Anapath -- Dr.

Saad Khairallah -- Session 1

- General pathology: this is the first contact you will have with diseases and pathological anatomy.
- Both histology and organ histology have already been studied.
- Histology is a science that simply studies from the microscopic point of view, the lesions that occur, the normal
aspects of different tissues and different organs.
o In general histology, we study the tissues (muscular, cartilaginous, bone, adipose, connective, epithelial, etc.)
o In the special histology, we study how these tissues will be arranged within the different organs and
therefore the structure and architecture of each organ is studied.
o So we won’t be looking at what is normal, instead we will look at what is considered abnormal in the
organism.
- We will begin with the general pathological anatomy, which is going to be an entirely global study of the various
disease processes, such as the general histology which was a global study about the tissues.
- So we will study:
o Inflammation which is a disease-causing process, which will affect the different tissues.
o Neoplasia which is also a morbid process that affects different tissues.
o Malformations
- All these processes are general processes and will be studied in a general way.
- Each of these processes will present particular lesions which will occur in the tissues and which will be
transported from one organ to another;
o Suppose, for example, tuberculosis as inflammation, and the lesions which will be induced by the Koch
Bacillus in the tissues will be studied in the general pathological anatomy; and then study in the special
pathological anatomy tuberculosis in the various organs (in the lungs, the digestive tract, the liver, the
urinary tract, the gynecological apparatus, etc.).
- NB: If you have ever studied and prepared your general pathological anatomy, by arriving at the special
pathological anatomy, you have only to translate what you have learned in the general pathological anatomy,
into the tissues, into the different organs.
- Tuberculosis, follicular lesions, and caseating follicles, which are the tuberculous follicles, are to be studied this
year, & are therefore to be studied in the general pathological anatomy. Then we go to the lungs, genitalia, etc.
o In tuberculosis of the genital tract, for example, there must be follicular and caseating follicular lesions, so it
will not be described because they have been studied in general pathological anatomy.
- From a general point of view, general pathological anatomy by definition will study diseases.
o Diseases, as a whole, signify any marked modification of the anatomical or functional states generally
accepted as normal.
o We have already seen in histology, the normal appearance of a specific organ, a certain tissue, etc.
o Any variation of this normal aspect is due to a disease, and the study of these variations will be part of the
pathological anatomy.
o Therefore pathological anatomy is the study of lesions.
- In pathological anatomy, we will study the lesions:
o with the naked eye, that is called a macroscopic study
o by the microscope so it is a microscopic study
o immunologically, immunohistochemically
o from the molecular point of view, structural biology of DNA
- So it's a discipline that is very broad and it deals with all the organs, all the specialties, and it's a discipline that
can be quite easy if you if we relate the general pathologic anatomy to the special pathologic anatomy.
- Therefore, pathological anatomy is the study of ultrastructural macroscopic and histological lesions (on the
electron microscope) and biomolecular (thus on the cellular DNA level in relation to the disease to organs,
tissues and cells).
- Pathological anatomy affects not only the tissues or organs, but also the cell, the nucleus, the cytoplasm, the
organelles, which will be modified by these diseases.
- It is cytopathology and histopathology, it is the microscopic appearance of the different lesions in the tissues.
- Thus the pathological anatomy is more general and more extensive than the field of simple histology.
- Two types of studies in pathological anatomy:
o General: it is the study of the lesions caused by the morbid processes in the tissues and the cells.
o Special or organs: it is the study of the alterations and modifications provoked by the morbid lesions at the
level of the organs themselves.

General pathological anatomy

 The most basic thing is to always have a tissue, and see how this tissue should be treated.
 The ultimate goal is to find on the tissues that there are specific lesions, and once found, it is said that such a
disease has caused such a lesion.
 The final goal is diagnosis.
 Throughout the medical profession, you are approaching a diagnosis, assuming a differential diagnosis, but we
can never reach a safe and certain diagnosis if there is no pathological anatomy.

Samples:

1. Simple biopsies (incisional or core biopsy):

o Special terminology
o Partial sampling that is done on a lesion always determined, ie does not cover the entire tissue
o Examples:
 By endoscopy, you remove a small fragment of a tumor in the stomach.
 You have a large tumor in the skin, you make a small resection, keeping the rest of the tumor at the level
of the skin.

2. Excisional biopsies:
o There is a small lesion that is removed directly by a biopsy
o Suppose you have a small nevus (chemiye) on the skin, you do a surgery and you completely remove the
nevus.

3. Puncture (needle aspiration biopsies):

o A sampling that is done via a needle (which may be small for blood or intra-muscular specimens) or much
larger trocars which are made up of an obturator (may be a metal or plastic sharpened or non-bladed tip), a
cannula (basically a hollow tube), and a seal.
o So the needle or the trocars are inserted and the material is collected inside. There are 2 types of material
collected:
 Either liquid and cytological Cells
 A tissue is collected - a core of tissue : Biopsy

4. Surgical procedure:
o Here, we have an affected organ where we remove it completely or partially. It is generally a large organ of
several centimeters in size that cannot be reached by puncture or biopsy, and requires surgery.
o Ex: appendectomy, cholecystectomy, gastric resection, radical mastectomy, etc.
o So when we make take surgical parts, it is either:
 An entire organ that is removed: appendectomy, cholecystectomy ...
 Or part of the organ: quadrantectomy (a quadrant of the breast is taken), partial gastrectomy, partial
colectomy ...
 And this is done according to the extent of the lesions and the type of diseases.

5. Necropsy (autopsy):
o Performed after the death of the individual either for:
 Scientist to know what kind of diseases, lesions
 Medico-legal to know the cause of death
Once we have received the sample, there is no way to see it directly on the microscope, so there are procedures that
had to be done before we get a blade and we study it:
I. Macroscopic examination: look at the sample and see what it is; if it is a small biopsy, a carrot, a biopsy
fragment, a tumor (ulcerative, necrotic, budded)
II. Microscopic study (more difficult) as it requires several steps to bring a tissue to be studied under a
microscope. One cannot put a gallbladder or appendix under the microscope to do the study; there is a process
to be done, it is the same technique used in histology. Among these techniques, the main one is Paraffin
Inclusion. So we will remove a small sample (1 cm) at the level of this lesion, tumor... Let's cut it to study it
under the microscope. This cut is done by these several following technical steps:
1. Dehydrate the tissue
2. Include Paraffin inside this tissue
3. This tissue must be placed inside a paraffin block
4. Cut this block with a Microtome: Cuts of 4 microns thick (hypertensive microtomes).
5. Then spread these sections on slides to be colored.
Nothing appears under a microscope if there is no coloring. Coloring is a very important technique that will
enable us to create contrasts in the different structures. Contrast and intensity, will make this tissue to be
apparent to the microscope.
6. Once this tissue is spread, it is possible to make several types of coloration. The purpose of coloring is to
recognize the tissue. There are 2 types of colorations:
o Routine colorations that will allow us to diagnose the tissue. It is a coloration that is used on all the
samples, in order to simply visualize the nuclei, the cytoplasm, the surrounding connective tissues and
the architecture of the organ. It's just an approach to be able to distinguish the type of tissue at the
sampling level. We will hear of this routine staining in surgery and anatomical pathology which is the
Hematin-Eosin-Saffron (HES) stain: These are the 3 colors that will be used:
 The hematin will color the nuclei in dark violet or black.
 Eosin will color the cell cytoplasm in red.
 The saffron will color the connective tissues in yellow.
So, any tissue will contain cells (with their nucleus, cytoplasm and connective tissue support) you will be
able to point out and say for ex: here we see cells arranged in strips spanning the lengths of the slide.
o Special colorations: Unlike routine colorations, these special colors have a specific purpose. So they are
not as commonly used as the HES. One must suspect something in a tissue and try to confirm it and
look for it by these colorations.
 They are part of the histo-chemistry because these dyes will use particular properties of substance
inside the tissues to reveal them. So, it is an association of a coloration (dye) that will be revealed
by a chemical reaction with a substance present inside the tissue (so we must first suspect what can
be as substance to look for in the tissue and there will be used a special staining to be able to prove
that this substance exists).
- PAS reveals the presence of the polysaccharide of mucin and glycogen
- Alcian blue is positive on mucus and mucin
- The Masson trichrome coloring connective tissue in green; about the equivalent of HES but
much more contrast
- Fontana for melanin pigments in tissues
- The coloration of Perl’s for iron
- The red combo
- Sudan red for adipose tissue
 Histo-enzymology: Use enzymatic reactions
 Immunohistochemistry: It detects substances in small quantities that would be invisible in special
staining like proteins, nuclear and membrane receptors.
- Uses Ag-Ab complexes on histo sections (400 Abs are used >> 400 structures can be looked for)
- Ex: protein S100 will be found in the tissue; so we will use Anti-S100 >> they are incubated on
the fabric >> the tissue is incubated in a solution of Anti-S100 >> Wash. If there is the protein:
an Ag-Ab reaction is established, the dye fixed on the Anti-S100 will be present on the tissue.
Otherwise: no reaction; after washing the dye will disappear and the staining will be negative
  Molecular Biology:
- ISH (FISH-CISH-SISH-BDISH); PCR
- It is a technique that goes to the cellular gene and search for a modification at the level of DNA
or RNA due to the studied disease
- For example: colon cancer; the KRAS gene mutation is detected and detection is done to give
the treatment.
 Electronic Microscopy
- It is used less and less, it is used for research purposes
- It is a microscope that instead of using white light, it uses electron rays. It is able to discriminate
at very large magnifications ranging from 200000-300000 to 1000000 times of the different
structures inside of the tissues, whereas in Optical Microscopy we have 40 * 10 at most 400
times or if we have perforating microscopes we can go up to 1000 times.
- Here are some examples:
o Histology of the alveoli inside the normal lung.
o Tuberculous lesions: follicle that will be formed by particular macrophages, these are cells
called epithelioid cells, by lymphocytes and multi-nucleated giant cells. Thus, it may or may
not have a central necrosis called caseous necrosis. Here one sees central necrosis, caseous
necrosis and epithelioid cells. Therefore, particular macrophages, giant cells and
lymphocytes form the crown around the necrosis , and it is characteristic that this structure
can be found anywhere, and it is recognized that it is a caseo-follicular nodule >>> A
tuberculous caseo-follicular lesion.
o Special pathological anatomy: this lesion (B on the slide) transposed at the level of an
organ. As soon as we recognize these nodules in the lung, we will say that this lung is
suffering from tuberculosis.

Tissue Processing

 The tissue can be obtained by biopsy, autopsy ...

 From a histological point of view, here are the different stages of the histo-technique.
o The reception and identification of the sample
o Labeling of the specimen with numbering
o Fixation (detailed later)
 There is no tissue that can be studied in pathological anatomy if it is not fixated
 If you put a piece of any tissue free in air for an hour it will get damaged  Fixation is important. The
step of fixation is a step that is special at time of sampling. This fixative that will keep the tissue at the
time of sampling is formol (formalin) of concentration 10%. Once the sample is taken it must be put
directly into the formalin. The sooner it is put, the better the fixation.
 Fixation can range from some hours to several days, and even a specimen put into formalin can
hypothetically be preserved forever.
o Dehydration (detailed later): it is necessary to remove the water from the tissue so that the paraffin can
enter inside it.
o Clearing (detailed later): once the water has been removed from the interior of the tissue, in order to be able
to be penetrated by paraffin, a substance must be placed in the tissue which can dissolve this paraffin and
make it penetrate inside, it is benzene.
o Impregnation (detailed later): After dehydration, the tissue must be put into benzene and then into hot
paraffin; it is the impregnation in which the paraffin penetrates into the tissue.
o Embedding (detailed later): Once the tissue is imbedded in paraffin, it is put into a mold filled with molten
wax, then paraffin is blocked.
o Suction cutting
o Staining
o Mounting
Repetition on the slides (the various detailed steps)

 Fixation means putting the specimen inside the formalin, it can be preserved permanently. Then we put the
sample inside the jar with formalin and then we put them in a cassette which will be put in a basket and the
whole steps after that will be automated: it is done in a machine that will take the sample from one medium to
another.

 Once they are in the cassette, the step of dehydration will start. The principle of dehydration is to remove the
water from the tissue, and to remove the water from the tissue we use alcohol, alcohol at 60 then 70 then 80
degrees up to 100 degrees, and so as the sample is moved from one alcohol concentration bath to the other,
more water will be removed. Reaching 100 degrees alcohol, there is no more water at all in the tissue. And this
passage in the different concentrations always takes place in the same machine.

 So, alcohol will replace water, and in the Clearing stage, the alcohol itself will be replaced by Xylenes.

 Slides: It is a machine that takes the sampling from one side and passes it from one bath to another, the duration
of the baths is programmed. It passes the sample into baths of alcohol then of xylene and then a bath of paraffin
at the end and one will have at the end a sample impregnated with paraffin.

 Once paraffin is obtained, the paraffin will penetrate the sample dissolved by xylene. We will take this sample
which was in the cassette, we will put it inside a mold. We'll cover it with that and put some paraffin on it. This is
called paraffin blocks.

 The paraffin block is fixed on this machine, and the microtome will raise the block and lower it on the knife.
Every time it raises the block it will advance by 4 microns in thickness and it will lower it on the knife. The
microtome will raise the block prepared again, advance it by 4 microns and we will have 4 micron cuts on this
machine.

 This cut will be spread on a slide, this is the spreading period.

 Then it will be colored with different dyes that are used.

 So here is also an automaton, a machine that passes the slide. The slide is placed in a basket as for the other
machine. And this machine will carry these slides from one dye bath to another bath.
So, 4 micron cuts are obtained, colored with the hematein. The coverslip is placed on top of the slide so that it
can be conserved. (From the internet: hematein is a dye of formula C16H1206 extracted from hematoxylin)