

Let's go. Let's follow the process--

DAVID MALE

So this is where we start?

PETER MOONEY

--starting where they book in.

DAVID MALE

OK. Good. So this is where you receive your specimens here?

PETER MOONEY

It is. Yep.

DAVID MALE

And then what you do with them?

PETER MOONEY

Well, this is our specimen reception area. So we get all the specimens from theatres and clinics, as I talked earlier. The
first thing we have to do is check that the request card has got exactly the same details as the specimen cup. It's vital.
If there's any discrepancy, it goes back to where it came from so it's done properly.

The specimens all arrive in various sized pots. You see here very small ones. There's a tiny little biopsy in the bottom of
that one.

DAVID MALE

Oh, yes.

PETER MOONEY

And we have much larger pots, going up to five litres depending on what type of specimen it is. They are obviously from theatres,
specimens, larger specimens. These tend to be from GPs or clinics.

DAVID MALE

OK. So what kind of specimen was that one?

PETER MOONEY

Well, that one there is a nesophogial biopsy.

DAVID MALE

OK.

PETER MOONEY

You can see how small it is.

DAVID MALE

Yes, it's tiny.

PETER MOONEY

Yeah.

DAVID MALE

And the large ones, what kind of specimens are they?

PETER MOONEY

In this one, there's a uterus and fallopian tubes. They're both uterus and fallopian tubes. But we see breast tissue, colon
tissue, kidney. You name it all, we'll get it and we'll have a look at it. The thing to say of course is, these pots contain
formaldehyde. Formaldehyde is a bit toxic. It's not very nice. So whenever we open the pots, we always do it on downdraft
tables, which I'll show you later. And we monitor the levels in the lab on a regular basis.

So what happens then if we're quite happy with the specimen? It's booked in on the computer system. This is a pan pathology
computer system, which the chemists have, the haematologists have, transfusion. And Rosia is booking a specimen now. And it
will be given a unique lab number. The name then stops.

We don't relate to the name. We relate to the lab number. So it will be 16h, whatever the number is.

DAVID MALE

And does this go towards a central database then?

PETER MOONEY

It does. All the other patient's histories, only if the patient's been here before. It's all held centrally. Yeah. Yeah. When
it's booked in, we get some labels come here. We get one for the pot, one for the card. And at the same time there's a cassette
produced, which are these funny-colored things on the top of here.

DAVID MALE

Is there any difference between the colours on these?

PETER MOONEY

Yes, there is. The cassette, which is going to have the tissue in it eventually, has the lab number on, which is there, and
what's in the tissue, what's in the pot. The colours denote urgency. If you come in, if your GP has referred you to the hospital
because they're worried about a condition you have, and that it could be serious, we treat those as urgent. So a prostate
biopsy, a breast biopsy, anything like that that somebody thinks that will treat very urgent--

DAVID MALE

So you would be looking for a diagnosis of cancer in this case?

PETER MOONEY

Yes. Yes. So the red cassettes denote urgency. There is a reason why these large specimens are urgent. Clinicians suspect
something.

DAVID MALE

OK.

PETER MOONEY

OK. These ones are-- this is a skin lesion. The patient may well have had it for 20 years and decided to go to a GP.

DAVID MALE

Less urgent.

PETER MOONEY

Less urgent. Yes. Yes.

DAVID MALE

OK. So you've booked your specimens in. And now what are you going to do with them.

PETER MOONEY

Well, this is the first real step in the process, David. What we do is we examine the specimens macroscopically. The specimens
are arriving throughout the day. And we have biomedical scientists look at specimens, as well as pathologists.

And we make a microscopic description, and we dictate it. The person will be wearing headphones. And we have secretarial staff
upstairs typing as they speak, more or less. The tissue is put in the cassettes. Now, it depends, again, on the size of the
tissue. The one we showed earlier, which was a tiny, little about a millimetre, two millimeter-sized esophageal biopsy, that
would be put in the cassette with a lid put on it.

DAVID MALE

As is?

PETER MOONEY

As is.

DAVID MALE

Yes.

PETER MOONEY

The much larger specimens, of course, we couldn't possibly sample it all. So if it's got a tumour, we'd sample the tumour,
the resection margins. And that's very much an experiential thing.

DAVID MALE

So it's really important to get exactly the right bit of the tissue to look at?

PETER MOONEY

Absolutely. Yes. Sampling tumours limits every section, which is very much an experienced pathologist would do that.

DAVID MALE

So it's the pathologist that will be looking at the initial cutting of--

PETER MOONEY

Of the largest specimens. Absolutely. More and more labs are trying to train bio-medical scientists to do this. But it's a
lot of training involved.

DAVID MALE

Very skilled.

PETER MOONEY

Very skilled. Yes. Yes. So it's very important that the block of sample tissue that goes in the cassette is what's going to
be seen down the microscope later on.

DAVID MALE

OK.

PETER MOONEY

You can always go back to the specimen, of course, and take more. But, of course, that will slow the process up.

DAVID MALE

Of course.

PETER MOONEY

It's better to get it right the first time.

DAVID MALE

OK. So we can we go and have a look at the next stage now?

PETER MOONEY

Yes, of course.

DAVID MALE

Right. Let's go. So you've got your samples now on the cassettes. What happens to them here?

PETER MOONEY

Well, this is the tissue processing stage. What we're trying to do is to try to get a very thin section of tissue onto a microscope
slide so we can look down a microscope. To do that, we need a nice, firm piece of tissue.

We're replacing all the water in the tissue-- the tissue is about 80 per cent water-- with wax, a bit like candle wax. It's
called fibre wax, slightly specialised wax. Quite a slow process, usually overnight. Most of the specimens go overnight.

So as I alluded to earlier with haematology chemistry, we get results very quickly. Histology is much slower because the tissue
cassettes are going in here. And overnight, these reagents, IMS, and distilled methylated spirit, and xylene, and finally,
wax, will visit the reaction chamber. And over that length of time, by 8 o'clock tomorrow morning, all the water will have
been removed from the tissue, and it will be replaced by wax.

DAVID MALE

So obviously, the whole of that process is effectively automated by this machine.

PETER MOONEY

It is. Yes.

DAVID MALE

You just have to put the material in.

PETER MOONEY

Put the material in. Press the button.

DAVID MALE

And then it comes out first thing the next morning.

PETER MOONEY

The following morning at 8 o'clock, or whenever, you look in there. You think nothing's different. Only the tissue, instead
of sitting in formalin, is sitting in wax.

DAVID MALE

OK. And then where do you take the tissue?

PETER MOONEY

From there we take it for embedding.

DAVID MALE

OK. And that's back in the main lab.

PETER MOONEY

That's back in the main lab.

DAVID MALE

OK. Let's go and have a look at that then. So this next process is embedding here. What happens at the station?

PETER MOONEY

We're following the processing, which has happened overnight. The following morning all the cassettes are taken out of the
machine and embedded in moulds, so various sized moulds. And what Liz is doing at the moment is she's taking a mould, put
the tissue in the mould. In here, there's molten wax. And this is a dispenser, which dispenses wax.

This stage is very important. However the tissue's being cut, whether it's a skin biopsy, there's a transverse section, it
must be oriented correctly. Because the way the tissue's orientated in that mould will dictate what it looks like on a microscope
slide.

DAVID MALE

And that determines whether the pathologist can see what they're really interested in seeing at this stage.

PETER MOONEY

Exactly. Yes. And you have to get it right first time. If it's a piece of skin, and it's been poorly orientated, and it's
been cut, you could try to reorient it in the wax. But chances are that's been cut away.

DAVID MALE

So it's almost back to square one at that stage if it's not right.

PETER MOONEY

Yes. But you're always aware that this is one only sample. It's not like a blood sample. You can't go back to that patient
for a blood sample. This is a once only lesion, possibly. So this stage is very important.

So this is a case in point. Liz is being very careful to orientate the sample the correct way around. It's a piece of skin
with the epidermis and the dermis. So when you look at the microscope slide, it's the right way around.

So this is topped up with wax. And this is a cold plate to solidify the wax.

DAVID MALE

OK. So that was a liquid one, and this is now a solid one?

PETER MOONEY

So this one was done a little while ago. And it may come out the mould looking like that.

DAVID MALE

OK.

PETER MOONEY

OK? So we got the details along there. We've got the tissue embedded in there.

DAVID MALE

And that will go on now for sectioning.

PETER MOONEY

It will do. Yes, which is the next stage.

DAVID MALE

OK. Let's go and take a look at that then. So you've got the specimens now in their wax blocks. And they're going to be sectioned
on the microtome.

PETER MOONEY

That's right. A microtome. Is where the sections are cut from. Every time Vickie turns the handle, it advances the cassette
by three thousandth of a millimetre. That's about a cell thick.

So it's very important the sections are thin. You don't want to be looking down a microscope and seeing a thick section with
cells piled on top of each other. It's got to be nice and thin.

So again, quite labour intensive, like the embedding stage, and quite skilled. Often people refer to histology more of an
art than a science because it takes practise and experience. What Vickie's done is she's taken a ribbon of tissue, floated
it on some warm, distilled water, picked it up with a microscope slide.

DAVID MALE

How many sections would be taken from a single specimen, normally?

PETER MOONEY

If it's a large block, we'll just take the one section. If it's a smaller piece of tissue, like the esophageal biopsy we looked
at earlier, you'd take three sections at different levels through the tissue. Because it's so small, you can't judge by eye
that you've actually caught everything. There maybe something lurking further down, so we do three levels.

DAVID MALE

Again, it's just to hit exactly the right place in the tissue so that you don't miss a lesion or something really important.

PETER MOONEY

Yeah. Yeah.

DAVID MALE

OK. So we've now got these sections on the slides. What happens to them now?

PETER MOONEY

When the water's drained from them, it will be put in an oven and the wax will be melted onto the slide to make sure the tissue
sticks to the slide. And that's the end of this process. And then it will be staining after that.

DAVID MALE

OK. Can we go and look at some staining machines?

PETER MOONEY

Yeah.

DAVID MALE

OK. Let's go.

PETER MOONEY

So the next stage, we've got the sections on the glass slide. But, of course, we can't see anything down the microscope. So
now we have to stain the tissue. Now, this is a multi-stainer. It will do various stains on here. But the one we're interested
in is called a hemotoxylin and eosin, or H&E, we will refer to it.

It's a purple stain and a pink stain. And the purple stain stains the cell nucleus purple. And the cytoplasm stains pink.
The machine's preprogramed, so it knows what to do.

We have various coloured clips. So if we put a red clip on it, the machine knows to stain it with H&E stains. We have other
staining techniques.

DAVID MALE

Oh, it's a coding system or it.

PETER MOONEY

It's a coding system so it recognises it. So basically, you pop it in the machine, close the drawer, an arm will come across,
and what this machine do is doing the reverse of the processing machine. It's taking all the wax off the tissue, and it's
replacing it with water. And it's put these stains in the relevant places. So it goes in to xylene, then alcohol, then water.
The tissue's stained, and then it's cover slipped.

DAVID MALE

So there are a lot of different things in there. Does this machine do things other than hemotoxylin and eosin?

PETER MOONEY

There's a stain called periodic acid Schiff, and we use that. We use it for staining immuno sections, immunohistochemistry
sections, so we can set it up to how we want when we want easily.

DAVID MALE

Is all your staining done automatically now or do you do any hand staining?

PETER MOONEY

No. We do hand staining as well. This is the automated section. But we do do, on this bench, we do do special stains. So you
can see all these bottles of tinctorial stains. We can stain for bacteria, for acid-fast bacilli, amyloid fat, lots of different
elements whether it is--

DAVID MALE

Those would be much less commonly done. It's normally H&E isn't it?

PETER MOONEY

H&E, yes, much, most of it's common. If a pathologist looks down the microscope, and they see something that might be tuberculous,
they would ask for a Ziehl-Neelsen stain.

DAVID MALE

So how long does this machine take to do the H&E staining?

PETER MOONEY

It takes about 30 minutes or so to do a H&E as it goes to the ends up in xylene, and then it has a cover slip put on. Actually,
there's one happening at the moment where the slide is being raised up. It's had some mountant and a thin cover slip put on
it. And this is done automatically.

It is very important at this stage not to get any air bubbles under the cover slip because down a microscope, the air bubbles
look huge. So this machine achieves that without air bubbles.

DAVID MALE

So, and it's assembling them on a rack here ready to go to microscopy?

PETER MOONEY

That's right.

DAVID MALE

OK, Peter, so you've got your stained sections now. And you need to marry them up to the original blocks from which they came.

PETER MOONEY

That's right. This is a very important step. We have to ensure that the tissue on the glass slide marries up exactly with
the tissue in the block. So we check the numbers. We do a visual check at this stage that it's correct.

Throughout the process in the lab, of course, the card has been annotated with initials of all the members of staff to ensure
that the tissue is correct, the names are correct, the numbers are correct.

DAVID MALE

So every single stage you've got checks going through to make sure that you've got absolutely the right tissue and the right
sections.

PETER MOONEY

Absolutely. Absolutely vital, because it we make an error, the worst error would be to send a report out on a John Smith when
it really belongs to a Jane Smith.

DAVID MALE

A wrong diagnosis, in other words.

PETER MOONEY

Absolutely. So we have as many checks as possible built into the system to ensure that doesn't happen.

DAVID MALE

OK. So it's a very rigorous quality control right the way through.

PETER MOONEY

Absolutely. Yes. Yes. both microscopically, like we're doing here, and later microscopically.

DAVID MALE

So presumably, the first thing is that you check that you've got the right tissue and everything else. And you do that on
a microscope in the lab here?

PETER MOONEY

That's right. We'll do a visual check here to make sure that the shape of the tissue, if you like, resembles the shape of
the block.

DAVID MALE

OK.

PETER MOONEY

But then we'll do a microscopic quality control as our next stage.

DAVID MALE

So this is the final stage of your quality control. You're starting to look at the sections under the microscope now.

PETER MOONEY

Yes, another important step. What Kimmy's doing at the moment, she's checking the quality of the section. We check beforehand
that the numbers all match and the names all match with the card. She's done that again. It's always a different person.

She's checking down the microscope for the quality, that the staining is correct, that the quality of the section that was
cut earlier is correct. There are no scores in it because a score down a microscope looks very big, that it's representative
of all the section, and if it's a piece of skin that it isn't being superficial and there's bits missing. So we're looking
at the qualities.

Kim has checked that, decided it can go out to the pathologist. And initialled the card. So that's not ready for the patho--

DAVID MALE

OK. So that's the end of your process here and you are satisfied that this is ready for diagnostic pathology?

PETER MOONEY

Absolutely. Yes.

DAVID MALE

OK. It's an absolutely fascinating process. I've been really interested seeing this. So thank you very much, Peter, for showing
this, and all your staff too.

PETER MOONEY

Yeah, of course, great pleasure. Thank you.

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Transcript

NARRATOR: In this video, I will show you how to use the virtual microscope to look at different tissues. The microscope emulates
many of the functions of a conventional light microscope, including the ability to select different slides, you can also change
objectives, which alters the magnification of the section, you can focus the image and adjust the level of illumination, and
there are a variety of other features that are found on a real microscope, which we will introduce later on. But the most
useful feature of this virtual microscope is the descriptions of the tissues and diseases which are presented alongside the
images. Clicking on highlighted items in the description will take you directly to what you're looking for. This is like having
an instructor just over your shoulder.

In this course we have allowed about five minutes to look at each of the sections, but the amount of time actually spent is
entirely up to you. Now let's take a look at the basic functions of the microscope. When you open up the microscope, you see
a screen like this. In the centre is the microscope itself. On the left are its controls. On the right you have the slide
sets and the descriptions of each section.

I will begin by opening the slide catalogue. It contains collections of slides of different tissues, and I will start by selecting
blood. I can now see the thumbnail images of the 20 slides in this connection. The mouse pointer gives a short description
of each slide as you hover over it. In this case, I'm going to select a normal blood smear and click on it to load it onto
the microscope stage.

This area here shows the stage view, what the slide looks like on the microscope stage. And the circular window at the top
shows what you would see looking down at the section. This is the eyepiece view. At the moment we cannot see very much because
the magnification is too low. So I'm going to change to a 10 by objective by clicking here. You have a selection of objectives
available.

Now you can see some cells in the eyepiece window, and they are in fact mostly red blood cells. Look again at the stage view
and you'll see that this red circle defines the area that you are observing through the eyepiece. I can scan across the section
by clicking on the four arrows around the stage view. On a real microscope the slide may be moved manually or the stage may
be motorised.

I'm going to go to the highest magnification now, times 40. And the area that I'm viewing is correspondingly reduced. I can
adjust the focus of the microscope by clicking here. The controls to the left of the wheel are the course focus. And the controls
to the right of the wheel are the fine focus. I can also adjust the illumination, which is equivalent to changing the power
from the lamp. So I adjust to the right level of lighting and I'm now adjusting so that my view is in focus.

Finally, let's look at the description of the slide. It says red blood cells, or erythroctyes, are the predominant cell in
this blood smear. White blood cells, or leukocytes, are also present, and they include the cells called neutrophils. If I
want to see what a neutrophil looks like, I just click on the blue text and the stage immediately jumps to an example of the
neutrophil, which is now in the centre of the eyepiece viewing field. Using these active descriptions will give you a lot
of information on the slide, and will help confirm that you are looking at the right cells. There are other functions on the
microscope, too, but we will return to those later.

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Transcript

DAVID MALE

In this video, I'll be showing you some more functions of the virtual microscope, and also how to calibrate a microscope using
a standard etched slide. This information allows you to measure the size of cells and structures on the sections.

On a real microscope, the magnification is normally described as the magnification of the objective multiplied by the magnification
of the eyepiece. For example, using a times 10 objective and a times 10 eyepiece gives a times 100 magnification. But this
assumes that you're looking at the section from 30 centimetres away, which is about as close as most people can normally accommodate.

On a virtual microscope, the size of the image will depend on the objective used to produce the image, the camera attached
to the microscope, and the size of the screen used to view the image, which could be anything from a mobile phone to a cinema
screen. Moreover, the perceived magnification also depends on how far a person is from the screen.

So stating a magnification for a virtual microscope is fairly meaningless. It depends entirely on the device being used to
view it. What you really want to know is how big the original item is. And that is one reason for calibrating the microscope.

As well as showing the additional microscope functions, there are some shortcuts which can be done on the virtual microscope,
but not on a real microscope. So let's take a look at some of those now.

I have the normal blood smear loaded on the stage again. First, look at this tool icon. If you check the box, sections will
automatically be loaded in focus. Not so realistic, but it will save you time. Another shortcut is on the stage view. If you
click and drag the red circle, you'll be able to move around the section much more quickly than if you just used the stage
movement arrows.

Another bit of information is given by the x- and y-coordinate positions corresponding to the centre of the eyepiece view.
In this case, the centre of the view is position x equals 4,445, y equals 3,958. This is useful if you want to describe exactly
where something is on the section.

Just above our two boxes, suppose that someone asks you to describe the leukocyte located at position 4,190, 2,400. Then you'd
just type those coordinates into the boxes and click the Go button. The microscope takes you immediately to the correct point.

If you now change the objectives, you can see that the cell at that point is a neutrophil. You might want to take a picture
of it. In this case, you need to click on the Camera icon, which will grab the eyepiece image. Depending on the device you
are using, there are various options to copy, print, or save the image.

Returning to the microscope again, if you want to count cells, the Grid control will put an overlay onto the section, which
makes it easier to count the number of cells or other features in a defined area. When doing this, it is conventional to count
in anything that overlaps the top or left hand side of a square, and discount anything overlapping the right or bottom sides.

I'll take the grid off now, as I want to show you how to calibrate the microscope. To do this, I first need to load a standard
etched slide onto the stage. This slide has been photographed on the real microscope using exactly the same objective and
camera as was used on that microscope to take images of the other section shown here. The spaces on this slide are 1 millimetre
apart. The spacings between the numbers 5 and 6 are 0.1 millimetre apart.

Let's suppose that I want to calibrate the times 20 objective on the virtual microscope. First, I add a fixed graticule to
the eyepiece image. On a real microscope, the graticule would be part of the eyepiece. Now I'm switching to the times 20 objective.
I'm going to align the marks on the graticule with the marks on the slide. I can now see that 1, 2, 3, 4, 5, 6, 7.5 marks
on the graticule correspond with 0.1 millimetre. This means that with this objective, one whole division on the graticule
equals 0.1 millimetre divided by 7.5, which equals 0.013 millimetres or 13 micrometres.

Armed with this information, I can now work out the size of a cell. Let's have another look at that neutrophil, which is at
position 4,190, 2,400. I must look at it on the same objective as I used for calibration-- times 20. And you can see that
the cell just about occupies one division of the graticule, which implies that it is approximately 13 micrometres in diameter.
For convenience, both the grid and the graticule can be rotated.

Remember that calibration has to be done for each objective. When micrographs are produced, it is conventional to add a scale
bar. In this case, I'm adding a scale bar corresponding to 50 micrometres to the image. If the scale bar is part of the image,
then any change in the size of the image will change the length of the scale bar, so the size of the cells is unambiguous.
Finally, I'm going to take the graticule off the image. And now you should try to calibrate the other objectives yourself.

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PROFESSOR DAVID MALE

In this short video, I will be explaining how to do a differential leukocyte count on normal blood. Specifically, I am looking
for neutrophils, lymphocytes, monocytes, eosinophilic, and basophils.

I have the normal blood smear loaded on the microscope stage. I'm going to use the times 20 objective because that allows
me to distinguish the cell types, and gives me a reasonable area to look at. I'm also going to add the grid to help with counting.
And I'm going to start near the top left of the section.

Here, I can see one, two neutrophils, and one lymphocyte. I'm going to scan across the section, looking specifically for cells
that lie between the upper and lower heavier grid lines. Another neutrophil. Two more neutrophils.

This one is a monocyte. There's a small lymphocyte. Once I reach the end, I'm going to drop down to a lower line and scan
back, making sure not to count the same cells twice. And carrying on as before. There's a lymphocyte, and an eosinophil, and
so on, until I've counted 100 cells.

Now at this stage, I would tally up the cells of each time. And as you can see, it falls within the normal range.

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DAVID MALE

In the first part of the course, we showed you how to use a microscope, and how to recognise a variety of different cell types
from blood under the microscope. In this part, we're going to introduce you to a variety of additional different types of
human tissue. Often to help recognise structures within tissues, histologists use specialised stains on their sections. And
we'll be showing you some examples of that, too. One of the ideas that I want to get over to you this week is that there is
a range of normality within tissues.

So for example, the skin on my face, or the skin on my head, or the skin on my finger are all different, and will appear different
underneath the microscope. But they are all quite normal. If, for example, I was a guitar player, I might have calluses on
my fingers. But they would still be within the range of normality.

By the end of this week, you should be able to recognise a variety of different human tissues just from their histological
appearance.

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PROFESSOR DAVID MALE

Cell types. In this video, I will look at some of the sections that you will be examining during the course. And I'll be looking
for different types of cell. I'm not aiming to examine every detail of the sections. You'll have time to do that later. And
the legends on the sections will help you to identify specific features.

I will be looking for different types of epithelium in the stomach and lung, endothelium in different blood vessels, smooth
muscle in the wall of the stomach and an artery. And you will also see examples of collagenous tissue in different organs.

I'll start by looking at the section of stomach, which is slide number three. There is a layer of epithelial cells lining
the inside of the stomach. If I look on the times 20 magnification, you can see that this is a single layer of columnar epithelium.

Because of the way a section is cut, the appearance of the cells may not always correspond to the classical columnar form,
as you can see here. And if I move across, also here.

If I move to the next layer of cells, the submucosa, and I return to the times 20 magnification, you'll see an example of
collagenous tissue. There are relatively few cells and a large amount of extracellular space, of which a large proportion
is collagen secreted by the cells in this layer.

Further out again is a layer of smooth muscle cells. These cells are densely packed. And they have elongated nuclei. And they
run in lines corresponding to the direction of contraction of the muscle. The smooth muscle in the gut propels food through
the system.

I'm now going to look at a blood vessel, in this case, the cross-section of a small artery. There's a single layer of endothelial
cells lining the inside of this vessel. The cells and their nuclei are very thin and flattened.

The artery also has smooth muscle in its wall, which resists the pressure from the blood flow and partly controls the amount
of blood that can pass through the lumen of the vessel. Around the edge of the vessel, there is a layer of collagenous tissue
referred to as the adventitia.

Finally, I'm going to look at a section of lung. The outside of the lung has an outer surface called the pleura. It consists
primarily of collagenous tissue, which you can see at the high magnification. The main cell types in the lung are epithelial
cells called pneumocytes, which are involved in gas exchange. There are some examples here. There are also many capillary
blood vessels and small arteries called arterials.

As you start to explore the sections, you will find common patterns of cellular organisation in different tissues and organs.

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DAVID MALE

The digital microscope is a virtual representation of a real microscope, this microscope here. And we have a large collection
of slides, which we've collected from various pathology departments and hospitals and histology units all around the country,
specialists units. And then they have given us the opportunity to create very large images. It's very, very like Google Earth
type of images, very big scans, which we can effectively zoom across and scan into, which recreates what is going on in a
real microscope here.

And we have a collection of around about 300 slides that we've obtained in this way. And we make them starting from a unit,
a little section like this which goes onto the microscope. And then there's a sort of fairly complicated set up. It's a high-end
microscope. It costs about 50,000 or 60,000 pounds. But it does give students the opportunity to look at material that they
would not otherwise be able to see from specialist units and also on the kind of microscope that they would never, ever be
able to get their hands on.

Initially, we designed the resource for undergraduate students and particularly sort of first and second year of their studies.
They would be medical students or zoology students, this kind of person. It's also for training hospital laboratory technicians.
And we're starting to put in an a-level teaching set as well at the moment, which is, obviously, a little simpler, but also
includes some plant materials to go with it.

At the other end, we have very specialist sets of pathology slides. So, for example, there's a set from the Institute of Ophthalmology,
a set of eye slides, very particular kind of diseases there. And this kind of material would be suitable for really up to
a hospital consultant level.

We have a series of 20 units, which are listed here in the catalogues. So for example, you'll see that one of the sets is
a set of skin slides. And if I pull that up by clicking on it like that, you'll see that there's something like 15 or 16 slides
in there of different skin pathologies.

When you want to look at one of these slides, you just load it up by clicking on it. And this loads it onto the microscope
stage here. And then we get a larger view of it over here. And the digital microscope allows us to move around so we can scan
across the slide like this. We can zoom in by changing, effectively, the objective that we're using here. And you can see
now we've got a larger image of it on the screen there.

We can do a whole number of things like add grids onto it or graticules. We can change the focus of it. It emulates the workings
of a real microscope in changing the focus and also the lighting of the slide.

And the most useful thing of all are these legends here. So for example, if we click on this one, which says we're going to
a sweat gland, an eccrine sweat gland, then the microscope will immediately jump to that particular position. So that it effectively
replaces the function of a tutor that you might have looking over your shoulder at what you're looking at down a real microscope.

You can go to a particular area on the slide by typing in its coordinates. And when you press go, it will immediately jump
to a particular area, and then you can ask the student what they are seeing at that particular point on the slide.

We also have a camera button. And so, for example, a student could take a picture of what they're seeing and what magnification
they're using and insert this little piece of picture into an assignment of some sort, say, some description of a pathology
or whatever. And then that can be used as part of an assessment.

As we started to talk to people we found people coming to us with their own specialist collections and saying, actually we've
got an excellent set of liver slides or an excellent set of kidney slides. And we'd really like to include this material.
So we are open to working with various partners partly to expand the collection and improve it, but also to help create teaching
material that they can work with and will be useful for them.

TEXT ON SCREEN

The slide collection includes 320 sections with a set of normal tissues and 12 collections based on tissue type and pathology.
Slides and images were provied by De Montfort University, UCLH London, The Institute of Opthamology, Cambridge University,
Milton Keynes NHS Trust, Leicester General Infirmary, KCL London. The microscope was developed by a team at The Open University,
directed by Professor David Male and it was funded by a project grant from JISC.

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DAVID MALE

This short video gives you some guidance on how to examine a histological specimen. You will have noticed that when a section
is loaded onto the microscope, it always resets to the lowest magnification with the times 2 objective. There are a number
of good reasons for starting with the lowest magnification. The first and the main reason is that it is important to get a
good overview of the section before starting to examine it in detail.

Although it is tempting to jump straight to the high magnification, many structures cannot be seen at high magnification.
Moreover, the critical features of a tissue may only be visible in part of the section.

Let's take the pancreas as an example. At a low magnification, the islets of Langerhans, which contain the insulin producing
cells, can readily be distinguished here. But if I had started using a high magnification on the centre of the section, I
would completely have missed them.

Another reason for starting with a low magnification is to see the overall arrangement of the tissue. In this section of lymph
node, the developing follicles of lymphocytes can be seen distributed around the edge of the node. And the overall organisation
of the tissue is clear. However, you'll see that it is quite difficult to distinguish these structures if you just scan across
them at high power, even if you do so systematically as I'm doing so now. It's really a question of not being able to see
the wood for the trees.

Two other practical reasons for starting with low power are, firstly, that the low power objective is further away from the
slide than a high power objective, so there is less chance of damaging the slide by pushing the objective into the slide while
focusing. And, secondly, that the depth of field is greater with low power objectives, so it is easier to find the approximate
focus position for that particular slide.

Once you have identified an area of interest, systematically scan back and forth across it using an objective which is appropriate.
For example, if you need to distinguish subcellular structures, then a high magnification is needed. But if the aim is to
identify a mass of tumour cells, then the times 4 or times 10 objective might be more appropriate.

In this case, I'm going to look at the thymus for a structure called the Hassall's corpuscles. Nothing much can be distinguished
on low magnification, but the distinctive structures are readily visible if you use the times 20 objective. You can see some
here and here.

It is often a good idea to flip between normal tissues and an abnormal or diseased tissue. This makes it easier to identify
the abnormality. Later in the course, you'll be looking at some diseased tissues and this type of systematic approach will
be very helpful. Aim to develop good practice as you examine the sections.

To summarise, start with a low power objective and adjust the light if necessary. Examine the overall structure of the tissue.
And identify key features or abnormalities. Use appropriate high power objectives for the structures being examined. And scan
systematically.

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DAVID MALE

In the second part of the course, we introduced you to a number of different human tissues, including the skin, the gut, blood
vessels, pancreas, and so on. In this part, we're going to introduce you to a further series of tissues, but, in particular,
we are going to relate the structure of the tissues to their function. We've chosen a number of functions, including excretion,
illustrated by the kidney; communication, illustrated by the nervous system; strength, illustrated by bone and cartilage.

The relationship of the structure of a tissue to its function is very important for understanding a diagnosis of disease.
For example, if heart muscle becomes expanded, it may be because the person has been training or it may be because their heart
is overloaded and there is some specific pathology taking place. Understanding these relationships is really important for
understanding the basis of disease. And that is where we will be going on the next part of the course.

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DAVID MALE

Histology of the kidney. In this video, I'll be taking you on a short histological tour of the kidney, pointing out some of
the parts of the nephron, a structure that philtres the blood and ultimately produces urine.

Here's a diagram of a nephron. Each nephron acts as a urine producing unit. And there are many thousands of them in a single
kidney. The blood is initially filtered in the capillaries of the Bowman's capsule. It's located in the cortex of the kidney--
the outer layer. The filtrate first enters the proximal convoluted tubules and then passes down into the loop of Henle in
the medulla before returning to the distal convoluted tubules in the cortex.

As it passes through the nephron, some salts and small molecules and water are reabsorbed, while other salts and small molecules
may be secreted into the tubules for excretion. The processed filtrate, which is now urine, finally passes into the medullary
collecting ducts and enters the renal pelvis.

The kidney section in the virtual microscope includes the cortex-- the outer region of the kidney-- and some parts of the
medulla are also visible in the section. The appearance under the microscope is not nearly as neat as this diagram. Let's
take a look.

The kidney is slide 9 in the week 3 set. And I have already loaded it onto the microscope stage. It shows a section which
extends from the capsule of the kidney at the right hand end of the section. And it includes the full depth of the cortex
and part of the medulla, starting about here.

Let's look first for a Bowman's capsule. There are two nice examples here. I've got to switch to the times 10 objective. Let's
look first at this Bowman's capsule. The tuft of capillaries, the glomerulus, is in the centre of the capsule. And it's supplied
with blood by the renal arterioles, which enter here. In this example, it is possible to see how the arterioles enter the
capsule, but that is not always the case. It depends on where the section runs through the capsule.

Also visible here is the juxtaglomerular apparatus, which regulates renal blood flow and the glomerular filtration rate. The
epithelial cells lining the Bowman's capsule are also very visible, although it's difficult in this instance to see where
the capsule opens into the proximal convoluted tubules, usually on the opposite side to where the blood vessels enter.

If I now move to the outer part of the cortex, you can see two different sorts of tubules. The proximal tubules have thicker
walls and a smaller lumen, while the distal tubules have thinner walls and a more obvious lumen.

Also visible in this section are some capillaries with flattened endothelial cells. Erythrocytes can help to identify blood
vessels, but in this case, the organ was perfused to remove blood before sectioning, so you won't see them here.

Finally, I'm going to look at the medulla. I will switch back to the times 4 objective. And, now, as I move towards the medulla,
you can start to see the loops of Henle becoming more apparent. I'll go to the times 10 objective so that you can see them
a little better. They're interspersed with, and run parallel with, the medullary collecting ducts. So it can be difficult
to distinguish them. But the ducts are lined with clear-celled cuboidal epithelium, while the epithelium on the loops of Henle
is usually more flattened.

I'll move down towards the bottom of the section here and you can start to see some of the medullary collecting ducts. You
may also see some blood vessels in the tissue. There is an example of a venule up here.

It's worth taking some time to look at the different structures of the normal kidney, each one performing its own function
in the production of urine and the overall function of excretion.

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DAVID MALE:

Now that you can identify a number of different human tissues under the microscope, we're going to start introducing some
pathological sections. We've chosen a number of themes to look at this week-- infection and inflammation. For example, this
is going to be illustrated by tuberculosis in the lung.

Another theme is cell death and degeneration. And there you'll be looking at Alzheimer's disease.

And, finally, tumours. Tumours are interesting because they may be a primary tumour, one that has originated from the tissue
that you're actually looking at, or a tumour may be carried from another site to the tissue that you've got under the microscope.
This is a secondary tumour. And the process by which it moves from one part of the body to another is called metastasis.

At the end of this course, we're going to ask you to look at a number of different pathological sections to identify the
abnormalities, to try to deduce the underlying causes, and, finally, to venture a diagnosis of what the disease might be.

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DAVID MALE

Histopathology for the diagnosis of disease. In this video, I will introduce some of the pathological specimens that you will
be examining. As with any histological section, you should start by scanning the section at low power to identify any major
anatomical abnormalities. The higher power objectives can be used to see changes in the individual cells or the extracellular
matrix.

Sections will often have normal and abnormal areas, which can be compared. At this stage, you may not remember what the normal
tissue looks like, so flip back to a normal tissue section for comparison.

When describing the appearance of a section, be absolutely clear and objective about what you see. Any interpretations can
come later.

In some cases, there are pairs of sections-- one with hematoxylin and eosin stain and one with an immunohistochemical stain,
which identifies specific features which may be critical for diagnosis.

When interpreting a section, consider whether any changes might be just a normal physiological change, for example, due to
age or hypoplasia, and don't over interpret what you see. Remember, it takes many years to train a consultant histopathologist.
Now, let's take a look at some sections.

In this case, I've loaded section number 7 from the cerebral cortex of someone who had dementia. It has been diagnosed as
Alzheimer's disease. But how do we know that?

At low power, you can see the edge of the cortex and the meninges with some blood vessels. The rest of the tissue is fairly
homogeneous.

I look at high power across the width of the section. I'm going to track to the left towards the edge of the cortex.

The nuclei of the neurons are small and condense, which suggests they have died, possibly by apoptosis. There is no significant
infiltration with inflammatory cells, leukocytes. But as I carry on, you can see areas with vacuoles where neurons have been
lost. The blood vessels appear relatively normal.

Just for comparison, I will look at a normal cortex, section 6. Although the staining here is rather different, you can see
that the neurons are denser and they don't have these small condensed nuclei. They look much more normal.

Back to section 7 again. One of the hallmarks of Alzheimer's disease are plaques in the cortex containing deposits of a protein,
A beta. But we can't seem them well on this H&E section. And this is where a histopathologist might ask for a specialised
stain.

And section 8 is an immunohistochemical stain for A beta. You can immediately see deposits of the protein, referred to as
beta amyloid, distributed throughout the cortex. If I increase the magnification, you can see that these deposits are extracellular.
And they are not particularly associated with blood vessels. So this strongly indicates a diagnosis of Alzheimer's disease.

When you look at these sections, try to think about the normal anatomy and function of the tissue, as this will help in interpreting
your observations.

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DAVID MALE

We hope you've enjoyed this course introducing histology and histopathology. This is just an introduction to a huge area,
but the skills that you have learned in microscopy and learning how to recognise different tissues in the abnormalities are
key skills that are required for this subject area. The sections that we have shown you are just a small portion of the collection
that we assembled for Jisc, who funded the development of the virtual microscope in these laboratories. In fact, this microscope
here is the one that we used to produce all the sections that you have been looking at.

If you want to take your studies further, then you should go to the open science laboratory where you will find the entire
slide collection that has been assembled for this. Good luck with it. And I hope you enjoy it further as you progress.

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