Basic histotechniques

Dr. Ajit Surya Singh

1. Basic Histopathological Techniques Dr . Ajit Kumar Singh 1 st year PGT MD (Laboratory medicine) Chittaranjan National Cancer Institute

2. PROTOCOLS FOLLOWED IN HISTOTECHNIQUES Receipt & Identification Labeling of the specimen with numbering Fixation Grossing Dehydration Clearing Infiltration Embedding Section cutting Staining Mounting

3. Receipt & Identification, Labeling of the specimen with numbering Patient information Clinical history Site of origin of specimen Lab number (unique identification)

4. Fixation Chemical process by which biological tissues are prevented from decay (autolysis or putrefaction) Preserves original shape, size & structure. Prevents autolysis (acts against hydrolytic enzymes) Stops the bacterial effect.

5. TIME OF FIXATION : Medawar (1941) t=(d/k)^2 d is depth reached by fixative k is Coefficient of Diffusability .79 10% formaldehyde 1.0 100% ethanol 1 hour – 0.8 mm penetration. 2 hours – 1.2 mm penetration. 4 hours – 1.6 mm penetration 25 hours – total 1 cm thick block (5 mm from each side) d= k√t

6. CHOICE OF FIXATIVE : Routine 10% buffered Formalin Bone marrow Zenker’s formal ( Helly’s fluid) biopsies GI biopsies Bouin’s fluid (Picric acid) Bone Formic Acid in 10% HCl Adrenal medulla Hollande’s Brain tissue Formalin ammonium bromide E/M specimen Gluteraldehyde ( preffered ) Zamboni’s Flemming’s Schaudinn’s 10 % formol alcohol

7. CHEMICAL FIXATIVES COAGULANT FIXATIVES ALCOHOL (PRECIPITANTS) ETHANOL, METHANOL TRICHLOROACETIC ACID ACETONE PICRIC ACID ACETIC ACID NON COAGULANT (CROSS LINKING ) FORMALDEHYDE GLUTERALDEHYDE OSMIUM TETROXIDE POTASSIUM DICHROMATE MERCURIC CHLORIDE ZINC CHLORIDE CHROMIUM TETROXIDE

8. FORMALDEHYDE MOA : In aqueous solution formaldehyde forms methylene hydrate Reacts with several side chains of proteins eg . Lysine , cysteine , histidine , arginine , tyrosine, and reactive hydroxyl group of serine and threonine

9. Penetrates tissue rapidly Slight shrinkage in tissue Preserves all structural details Reacts with phosphatidyl ethanolamine causing degradation but not with other lipids like cholesterol , cerebrosides , sulphatides and sphingomyelins Loss of carbohydrate is high yet it can be demonstrated satisfactorily.

10. The cross linkage does not harm protein structure, so antigenicity is not lost, hence Formalin is suitable for immunohistochemistry .

11. End point- change in colour of blood can be accepted as an end-point of formalin fixation FORMATION OF ACID FORMALIN HEMATIN MAKES BLOOD TURN TAN BROWN

12. GROSSING

13. Tissue Processing In order to cut thin sections of the tissues, it should have suitable hardness and consistency when presented to the microtome.

14. Tissue Processing Dehydration Clearing Infiltration Embedding

15. Dehydration It is removal of ‘free’ unbound water and aqueous fixatives from the tissue components. Done by passing the tissue through increasing concentrations of dehydrating agents. ( GRADED DEHYDRATION ) If concentration gradient is excessive/ drastic, diffusion currents across the cell membranes increase the possibility of cell distortion . Excessive dehydration -- tissue become hard, brittle and shrunken. Incomplete dehydration -- impair the penetration of the clearing reagents -- tissue remains soft and nonreceptive to infiltration.

16. Tissues are dehydrated by using increasing strength of alcohol ; i.e. 70%, 90% and 100%. The duration for which tissues are kept in each strength of alcohol depends upon the size of tissue, fixative used and type of tissue. Delicate tissue will get high degree of shrinkage by two great concentration of alcohol. ( start with less concentrations) The volume of alcohol should be 50-100 times that of tissue.

17. In automated processors– Anhydrous CuSO4 can be added to last jar of dehydrating agent. Indicates completion of dehydration.

18. Clearing Clearing reagents act as an intermediary b/w the dehydration and infiltration solutions . They should be miscible with both solutions . As paraffin wax is not alcohol soluble, we replace alcohol with a substance in which wax is soluble. When the dehydrating agent has been entirely replaced by most of these solvents the tissue has a translucent appearance : hence the term ‘clearing agent’.

19. Most clearing agents are aromatic hydrocarbons or short-chain aliphatic hydrocarbons. Most clearing agents are flammable liquids , which warrant caution in their use Clearing agents with a low boiling point are generally more readily replaced by paraffin wax.

20. Criteria for suitable clearing agent: Rapid removal of dehydrating agent Ease of removal of melted paraffin Minimal tissue damage Low toxicity Cost

21. CLEARING AGENTS Xylene - Routine Recyclable It is suitable for clearing blocks that are <5 mm in thickness Overexposure -- hardening of tissues. Toluene- flammable and volatile Chloroform- toxic Methyl benzoate and methyl salicylate Citrus fruit oils- pungent odour

22. In infiltration , tissue is impregnated with a medium, forming a matrix within the cellular spaces and preventing distortion of the tissue structure during microtomy . Embedding is enclosing of properly processed correctly oriented specimens in a medium that provides external support. INFILTRATION AND EMBEDDING

23. Tissue that come off the tissue processor are still in the cassettes and must be manually put into blocks by technician who must pick the tissues out of the cassette and pour molten paraffin over them. This embedding is very important because the tissues must be aligned or oriented properly in the block of paraffin. INFILTRATION AND EMBEDDING

24. Commercial embedding centres , which combine a heated mould store, a molten wax reservoir/dispenser and a cold plate, are readily available Paraffin wax is dispensed automatically from a nozzle into a suitably sized mold . The tissue is oriented in the mold ; a cassette is attached, producing a flat block face with parallel sides Once the wax has solidified, the tissue blocks may be gently removed from their moulds and prepared for microtomy .

25. Reagent minutes 70% Formol alcohol 60 80% Alcohol 60 90% Alcohol 60 100% Alcohol 60 100% Alcohol 60 Copper Alcohol 60 Copper Alcohol 60 Aniline 360 Xylene 60 Xylene 60 Paraffin wax 90 Paraffin wax 90

26. Microtomy Process in which tissue is sectioned and attached to a surface for further microscopic examination. The basic instrument used in microtomy is the microtome Advancing mechanism moves the object (paraffin block) for a predetermined distance until it is in contact with the cutting tool (knife or blade). The specimen moves vertically past the cutting surface and a tissue section is produced. Types of microtome used: Rotary Base sledge Rotary rocking Sliding Ultramicrotomes

27. PARAFFIN SECTION CUTTING Equipments required: Floatation Bath Slide drying oven or hot plate Fine pointed or curved forceps Sable or camel haired brush Scalpel Slide rack Clean slides Teasing needle Ice tray Chemical resistant pencil or pen

28. Floatation bath: Used for floating out tissue ribbons after sectioning Temperature in the bath should be 10°C below the melting point of the paraffin to be sectioned Prevent water bubbles from being trapped under the section ( use distilled water) Add alcohol/ drop of detergent- ↓surface tension- allowing section to flatten out with ease

29. Brush and Forceps : Removal of folds, creases and bubbles formed during floatation of section on water bath. Manipulating the section as it passes across the edge of the blade.

30. Drying oven/ Hot plate: Keeps warm air circulating around the slides Temp should be ~ melting point of paraffin Special care- CNS (↓ T to prevent splitting and cracking of tissues)

31. Slides : 75mm*25 mm; 1-1.2 mm thick Larger slides for brain and eyes Positively charge or pre treated with an adhesive resist detachment of the tissue from the slide during staining

32. Protein adhesives- albumin , gelatin, starch Prone to bacterial overgrowth and heavy staining Poly L Lysine : 0.1% solution; further diluted for use, 1 in 10 with distilled water Diminish with time APES (3 – Aminopropyltriethoxysilane ): cytology, esp specimens that may be bloody or contain proteinacious material

33. Charged or plus slides : Manufactured with permanent positive charge Coating the slides with a basic polymer in which a chemical reaction occurs leaving the amino groups linked by covalent bonds to the silicon atoms of the glass. Superior in their resistance to cell and tissue loss during staining or pre treatment such as enzyme and antigen retrieval.

34. Cutting section Trimming of tissue blocks Blocks are arranged on a cooling device , to cool both tissue and the paraffin. A small amount of water is adsorbed into the tissue causing slight swelling, making sectioning easier. Oversoaking may cause expansion and distortion of the tissue section. Proper processing eliminates the need to pre soak blocks. 3-4 microns Smooth slow stroke

35. Floating out sections Must be smooth with the trailing end of the ribbon making contact with the water first. Slight drag produced when the rest of the ribbon is laid on the water is sufficient to remove any folds that occur. Teasing with forceps 30 seconds- time for a ribbon to flatten; prolonged time causes excessive expansion, distorting the tissue

36. Drying sections: Temperature should be at the melting point of paraffin It is important to eliminate overheating during the slide drying stage, as cellular details may be compromised. For delicate tissues reduce temperature for prolonged time

37. Cutting hard tissues : The reason for cutting difficulties is more likely poor fixation or over-processing . Prolonged soaking of the block Exposing the block surface to running tap water for 30 minutes Slight reduction in knife slant

38. Decalcification Calcium deposits are hard and cannot be cut by microtome. Calcium is removed prior to embedding to allow sectioning. This is done by HNO3, HCl - strong and rapid action (on cortical bone), but also damage cell morphology. Not done for bone marrow. Acetic acid and Formic acid are used for bone marrow but are slow acting Formic acid in 10% HCl concentration all rounder EDTA is also used for decal

39. Staining Hematoxylin (basic dye) and Eosin (acidic dye)

40. Hematoxylin Hematoxylin is the most widely used stain Hematoxylin is extracted from the heartwood [logwood] of the tree Hematoxylon campechianum , with hot water & then precipitated out from the aq. sol. of urea Was originated Mexican state of Campeche But is now cultivated in the West Indies

41. Hematoxylin Haematoxylin by itself does not have staining property Oxidation product “ haematin ”- the actual staining compound Synthetic dyes have been recommended as replacements Celestine blue (CI 51050) Gallocyanine  (CI 51030) Gallein  (CI 45445) Erichrome cyanine R (also called chromoxane cyanine R) Solochrome cyanine (CI 43820).

42. The process of oxidizing haematoxylin to haematin is called RIPENING Methods of ripening Natural oxidation Chemical oxidation By exposure to natural light and air By adding sodium iodide (Mayer’s haematoxylin) or mercuric oxide (Harris’s haematoxylin) Slow process ( 6 to 8 weeks) Ready to use instantly Retains stain for very long time since it is completely oxidized. Short lived staining since the continuing oxidation destroys much of haematin to a colourless component. Eg : Ehrlich’s haematoxylin Delafield’S haematoxylin Eg : Mayer’s haematoxylin Harris’s haematoxylin

43. Mordant Haematin is anionic and has a poor affinity for tissues. So, nuclear staining needs a MORDANT to be added The mordant /metal cation confers a net positive charge to the dye mordant complex & enables it to bind to anionic tissue sites such as nuclear chromatin The cations used as mordants are: Aluminium Iron Tungsten Lead ( argyrophyl cells) Molybdenum

44. Blueing Nucleus is stained red colour by all stain which is converted to blue-black when the section is washed in a weak alkali solution Bluing agents: Running tap water Hot water 2% NaHCo 3 1% LiCo 3 Scott’s tap water Marble chips 1% ammonia vapor Aluminium solutions

45. Differentiation Removing the dye molecules from cytoplasm while keeping the nucleic acid complex intact 1% Hcl

46. Eosin Most suitable stain to combine with an alum hematoxylin It has good ability to distinguish b/w the cytoplasm of different types of cells & b/w different types of connective tissue fibers & matrices by staining them different shades of red & pink Eosins are xanthene dyes Commercially available types are ---Eosin Y (Eosin yellow ,eosin water soluble) ---Ethyl eosin ( Eosin S, alcohol soluble-not used ) ---Eosin B (Eosin bluish- used in hematology )

47. Eosin Y is the most widely used **Both water & alcohol soluble **Used as 0.5% or 1% solution in distilled water **A crystal of thymol is added to inhibit fungi ** Acetic acid is added to sharpen the stain Eosin B & ethyl eosin- used rarely Alternative red dyes of Eosin- Phloxine & Biebrich scarlet

48. Counterstaining with eosin changes the colour of haematoxylin alum-stained nuclei from blue to purplish This additive colour change may be due to attraction of eosin anions to positively charged amino acid side chains of basic nucleoproteins

49. Staining Deparaffinise the section by Xylene Bring the section to water (HYDRATION) [ 90% alc ---50 % alc. -- water ] Remove fixation pigment if any Stain in hematoxylin x 10 min Wash in running water x 5 min

50. Staining 6. Differentiate in 1 % acid – alcohol (1% Hcl in 70% alcohol) x 5-10 sec 7. Blued in tap water x 5min or less 8. Counterstain in 1% Eosin Y x 10 min 9. Wash in running water x 1-5 min 10. Dehydrate through alcohol 90% alcohol –10 sec Absolute alcohol --10 sec

51. RESULTS :-- NUCLEI------------------------- Blue / Black CYTOPLASM-----------------Varying shades of pink MUSCLE FIBRES----------- Deep pink / Red RED BLOOD CELLS------- RED / Orange

52. Mounting Slides cleared with Xylene & mounted in DPX along with cover slip D - Distyrene ( Polysterene ) P - Plasticizer ( Tricresyl phosphate) X - Xylene

53. Thank you