Tissue fixation and fixatives in histopathology

1. Tissue Fixation & Fixatives
Presenter : Dr. Santhipriya G.
Moderator : Dr. Aarthi C A.
22/9/2017 1Seminar

2. •Definition
•Aims of fixation
•Procedure
•Types of Fixation
•Classification of fixatives
•Factors affecting quality of fixation
•Fixation for selected individual tissues
•Decalcification
•Basic museum techniques
•Conclusion
Contents
22/9/2017 2Seminar

3. What is a fixative?
• A Substance which prevents post mortem changes and
preserves the morphological and chemical characteristics of
cells and tissues.
• Fixation is the process by which the constituents of the cells ,
and therefore of the tissues, are fixed in a physical , and partly
also in chemical state so that they will withstand subsequent
treatment with various reagents with a minimum loss of
architecture, significant distortion or decomposition.
22/9/2017 3Seminar

4. Aims of fixation
• To preserve the tissues as close to their living state as
possible
• To prevent autolysis and bacterial attack
• To prevent tissues from changing their shape and size
during processing
• To harden the tissues
• To allow clear staining of section subsequently
• To improve the optical differentiation of cells and
tissues
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5. Principle of fixation
• Denaturation and coagulation of protein in the tissues
• Have a property of forming cross links between
proteins, thereby forming a gel, keeping everything in
their in vivo relation to each other
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6. Procedure
A large specimen is received
It is cut to smaller pieces
For L/M it is
dipped in formalin
For E/M is dipped in
gluteraldehyde and
then osmium tetroxide
Takes 24 hours
22/9/2017 6Seminar

7. QUALITIES OF IDEAL FIXATIVE
• Should have good tissue penetration
• Should stabilize the tissue
• Should preserve cellular constituents
• Should increase tissue consistency
• Should confer optical differentiation for better
visualization
• Should maintain its chemical composition
• Should be cheap, nontoxic, noninflammable,
nonirritant and easy to prepare
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8. Methods of fixation
1. Heat fixation
2. Perfusion fixation
3. Immersion fixation
4. Vapour method
5. Coating/Spray fixation
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9. Heat fixation
• After a smear has been
allowed to dry at room
temperature, the slide is
gripped by tongs or a
clothespin and passed
through the flame of a
Bunsen burner several
times to heat-kill and
adhere the organism to
the slide
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10. • Generally preserves overall morphology but not
intenal structures
• Routinely used with bacteria and archaea.
• Will shrink or destroy the capsule(glycocalyx) and
cannot be seen in stains
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11. Perfusion fixation
• Fixation via blood flow:
injected in the heart
with the injected
volume matching
cardiac output
• The fixative spreads
through the entire body,
and the tissue doesn't
die until it is fixed
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12. • Advantage:preserves perfect morphology
• Disadvantage:subject dies and the cost is high,
because of the volume of fixative needed for larger
organisms
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13. Immersion Method
•Most common method of
fixation.
•Samples of tissue is immersed
in fixative solution of volume
at a minimum of 20 times
greater than the volume of the
tissue to be fixed
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14. Coating/Spray fixation
• Sprayed on a freshly prepared smear.
• Or applied with a dropper.
• Use of bottles and fixing solutions not required.
• Dual action:
 Fixation.
 Protective coating.
22/9/2017 14Seminar

15. Types of Coating fixatives:
Polyethylene Glycol (Carbowax) Fixative:
• 95% ethyl alcohol 50ml
• Ether 50ml
• Polyethylene Glycol 5g
Diaphane Fixative:
• 3parts 95% ethyl alcohol
• 2parts Diaphane
22/9/2017 15Seminar

16. Vapour fixative
• Vapour fixatives may be used to fix crystat - cut
section or blocks of frozen dried tissue.
• Formaldehyde vapour generated from heated
paraformaldehyde at 50-800c for 2hrs
• Acetaldehyde at 800c for 1-4 hrs
• Gluteraldehyde at 800c for 2min - 4hrs
• Using this method we have produced sections
showing excellent preservation of glycogen with a
very good morphological details
22/9/2017 16Seminar

17. Classification of Fixatives
Based upon use of fixative:
1. Microanatomical fixatives:
These are used to preserve the anatomy of the tissue.
2. Cytological fixatives:
These are used to fix intracellular structures.
3. Histochemical fixatives :
These are used to demonstrate the chemical constituents of
the cell.
22/9/2017 17Seminar

18. Classification of Fixatives
Based upon mechanism of action:
1. Physical:
Heat
Microwave
Freeze-drying
2. Chemical:
Coagulant Fixatives
Dehydrant Coagulant Fixatives
Non-coagulant cross-linking fixatives
22/9/2017 18Seminar

19. Chemical Fixatives
Simple Fixatives Compound Fixatives
 Formalin
 Mercuric chloride
 Osmium tetroxide Microanatomical Cytological Hematological
 Picric acid Formal Saline Methanol
 Acetone Neutral buffer Formalin
 Ethyl alchohol Zenker’s fluid
Nuclear Cytoplasmic
Carnoy’s Fluid Champy’s Fluid
Clarke’s Fluid Regaud’s Fluid
22/9/2017 19Seminar

20.  The most widely used fixative .
 It is prepared by mixing 40 % Formaldehyde gas in
100 w/v of distilled water.
 The resultant mixture is 100 % Formalin.
 Routinely, 10 % formalin is used which is prepared by
mixing 10 ml of 100 % formalin in 90 ml of distilled
water.
Formalin
22/9/2017 20Seminar

21. MECHANISM OF ACTION
 It forms cross links (methylene bridges) between amino acids
of proteins thereby making them insoluble.
 Many of these changes are reversible with water.
 These reactions naturally alters cell physiochemistry &
reactivity of tissue to certain histochemical stains.
22/9/2017 21Seminar

22.  ADVANTAGES :
1. Rapid penetration
2. Easy availability & cheap
3. Does not overharden the tissue
4. Fixes lipids for frozen sections
 DISADVANTAGES:
1. Irritant to the nose , eyes and mucous membranes
2. Formation of precipitate of paraformaldehyde - which can be
prevented by adding 11- 16 % methanol.
3. Formation of brown-black formalin pigment
4. Carcinogenic.
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23. Formalin pigment
22/9/2017 23Seminar

24. ROUTINE FORMALIN FIXATIVES:
 10% formal saline: Most commonly used fixative
Water (distilled) 900ml
Sodium chloride 9gm
Formalin 100ml
• 10% buffered neutral formalin: (better than formal saline)
Water (distilled) 900ml
Formalin 100ml
NaH2PO4 4gm
Na2HPO4 6.5gm
22/9/2017 24Seminar

25. Alcohol fixatives
• Are recommended for the preservation of glycogen
• Carnoy's fixative(rapid in action,may be used for
urgent biopsy)
Absolute ethanol 60ml
chloroform 30ml
Glacial acetic acid 10ml
• Nuclear fixative
22/9/2017 25Seminar

26. Picric acid fixatives
• It react with histones and basic proteins and forms
crystalline picrates with amino acids.
• It preserves glycogen well, but caused considerable
shrinkage of tissues
• Owing to its explosive nature when dry, it must be
kept under a layer of water
• Tissue cannot be kept in picric acid for more than
24hrs
• Nuclear fixative
22/9/2017 26Seminar

27. • Rossman's fluid
100% ethanol 10ml
picric acid 90ml
formalin 10ml
• Gendre's fluid
90% ethanol 5ml
picric acid 80ml
40% formaldehyde 15ml
 Bouin’s fluid (formalin-picric-acetic)
picric acid 75ml
Formalin 25ml
Acetic acid 5ml
fix for 12-24hrs
wash several days in 95%
ethnol
fix for 4hrs
wash 80%, 95% and
100% ethanol
fix few to 18hrs
GI Biopsies
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28. Mercuric chloride fixatives
• Recommended for fixing fetal brains
• Penetrates poorly and produces shrinkage of tissues
• Mercury pigment must be removed with lugol's iodine
22/9/2017 28Seminar

29.  Zenker’s fluid:
Mercuric chloride 5gm
Potassium dichromate 2.5gm
Sodium sulphate 1gm
Distilled water 100ml
Acetic acid 5ml
 Advantages - even penetration, rapid fixation
 Disadvantages - pigmentation, tissue must be washed in
running water to remove excess dichromate
 Choive of fixative for Bone Marrow Tissue
22/9/2017 29Seminar

30.  Helly's fluid
Formaline is added instead of acetic acid to zenkers fluid
Distilled water 1000ml
Potassium dichromate 25g
Sodium sulfate 10g
Mercuric chloride 50g
Formaline 5ml
 Advantage - excellent microanatomical fixative especially for
bone marrow, spleen and kidney
22/9/2017 30Seminar

31. Coagulant fixatives
Acetic acid : Glacial acetic acid
Advantages :
 Best fixative for nuclei.
 Counteracts the shrinking effect of others.
Disadvantages :
 Pronounced swelling of collagen fibers
 Distorts mitochondria & Golgi body
Acetone :
Advantage:
 Best fixative for certain enzymes (acid phosphatase & lipase).
22/9/2017 31Seminar

32. Ethyl alcohol :
Advantages :
 Best for alkaline phosphatase & lipase
 Coagulates protein
Disadvantages:
 Powerful dehydrating agent- hardening & shrinkage
 Improper fixation of chromatin
 Distorts mitochondria & Golgi
22/9/2017 32Seminar

33. Chromic acid:
Advantage :
 Good fixative for carbohydrate
Disadvantage:
 Powerful oxidising & rapid hardening with brittleness
 Washing with tap water for several hours required after
dehydration to avoid insoluble precipitate formation.
22/9/2017 33Seminar

34. Picric acid:
Advantages :
 Good fixative for proteins, carbohydrates & glycogen
 Staining becomes better
 Enhances results with cytoplasmic stains
Disadvantages :
 Much shrinkage & less hardening
Mercuric chloride:
Advantages :
 Good fixative for proteins
 It shrinks but doesn’t distort tissue
 Fixes both nucleus & cytoplasm
 Act as secondary fixative
Disadvantages :
 Mercury pigments (greenish brown).22/9/2017 34Seminar

35. Mercury pigments
22/9/2017 35Seminar

36. Non coagulant fixatives
Potassium dichromate :
Advantages :
• Excellent fixation of mitochondria, phospholipids & myelin
• Iron-containing pigments better fixed
Disadvntages : -
• Chromatin gets dissolved
• Mitochondria gets thickened
• Formation of insoluble precipitants
• Brittleness of tissues
22/9/2017 36Seminar

37. Glutaraldehyde:
• Most highly cross-linking of all the aldehydes. GTA fixation
is irreversible.
• Is the most widely applied fixative in both scanning and
transmission electron microscopy.
22/9/2017 37Seminar

38. Factors affecting quality of fixation
1 - Temperature
• It will increase the rate of penetration
• It will also increase the rate of autolysis and diffusion of cellular
components.
• Affects the morphology of the tissue.
2- Size
• Ideal size of the tissue should be 3mm.
3 - Volume ratio
• Volume of fixative is at least 15 to 20 times greater than volume of
tissue.
22/9/2017 38Seminar

39. 4 – Time
• Minimum fixation time for 5mm tissue is 12hrs.
• For electron microscopy sliced tissue is preserved for
3 hrs in 3% glutaraldehyde.
5-- Buffers & pH
• Chemical fixation is a complex set of oxidative and reductive
reactions, thus is constantly changing.
• At a specific pH, all proteins have a point, the isoelectric point
(IEP) where the numbers of + and - charges are equal. Fixation is
most effective at the IEP.
22/9/2017 39Seminar

40. 6 - Osmolality :
• The addition of a buffer to the fixative solution may alter the
osmotic pressure exerted by the solution.
• Hypertonic solutions give rise to cell shrinkage whereas hypotonic
and isotonic fixatives result in cell swelling and poor fixation.
• With electron microscopy, the best results are obtained using
slightly hypertonic solutions (isotonic solutions being 340 mOsm)
adjusted using sucrose.
22/9/2017 40Seminar

41. 7 - Penetration
• Fixatives penetrate the tissue at different rates.
• The tissue is fixed starting at the periphery of the
tissue and working inward toward the center of the
tissue.
22/9/2017 41Seminar

42. Choice of fixatives
Solutions Colors Tissues
Zenker’s fluid Orange Bone Marrow Biopsies
Helly’s fluid Orange Bone Marrow Aspirates
B-5 Transparent Bone Marrow Cores and Tumors
Bouin’s fluid Yellow GI Biopsies
Hollande’s fluid Green Gastrointestinal biopsies &
endocrine tissue
Orth’s fluid Orange Decals and
Bones
Adrenal Medulla
22/9/2017 42Seminar

43. Solutions Colors Tissues
Zamboni’s Yellow EM Fixatives
Carnoy’s Clear Nuclear Fixatives
10% Formalin Clear Routine
10% Formal saline Clear Routine
Neutral buffer formalin Clear Prevent Pigments
Formalin ammonium
Bromide
Clear Brain Tissues
10% Formal Alcohol
Clear EM Specimen
Flemming’s Clear EM Specimen
Gluteraldehyde Clear EM Specimen
Schaudinn’s
Clear EM Specimen
22/9/2017 43Seminar

44. Fixation for selected individual tissues
• Eyes - 48hours - NBF
• Brain - 2weeks - NBF
• Lungs - 2hours - inflate fixed in NBF
• Lymphoid Tissue
• Testis
• Muscle biopsies
• Renal biopsies – Carson’s modified Millonig’s fixative
Routinely in NBF
22/9/2017 44Seminar

45. DECALCIFICATION
• Formic acid, Nitric acid
• For bony tissue, and also for any calcified tissue
• Formic acid
90% formic acid
2 to 4 mm thick blocks
1 to 7 days depending on concentration of acid
• It spares hemosiderin
• Better preservation of tissue architecture
22/9/2017 45Seminar

46. Nitric acid(HNO3)
• 1 to 3 days
• Tests for completion of decalcification - Touch,
pliability, and resistance to fingernail, by needling,
x-ray, chemical method(ammonium oxalate)
22/9/2017 46Seminar

47. Basic Museum Techniques
1.Reception
2.Preparation
3.Fixation
4.Restoration
5.Preservation
6.Presentation
22/9/2017 47Seminar

48. • The fixatives used in museums all over the world are
based on formaline fixative technique, and are
derived from Kaiserling technique and his
modifications.
• Kaiserling recommended that the initial fixation be a
neutral formalin(KI) solution and then transferred to a
final preserving glycerin solution(KIII) for long term
display.
• Colour preservtion is also maintained with these
solutions
22/9/2017 48Seminar

49. Kaiserling Technique
• The specimen needs to be kept in a large enough container
which can accommodate specimen along with 3-4 times
volume of fixative.
• Specimen is stored in the Kaiserling I Solution for 1 month
depending on the size of the specimen. The specimen should
not rest on bottom or an artificial flat surface will be produced
on hardening due to fixation.
• Kaiserling I Solution:
Formalin 1L
Potassium acetate 45 g.
Potassium nitrate 25 g.
Distilled water Make up to 10 litres
22/9/2017 49Seminar

50. Restoration of specimen
• It is required to restore the specimens, as they lose their natural
color on fixation.
• The recommended method is the Kaiserling II method.
• It involves removing the specimen, washing it in running
water and transferring to 95% alcohol for 10 minutes to 1hour
depending on the size of specimen.
• The specimen is then kept and observed for color change for
around 1- 1.5 hrs. After this step, specimen is ready for
preservation.
22/9/2017 50Seminar

51. • Kaiserling II Solution:Alcohol 95%
Store specimen in this solution for 10 minutes to 1 hour
depending on size of specimen.
• Rejuvenator Solution:
Pyridine 100 ml
Sodium hydrosulphite 100 gm
Distilled water 4 litres
• Formalin decreases the natural colour of the specimen.
However, rejuvenator solution restores the colour.
22/9/2017 51Seminar

52. Preservation of specimen
• It is based on glycerine solution.
• Kaiserling III Solution:
Potassium acetate 1416 g.
Glycerine 4 litres
Distilled water Make up to 10 litres
• Thymol crystals added to prevent moulds.
• Leave the solution to stand for 2 – 3 days before using to
ensure proper mixing of chemicals.
• Add 1% pyridine as stabilizer. This solution acts as permanent
fixative.
• The specimen will initially float to surface but later sink to
bottom.
22/9/2017 52Seminar

53. Mounting the Specimens
• To support the specimen within its jar, it is attached
to the specimen plate or rectangular bent glass rods. It
can be done by tying the specimen with nylon
threads.
• Double knots should be made by threads, on the
specimen surface.
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54. 22/9/2017 54Seminar

55. Conclusion
 There is no universal fixative which will serve all
requirements.
 Each fixative has specific properties and disadvantages
 Careful consideration and selection of the appropriate fixing is
required.
 10% buffered formalin and 2.5% Gluteraldehyde are currently
the most widely used fixatives for routine light microscopy
and ultrastructural studies, but they too, have inherent
disadvantages.
 Increasing interest in tissue and cell constituents including
cellular proteins detectable by immunohistochemical
techniques imposes additional requirements for the
preservation of such substances.
22/9/2017 55Seminar

56. Reference
• Spencer L T, Bancroft J D. Tissue processing. In :
Suvarna S K, Layton C, Bancroft J D, editors.
Bancroft theory and pratice of histological
techniques. 7th ed. China. Elsevier; 2013. P 63-84.
• Mondal S K. Museum techniques. Mondal S K,
editors. Manual of Histological techniques.2nd ed.
India. Japee ;2011.P 127-30.
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57. Thank you !!
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