The document discusses the methods and processes involved in histopathology, detailing the history and classification of tissues, tissue processing techniques including fixation, dehydration, and embedding, as well as staining procedures for microscopic examination. It also covers the importance of histopathological evaluations in diagnosing diseases, including malignancies and infections, and outlines potential pitfalls in tissue processing and staining. A comprehensive overview of histological techniques such as using a microtome for sectioning and the use of staining methods like hematoxylin and eosin is provided.

1. Tissue Processing
in
Histopathology
Dr. Kamal U Zaidi

2. MARCELLO MALPHIGHI (1629-1684)
First to utilize microscope to study animal and plant structure and
histology (tissues structure)
NEHEMI GREW (1642-1712)
Compares plant and animal tissue in article before Royal Society
Tissue histology, describes parenchyma
Preamble

3. Histology, study the organization of tissues at all levels, from the
whole organ down to the molecular components of cells that are found
in most multicellular plants and animals.
Animal tissues are classified as epithelium, with closely spaced cells
and very little intercellular space; connective tissue, with large
amounts of intercellular material; muscle, specialized for contraction;
and nerve, specialized for conduction of electrical impulses. Blood is
also sometimes considered a separate tissue type.
Plants are composed of relatively undifferentiated tissue known as
meristematic tissue; storage tissue or parenchyma; vascular tissue;
photosynthetic tissue or chlorenchyma and support tissue or
sclerenchyma and collenchyma.
Preamble

4. DEVELOPMENTAL HISTOLOGY:- Tissue formation and
specialization in growing embryos
HISTOPHYSIOLOGY:- Relations between morphological changes
and physiological activity
HISTOCHEMISTRY:- Chemical composition of tissue structures.
GENETIC HISTOLOGICAL:- in-situ hybridization of DNA probes
that enable analysis of specific genetic sequences
IMMUNOHISTOCHEMISTRY:- Labeled antibodies that attach to
specific parts of specified molecules.
Preamble

5. Histological Tissue Processing

6. Tissues Collection
• The tissue is removed from the animal body or plant part
• Placed in a fixative which stabilizes the tissues to prevent decay.
• The most common fixative is formalin (10% formaldehyde in
water)
Surgery/Biopsy/Autopsy

7. Tissue cassettes used to hold samples of all sizes for histological processing.

8. Gross Examination
• Describing the specimen and placing all/parts of it into a
small plastic cassette which holds the tissue while it is
being processed to a paraffin block.
• Initially, the cassettes are placed into a fixative.

9. • Proper identification and orientation of the specimen.
• Unlabelled specimen should never be processed.
• A properly completed histopathology requisition form
containing patient’s name, age, sex, relevant clinical data,
surgical findings, nature of operation and name of tissue
submitted.
• Careful search and examination of all the tissue submitted
in order.
Gross Examination

10. Fixation
• To preserve tissues permanently in as life-like a state as
possible.
• Fixation should be carried out as soon as possible after
removal of the tissues or soon after death to prevent
autolysis.
• There is no perfect fixative.
• The fixatives depend on the type of tissue present and
features to be demonstrated.
• Five major groups of fixatives
– Aldehydes
– Mercurials
– Alcohols
– Oxidizing agents

11. • To prevent autolysis and bacterial attack.
• To fix the tissues so they will not change their volume
and shape during processing.
• To prepare tissue and leave it in a condition which allow
clear staining of sections.
• To leave tissue as close as their living state as possible,
and no small molecules should be lost.
Fixation

12. Processing of Tissue
Embed the tissue in a solid medium firm enough to support
the tissue and give it sufficient rigidity to enable thin
sections to be cut and yet soft enough not to damage the
knife or tissue.
Stages of processing
- Dehydration.
- Clearing.
- Infiltration & Impregnation
- Embedding.
- Sectioning
- Staining
- Mounting

13. Water is completely removed from fixed tissue.
Procedure
• Tissue blocks are place in capsules (Cassettes)
• These tissues are passed through a series of increasing
concentration of alcohol.
2 bath in 80 % alcohol for 1hour
1 bath in 90 % alcohol for 1hour
3 bath in 100 % alcohol for 1hour
Dehydration

14. Clearing
• During dehydration water in tissue has been replaced by
alcohol.
• alcohol should be replaced by paraffin wax.
• As paraffin wax is not alcohol soluble, we replace alcohol
with a substance in which wax is soluble.
• Reagents:
Xylene, Chloroform, Benzene, Carbon tetrachloride.
Procedure
• 2 bath in Clearing agent for 1hour

15. Automatic Tissue Processor
Procedure:
• Place the solution and paraffin in respective
beakers of the equipment.
• The timing leaver is set at zero and the machine
at started at require time
• The basket with the cassettes Automatically
change position and takes a bath in different
reagents kept in beakers in order to accomplish,
dehydration, clearing, infiltration. the final dip in
the warm paraffin.
• Cassettes are opened next day morning for
embedding.

16. Step Timing Solution Period
1 5:30 to 7:30 p.m 80% 2 hour
2 7:30 to 8:30 p.m 95% 1hour
3 8:30 to 9:30 p.m 95% 1 hour
4 9:30 to 10:30 p.m 100% 1 hour
5 10:30 to 11:30 p.m 100% 1 hour
6 11:30 to 12:30 a.m 100% 1 hour
7 12:30 to 1:30 a.m xylene 1 hour
8 1:30 to 3:30 a.m xylene 2 hour
9 3:30 to 5:30 a.m paraffin 2 hour
10 5:30 to 7:30 a.m paraffin 2 hour
11 7:30 to 9:30 a.m paraffin 2 hour
Automatic Tissue Processor

17. Tissue specimens are loaded onto a tissue processor for preparation for
embedding in paraffin and subsequent cutting on a microtome

18. Infiltration & Impregnation
Clearing agent is eliminated from the tissue by diffusion in the
surrounding melted wax (infiltration).
Diffuses in the tissue by replacing the xylene (Impregnation).
Procedure
• Replace xylene with paraffin
• 2 bath in melted paraffin 1hour
• The temperature of the paraffin oven
should be between 50-56◦C.
• Remove all bubbles

19. Casting / Blocking.
The infiltrated and impregnated tissue is placed in warm liquid
paraffin, which forms a firm block after cooling.
Embedding
Procedure
• Paraffin wax is first melt 56◦
C to 58◦
C
and filter through course filter paper.
• Fill mold with paraffin.
• Place tissue in mold.
• The mold is then placed in a container
of cold water or kept in refrigerator for
10 to 20 minutes.

20. Sample of decalcified bone that has been dehydrated, cleared of lipids,
and infiltrated with paraffin is embedded in paraffin for sectioning on a
microtome.

21. Sectioning
Procedure
• Trim the block with a knife until 1 to
3mm of paraffin remains all side of
the tissue.
• Fix the block in the block holder of
the microtome. Insert tightly a knife
in the knife holder with proper
position.
• Cut sufficient sections.
Microtome
Cut biological specimens into very thin sections for microscopic
examination.

22. Sledge Microtome Rotatory Microtome
Computer Microtome

23. Frozen Section
• The frozen section is a technique in which tissue is frozen rapidly to
the temperature of -20o
C and the sections are cut and stained.
• Tissue can be examined microscopically within 5-10 minutes of its
removal from the body.
• It reduces the time of processing from 18 hours to 5 minutes.
Procedure:
• Tissue is placed in a metallic block is covered with approximate
amount of isopentane.
• Isopentane has the property to freeze rapidly at -20o
C.
• Block-holder is placed over the freezing stage of cryostat close to
glass door of cryostat to maintain its temperature.

24. • Isopentane along with tissue is frozen within 1-2 minutes.
• Open the door of cryostat. Transfer the block holder to its stage and
fix it.
• Spray the block and knife with isopentane to maintain temperature.
• Trim the block with cutting mechanism adjusted at 25 micron
thickness.
• Before cutting the actual sections replace the “anti roll plate”
• Make 8-10 micron thick sections.
• Sections are transferred to slides, which are then rapidly taken to the
staining rack.
• Routinely frozen sections are stained with Haematoxylin and Eosin
stain

25. Cryostat MicrotomeHistochiller

27. Microtome to cut demineralized bone embedded in paraffin for
slide preparation and Paraffin sections, 5μ in thickness.

28. A. Basic dye: Hematoxylin
basophilic structures
B. Acid dye: Eosin
acidophilic (eosinophilic) structures
Staining

29. Deparaffinize the section
• Flame the slide on a burner and then place it in xylene for
3 to 4 times.
Hydration
Hydrate the section by passing it through decreasing
concentration alcohol bath and water
• bath in 100 % alcohol for 30 to 60 second
• bath in 90 % alcohol for 30 to 60 second
• bath in 80 % alcohol for 30 to 60 second
• bath in 70 % alcohol for 30 to 60 second
Staining Procedure
Staining

30. Staining
• Stain the section with hematoxylin solution for 3 to 5
minutes wash in runing tap water.
• quickly dip the slide in 0.5% HCL.
• Quickly rinse the slide in tap water 30 t0 60 second.
• Dip the slide several time in dilute ammonia water.
• Wash in tap water and then rinse 95% alcohol.
• Agitate the slide in eosin solution for 10 to 60 second drain
the staining solution.
Dehydration
• bath in 70 % alcohol for 30 to 60 second
• bath in 95 % alcohol for 30 to 60 second
• bath in 100 % alcohol for 30 to 60 second
Staining

31. Clearing
• Place the slide twice in xylene for 30 t0 60 second.
Mounting
• Drain the excess xylene and mount DPX or canada balsam
with a cover slip.
Staining

32. Microscopy
• Nuclei, fibrin, fibroglia and microglia : Blue
• Collagen : Yellow or Brownish Red
• Coarse elastic fibrils : Purplish tint

33. Fibroblasts with dark nuclei [A] are
seen here along with thick collagen
fibers [B], thin elastic fibers [C] and
very fine reticular fibers [D].
Microscopic view of a histological
specimen of human lung tissue stained
with hematoxylin and eosin.
Microscopy

34. Bright field images of eye lens of a diabetic patient. a) Stained with
eosin only b) stained with Hematoxylin only c) Stained with H &E d)
contained no stain
Nelson et al., Between Normal andMalignant Histology Tissues Using Fluorescence Lifetime Microscopy
Microscopy

35. Microscopy
• Analysis, the cellular morphology and
tissue distribution to deduce whether
tissue regions has abnormalities.
• Standard for clinical diagnosis of
cancers, as well as for identification of
prognostic and therapeutic targets.
Images are taken at low magnification and include many objects of
interest, such as cells and prominent cellular structures (e.g., nuclei).
These are widely distributed in the images and surrounded by different
neighboring tissues (for example, in the cervix, epithelium, and
stroma).

36. Pitfalls
1. Knife marks (scratches perpendicular to knife edge)
2. Compression (waves parallel to knife edge)
Poor sectioning

37. Pitfalls
1. Folds & tears
2. Excess albumin (stain)
Mounting sections

38. Staining
1. Inadequate rehydration (uneven staining)
2. Too dark or Too light (timing off)
3. Inadequate agitation
Pitfalls

39. Coverslipping
1. Bubbles
2. Excess Per mount
3. Two cover slips
Pitfalls

40. use
The histological evaluation of surgical biopsies from affected tissues
is a standard way of assessing pathological change and determining
treatment in many diseases.
• Malignancies
• Bacterial Infection
• Viral Diseases
• Fungal Infection

41. Histology of Thyroid Tumors. A. Follicular
adenoma: note the sharp separation of a
follicular tumor from the surrounding tissue
by a uniform fibrous capsule. B. Follicular
carcinoma with capsular penetration. C.
Papillary carcinoma metastatic to a lymph
node: typical appearance of papillary
carcinoma with complex and branching
papillae. D. Higher magnification showing
optical clear, overlapping and grooved (arrow)
nuclei. E. Tall cell variant papillary
carcinoma, lined by tall cells (arrow). F.
Undifferentiated carcinoma with elongated
tumor cells. G. Medullary carcinoma. H.
Papillary carcinoma with clear cell changes:
typical intranuclear inclusion (inset).

42. Osteomyelitis, changes evident by increased
white blood cells (black arrow) and fibrosis
(blue arrow), as shown in this histology slide of
the bone.
Fungal elements are seen within a dermal blood
vessel and invading the surrounding dermis
Herpes zoster skin infection. A large
intraepidermal vesicle (arrow) surrounded by
inflammatory cell infiltrate.

43. You