INTRODUCTION & FIXATION

Bachelor of medical laboratory science Lecture presentation about tissue fixation

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2. FIXATION The purpose of fixation is to preserve the tissue in such a way that it appears the same in a microscopic preparation as it appeared while living and functioning. We must acknowledge that this is an impossible aim, as the very act of fixation stops all functions and distorts in some fashion the appearance. Nevertheless , the aim should be to approach this ideal as closely as possible. 2

3. FIXATION A fixative is a substance which preserves the shape, structures, relationship and chemical constituents of tissues and cells after death. Therefore fixation is the process of maintaining tissue or cell morphology in a life like state as much as possible. N.B: Fixation helps prevent post mortem changes like autolysis and putrefaction. 3

4. A fresh, unfixed specimen after surgical removal. To prevent degeneration or drying-out the specimen should be fixed as soon as possible 4

5. TERMINOLOGIES USED Secondary fixation . After fixation in formal saline, the tissue is treated with an appropriate fixative e.g. zenkers formal and H eidenhains s usa . The second fixation or treatment improves the preservation and staining of specific tissue elements . Post fixation. Some proteins are masked by fatty substances which do not allow fixative to penetrate tissue. To be able to demonstrate such proteins, the tissue is fixed, dehydrated, cleared and then later fixed in ethanol to preserve the proteins. 5

6. Washing out. Washing of tissue in running tap water to remove the fixative used. This is because some fixatives if not washed out can react with the dehydrating agent during tissue processing e.g. potassium dichromate and osmium tetroxide can react with ethanol . Post chromatisation This refers to treatment of tissue with 3% potassium dichromate after normal fixation. This is important since it links specific tissue elements to the stains being applied e.g mitochondria. 6

7. , It can be done by treating tissue sections with 3% potassium dichromate for 12-24 hours. N.B: Post chromatization , also known as post chroming and usually applies to tissue fixed in formal saline . The purpose of post chroming is to mordant the tissue. However, post chroming is different from post mordanting as the latter is carried out after staining e.g. application of iodine in gram staining . 7

8. POST MORTEM CHANGES Changes that take place on a cell or tissue after death of an organism i.e . changes that take place on cell or tissue after tissue has been removed from a living person e.g. skin and breast. These changes include; autolysis and putrefaction. Autolysis refers to destruction of cell or tissue caused by enzymes produced by lysosomes which digest a way the different cellular components following death . Putrefaction refers to destruction of cell or tissue by normal flora found in normal and pathological conditions e.g. T.B found in pathological condition leading to production of gas leading to a filthy smell. 8

9. . N.B ; post mortem changes are most prominent in highly specialized tissue e.g. liver, intestinal tract, kidney and brain. 9

10. PURPOSES OF FIXATION To kill tissues by denaturing proteins(enzymes) so that postmortem activities such as putrefaction (bacterial attack) and autolysis(enzyme attack) can be prevented. Bacterial attack can be prevented by observing very strict antiseptic techniques while autolysis by altering the shape of the enzyme by fixatives which leads to destruction of tissue biological activity. Note: severely autolyzed tissues fail to stain. 10

11. CONT’… 2. Fixation helps stabilize tissue elements (maintains relationship between cells and extracellular substances such as connective tissue fibers and amorphous ground substances) so that the effect of any subsequent procedures will be minimal. A well fixed tissue is almost impervious/resistant to abuse during the processing and staining procedures . 11

12. … 3. It brings out differences in refractive index and to increase the visibility of or the contrast between different tissue elements . Therefore , enhancing differences in the refractive indexes of various tissue structures will increase the contrast between those structures. 12

13. … 4. Most staining is enhanced by fixation and frequently tissue that has not been fixed well stain poorly. 5. Fixatives also aid in rendering tissue constituents such as lipids and carbohydrates insoluble so that they are studied. 6. Fixation makes tissue firm so that gross dissection and collection of thin sections required for processing becomes much easier. 13

14. CRITERIA OF A GOOD FIXATIVE Be able to maintain cell or tissue morphology. Should not affect staining. Be able to evenly and quickly penetrate tissue. Aid attachment of the smear onto the slide to prevent washing off during staining. Ability to inactivate enzymes especially lysosomal enzymes to avoid autolysis. 14

15. . 6. Must prepare the tissue/cell for the reagents that would be applied during staining or after staining e.g xylene, alcohol and DPX. 7. Able to kill bacteria and molds to avoid putrefaction. 8. Be simple to prepare and economical in use. 9. Ability to make tissue firm so that grossing and collection of thin sections required for possessing become easier . 10 . Ability to act as a mordant which serves to link the dye to the tissue/cell. 15

16. HALLMARKS OF GOOD FIXATION A well fixed cell will show the following features; Nuclei with various crisp chromatin pattern and a crisp blue nuclear chromatin membrane. i.e. the nuclei should not show any smudginess, bubbling, or fading. Nuclei should not show fading . There should not be cell shrinkage Cell cytoplasm should be preserved and should stain well with eosin . Smudge bubbling 16

17. Crispy Chromatin 17

18. FACTORS THAT AFFECT THE RATE OF FIXATION 1.Temperature ; the temperature at which fixation is carried may affect tissue morphology. In general, increasing the temperature of the fixative up to approximately 45-55 C has little effect on tissue morphology. Note; Traditionally, refrigerator temperatures of 0°c-4 ° c was considered the ideal temperature for the fixation of specimens for electron microscopy. Formaldehyde fixation performed at room temperature instead of refrigerator temperature produces proper tissue preservation and less tissue effect. 18

19. Factors cont’ 2.Size This should be considered when the gross tissue specimen is placed in a fixative. Specimens such as segments of colon or small intestines should be surgically opened to expose all tissue layers before placed in the fixative. Solid organs e.g. breast, spleen and kidney should be covered with adequate fixative and bread-loafed to allow faster penetration of the fixative. 19

20. Factors Cont’ Volume ratio; the fixative volume should be at least 15-20x greater than the tissue volume. Fixative molecules are bound chemically to the tissue and thus the solution is gradually depleted of these molecules. Tissues also contain soluble salts that are dissolved by fixative solution. The 2 way exchange does not greatly alter the characteristics of the fixative if a large volume ratio is used. However if the volume of tissue is greater than that of the solution, the fixative composition can be altered leading to poor fixation. 20

21. … Time ; ideally, tissue should be placed in a fixative immediately after surgical removal and autopsies should be performed immediately after death. The more time that elapses between interruption of blood supply and fixation, the more postmortem changes occur. Note; Tissue that is not well fixed does not process well and subsequently will not stain well. Therefore, adequate fixation time is of primary importance in quality assurance. 21

22. … Choice of fixative; this depends on the investigation to be carried out e.g. if immunohistochemistry is to be done on tissues, the ideal fixation is achieved by rapid freezing. Penetration; fixatives solutions penetrate at vastly/massively different rates. Research shows that formaldehyde penetrates faster than any other fixative. 22

23. … Tissue storage : The method of wet tissue storage is very important because some tissues may be needed for additional studies otherwise if a tissue is not fixed and stored properly, additional studies may be impossible. Neutral buffered formalin has been seen as the best solution for tissue storage for long which is not true with many other fixatives. 23

24. … PH; the PH of the fixative is very important in preventing formation of pigments. Maintaining fixative PH. between 7.2-7.4 prevents formation of pigments in tissues which can lead to false positive results. Osmolality of fixative and ionic composition; The osmolality of the buffer and fixative is important; hypertonic and hypotonic solutions. The best morphological results are obtained with solutions that are slightly hypertonic 24

25. … Suspension; fixation properties are displayed in line with the way the tissue is suspended in the fixative. Agitation; increased agitation increases the rate at which tissue is fixed. Concentration of fixative ; Effectiveness and solubility primarily determine the appropriate concentration of fixatives. Concentrations of formalin above 10% tend to cause increased hardening and shrinkage. 25

26. Cont’ Additives use The addition of electrolytes such calcium chloride, potassium thiocyanate , ammonium sulfate, and potassium dihydrogen phosphate and non-electrolytes like sucrose and dextran in fixatives improves the morphology of the fixed tissue. T hese additives may react either directly with proteins/enzymes causing denaturation, or independently with the fixatives and cellular constituents. 26

27. CLASSIFICATION OF FIXATIVES Fixatives are classified according to their mode of action ALCOHOLS : These fix tissue by coagulating proteins forming a semi-solid structures that hold intracellular structures and prevent them from leaking out of the tissue. Examples include; 100% methanol and 95% ethanol. 27

28. CLASSIFICATION cont’ ALDEHYDES: These fix tissue by formation of cross linkages and metylene bridges. These appear like wire mesh and hold intracellular structures with in the cell. They are formed as a result of the interaction between the covalent bonding of the fixative and hydrogen bonding with in the proteneous structures of the tissue. Aldehydes also fix tissue by formation of methylene bridges (similar to cross linkages). 28

29. … They are responsible for masking epitopes in biological tissue and therefore tissue fixed with aldehydes need to be un masked before immunohistochemistry procedures are done. Examples of aldehydes include; formaldehyde/formalin , glutaraldehyde and glyoxal . 29

30. Note They usually cause extensive cross linking hence causing adverse effects in immunohistochemical methods. Glyoxal is used as a 40% aqueous solution and buffered at PH of 4.0 Do not cause formation of artifacts like smudgy nuclei and distorted staining as with formalin. Causes slight reduction of staining when tissue is stored for long in it. Most special stains are satisfactory after glyoxal fixation. 30

31. Formaldehyde solutions used in the lab 1. 10% neutral buffered formalin ; this is the most widely used solution for routine formalin fixation. Its a hypotonic solution with a PH of approximately 6.8. 2. 10% neutralized formalin ; though it has been widely used, its not recommended because the Solution becomes acidic after withdrawal from the storage bottle. 31

32. … 3. 10% formalin saline/formal saline ; this solution is isotonic exclusive of the formaldehyde but some times produces formalin pigments. 4. Calcium formalin ; recommended especially for the fixation and preservation of phospholipids in tissues. 5. Formalin ammonium bromide ; recommended for tissue specimens of the central nervous system. 32

33. … 6. Modified millonig formalin 7. Alcoholic formalin 8. Phosphate buffered paraformaldehyde 9. Acetate formalin 10. 10% aqueous formalin 33

34. WHY 10% FORMALIN IS ROUTINELY IN HISTOLOGY Cause less shrinkages to tissue as compared to other fixatives. It's chemical composition is stable and can be used for a long time. Relatively cheap and readily available as compared to other fixatives. Hardens tissue more than any other fixative except acetone and ethanol. Allows most staining protocols to be performed on tissue fixed in it . Quickly and evenly penetrates tissue compared to other fixatives, 34

35. DISADVANTAGES OF USING 10% FORMAL SALINE When used in addition with Nacl to achieve the correct osmolarity , the solution becomes acidic by reacting with atmospheric oxygen leading to formation of formic acid which leads to black acid hematin ( formalin pigment) This is common in tissue containing a lot of blood. The formalin pigments are formed when the PH of formalin drops below 6.0. 35

36. OXIDIZING AGENTS These also fix tissue by formation of cross links with the proteins in the tissue . However , they are not routinely used because they cause extensive denaturation of proteins. Examples include; Potassium dicromate and Osmium tetroxide . 36

37. Note Osmium tetroxide is primarily used for fixation of specimens for electron microscopy. Used in fixation and preservation of fats and lipids in tissues It penetrates only a few cell layers so the sections must be extremely thin. It cause tissue swelling which can be minimized by addition of calcium or sodium chloride in osmium containing fixatives. Potassium dichromate also preserves lipids but not at the same degree as osmium tetroxide. 37

38. PICRATES These are fixatives that contain picric acid. examples include; bouin’s and hollande’s solutions. N.B; Most picrates work on coagulation principle. Picrates like bouin’s solution stain everything they come into contact with yellow. However it can be removed with 50- 70% ethanol, lithium carbonate. separately or during the staining sequence . 38

39. CONT’ 3. Bouin’s solution is an excellent general fixative for connective tissues(glycogen) and hollande’s solution is excellent for gastrointestestinal biopsies and endocrine tissues. 4. Picrates leaves tissue very receptive to acid dyes like eosin and gives tissue a very good soft consistency. 5. The only disadvantage with picrates is either causing extreme shrinkage or allows extreme shrinkage to occur in the subsequent processing steps. 39

40. MERCURIALS These are fixatives that contain mercuric chloride e.g. Z enkers , Helly’s , schaudinn’s,ohlmacher’s , B arnoy-lebrun and B5 solutions. Note; 1. Fixatives containing mercuric chloride are mostly used on haemopoetic tissues like bone marrow, liver, spleen , lymph node and kidney. 40

41. MERCURIALS 2. Major advantage of using mercury as a fixative is that it leaves tissue highly receptive to staining. 3. Mercury is highly poisonous. Therefore, fixatives containing it are not routinely used. 4. Mode of action of mercurials is un known. 5.Presence of mercury in tissue inhibits its freezing . Therefore, frozen sections are difficult to prepare. 41

42. FIXATION ARTIFACTS Artifacts are structures or features in tissue that interfere with normal histological examination under the microscopic. Fixation artifacts are pigments that end up being deposited in tissues after using histological fixatives. Examples include; Formalin pigments, Pink disease artifacts, Chrome deposited Mercury chloride pigments. 42

43. FORMALIN PIGMENTS Also called acid formalin hematin . They appear as brown granules in tissue and the concentration of the pigments is highest in tissue containing blood vessels due to the presence of hemoglobin which reacts with formic acid. The formic acid is as a result of a reaction between formalin and atmospheric oxygen . Formalin pigments can be avoided by using buffered formalin at a PH of 7.0. the buffer breaks down formic acid to water and hydrogen ions . However, if they are already present in tissue, they can be removed using barrets method. 43

44. BARRET'S METHOD Take sections to xylene to remove cover slip and dpx Put in 100% alcohol to remove the xylene Hydrate to water. Rinse in absolute ethanol Flood the sections with saturated alcohol picric acid for 5-10 minutes. Wash with 95% ethanol to remove the yellow color of picric acid and control this microscopically. Wash with tape water. Stain as desired. 44

45. MERCURY CHLORIDE PIGMENTS This is a dark to brown pigment found in sections of tissue fixed using fixatives containing mercuric chloride. Found uniformly distributed in sections. It can be removed from tissues during tissue processing by adding 0.25% sodium iodine in the 80 % ethanol used for dehydration. Pigments are removed by converting mercuric chloride into mercuric iodine which is water soluble. 45

46. CHROME DEPOSITS These are found in tissue fixed using potassium dichromate. They are fine granules that are removed from tissue by washing sections in running tap water o r washing in 1% acid alcohol then water . 46

47. PINK DISEASE ARTIFACTS These are artifacts found in tissue rapidly fixed in buffered formal saline. This is common in lymphode and epithelial cells and characterized by the nucleous failing to stain with hematoxylin and instead taking up eosin . These artifacts are avoided by adding 2% acetic acid to the fixative or by adding 1% HCL to the absolute ethanol used to dehydrate sections. 47

48. TROUBLE SHOOTING IN FIXATION Autolysis caused by delayed fixation which is solved by; Placing specimens in fixatives solution as soon as possible and ensuring that the volume is 15-20x that of the tissue. Surgically opening large specimens such as segments of colon or small intestines to expose all layers such that the inner epithelial surfaces are covered by the fixatives. 48

49. . Slicing /bread loafing solid/large organs such as the kidney, spleen and breasts to ensure adequate fixation . Note; Autolysis may be shown by a loss or total disappearance of nuclear chromatin. Some cells may disappear such as epithelial cells in intestinal specimens or there may be cell shrinkage with artifactual space around the cells. 49

50. Incomplete fixation Because of rapid turnaround time deemed necessary, specimens are not usually fixed adequately. If tissue is not well fixed when processing has begun, fixation continues in the alcohol and the center of tissue will often be more eosinophillic than the peripheral. If signs of incomplete fixation is noted on H and E stained section, the following corrective actions should be taken; 50

51. Corrective actions Increase the time allowed in fixative solution Change to another fixative such as zinc formalin which still requires several hours for complete fixation or Glyoxal which is an extremely rapid fixative. Place formalin alcohol in the first 3 changes of the processing cycle to decrease fixation time and also begin dehydration. 51

52. Corrective actions Ensure that the gross sections are thin enough for good reagent penetration and the amount of fixative is 15-20x that of the tissue. Ensure that the formalin solution is not depleted because of overuse; change the solutions frequently. Use agitation of cassettes in fixatives holding solutions during or after grossing. 52

53. Ischemia time Cold ischemia time, is defined as the time from the removal of the tissue from the patient to the initiation of tissue fixation. This time should be shortened as much as possible, specifically, no more than 1h. 53

54. REVISION QUESTIONS D efine; fixation, coagulative fixative, non- coagulative fixative, additive fixative, non-additive fixative. List down the functions of fixation. Discuss the factors that affect the rate of tissue fixation. Describe the B arrets method of removing pigments from tissues. 54

55. . 5. Explain the mode of action of; Aldehydes fixatives Picrate fixatives Mercurial fixatives Alcohols 6. List down 4 reasons why 10% formalin is the routinely used fixative in a histopathology lab. 7.Discuss the classification of fixatives according to composition. 55

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