Processing of tissue

PROCESSING OF TISSUE

1. PROCESSING OF TISSUE Dr.T.Arivazhagan Post Graduate Department Of Pathology

2. INTRODUCTION It describes the steps required to take animal or human tissue from the fixation to the state where it completely infiltrated with a suitable histological wax and can be embedded and ready for section , cutting on the microtome.

3. Types Manual – Hand processing Automated – Machine processing Tissue transfer – Specimens are transferred Fluid transfer – Specimen held in a single container and the fluids are pumped in & out

4. Importance ? Inappropriate processing schedule or fundamental mistakes Production of improper section of tissue Not provide any useful microscopic information Specially in a situation where the whole tissue is processed

5. Steps? Obtaining the fresh specimen Fixation Dehydration Clearing PROCESSING Wax infiltration Embedding Blocking out Section cutting Routine staining

6. Obtaining specimen Separate room is required for specimen reception Dissection area must have Good lighting Good ventilation Non absorbent wipe clean surface Other equipment's Prior to fixation some tissue from the specimen need to reserve for other departmental assessment

7. Specimen dissection plan 1.Small samples: Rarely need dissection It may be placed in a nylon bag in order to prevent them of falling Eosin used as a marker Recommended to count the small tissue at the time dissection to verify prior to section cutting 2.Core biopsies: Tissue laid out in a longitudinal fashion so that majority of the tissue can be cut

8. 3.Skin biopsies: Sections are best managed in sequential / serial transverse section 4.Bowel specimens: Large specimens are sampled at multiple blocks Resected margins also included as a part of dissection Particular attention is given for lymph node 5.Gynecological specimen: Commonly cervical biopsy obtained Uterine specimens sampled in terms of cervix,endometrium,myometrium along with common benign lesions

9. 6.Breast specimens: Usually need for inked margins for orientation purpose Lymph node status also assessed

10. FIXATION Main aim of this step is preserving the cells & tissue components with minimal distortion of the tissue. Effects: Prevent the putrification & autolysis Hardness the tissue Make cells to insensitive to hyper & hypotonic solutions Act as a mordant Induce optical contrast for good morphologic examination

11. Ideal fixatives: Should be cheap & easily available Should be stable & safe to handle Should have rapid on of action Should cause minimal loss of tissue Should give even penetration Should retain the normal colour of the tissue Should not bind to the reactive groups

12. Types 1.Simple – Contain one substance 2.Compound – Contain 2 or more substance Also divided into; 1.Micro anatomical - Preserve the anatomy of the tissue 2.Cytological – Preserve the intra cellular constituents 3.Histochemical – Demonstration of histochemical constituents & enzymes

13. Formalin : 10% buffered formalin commonly used Commercially available as saturated solution of formaldehyde gas in water 40% formalin consider as 100% formalin 10 ml of commercially available formalin + 90ml of water Usually the volume of the fixatives should be at least 10 times of the tissue size 4mm thickness of tissue takes 6 to 8 hours to get fix.

14. Advantage Rapid penetration Retain the normal colour of the tissue Cheap & easily available Disadvantage Excessive hardening of the tissue Irritation to skin, mucous membrane

15. Other fixatives 1. Glutaraldehyde – used in electron microscopy 2. Mercuric chloride formalin Shrinks the tissue but not distort it Corrosive effect 3. Susa fixatives 4. Zenker’s fluid 5. Helly’s fluid Suitable for bone marrow ,spleen, lymph node,pancrease 6. Bouin’s fluid 7. Carnoy’s fluid 8. Sanfelice’s fixatives 9. Flemming’s fixatives 10. Orth’s fluid

16. DEHYDRATION Wax never penetrate the tissue in the presence of water It is possible only after the dehydration process So it is an essential preliminary process It is carryout by passing the tissue through a series of ascending grades of alcohol 70%,80%,90% & absolute alcohol

17. Reason – in order to prevent the distortion of tissue when transfer a tissue from an aqueous medium to absolute alcohol directly The frequency of alcohol renewal is based on the use of it Some times 10mm layer of anhydrous copper sulphate used as indicator of water.

18. Alternatives Methyl alcohol Isopropyl alcohol Acetone Pyridine Dioxane Cellosolve

19. CLEARING This is the process in which alcohol from the tissue and cells is removed and replaced by a fluid It also makes the tissue transparent Xylene is commonly used Rapid in action Readily eliminated in the paraffin oven 3mm tissue blocks cleared in 15 to 30 minutes Toxic & Highly inflammable

20. Other agents Toluene Carcinogenic Benzene Chloroform – Poisonous Cedar wood oil – Expensive & Very viscous

21. Chloroform Non inflammable Slow in action Little hardening effects So specially used in brain & bone tissue Cedar wood oil Makes tissue transparent Expensive Gently act in the tissue Sometimes it produce needle like crystals

22. WAX IMPREGNATION This is the process in which empty space in the tissue & cells are filled with wax after the clearing step This has been done in molten paraffin wax at melting point range from 54 to 62’c It allow the tissue to be harden from which section may be taken At 45 to 50’c - Soft in consistency At 55 to 60’c – Hard in consistency

23. Best quality of wax : Filtration done during the preparation of the process itself Nevertheless advisable refillter before it in use Addictive's also used to improve the consistency Paraffin wax with microcrystalline Paraffin wax with synthetic rubber

24. Tissue processor Open – Hydraulic 12 stations 10 stations are glassl steel J ars 2 stations are thermostatically controlled wax bath Formalin in 1 Dehydration in 6 Cleaning in 3 Impregnation in 2 Closed - Vacuum 1.5 hrs at each station & whole process takes about 18 hours Rapid processing completed in 2.5 hrs Each station at 10 – 20 minutes Adv - No hazard by toxic fumes - Shorten the processing time

25. EMBEDDING & BLOCKING Completely impregnated material with wax has been necessary to obtain a solid block containing tissue This is done by filling a mould of suitable wax Followed by orienting the specimen Ensure its being cut in the right plane Finally cooling the mass Metallic L ( LEUCKART’S) module commonly used Different coloured plastic modules also used.

26. Other methods Ester wax method: Diethylene glycol distearate + glycol monosterate + polyethylene glycol Has low melting point Water soluble wax method: Polyethylene glycol used Cellulose nitrate method: Celloidin +low viscosity nitrocellulose are using Reduction in shrinkage of tissue

27. 4.Celloidin paraffin double method: Cellulose nitrate + paraffin wax Used for bone , brain,muscle,animal tissue 5.Synthetic resin method:

28. SECTION CUTTING Microtomy in which tissue is sectioned and attached to a surface for further microscopic examination Microtome is the instrument basically used Thickness range from 1 micrometer to several hundred micrometer Common range is 3-8 micrometer Both paraffin & frozen sections are used

29. Types Rotary microtome Base sledge microtome Rotary rocking microtome Sliding microtome Ultra microtome

30. Rotary Microtome Most commonly used Basic mechanism is rotation of a fine advanced hand wheel by 360 degree Knife is fixed Tissue block is movable It may be manual, semi automated, fully automated Ability to cut thin 2 – 3 micrometer sections Honing & Stropping- Process of sharpened the knife

31. Sliding microtome Used in the cutting of cellulose nitrate embedded tissue Tissue block is fixed Knife is movable

32. Base sledge microtome Used for very hard tissue or large blocks Ex: brain & heart tissue Base is moved to and fro run against the knife to produce section

33. Rotary rocking microtome Commonly used in cryostats Knife is immovable Tissue blocks is held in a spring bearing rocking arm ULTRA MICROTOME: Used exclusively for electron microscope

34. Types of knives A- Biconcave B- Planoconcave C- Wedge shaped D-Tool edge

35. Freezing microtome Designed for the preparation of frozen section of fixed or unfixed tissue It does not need preliminary embedding step The stage is connected to an cylinder which has co2 for rapid cooling

36. FROZEN SECTION Tissue become firm Tissue is rapidly frozen Ice acting as embedding medium Sections are produced with out using of dehydration,clearing,wax embedding steps

37. Uses Rapid production of sections Immunofluorescent methodology IHC Silver demonstration methods in neuropathology

38. Merits Quick diagnostic method Every type of staining can be used Minimal shrinkage of tissue Lipids & enzymes also can be demonstrated Demerits Difficult to cut serial sections Not possible to maintain tissue blocks for future use Thick sections Structural details tend to be distorted due to lack of embedding medium

39. Types Freezing microtome using co2 gas Refrigerated microtome ( Cryostat)

40. Equipment required Floating water bath Slide drying hot plate Fine pointed forceps Camel haired brush Scalpel 6. Slide rack 7. Clean slides 8. Teasing needle 9. Ice tray 10. Chemical resistant pencil

41. Water bath Temperature is below 10’c of that of melting point of paraffin Removes the folds in the section Section adhesives: 1. Egg albumin 2. Gelatin 3. Poly L Lysine 4. 3 Amino propyl triethoxysilane Slide Routine 76x25mm 1x1.2 mm thickness Large slides also available for special tissues such as eyes & brains

42. Staining method Routine staining done with Hematoxylin & Eosin Procedure : Section first deparaffinised by using xylene Hematoxylin is a water based dye So sections are rehydrated before staining by descending grade alcohol

43. Place the slide in hematoxylin stain for 8-10minutes Rinse in water Differentiation is done by 1% alcohol for 10 seconds Rinse in water Blueing done by putting the section in scotts tap water(sodium bicarbonate & magnesium sulphate) for 2-10 minutes Counter stain with 1%aquous solution of eosin for 30 sec

44. One dip in tap water Before mounting sections have to be dehydrated by ascending grade alcohol Finally cleared in xylene Mount in DPX( dextrene polystyrene xylene) or Canada balsam Result : Nuclei – blue Cytoplasm – pink RBC,keratin

45. EOSIN Most suitable stain to combine with an alum hematoxylin Ability to give a proper differentiation between cytoplasm of different types of cells Crystal of thymol added to inhibit the growth of fungi Little amount of acetic acid with sharpen the staining

46. Types : Eosin Y - Water soluble - Widely used Ethyl eosin – Alcohol soluble Eosin B Substitutes: Phloxine Biebrich scarlet

47. HEMATOXYLIN Natural dye obtained from log wood of tree HEMATOXYLON CAMPECHIANUM Hematoxylin – inactivate product Oxidized to an active product as hematin This process know as ripening Done by naturally in sunlight or chemically by addition of sodium iodate mercuric oxide,kmno4 Mordant – helps in penetration the stain particles to the tissue

48. Types Based on the mordant; Alum hematoxylin Iron hematoxylin Tungsten hematoxylin Molybdenum hematoxylin Lead hematoxylin Hematoxylin with out mordant

49. Hematoxylin preparation Reagents required: Hematoxylin powder Potash alum Mercuric oxide Ethanol Distilled water

50. Steps Weight hematoxylin 4g for 900 ml solution Weight mercuric oxide 2g for 900ml solution Weight potash 1oogms for 900 ml solution

51. 60ml of ethanol 800 ml of boiled water + potash alum till it is dissolved Mix 4g of hematoxylin in 60 ml of ethanol and shake well to dissolve it Mix potash alum dissolved solution to hematoxylin dissolved solution Heat the solution & add mercuric oxide Keep on heating till pinkish colour appear Then cool it Stain ready to use