Histo Techniques.pptx......................

Main point about the histotechniques and special staining

1. HISTO TECHNIQUES Presented by: Dr. Urbi Satpati Guide: Dr. Mohsin Sayed

2. HISTOPATHOLOGY Microscopic examination of biologically disease tissue to observe the appearance of tissue in very fine detail. Histopathology = Histo + Patho + Logy Tissue Suffering/Disease Study

3. SPECIMEN IDENTIFICATION AND RECEIVING Check label (name, age, registration number, nature of tissue, clinical diagnosis and the requisition form is also duly filled) Check if specimen is in proper fixative. Make entry in register by giving the specimen a unique pathology number and the same labelled over the specimen bottle.

4. TISSUE PROCESSING PRINCIPLE- The treatment of tissues through series of reagents and subsequent embedding in a medium, e.g. paraffin wax (which is similar in density to tissue thus enabling smooth sectioning) is termed as ‘tissue processing. The aim of tissue processing should be minimal morphological cell distortion. Tissue processing occurs due to diffusion of various substances/fluids into and out of stabilized porous tissues. The diffusion process results from the thermodynamic tendency of processing reagents to equalize concentrations inside and outside the bits of tissue.

5. TISSUE SAMPLING Tissue blocks for processing should be thin; usually 1–2 mm thick for urgent processing of specimens and 3–5 mm for routine material to be processed overnight. Specimens should not be tightly packed into processing cassettes or containers but should have sufficient free space to facilitate fluid exchange. Small specimens and tissue fragments, e.g. endometrial biopsies, endoscopic biopsies, etc. are processed, wrapped in lens tissue and placed in fine mesh containers

6. Stage of tissue processing FIXATION GROSSING DEHYDRATION CLEARING IMPREGNATION EMBEDDING

7. Factors affecting processing 1) Specimen size: Thicker the specimen longer the impregnation time, ideal thickness(3-4mm) 2) Agitation: rate of agitation is important ,as too low rate is ineffective while too high rate may cause damage to soft and friable tissue . 3) Heat : increases rate of penetration & exchange of fluids and chilling decreases it .Overheating of tissue cause brittleness. 4) Viscosity : affects the rate at which fluids are capable of penetrating tissues. The larger the molecules ,the higher are the viscosity and the slower the rate of penetration . Cedar wood oil & molten wax are of high viscosity, require longer immersion time. 5) VACUUM : vacuum considerably reduce the impregnation time

8. FIXATION Fixation is a process by which the constituents of cells and tissue are fixed in physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds called Fixatives Fixative : Tissue = 10:1 Inadequate fixation - Zonal fixation, Altered morphology, Affect staining Average penetration rate – 1mm per 1 hrs (alcoholic formalin increase penetration rate) Over fixation – corrected by conc ammonia plus 20% chloral hydrate.

9. Aim Of Fixation Inhibition of autolysis and putrefaction Preservation Hardening Solidification of colloid material Optical differentiation

10. MECHANISM OF ACTION : Most fixatives act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents. No fixative will penetrate a piece of tissue thicker than 1cm.

11. PROPERTIES OF AN IDEAL FIXATIVE: 1.Prevents autolysis and bacterial contamination. 2.Preserves tissue in their natural state and fix all components. 3.Preserves tissue volume. 4.Avoid excessive hardening of tissue. 5.Allows enhanced staining of tissue. 6.Should be non-toxic, non-allergic and not very expensive

12. Process of Fixation Physical Chemical Others Heat 1) Immersion Coating Method Microwaving 2) Perfusion fixation Cryopreservation/freeze-drying

13. 1) Heat Fixation :– It is the usual mode of preparing bacteriological smears. Preserves overall morphology, but not internal structures. Heat denaturates the proteolytic enzymes and prevents autolysis. Tissue is placed in 20-30 ml of fluid and heated below the boiling point over the spirit flame for 1 min or until the tissue floats on the surface. Then it is cooled immediately and the tissue is taken for processing.

14. 2) Microwave :- Rapid fixation It can reduce time of fixation of some gross specimens and histological sections from more than 12 hrs to <20 mins. More safe (as fixation in formalin produces many dangerous vapour ; microwave processing system designed to handle those vapours ). Recently, commercial glyoxal- based fixative which do not form vapours when heated at 55 C have been used for microwaving.

15. 3) Freeze- drying It is a useful technique for studying soluble materials and small molecules; tissue cut into sections Immersed in liquid N2 and water is removed in a vacuum chamber at -40 degree Celsius Post fixed with formaldehyde vapour Bringing the temp. gradually to 4 degree Celsius to complete fixation In substitution, we can use alcohol/ acetone in -40 degree Celsius. Freeze drying method mainly used for research purpose and rarely used for clinical lab.

16. 4) Immersion Fixation M/c method, where tissues are submerged in a fixative solution (ex: formalin) 5) Perfusion Fixative A fixative solution is introduced into the body through the blood vessels often used for larger specimens like brains. 6) Coating Methods Used for cytology samples, where a spray fixative is applied to the smear for protection and transportation.

17. CHEMICAL FIXATION FORMALDEHYDE (ALDEHYDES) Commercially available as formalin (40% formaldehyde dissolved in water). Various forms in use are: Formal(10% formalin) saline – most common used, contains: Formaldehyde(37%) :100 ml Water :900 ml Sodium chloride :9 g

18. B) Formal (10% Formalin) buffered saline Distilled water :900 ml Formaldehyde(37%):100 ml Sodium chloride :9 g Sodium phosphate :12 g C ) 10% neutral buffered formalin(NBF): Formaldehyde(37%) :100 ml Distilled/tap water :900 ml Sodium phosphate monobasic monohydrate :4g Sodium phosphate dibasic anhydrous :6.5 g

19. D ) formal calcium acetate : Tap water : 900 ml Formalin : 100 ml Calcium acetate : 20g Principle – Aldehyde groups in formalin form complexes by forming methylene bridges between protein molecules which then react with several side chains of proteins to form reactive hydroxymethyl side groups

20. Fixation time and temperature – an average tissue requires 10-20 times its volume of formalin/ normal buffers formalin for adequate fixation and immersion for 24 hr at room temp. if the volume of tissues is small a mini period of 8-12 hr is used. If the temp is raised to 45 degree Celsius, fixation time is shortened by 25-40%. Advantage Easily available and cheap Good penetration and fixation Preservation of fats, myelin, nerve fibres , amyloid and hemosiderin Does not cause excessive tissue hardening

21. Disadvantages It is slow in action (penetration of 1 mm in 1 hr ) On storage it becomes cloudy due to formation of paraformaldehyde Unsuitable for demonstration of fats and enzymes Has desaturating effect on proteins

22. Glutaraldehyde mainly used for electron microscopy. stabilize the tissue by cross linking. Extensive cross linking results in better preservation of tissue. Any tissue fixed by glutereldehyde must be small in size(max. 0.5mm) lipid are not fixed by glutaraldehyde & excreted during dehydration. So secondary fixation required

23. Advantage Disadvantage It is dialdehyde, so more cross linking occur, this more stabilize the tissue. Causes less shrinkage. Does not cause dermatitis More expensive Less stable Penetration is slower

24. Osmium tetraoxide 2% solution with ph of 7.2-7.4 is a standard solution used for fixation purpose Principle – osmium tetra oxide cross – links with proteins, reacts with unsaturated lipids and forms monoester as well as diester linkages in the tissues. It rapidly fixes the tissue and stains tissue structure due to formation of hexavalent osmium in proportion to the content of reactive and reducing groups.

25. Advantag e – Suitable for demonstration of lipids Good preservation of golgi bodies and mitochondria Disadvantage – Expensive Harmful to eyes and throat slow penetration

26. Flemming’s fluid 1% aqueous chromic acid : 15 ml 2% osmium tetraoxide : 4 ml Glacial acetic acid :1 ml Zenker’s fluid Distilled water : 100 ml Mercuric chloride : 5 g Potassium dichromate : 2.5 g Sodium sulfate : 1 g B5 fixative Mercuric chloride 12gm sodium acetate 2.5gm distilled water 200ml add 2ml of 37% formaldehyde in 20ml of above solution just before use. > Frequently used for BM, lymph node ,spleen & other haemopoietic tissue.

27. SPECIAL FIXATIVES Dichromate and chromic acid fixation – mitochondria are well preserved . Fixatives for DNA,RNA and protein analysis-HOPE( glutamic acid buffer mediated Organic Solvent Protection Effect ) , DSP( dithiobissuccinimidyl propionate ), Zinc based. Metallic ions as fixative supplement. Bouin’s fluid- for fixation of Delicate tissues (like testes). Zenker’s fluid – used for study of spleen and bone marrow. B5 fixative is used for LN and bone marrow biopsy.

28. Factors affecting fixation Buffer & pH Temperature Penetration capacity Volume change Osmolarity of fixative solution Concentration of fixative Duration of fixation & size of specimen Additive (calcium chloride, potassium thiocynate , ammonim sulfate, sucrose& dextran etc.)

29. GROSSING

30. DECALCIFICATION The process of removing calcium salts from the tissue to make it amenable for sectioning is known as decalcification. Failure to remove calcium from the tissues will result in torn and ragged sections, and damage to the edge of the microtome knife. The major bulk of the bone is approximately 70% mineral and 30% organic component by weight. The mineralized bone tissue is composed of calcium hydroxyapatite, calcium carbonate in organic collagen matrix and ground substance.

31. Stages of decalcifications:- 1)Selection of tissue 2)Fixation 3)Decalcification 4)Neutralization of acid 5)Thorough washing.

32. A) Selection of tissue Specimens arriving in laboratory vary in size Thin slices of bone obtained by fine toothed bone saw.(Tendons should be removed before sawing) Cut surface should be retrimmed . The first cut through midplane then 4-5mm thick slabs are cut parallel to the first cut. Ideally thickness for decalcification should be 3-4mm. For selection, sample blocks with least mineralisation should be selected for quick diagnosis.

33. B) Fixation:- Tissue must be fixed adequately before decalcification. Selected bone placed in 10% neutral buffered formalin for 24-48hrs. Bone marrow is best fixed in Zenkers fluid. C) Decalcification:- To obtain satisfactory sections of bone, inorganic calcium must be removed from organic collagen matrix, calcified cartilage. Volume of decalcifying fluid- 100 times that of tissue. Solution should be changed frequently because calcium ions cause saturation of the surrounding solution.

34. D) Acid neutralization:- Mainly by weak alkali as 5 g/dl Li or sodium salphate . E) Washing :- 2-4 hours in alcohol or overnight in water to remove alkali after neutralization.

35. Methods of decalcification Acid Decalcification Chelating agents Ion-exchange resins Electric ionization Surface decalcification

36. 1) Acid decalcification:- Strong inorganic acids- 1) Nitric acid (5-10%) Nitric acid: 5-10ml Distilled water: make upto 100ml This solution gives quickest results and can be used for large and heavily mineralized cortical bone specimens. Length of time- 24hrs to 2-3 days DNA and RNA are readily hydrolyzed . If overdecalcified – poor nuclear staining Nitric acid sometimes cause yellowing of specimen.

37. 2) Formalin-nitric acid (10%) i ) Nitric acid- 10ml ii) formalin- 10ml iii) Distilled water- 80ml Formalin is added to protect tissue from maceration and swelling. Excellent for calcified arteries and glands, thyroid and chitinous material. Also good for urgent biopsies as needle or small specimen biopsies. Disadvantage- nuclear staining not good 3) OTHER - perenyl’s fluid

38. Weak Organic Acids A. Formic acid (5-10%) i ) formic acid – 5-10ml ii) Distilled water – make upto 100ml Suitable for most routine surgical specimens. Gentle on tissue than nitric acid and safer to handle. B. Formalin-formic acid (10%) ( Goodling and Stewarts fluid) formic acid- 5-10ml formalin- 5ml Distilled water- make upto 100ml It simultaneously fixes and decalcifies the tissue. Time-1-10 days

39. 2) Ion exchanging resins :- Eg , Ammonium salt of sulphated polyestene Advatanges :- faster decalcification, tissue preserved, longer used 3) Chelating agents :- Eg , EDTA Used for bone marrow biopsy

40. 4) Electrolyte Method:- Used 10% Formic acid and 8% HCL. Attract Chloride ion by addition to solution and supply current. Temperature :- 35-45 Degree C. 5) Surface decalcifications :- Surface of block is trimmed then block is placed in solution of 1% HCl face downwards for 15-60 mints.

41. END POINT OF DECALCIFICATION 1. Specimen radiograph Fine detail specimen radiograph (X-Ray)- Most accurate Microradiography 2. Chemical testing 3 . Physical testing - Less accurate and damages the tissue

42. Specimen radiograph 1. FINE DETAIL SPECIMEN / X-RAY EXAMINATION Most sensitive test. Areas of mineralization and tiny calcification easily identified. Give extent and nature of lesion. Confirms the presence of foreign material eg. Prosthetic devices, metal piece. 2. MICRORADIOGRAPGY High resolution fine detail contract x-ray of thinner sections of bone. Application in bone metabolic diseases

43. CHEMICAL TEST Simple and reliable method. Working solutions- Ammonium hydroxide stock 5%. Ammonium oxalate stock 5%. PROCEDURE - Insert pipette into decalcifying solution containing specimen. Withdraw 5 ml of the solution from the bottom of container which has been in contact with the specimen and place it in a test tube. Add a piece of litmus paper. Add 5 ml of working solution. Mix well till litmus paper turns blue

44. INTERPRETATION - If a precipitate forms calcium is present(because of calcium oxalate) Decalcification is complete when no precipitate is observed on two consecutive days of testing. Once decalcification is complete tissue is washed in running tap water. BUBBLE TEST- On adding a strong acid, gas bubble forms on the bone surface as calcium carbonate combines with acid to from CO2. PHYSICAL TEST- Banding the specimen or inserting a pin directly into tissue. Disadvantage- tears and false positive microfractures.

45. Factors affecting the rate of decalcification Age- Immature cortical bone decalcify faster than mature. Concentration of agent- Increased concentration of acid causes faster decalcification but harmful. Temperature- Increased temp causes faster decalcification but has maceration effect. Agitation- Agitation hastens the exchange of fluid with in tissue. Thickness of tissue- thinner tissue bit takes less time

46. Tissue processing

47. TYPE OF TISSUE PROCESSING Manual tissue processor Automated tissue processor Microwave tissue processor

48. 1) AUTOMATED TISSUE PROCESSOR : The basic principle of tissue processor is to transfer the tissue in different fluid for a specified time in a desired environment. Advantages :- Saves technician tim Gives reliable quality control Saves labor .

49. 2)MANUAL TISSUE PROCESSING:- Rarely used now a days. Manual tissue processing is usually undertaken for the following reasons: 1. Power failure or breakdown of a tissue processor. 2. A requirement for a non-standard processing schedule as for: a. Rapid processing of an urgent specimen. b. Delicate material. c. Very large or thick tissue blocks. d. Hard, dense tissues. e. Special diagnostic, teaching or research applications. f. Small scale processing requirements.

50. 3) MICROWAVE TISSUE PROCESSOR:- Processing completed in 2hr, for large tissue process completed in 3-4hr. Capacity up to 30 cassettes. Automatic temp control. Single dehydration/clearing agent used. Organic solvent and acid resistant cabinet coating. Built in heavy duty exhaust fan for extraction of vapors to fume hood.

51. Tissue processor types:- Tissue transfer processor Fluid transfer processor In this system the bucket of tissue is transferred from one carousel to other after a specified time T issue is kept in the container, and the container is periodically filled with particular fluid

52. STAINLESS STEEL BASKETS JARS

53. 99% alcohol 6 pm 99% alcohol 6:30 pm 70% alcohol 5 pm 95% alcohol 5:30 pm 99%alcohol 7:30 pm 99% alcohol 8:30 pm Xylene 9:30 pm Xylene 10:30 pm Xylene 12 am Parafin wax 1:30 am 99% alcohol 7:30 pm 99% alcohol 8:30 pm 95% alcohol 5:30 pm 70% alcohol 5 pm

54. Step of Processing Daytime Overnight Fixation 60 mints 120 mints 70% Reagent Ethanol 10 mints 30 mints 95% Reagent Ethanol 10 mints 30 mints 99%Reagent Ethanol 10 mints 30 mints 99%Reagent Ethanol 10 mints 60 mints 99% Reagent Ethanol 10 mints 60 mints 99% Reagent Ethanol 10 mints 60 mints xylene 10 mints 60 mints xylene 10 mints 90 mints xylene 10 mints 90 mints Paraffin wax 10 mints 60 mints Paraffin wax 10 mints 90 mints Paraffin wax 10 mints 90 mints Paraffin wax 15 mints 90 mints Embed - - Total time 3 hrs 15 mints 14 hrs Duration

55. GENERAL CONSIDERATIONS DURING PROCESSING 1. Baskets and metal cassettes should be clean and wax-free type. 2. Tissues should not be packed too tightly in baskets so as to impede fluid exchange. 3. Processors must be free of spilt fluids and wax accumulations to reduce hazards and ensure reliability. 4. Fluid levels must be higher than the specimen containers. 5. Timing and delay mechanisms must be correctly set and checked against the appropriate processing schedule. 6. A processor log should be kept in which the number of specimens processed, processing reagent changes, temperature checks on the wax baths and completion of the routine maintenance schedule are recorded as an integral part of the laboratory quality assurance program.

56. DEHYDRATION Displaces residual fixative as well as cellular free water as supporting medium is not miscible with water Correct dehydration – remove only free water leaving bound water intact. Graded alcohol is used since sharp difference in concentration of dehydrating agent may damage delicate tissue.

57. Heat or increased time in higher grade alcohol removes bound water , producing processing artifact like shrinkage , ‘parched earth’ effect and abnormal staining. Incomplete dehydration- impair penetration of clearing reagents leaving tissues non-receptive to paraffin wax infiltration. Routine laboratory – 70 ,90 and 100% alcohol is used Common dehydrating agents – Ethyl alcohol,methylated spirit,methanol,butyl alcohol,isopropyl alcohol

58. CLEARING Removal of dehydrants creating space for paraffin infiltration. Clearing agent – miscible with both dehydrants and wax and also dissolve lipids. Most common – Xylene Xylene – highly lipid soluble but causes drying by removing bound water.

59. Ideal clearing agent - Low viscosity and high penetration rate -Low melting point -Miscible with both alcohol and molten wax -No tissue damage -Less toxic -Less inflammable -Economic End point detection – tissue becomes transparent Prolonged exposure to clearing agent: The brittle and more friable tissue Different clearing agents: Xylene, toluene, chloroform, amyl nitrate, cedarwood oil and limonen

60. INFILTRATION AIM : to support the tissue , allowing easy sectioning PRINCIPLE : Clearing agent is removed by the process of diffusion, and the tissue space is now infiltrated with the embedding media. Less infiltration – soft tissues ; makes it difficult to section. Too much time in high temperature wax - brittle tissue.

61. Ideal impregnating medium : Miscible with clearing agent Liquid in higher temperature and solid in room temperature Homogenous and stable Non-toxic and cheap T ransparent Fit for sectioning the tissue Most popular universally used impregnating medium – Paraffin Wax (melting point is 50-60 degree celcius )

62. Other media : MODIFIED PARAFFIN WAX:- Epoxy resin 1) Paraplast Acrylic medium 2)Ester wax Agar gel 3)Water soluble waxes or PEG Gelatin Celloidin

63. EMBEDDING Creating tissue blocks by using external support medium to enable microtomy .

64. Leuckhard Embedding Moulds (L- mould ) Leuckhard embedding moulds have two arms. One arm of the L is longer than the other arm. The two L-shaped arms are adjusted to make a convenient size for block. Adequate lubricant such as glycerine is applied to the L arms and metal plate for easy removal of the tissue.

65. MOULDS CASETTE Tissue is oriented in mould , fixed to bottom of mould , a cassette kept on top of mould , wax is filled into it and kept on cold plate .

66. ORIENTATION OF DIFFERENT SPECIMENS

67. TISSUE BLOCK

68. SECTION CUTTING (MICROTOMY) Tissue is sectioned and attached to surface of glass slide for further microscopic examination Types : 1. Rotary 2. Base sledge 3 . Rotary rocking 4. Sliding 5 . Ultra microtome Types of knives : 1. Disposable blades -commonly used 2 . Glass and diamond knives - electron microscopy and plastic resin embedded blocks

69. R otary microtome Most popular – routinely used C an cut almost all type of tissues K nife held stationary and tissue block moves C uts accurate sections at 0.5 to 2 micrometer P roduce ribbons of serial sections

70. S liding microtome K nife is stationary and tissue block slides under it. U sed in cutting celloidin embedded blocks usually for research purposes.

71. B ase sledge microtome T issue block held stationary and knife slides across the top F or large blocks, hard tissues, whole mounts U sed in neuropathology and ophthalmic pathology

72. R otary rocking micotome C ommonly used in cryostats

73. U ltramicrotome U sed exclusively for electron microscopy U ltra thin sections G lass or diamond knifes are used

74. M icrotome knives

75. K nife sharpening M anual – Honing and Stropping A utomated machine Honing – sharpening with the help of stome like Carborandum stone , Arkansas stone, Belgian black , Belgian yellow. Stropping – final step in sharpening dome before cutting or while cutting sections

76. HONING STONE - CARBORANDUM LEATHER STROP

77. EUIPMENT REQUIRED Ice tray or cooling platform. Fine pointed or curved forceps. Sable or camel haired brush. Teasing needle. Flotation (water) bath. Clean slides. Slide rack. Slide drying oven or hot plate. Chemical-resistant pencil or pen. Electronic slide labeling instrument.

78. ICE TRAY / COOLING PLATFORM Cools both tissue and wax giving tham similar consistency.

79. M icrotome R outine sections cut at 4-5 micrometer W hen a ribbon of 6-8 serial sections is cut, first section is held by forceps and last section eased from knife edge by brush or needle

80. F loatation bath T hermostatically controlled water bath T emperature should be 10 degree celcius below the meling point of wax S ection flatten out on the surface of water Sections are gently picked up on clean slides already smeared with an adhesive , Mayer’s egg – albumin, glycerol

81. D rying of tissue on slide T emperature approximately same as melting point of paraffin wax W ax on the slide is removed during heating D rying complete in 30 minutes

82. STAINING

83. STAINING Routinely used – Hematoxylin and Eosin stain. Hematoxylin stains acidic molecules in shades of blue(Nuclear stain – Blue) Eosin stains basic materials shades of red, pink and orange (Cytoplasmic stain – Pink)

84. Hematoxylin is extracted from heart wood of tree Hematoxyl um campechianum . Hematoxylin itself is not a stain. Oxidation produ ct Hematein is a natural dye. 1. Natural oxidation – ripening, by exposure to light & air e.g. Ehrlich’s and Delafield’s hematoxylin solutions. 2. Chemical oxidation Sodium iodate (Mayer’s hematoxylin ) Mercuric oxide (Harris’ hematoxylin )

85. Bluing Converts the soluble red color of hematoxylin into Insoluble blue color. It is pH dependent and occurs in alkaline solution Eg : 1) Scott’s Tap water 2) Ammonia water 3)Dilute lithium carbonate

86. Mordants Chemical used to improve ability of dye to adhere to a tissue Examples – 1) Alum 2) salts of aluminium ,copper ,iron 3) Oxalic acid 4) Tannic acid

87. Routine Hematoxylin & Eosin staining (for Histopath sections ) Heat the slides for approx. 1 hr. Keep in Xylene 1 for 5 mins. Keep in Xylene 2 for 5 mins. Air dry Keep in Alcohol 1 for 5 mins. Keep in Alcohol 2 for 5 mins. Keep in running water for 15 mins. Keep in Hematoxylin for 30 mins. Keep under running water for 5 mins. 1-2 dips in 1% Acid alcohol. Keep under running water for 30 mins. Kep in 2% Eosin for 2 mins. Dehydrate in Alcohol 1-1 dip. Dehydrate in Alcohol 2-1 dip Air dry. Keep in Xylene for 1 min. Air dry. Mount in DPX Mountant .

88. AFB Staining Ziehl- Neelsen Stain for Histopathology Staining Deparaffinize the smear, Xylene (10 min.), Alcohol (10 min.). Keep in running tap water for 15 mins. Air dry. Flood the smear with Carbol Fuchsin. Heat smear till steam comes. Keep the stain for 10 mins (if not heated then keep the stain for 25 mins) Wash in water. Flood smear with 25% H2SO4 for 3 mins till color gets yellow. Wash in water till color turns light pink. Counter stain with 1% Methylene blue for 1 min. Wash with water. Air dry. Keep in Xylene for 1 min. Mount in DPX mountant

89. PAS Staining Deparaffinize Xylene for 5 mins x 2 changes. Take to Alcohol for 5 mins x 2 changes. Keep sections in running tap water for 15 mins. Periodic acid for 5-10 mins Wash thoroughly in tap water, rinse in distilled water. Pour Schiff’s solution on smear for 15 mins. Keep slide in Coplin jar along with Schiff’s solution & add water to it slowly color turns pink. Keep for 15 mins. Rinse in water. Counter stain Haematoxylene for 1 min. Keep in running tap water for 10 mins for bluing. Keep in Xylene for 1 min. Mount in DPX mountant .

90. Staining types Progressive Regressive Staining is not followed by a differentiator which removes excess stain Deliberately over stained and then differentiated until end point is reached Examples of differentiator – Acetic acid , Hydrochloric acid, acid alcohol

91. Mounting

92. MOUNTING Last step DPX – Distrene , Plasticiser , Xylene DPX is colourless , neutral substance in which all standard stains are preserved It is common plastic , polystyrene mixed with hydrocarbon solvent ( like xylene)

93. Special stains

94. GROUP STAIN CARBOHYDRATE PERRIODIC ACIS SCHIFF(PAS) MUCICARMINE ALCIAN BLUE AMYLOID CONGO RED THIOFLAVINS CONNECTIVE TISSSUE MASSONS TRICHROME GOMORIS TRICHROME-BLUE AND GREEN ELASTIC STAIN/VERHOEF VAN GIESON STAIN RETICULIN STAIN TOLUIDINE BLUE SAFRANIN O NUCLEIC ACID FEULGEN STAIN MICROORGANISMS GRAM AFB GROCOTT METHANAMINE SILVER (GMS) PAS

95. GROUP STAIN FAT OIL RED O MINERALS PERLS IRON STAIN VON KOSSA AND ALIZARIN RED S RUBEANIC ACID AND RHODANINE URIC ACID METHENAMINE SILVER MELANIN FONTANA MASSON SCHMORLS METHOD LIPOCHROME SUDAN BLACK B ZIEL NEELSON SCHMORLS HEMATOLOGY GIEMSA MYELOPEROXIDASE(MPO) LEUKOCYTE ALKALINE PHOSPHATASE(LAP) TARTARATE-RESISTANT ACID PHOSPHATASE(TRAP)

96. RETICULIN STAIN PRINCIPLE- Oxidation-Reduction of silver technique. APPEARANCE - Reticulin fibre – BLACK Nuclei – GREY Other elements – PINK USES - stain reticulin fiber CONDITIONS – Fibrosis,cirrhosis of liver Aplastic anemia myelofibrosis

97. OIL RED O STAIN PRINCIPAL - Fat soluble but water insoluble APPEARANCE - Lipid – RED Nuclei – BLUE USES -stain neutral lipids CONDITION -1.Fatty liver 2.Lipid storage disease

98. ALCIAN BLUE PRINCIPLE - Salt linkage with acidic group APPEARANCE - Mucin – BLUE USES - Connective tissue and epithelial mucopolysaccharids . CONDITIONS - 1.Carcinoma of stomach 2.Carcinoma colon/rectum

99. MUCICARMINE PRINCIPLE - electrostatic interaction between cationic carmine- aluminium complex to acid mucin . APPEARANCE - Cytoplasm of mucin producing cells are deep pink Nucleus – blue Other elements – yellow USES - stain mucin CONDITIONS - 1.Adenocarcinoma 2.Signet ring cell carcinoma 3.Cryptococcus neoformans

100. FONTANA MASSON PRINCIPLE - Reduction of silver APPEARANCE - Stains BLACK U SES - Argentaffin , chromaffin,melanin and some lipochrome pigment stain black CONDITIONS - 1.neuroendocrine tumor 2.melanoma 3. fungal infection lik cryptococcosis

101. PERL’S STAIN PRINCIPLE -Iron- prussian blue reaction APPEARANCE - Iron – Prussian blue USES - detect iron and hemorrhage ( hemsiderin have ferric ion) CONDITIONS - 1.Hemochromatosis 2.Chronic venous congestion of the lung

102. MASSON’S TRICHROME STAIN PRINCIPLE -electrostatic attachment between the dye and tissue. Selective sequential staining. APPEARANCE - Erythrocytes –orange scarlet Connective tissue –blue. Muscle- red. Nuclei-grey to black. USES - mainly for collagen fibers ( eg , differentiate myosarcoma from fibrosarcoma )

103. VERHOEFF VAN GIESON Elastic fibres – BLACK

104. MYELOPEROXIASE Myeloid cells – Red brown granules

105. REFERENCE BANCROFT’S THEORY AND PRACTICE OF HISTOLOGICAL TECHNIQUES-EIGHTH EDITION HISTOLOGICAL TECHNIQUES-K.LAKSHMINARAYAN

106. Thank you..