This document provides an overview of histopathology and cytopathology procedures. It discusses the key steps in tissue processing which include fixation, dehydration, clearing, embedding, sectioning and staining. Common stains used are hematoxylin and eosin. The document also describes different biopsy and cytology techniques as well as the use of immunohistochemistry, frozen sections and microtomes.

1. Item no. 01
Introduction. Preservation and
transportation of specimen and
tissue processing
Dr. A K M Maruf Raza
Associate Professor of Pathology

2. Introduction to Pathology
• Pathology is the study of the:
structural, biochemical, and functional changes
in cells, tissues and organs that underlie
disease.

3. Pathology is divided into:
General pathology :
• General pathology is concerned with the common
reactions of cells and tissues to injurious stimuli.
Systemic pathology :
• Concerned with the alterations and underlying
mechanisms in organ specific diseases

4. The four aspects of a disease process :
I. Etiology or Cause
II. Pathogenesis
III. Morphologic change
IV. Functional Derangements and Clinical
Manifestations

5. Etiology or Cause
Etiology or Cause can all be grouped into two
classes:
• Genetic (e.g., inherited mutations and disease-
associated gene variants, or polymorphisms).
• Acquired (e.g., infectious, nutritional, chemical,
physical).

6. Pathogenesis
Pathogenesis refers to:
The cellular, biochemical and molecular
events that occurs in cells or tissues to an
injurious agent.

7. Morphologic change
Morphologic changes refer to:
The structural changes in cells or tissues that
are characteristic of a disease process.

8. Functional Derangements and Clinical
Manifestations
The end results of etiology, pathogenesis and
structural changes are:
i. functional abnormalities leading to the
clinical manifestations (symptoms and
signs) of disease.
ii. clinical course and outcome.

9. Histopathology
Histopathology:
Microscopic examination of stained tissue
sections for diagnosis of disease is known
as histopathology.

10. Biopsy
Biopsy:
• It is the process of removal of tissue from the living
body for the diagnosis of disease.
Biopsy is done for :
• Confirmation of clinical diagnosis.
• Exclusion of differential diagnosis.
• To assess excision margin clearance in case of
malignancy.
• Grading of malignancy.
• Staging of malignancy.
• Assessment of prognosis.

11. Procedure in Histopathology/
Tissue processing in
Histopathology

12. Procedures in Histopathology
1. Tissue taken from the body as Biopsy by the
Surgeon.
2. The tissue is labeled properly giving details
like name, age and sex in a requisition form,
identity of the tissue, provisional diagnosis
and other investigation findings.

13. Procedures in Histopathology
3. Tissue then put in a container with 10%
formalin and send to the Pathology department
for Histopathological examination.
4. The container containing tissue must be
properly labeled and related clinical
information should be given in a requisition
form.

14. Procedures in Histopathology
5. In pathology department, tissue is registered
in register book and the tissue sample is
transfer to the Gross examination table.
6. The histopathologist examines the whole
tissue, gives description of it, mentioning any
abnormalities in the tissue and takes tissue
blocks of 2-3 millimeters thickness for
processing of tissue.

15. Procedures in Histopathology
7. Then the tissue block is taken for Tissue
Processing and staining. The basic steps of
tissue processing includes:
1. Fixation by 10% formalin.
2. Dehydration by Ethanol.
3. Clearing by Xylene.
4. Tissue Embedding by paraffin.
5. Tissue sectioning by Microtome (3 to 6
μm thickness).
6. Staining (Hematoxillin and Eosine).
7. Cover-slip /Mounting of slides.

16. Fixative
Routine fixative:
• 10% formalin.
• 10% formalin is the most commonly used
routine fixative.
• Volume used Should be at least a 10:1 ratio
of fixative to tissue.
• The commercially available formalin is 40%
formaldehyde gas dissolved in water.

17. Fixative
Other fixatives :
(a) Zenkers fluid: It is preferred for fixing
lymphnode, liver, spleen and bone-marrow.
(b) Bouin’s solution : For preservation of Testis
tissue.
(c) Carnoys fixative: For rapid fixation within 6
hours.
(d) Clarke’s solution.
• For electron microscopy fixatives used are:
– Glutaraldehyde.
• For frozen section no fixative is needed.

18. Aim of fixation:
I. Enzymatic digestive processes (autolysis) are
stopped thus the normal tissue structure is
preserved.
I. To facilitate staining of tissue.
I. Tissue become hard after fixation which helps
in microtome sectioning.
II. Aid in visual differentiation of structure of
tissue.

19. Stains used in Histopathology slide
• Hematoxylin and eosin (H&E) stain:
• It is the most common stain used for
histopathology. It is a combination of two stain,
hematoxillin, eosin.
• Hematoxylin has affinity for the nucleic acids
of the nucleus and stains the nucleus
appearing blue/ Violet in slide.
• Eosin stains the cytoplasmic proteins of cells
and appears pink to Red in slide.

20. Other Histologic Stains
1. Periodic acid–Schiff (PAS) stain (Mucin,
Basement membrane)
2. Reticulin stain
3. Masson trichrome stain.
4. Oil red O stain (For fat and Lipid)
5. Congo red stain ( For Amyloid)

21. Cytopathology
• Definition: Cytopathology is the study of
cells of different parts of body under
microscope to diagnose different disease.
• It is rapid, minimal invasive and inexpensive
diagnostic procedure.
• But gold standard of tumour diagnosis is
Histopathological examination.

22. Cytopathological Method/Sample
1. Aspiration cytology (FNAC).
2. Exfoliative cytology (Paps Smear).
3. Brush Cytology (Bronchial brushing, Stomatch
brushing).
4. Imprint Cytology.
5. Fluid Cytology for malignant cells (Pleural
fluid, Ascitic fluid).

23. Aspiration cytology
• This is the analysis of cells taken by aspiration
by a needle from an organ or a mass of a
organ.
• This procedure is called Fine Needle Aspiration
cytology (FNAC).
• A needle is inserted into the area of the body to
aspirate the material.
• FNAC sometimes done with the use of imaging
(e.g. ultrasound guided or CT guided FNAC).

24. Exfoliative cytology
• This is the analysis of cells that are shed from
body surfaces.
• Examples include the lining cells of the uterine
cervix, Sputum, cells of urinary bladder.
• The analysis of cells from the cervix is called
cervical or Papanicolaou smear (Pap smear).

25. Fixatives used in Cytopathology
I. 95% Ethyl Alcohol (Ethanol).
II. Propanol.
III. 100% Methanol.

26. Stains used in Cytopathology
I. Papanicolaou Stain (Paps stain).
II. May-Grunwald-Giemsa (MGG) Stain.
III. Haematoxylin and Eosin (H&E) stain.

27. Immunohistochemistry
• Immunohistochemistry is a special
histopathological technique.
• It is the additional investigative procedure for
routine histopathology.
• Immunohistochemistry used to detect antigens
within tissues.

28. • IHC help in accurate diagnosis of malignancies
where routine histopathology fails.
• IHC can help determine the origin of the
tumour.
• IHC also plays role in treatment and prognostic
evaluation. Estrogen and progesterone
receptors (ER, PR) in breast cancer. ER, PR
positive breast tumour has better prognosis.

29. Frozen Section
• It is a specialized histopathological procedure
in order to make a rapid diagnosis of a tumour
mass during surgery.
• The procedure usually takes only few minutes
and result is communicated to the surgeon
over telephone.
• Frozen sections are performed with an
instrument called cryostat (Temperature is -20
to -30 celsius).

30. Frozen Section
• In frozen section tissue is send fresh without
any fixative.
Uses:
• Rapid diagnosis :To detect whether a breast
nodule is benign or malignant to decide
lumpectomy or mastectomy.
• To see excision margin whether it is free or
invaded by tumor.
• Diagnosis of hirusprung disease in neonates
and children.

31. Microtome
Microtome is an instrument used in histopathology
to make thin tissue sections for microscopic
study.
Types:
• Rotary microtome
• Sliding microtome
• Freezing/ cryo microtome
• By microtome tissue sections are made
usually in 3-6 micron thickness.