fixation and fixatives for mlt students.

Fixatives for pathologist and mlt students

1. FIXATION AND FIXATIVES Dr.T.Sujitha 1 st year PG Moderator : Dr.M.Ananth Satyanarayana

2. INTRODUCTION Fixation is known as a physiochemical process in which cells or tissues are fixed physically and chemically. As a result , the tissue or cell can combat the successive treatment by various reagents with negligible disfigurement of morphology. For the purpose of tissue processing in the histopathology, fixation of tissue is considered as necessary and essential step.

3. AIMS OF FIXATION Inhibition of autolysis : Dissolving of cells by enzymatic action Inhibition of putrefaction : Breakdown of tissue by bacterial action Preservation of tissue constituents in a life like manner or nearest to its original state in living system Hardening affect of fixatives make tissues firm so that gross cutting becomes much easier and allow easy manipulation of soft tissues

4. Optical differentiation : Fixatives help make the tissue more easily permeable for subsequent reagents and play a role in emphasizing dissimilarity in refractive index and thus help in increased visibility of different elements of tissue Solidification of colloid material :Fixation has the effect of convertiing normal semifluid consistency (sol) to irreversible semisolid consistency (gel)

5. IDEAL FIXATIVE Should prevent autolysis and putrefaction Should be stable and safe Rapid in action Give even penetration Cheap and available Minimal loss of tissue Should retain normal colour of tissue Should not impart its own colour to tissues Should not bind to reactive groups meant for dyes

6. Factors affecting fixation pH of fixative : Neutral pH is preferrable in most of the tissues Optimum pH is 6 to 8 Fixative should be chosen as close as the tissue pH. Eg:Optimum preservation of gastric mucosa is obtained at pH of 5.5 Chromograffin reaction for catecholemines granules (adrenaline , noradrenaline ) is stable at pH of 6.5

7. Buffers are used to bring the ph of tissues to optimal range. Eg : phosphate S- colloidine veronal acetate bicarbonate Tris and cacodylate etc.

8. Temperature : Room temperature is suitable for routine work (general surgical specimens) High temperature facilitates fixation Low temperature (0 -4C) is suitable for enzyme histochemistry

9. Duration of fixation : Depth of penetration of fixative is directly proportional to the square root of time of fixation Formalin fixes 1mm per hour For small tissues 6 hr in formalin is optimum For larger tissue, 24 hrs is optimum time As this process is relatively slow, tissues selected should be sufficiently thin to be adequately fixed in a reasonable time.Best thickness for routine use is 3-5 mm

10. Osmolarity of fixative Hypertonic solution – cell shrinkage Hypotonic - cell swelling Best osmolarity is mild hypertonic – 400 – 450 mOsm Concentration of fixative Mild lower concentration with neutral ph is preferable Very low concentration prolong time of fixation Higher concentrations causes rapid fixation with undesirable effect

11. Methods of fixation CHEMICAL Immersion Infusion Coating or spray fixation Vapor fixation PHYSICAL Heat Microwave fixation Freeze drying

12. Classification of fixatives based on chemical composition Physical agents : Heat, Microwaves Aldehydes : Formaldehyde, Acrolein , Glutaraldehyde Coagulants : Methyl alcohol, Ethyl alcohol, Acetic acid Oxidising agents : Osmium tetroxide Miscellaneous : Picric acid, Mercuric chloride

13. Classification based on number of structures fixed Simple Fixatives : Formaldehyde, Picric acid, Osmium tetroxide , Glutaraldehyde , ethyl alcohol Compound Fixatives : Bouins fluid, Formal saline, Zenker’s fluid, Carnoys solution

14. Formaldehyde is a gas which is soluble in water to approximately 40% by weight. This saturated solution is commercially available as 40% formaldehyde or formalin FORMALIN – 40% Formaldehyde 10% FORMALIN – Formalin(40% formaldehyde) – 10ml Water - 90ml Formalin is the most widely used fixative Formaldehyde

15. After continuous storage for long periods, accumulations of white deposits are observed in the solution. These are the precipitates of paraformaldehyde. By storing formalin at low temperature, these white deposits can be avoided. Also, 10% methanol may be added into the formalin to minimize the polymerization reaction that produces paraformaldehyde precipitate.

16. GLUTARALDEHYDE Glutaraldehyde was found as particular fixative for ultrastructural researches. Although penetration rate of glutaraldehyde is found to be slower when compared with formaldehyde, glutaraldehyde is more effective cross-linker for proteins than formaldehyde and it also inhibits enzyme activity Glutaraldehyde preserves the ultrastructure of the tissue, thereby it is used in electron microscopy studies

17. Due to poor penetration and overhardening properties, it is not used as tissue fixatives for light microscopy. On exposure to oxygen, glutaraldehyde becomes unstable and breaks down with decrease in pH. Glutaraldehyde can act as sensitizer, and its exposure may result in respiratory tract, skin, and digestive tract irritation

18. OSMIUM TETROXIDE Osmium tetroxide is type of fixative that is water soluble and also soluble in nonpolar solvents For electron microscopic studies, osmium tetroxide is used as secondary fixative Osmium tetroxide is also helpful for staining of lipids in frozen sections. It is observed that fixation by osmium tetroxide causes swelling in tissue, which can be decreased by adding sodium chloride or calcium chloride to fixatives. Continued exposure to osmium tetroxide vapors can cause deposition into cornea , which eventually leads to blindness .

19. MERCURIC CHLORIDE Mercuric chloride was favored for its ability to enhance the staining properties of tissues, particularly with the trichrome stains. However, it is now rarely used in the clinical laboratory due to the health and safety issues Nowadays, mercurial fixatives are not routinely used except for fixation of hematopoietic tissues These fixatives penetrate slowly, so specimens must be thin Mercury-based fixatives are toxic and all should be handled with care. They should not be allowed to come into contact with metal, and should be dissolved in distilled water to prevent the precipitation of mercury salts

20. Mercury-containing chemicals also pose an environmental disposal problem A further major disadvantage of mercuric chloride fixation is the formation of intense black precipitates of mercuric pigment in the tissue which gives them inferior value for immunohistochemical and molecular studies . In recently fixed tissues, these precipitates can be removed by a Lugol’s iodine step in the staining procedure, followed by bleaching of the section in sodium hypochlorite solution A potential replacement for mercuric chloride is zinc sulfate.

21. GLYOXAL Glyoxal was first advocated in 1943. They are also known as ethanedial or oxalaldehyde . Glyoxal is considered as alternative fixative to formalin because it is a dialdehyde in nature Glyoxal fixed tissues may demonstrate precise cellular details, lysed erythrocytes, and disintegrated microcalcifications . For microwave fixation, glyoxal is the chief component used in the fixatives. It does not produce vapors at room temperature , so it is considered as less dangerous in use than formaldehyde.

22. PICRIC ACID Picric acid is an example of a coagulant fixative. It forms picrates with basic protein groups, which causes coagulation . For the purpose of demonstration of DNA or RNA, picric acid fixatives are not used as picric acid and can hydrolyze nucleic acids Although picric acid is not able to fix most carbohydrates and lipids, picric acid is the most advised fixative to preserve glycogen. Brighter staining is seen by picric acid fixatives

23. Picric acid is an acidic solution. Therefore, sometimes it gets washed out by alcohol. To avoid this, lithium carbonate is added, which acts as a neutralizer. If picric acid is present in the tissue or not completely removed, distortion or obliteration of cellular structures will occur as outcome

24. ETHANOL AND METHANOL Fixation initiates at 50 to 60% concentration for ethanol and greater than 80% concentration for methanol They are known to be coagulants that cause protein denaturation. They cause interruption in hydrogen and hydrophobic bonding by substituting water in tissue environment, which results in change in tertiary structure

25. Ethanol causes mispresentation of cytoplasmic as well as nuclear details, but sometimes it can be used for preservation of glycogen. Methanol is more commonly used for fixation of exfoliative cytology smears and blood films

26. ACETONE Acetone is another fixative agent used in histopathology. It acts as an efficacious lipid solvent that results in tissue brittleness. Apart from tissue fixation, they are primarily used as an agent for dehydration in tissue processing. Because of extremely volatile as well as flammable nature, they are not recommended for use in automatic tissue processor.

27. ACETIC ACID Acetic acid is considered as a noncoagulative fixative agent. It acts by causing nuclear proteins coagulation. It stabilizes and assists to prevent nucleic acids loss. Acetic acid, when combined with ethanol, is used as an effective cytological fixative that helps in conservation of nucleic acids, but if it is used singly, it results in swelling of cells. Time required for fixation by acetic acid is less as penetration of acetic acid is faster into tissues

28. ACROLEIN Acrolein provides magnificent preservation of structural detail and conserves the virus antigenicity. It is also known as acrylic aldehyde. Acrolein is not commonly used because it is unstable at alkaline pH and forms insoluble polymers. Acrolein is highly reactive and is found to penetrate tissues rapidly. Acrolein fixatives are chiefly used in ENZYME HISTOCHEMISTRY.

29. GENIPIN Genipin is a glycone derivative and is outcome of geniposide . Genipin is considered as a cross-linking agent used for biological tissue fixation Genipin demonstrates its cross-linking properties at pH 7.4–8.5. It is a known cross-linking agent and has proved its potential in various biomedical application such as dentistry, articular cartilage tissue engineering applications, nerve regeneration, and so on.

30. POTASSIUM DICHROMATE Potassium dichromate is also a noncoagulant fixative, but if used in combination with acid solution, it acts as a coagulant fixative. It is seldom used alone for fixation because chromate ions will link with few lipids and makes them insoluble . By increasing the amount of reactive basic groups, the affinity of tissues for eosin staining will boost up. It conserves mitochondria but dissolves DNA

31. Based on type of structures fixed Histochemical fixatives : Formaldehyde, Glutaraldehyde, Vapor fixatives Microanatomical fixatives : Bouin’s fluid, 10% formalin, Zenker’s fluid, formal calcium, Helly’s fluid, Rossman’s fluid Cytologic fixatives : Champy’s fluid, glacial acetic acid, alcohol , formal saline, Carnoy’s fluid, Clarke’s fluid, Flemming’s fluid

32. Microanatomical fixatives These are used when it is desired to preserve the anatomy of tissue, with the correct relation ship of tissue layers and large aggregates of cells. Fixatives from routine use are from this group FORMAL SALINE Formalin(40% formaldehyde) – 10ml Sodium chloride - 0.9 gm Water- 100ml It is the most common and widely used fixative. Tissues can be left in it for long periods without excessive hardness or damage, may be sectioned easily after one year

33. Tissues fixed for a long time in formal saline, occasionally contain a pigment – Formalin pigment Formalin pigment is insoluble brownish black granular pigment due to reaction of formalin pigment with hemoglobin derivatives This can be removed in sections before staining by treatment with picric alcohol or 1% alcoholic solution of sodium hydroxide The formation of this pigment is prevented by neutralizing or buffering the formal saline

34. FORMAL CALCIUM Formalin - 10ml Calcium chloride – 1gm Water – 10ml This fixative has most of the advantages of buffered formal saline in addition to preserving phospholoipids It should not be used to fix material in which calcium or alkaline phosphatase are to be demonstrated because of its calcium content Fixation time is same as formal saline

35. HEIDENHAINS SUSA: Mercuric chloride - 4.5g Sodium chloride - 0.5g Trichloroacetic acid - 2g Acetic acid - 4 ml Formalin - 20 ml Distilled water – to 100 ml

36. This is probably the best fixative available for routine biopsy work , allowing brilliant staining with good cytological detail. It is well balanced and gives rapid and even penetration with a minimum of shrinkage. It is an intolerant fixative, tissues left over in it over 24 hrs are bleached and excessively hardened. Tissues exceeding 7-8 mm thickness are fixed in 12 -24 hrs Small pieces , not thicker than 3mm are fixed in 2-3 hrs.

37. ZENKER’S FLUID Mercuric chloride Potassiumdichromate Sodium sulphate Water Add glacial acetic acid immediately before use HELLY’S FLUID – ZENKER FORMAL Replace glacial acetic acid with formalin immediately before use It is also known as Spuler’s or Maximow’s fluid It is excellent fixative for bonemarrow ,spleen and blood containing organs

38. BOUIN’S FLUID Contains :Picric acid -70ml, formalin, glacial acetic acid tissue fixed in it gives brilliant staining by trichrome methods. Excess picric acid imparts yellow colour to the tissues, should be removed from the section by treatment with alcohol or prolonged washing Bouins fluid can be used to demonstrate glycogen GENDRE’S FLUID Contains picric acid- 80ml, formalin, glacial acetic acid Gendre’s fluid is better fixative for glycogen due to increased concentration of picric acid

39. NUCLEAR Carnoy’s fluid Newcomer’s fluid Flemming’s fluid CYTOPLASMIC Champy’s fluid Regaud’s fluid Muller’s fluid Formal saline Formal calcium Schaudinns fluid CYTOLOGIC FIXATIVES

40. CARNOY’S FLUID Contains absolute alcohol, chloroform, glacial acetic acid It penetrates very rapidly and gives excellent nuclear fixation with preservation of Nissl substance and glycogen Being a rapid fixative it is sometimes used when urgent diagnosis is needed Fixation is usually complete in 1-2 hrs, small pieces 2-3 mm thickness are fixed in 15 min

41. NEW COMER’S FLUID Iso propanol – 60ml Propionic acid – 30 ml Petroleum ether -10ml Acetone - 10ml Dioxane – 10ml This fixative , which penetrates rapidly was devised for the fixation of chromosomes and preserves chromatin better than carnoy’s fluid

42. FLEMMING’S FLUID 1% aqueous chromic acid – 15ml 2% aqueous osmium tetroxide -4 ml Glacial acetic acid – 1ml Rarely used because of its poor and uneven penetration Can be used as cytoplasmic fixative In common with all osmium tetroxide fixatives, tissue must be small (not more than 2mm in thickness)

43. CHAMPY’S FLUID 3% potassium dichromate – 7ml 1% chromic acid – 7ml 2% osmium tetroxide – 4ml Champy’s fluid preserves mitochondria, fat, yolk and lipids REGAUD’S FLUID 3% potassium dichromate – 80ml Formalin (40% formaldehyde) – 20ml Good fixative for mitochondria Chromaffin tissue is well demonstrated

44. MULLER’S FLUID Potassium dichromate – 2.5gm Sodium sulphate -1gm Distilled water – 100ml Rarely used as a fixative except for bone specimens SCHAUDINN’S FLUID Mercuric chloride – 2parts Absolute alcohol – 1part This fixative has been popular for many years as a cytoplasmic fixative for wet smears

45. POST CHROMATIZATION It is the treatment of tissues with 3% potassium dichromate following normal fixation This technique is employed to mordant tissues, particularly mitochondria and gives improved preservation and staining of these elements.

46. SECONDARY FIXATIOIN Wallington advocated basic fixation in formal saline followed by treatment with a fixative appropriate to the tissue concerned. The so called soft fixation of formalin is overcome by subsequent treatment with formal mercuric chloride, or zenker formal for 4hrs. Staining and preservation of tissue constituents is improved by this technique, but not recommended as a routine procedure

47. FIXATION ARTEFACTS Formalin pigment : insoluble brownish black granular retractile pigment due to reaction of formalin with hemoglobin derivatives. Treat with1.8 % picric acid in absolute ethyl alcohol for 15 min. Mercury pigments : darkbrown irregular deposit throughout the tissue. Removal : application of iodine converts it into mercuric iodide which is removed by sodium thiosulphate

48. Fuzzy staining : the nuclear and cytoplasmic details are obscured and section looks fuzzy and hazy. Cause- improper fixation either due to insufficiant fixative or too little time in fixative. Prolonged fixation cause shrinkage of tissue followed by seperation . The tissue may show holes or empty spaces within it

49. Dichromate deposit : if the tissue is not properly washed after dichromate fixation, then chromium salt may form. This chrome Salt react with alcohol, and insoluble yellow brown precipitate may appear. Removal by 1% hypochloric acid in 70% alcohol for 30 min.

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