Histo techniques -Presentation (1).pptx New latest ppt

Histo Technologies

1. Histotechniques Moderator: Dr. Anupama Jha Prepared by: Dr. Kamaljeet

2. Overview Definition Specimen receiving Grossing Fixation Tissue processing Embedding Microtomy

3. Definitions
Histology- greek - histos + logia (A.F.J.K. MAYER 1819) – Study of microscopic anatomy of cells and tissues Histopathology – Microscopic study of diseased tissue Histotechnology – processing of tissues in such a manner as to enable microscopy/ study of the tissue

4. AIM Obtaining thin enough sections of the tissue so as to enable light to pass through it

5. SPECIMEN RECEIVING Documented policy defining the minimum demographic details of the patient
Spill kit -5% hypochrorite , gauze piece, yellow bag, gloves
Sample transportation – covered containers, in 10% buffered formalin, chutes
Unique identification number Strict Sample rejection criteria

6. “FIX AT A POINT IN TIME”
• Viable tissue is dynamic
• Process by which constituents of cells are fixed in a chemical so that they will withstand subsequent treatment with various reagents with minimum distortion or decomposition and keep the tissue in as life like manner as possible • Ideal fixative does not exist
- Prevent degeneration and autolysis
- Harden to enable cutting
- Not distort the cellular constituents
- Support chemicals used in processing

7. TYPES OF FIXATION Immersion
Perfusion brain, spinal cord, lungs, embalming
Vapour
Spray
Microwave fixation/Stabilization Freeze drying

8. MICROWAVE PRINCIPLE Water molecules are dipolar
Microwave produces alternating electromagnetic fields
Water molecules rotate and this energy is dissipated as heat
This heat speeds up the process of fixation, dehydration, clearing and staining

9. FREEZE DRYING Fresh tissue immersed in isopentane cooled by liquid nitrogen at -160° to -180° - Quenching
Tissue water removed under vacuum, absorbed by phosphurous pentoxide – sublimation
Put in embedding medium
Little chemical alteration, no loss of glycogen
Modification of this technique useful in enzyme histochemistry

10. A new approach to enzyme histochemical analysis of biopsy specimens Standard histological processing abolishes the activity of most enzymes
Freeze drying is the optimal method of tissue preservation maintaining tissue components in their native state. Conventionally, however, the freeze dried tissue specimens are fixed and embedded in wax after freeze drying.
Glycol methacrylate resin has been widely used as an alternative embedding medium to wax and the activities of a limited number of enzymes have been shown in tissue fixed in aldehyde and embedded in resin. Enzyme histochemistry performed on these resin sections gives good results.

11. SIMPLE FIXATIVE COMPOUND FIXATIVE
Formal dehyde zenker’s fluid Glutaryl dehyde Bouin’s fluid Ethyl alcohol PHYSICAL METHODS CHEMICAL METHODS
Heating Aldehydes – Formaldehyde
Microwaving Glutaraldehyde Freeze-drying Alcohol-Methyl alcohol, ethyl alcohol Methacarn Glacial acetic acid Bouin’s fluid
Osmium tetroxide

12. Formulae Formal Saline 40% Formaldehyde 100 ml
NACL 9 gm
Tap Water 900 ml 10% Buffered Formalin 40% Formaldehyde 10 ml
Sodium Hydrogen phosphate 0.4gm
Disodium Hydrogen phosphate 0.65gm
Tap Water 90 ml Buffered formalin prevents formation of pigment acid formaldehyde hematin formed from hemoglobin at acidic pH.

13. Formulae Zenker’s fluid
* Distilled Water 1000 ml
* Hg C12 50 gms * K-Dichromate 25 gms * NaSO4 10 gms It provides excellent fixation of nuclear chromatin, connective tissue fibres and some cytoplasmic features but does not preserve delicate cytoplasmic organelles such as mitochondria.

Currently out of fashion because of the toxicity.

14. Bouin’s fluid Sat aq. Picric acid 75ml 40% formaldehyde 25 ml Glacial Acetic acid 5 ml • Bouin’s is generally used for testicular biopsy fixation because it preserves nuclei and chromosomes especially well during meiosis.

15. Glutaraldehyde Used for electron microscopy with osmium tetroxide Glutaraldehyde produces nuclear and cytoplasmic shrinkage while osmium produces swelling and balances it Formalin is not useful for EM because methanol which is added to commercial preparations has denaturing action on tissue components.

16. Tissue preparation for immunocytochemistry The use of 10% NBF, 10% zinc formalin, or 10% formal saline is recommended, particularly for demonstrating kappa light chains on mantle and follicle centre cells.

A fixation time of 12 hours in 10% NBF showed optimum staining of all antigens.

A highly acidic formalin of pH 3.0 produced the best immunostaining but in our experience at the expense of morphology. The use of formalin at pH 5.0 is therefore recommended giving good morphology and immunoreactivity .

17. Factors affecting fixation 1. Hydrogen ion Concentration & Buffers 1. Satisfactory fixation occurs at pH between 6 and 8.
2. Buffers like phosphate, bicarbonate, s- collidine and cacodylate are chosen. 2. Temperature 1. Higher temp causes faster fixation
2. Lower temperature preserves tissue better
3. A tissue of 4mm thickness will be adequately fixed in NBF 10 to 20 times the volume in about 8 hours at room temperature. Fixation time is shortened by 25-40% if temperature is raised to 40°c.
4. Faster fixation is obtained through agitation.

18. 3. Penetration of fixatives Theorotical rate of penetration is one mm in one hour

4. Osmolality of fixatives

Slightly hypertonic solution (400-450 mos ) ideally. Hypertonicity causes cell shrinkage

5. Concentration of the fixative

19. 6. Duration of Fixation ideal is 8 hrs over fixation causes hardening of tissue and loss of antigens 7. Volume change – Tissue fixed in formaldehyde and embedded in paraffin wax shrinks by 33%

20. Fixation Artefacts Changes in volume of tissue
Formalin pigment
Diffusion of unfixed material
Chemical changes

21. Decalcification • Section from bone /heavy mineralised tissue difficult to obtain

Methods
1. Acid decalcification – acq nitric acid, formic acid, - HCI, trichloroacetic acid, Gooding and stewart’s fluid

Ideal time is 24-48 hrs

2. Other fluids – Von Ebner’s fluid, Perenyl’s fluid

3. Chelating agents – EDTA solution, ideal agent for

EM because minimum artefacts

22. 4. Ion exchange resins – polystyrene 5. Electrolytic method – Electrolytic solution of HCI and formic acid used

6. Ultrasonic method

7. Surface decalcification – Achieved by inverting paraffin block in 5% HCI for one hour, top 30 micron is decalcified.

23. Factors affecting rate of decalcification
1. Concentration of active reagent

2. Temperature

3. Agitation

4. Density of bone

5. Thickness of the bit

24. Assesment of decalcification Needling, knifing, finger nailing X ray examination is the best. Chemical method using ammonia

25. Trimming Requirements Adequate light
Sharp instruments
Adequate water flow
Wooden board Cassettes Labels and lead pencil Description Sections Optimum size 2x2x0.3 cm
Optimum thickness 3-4 mm
In case of bony fragment, decalcification Cassettes

26. Tissue Processing

27. Tissue Processing Principle : The aim of tissue processing is to 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 Dehydration – to remove fixatives and water from tissue.
Clearing – replacing dehydrating fluid with a fluid that is totally miscible with dehydrating fluid and embedding medium
Impregnation – replacing clearing agent with the embedding medium
Embedding

28. Dehydration Hydrophilic agent is used which attracts water from tissue
Used in increasing strengths
70% is the first solution
Anhydrous copper sulfate used in last alcohol as it absorbs water from alcohol Examples Ethanol
Methanol
Methylated spirit
Isopropyl alcohol

29. Clearing Hydrocarbons which have refractive indices similar to tissue proteins, end point noted by transparent appearance of tissues. Criteria Miscible with both dehydrating and embeding agent
Speedy removal of dehydrating agents
Easy removal by paraffin Examples Xylene
Toluene
Chloroform
Citrus fruit oils
Cedar wood oil

30. Impregnation Paraffin wax – the prefered medium,
Melting point→ 52-56° C
Plastic point – 10° below melting point Paraplast – pellet form, melts rapidly Alternate embedding media Resins
Agar Gelatin Celloidin

31. Automated tissue processing

32. Important points Bone, skin and CNS tissues require three changes of wax

Two changes of wax suffice for other organs

33. Embedding Requirements Embedding media
Cold plate / ice plate
Wax Dispenser
L- molds / ice trays / paper blocks/tissue tek system Wooden blocks
Forceps Spatula
Typed labels

34. Embedding media Water insoluble media - Paraffin wax
- Carbowax - Plastic embedding medium – Butyl methacrylate –
-Acrylic
-Epoxy resin
- Epon - Maraglas 655
- Polyester resin Water soluble media - Durcupan - Aquon - Glycol methacrylate

35. Important points

• Size of the mould should be such that there is a margin of 1-2 mm around the tissue

• Embed all bits of skin / cervical cone only few mm apart in the same block with parallel epithelial edges

• Endometrial samples and cell blocks are best kept at centre of block and tamped to the bottom

• Stand mount – cyst walls, gall bladder walls, ovarian wedges and skin sections – required edge Pathkept at the bottom

36. • Donot embed small and large specimen in the same block, smaller may be lost in obtaining a better section of the larger one

• Muscle biopsies sectioned both longitudinally and transversely

• Tubular structures are cut transversely

37. Paraffin wax additives • Increase hardness or alter structure to give proper sectioning

• Examples
- Ceresin
- Bees wax
- Bay berry wax
- Micro crystalline waxes

38. Special embedding techniques • Double embedding - Impregnated in celloidin , blocked in paraffin wax
- Used in making blocks of tissues with varying consistency such as eyes where retina is easily detached Tissue mat and Bioloid - Thin walled and circular sections maintain their original shape due to elasticity of the media

39. Paraffin section cutting Requirements: Microtomes
Disposable blades Water bath
Fine forceps
Small squirrel hair brush
Slide rack
Clean slides
Ice tray
Diamond pencil

40. Microtomes Precision instruments that cut sections from the paraffin blocks, thin enough for examination under microscope.

41. Honing Abrasive powders Diamond Carbodundum (silicon carbide)
Aluminium oxide
Iron oxide
Ceric oxide Chromium oxide

42. Section adhesion Adhesives used - Poly L-lysine
- APES
- Mayer’s Egg Albumin
- Gelatin - Starch
- Resin
- Sodium silicate
- Celloidin

43. Difficulties commonly encountered in sectioning 1. Failure of block to ribbon
Too hard paraffin
Knife

2. Uneven Ribbons
Irregular knife edge
Knife not parallel to the block
Impure paraffin

3. Alternate thick and thin Ribbons
Too soft wax Loose block or blade
Faulty microtome

44. Frozen sections & Cryostat
Cryostat is a special microtome refrigerated to -20°C, tissue placed in it has water which is converted to ice, thus making the tissue hard and providing a self embedding medium

➤ Applications:
Rapid diagnosis
Assessing margins
Confirm/refute metastasis
Demonstration of fat which is lost during routine processing