Introduction to Histopathology

Dr- Yaxya Khadar Ciisa

B.Sc. , M.Sc.
 History
 The specialty of histopathology technique dates back to 1838, when
Johannes Miiller published his book, On the Nature and Structure
Characteristics of Cancer, the first book on histopathology.
 The first compound microscope had been constructed earlier in 1591
but suffered from severe optical problems.
 In 1673 Anton van Leeuwenhoek started the development of simple
microscopes with single lenses.
 Cont…
 The first microtome suitable for sectioning animal tissues was
constructed in 1848.
 Paraffin wax for infiltration and support during sectioning was
introduced during the mid1800s.
 Different laboratory chemicals were investigated for use as fixatives.
Formalin, widely used today, was first used in 1893.
 Automated tissue processors replaced hand processing starting in
1945, and cryostats were first manufactured in 1951.
 Histology
 The name "Histology" is derived from the Greek word for a tissue
"Histos", and "-logos" = the study of
 Is a study of tissues which include the examination of tissue structure,
relationship of different types of tissues.
 Other definitions:

 Is a study of normal tissues of the body.

 Is a study of tissue sectioned as a thin slice, using a microtome. It can

be described as microscopic anatomy.
 Histopathology
 Histopathology is the study of the diseased tissue.

 Histopathology, the microscopic study of diseased tissue, is an

important tool in anatomical pathology, since accurate diagnosis of
cancer and other diseases usually requires histopathological
examination of samples.
 Cytology
 Is detailed study of individual cells.

 Based on usage it can refer to:

 Cytopathology - the study of cellular disease and the use of cellular

changes for the diagnosis of disease.
 Cell biology - the study of (normal) cellular anatomy, function and
chemistry.
 Histopathology
 Histopathology is the study of the diseased tissue.

 Histopathology, the microscopic study of diseased tissue, is an

important tool in anatomical pathology, since accurate diagnosis of
cancer and other diseases usually requires histopathological
examination of samples.
 Histopathological techniques
 Branch of biology concerned with the demonstration of tissue
structure in the disease.
 Small amount of tissue is required to demonstrate morphology by
stains.
 Cont…
 Histopathological examination is used to provides diagnostic
information that is important for the diagnosis of the disease to
determine treatment plan.
 Tissue for study is obtained from:

 Biosy: Obtained during life either by open surgery (Excisional

or incisional) or via endoscopy.
 Autopsy: Obtained from the body after death.
 Methods of tissue examination (preparation
 Fresh cells and tissues.
 Vital staining.
 Cytological techniques.
 Sectional methods.
 Fresh cells and tissues
A) Cells suspended in fluid (e.g. blood or lymph), may be seen by: direct

examination in a drop of the fluid which may need dilution with normal

saline or isotonic solution (8.5 g/dl) conc.

B) cells found in loose connective tissue such as (subcutaneous C.T):

Examined directly if the tissue is thin. if thick, cells can be separated from

each other in normal saline by a method called dissociation or teasing.

 Vital staining
 Certain parts of living cells can be stained.
 Cells are dissociated in the staining solution which called supravital staining.
 Or by injection of the dye into the living organism which called intravital staining.
 These methods demonstrate part of living cells.
 Mitochondria, vacuoles found in cytoplasm.
 Nucleus doesn’t take the vital stain.
 Nuclear membrane in living cells prevent the entrance of the dye inside the
nucleus.
 Permeability of the nuclear membrane to dye is an indication of cell death.
 Cytological techniques
 Fluid containing cells, tiny fragments of tissue such as bone
marrow.
 Smeared on slide .

 Adherent cells fixed (to preserve their appearance).

 Smear is stained (to demonstrate cell structure).

 Mounted in a medium such as DPX.

 Finding of abnormal cells in body fluid or secretion may be

suggested or diagnostic of malignancy.
 Sectional methods
 Cutting of specimen into very thin section have advantages:
 Preserves the architecture of the tissue and the relationship of the cells to each
others.
 Three dimensional structure of the cells are appear.
 Most routine work and research is done by sectional method.
 Thick sections of fresh or fixed tissues may be cut freehand with a sharp knife (some
value for rapid diagnosis).
 Sectional method depend upon conversion of the tissue into a uniform consistency by
either freezing, infiltrating and embedding in paraffin wax, colloidin, or synthetic
reins.
 Most histological sections are 3-5 µ.
 Work flow in histopathology lab and instruments
 Reception

 Selection preparation

 processing

 Embedding

 Sectioning

 Staining

 Cover slipping

 Filing
 Reception
 (receiving the specimen either biopsy or blocks)
 Any specimen should be received with histopathology request form.
o Histopathology request form Should include:
 Name of patient relevant to name written on the specimen label.
 Site of the specimen compared with the site written and the specimen label.
 Age, sex, residence, date, phone No, consultant name and any clinical
marks.
 Fixed in 10% formalin.
 Specimen received should be labeled and the lab label should include:
 (A) lab. No. (B) the year e.g. (13/013).
 Specimen should be placed on the selection room and the form should be in
the selection file.
 Selection preparation
 Kidney shape disk. Different size of knifes, Gloves, Gauze and cotton, forceps, A
wooden piece, Saw, pencil, ruler, cassettes, 10% formalin, request form.
 Macro examination.

 Fixed or not.

 Shape of organ.

 Length.

 Soft or firm.

 Report any abnormality.

 Record how many pieces taken describe them, how many blocks.

• All these written on request form back.

Gross examination
 processing
 The most common technique is wax processing. The samples are
immersed in multiple baths of progressively more concentrated
ethanol to dehydrate the tissue, followed by a clearing agent, such
as xylene or Histoclear, and finally hot molten paraffin wax
(impregnation). )12- to 16-hour( During this process, paraffin wax
replaces the xylene.
Tissue processor
processing machine: Processing schedule:Work programmed
 Embedding
 Soft, moist tissues are turned into a hard paraffin block, which is then placed in a
mould containing more molten wax (embedded) and allowed to cool and harden.
 Embedding can also be accomplished using frozen, non-fixed tissue in a freezing
medium.
 This freezing medium is liquid at room temperature but when cooled will solidify.
 Non-fixed tissue allows for procedures such as in situ hybridizations for specific
mRNAs that would have been destroyed during the fixing process.
 Embedding preparation: (Melting paraffin wax, Moulds, Pencils, Forceps and
Cooling system).
Tissue embedding
 Sectioning
 The tissue is then sectioned into very thin (2–8 micrometer)
sections using a microtome. These slices, usually thinner than the
average cell, are then placed on a glass slide for staining.
 Frozen tissue embedded in a freezing medium is cut on a
microtome in a cooled machine called a cryostat.
 Preparation of sectioning: (Microtome and microtome knifes,Water
bath, Hot air oven, Slides, Forceps, Brush, Pencil, Adhesive
media,70% alcohol and Refrigerator or cooling system).
Sectioning with microtome
 Staining
 The embedding process must be reversed in order to get the paraffin wax out
of the tissue and allow water soluble dyes to penetrate the sections.
 Slides are "deparaffinized" by running them through xylene (or substitutes)
to alcohols to water.
 There are no stains that can be done on tissues containing paraffin wax.
 Frozen sections are stained by hand, because this is faster for one or a few
individual sections. The stain is a "progressive" stain in which the section is
left in contact with the stain.
 Routine staining:
• This is done to give contrast to the tissue being examined, as
without staining it is very difficult to see differences in cell
morphology.
• Hematoxylin and eosin (H&E) are the most commonly used stains
in histology and histopathology.
• Hematoxylin colours nuclei blue; eosin colours the cytoplasm pink.
To see the tissue under a microscope.
 Cover slipping
 The stained section on the slide must be covered with a thin piece

plastic or glass to protect the tissue from being scratched

 To provide better optical quality for viewing under the microscope

 Preserve the tissue section for years.

 permanent resinous substance beneath the glass coverslip, or a

plastic film, can be placed over the section.

Cover slipping
 Filing
 Blocks.
 Un stained slides.
 Stained slides.
 Result sheet.
END