Principles, Methods and Types of Fixatives

Principles, Methods and Types of Fixatives

1. By: Ms . Hafsa Sh. Hussein Histopathology & Cytopathology Lecturer Lecture 2: Principles, Methods and Types of Fixatives Faculty of Laboratory, EAU.

2. Once the tissue is removed from the body it will go through a process of self-destruction. This process is known as autolysis . If tissue is left without any preservation, then a Microbial attack will occur, the process is known as putrefaction . Why fixation is very important ??

3. In the fields of histology , pathology , and cell biology , fixation is a chemical process by which biological tissues are preserved from decay , either through autolysis or putrefaction . Fixation terminates any ongoing biochemical reactions , and may also increases the mechanical strength or stability of the treated tissues . Introduction

4. The basic aims of fixation are the following: To preserve the tissue nearest to its living state To prevent any change in shape and size of the tissue at the time of processing To prevent any autolysis To make the tissue firm to hard To prevent any bacterial growth in the tissue To make it possible to have clear stain To have better optical quality of the cells Purpose of Fixation :

5. Prevention of autolysis of the cells or tissue and prevention of decomposition of the tissue by bacteria. Allow long storage of tissues Characteristics of a good fixative:

6. Maintaining the volume and shape of the cell as far as possible It must kill the cell quickly without shrinkage or swelling

7. Consistently high-quality staining particularly routine stain. Must allow staining

8. Rapid action. It must penetrate the tissue rapidly Cheap Non-toxic

9. 1. Volume changes: Fixatives may change the volume of the cells. Osmium tetroxide cause cell swelling. Volume change may be due to: Altered membrane permeability , Inhibition of the enzymes responsible for respiration and change of transport Na+ ions. Fixation Changes Tissue in many ways..

10. 2. Hardening of tissue : The fixation changes the consistency of the tissue .

11. 3. Interference of staining : Fixation may cause hindrance of staining of enzymes. Formaldehyde inactivates 80% of ribonuclease enzyme ( RNAse ). osmium tetroxide inhibits haematoxylin and eosin staining.

12. 4 . Changes of optical density by fixation : The fixation may cause changes in the nuclei. The nuclei may look like condensed and hyperchromatic. Change of optical density of the nuclei

13. The fixative can be classified on the basis of the following criteria : Nature of fixation Immersion Coating Vapor Freeze – Drying Microwave Types of Fixation:

14. B. Chemical properties Aldehyde Oxidizing agent Protein denaturing Cross – Linkage

15. C. Component present Only one chemical present or More than one

16. D. Action on tissue protein Coagulalative Non – coagulative

17. Immersion fixation Coating fixation Vapour fixation Perfusion fixation Freeze – dry fixation Microwave fixation Heat fixation Methods (Natures) of fixation

18. The whole specimen is immersed in the liquid fixative Examples: Tissue samples are immersed in 10% neutral buffered formalin or Cytology smear immersed in 95% ethyl alcohol . The most used fixation in the laboratories. (1) Immersion fixation

19. Spray fixation The spray fixative usually consists of alcohol and wax . This is commonly used in the cytology samples . The spray fixative is used for easy transportation of the slide. (2) Coating fixation

20. Advantages: Fixation of the cells To impart a protective covering over the smear No need to carry liquid fixative in bottle or jar

21. Notes : The spraying over the smear should be smooth and steady, and the optimum distance of 10–12 inches should be maintained between the nozzle of the spray and the smear. The spray fixative usually consists of alcohol and wax. Therefore , this wax should be removed before the staining procedure.

22. Both Histo logy & Cytology samples. The vapour of chemical is used to fix either a smear or tissue section . The vapor converts the soluble material to insoluble material, and these materials are retained when the smear comes in contact with liquid solution. (3) V apour fixation

23. Vapour Fixative examples: Formaldehyde. Osmium tetroxide. Glutaraldehyde. Ethyl alcohol.

24. The fixative solution is infused in the arterial system of the animal , and the whole animal is fixed . The organ such as the brain or spinal cord can also be fixed by perfusion fixation . Mainly used in research purpose . (4) Perfusion fixation

25. Used for research purpose This technique is useful to study: soluble material very small molecules. (5) Freeze – drying fixation

26. The tissue is cut into thin sections. Immersed in liquid coolant “ Quenching ”. liquid nitrogen , Propane & Isopentane . Then rapidly frozen into a very low temperature. keeping it in close contact with chilled metal . How to perform Freeze – Drying fixation ?

27. Then ice within the tissue is removed with the help of vacuum chamber in higher temperature (−30 °C).

28. Advantages : Excellent for enzyme study No changing proteins No shrinkage tissue Preservation of glycogen

29. Microwave is a type of electromagnetic wave. Its frequencies between 300 MHz and 300 GHz . Its wavelength varies from centimetre to nanometre . (6) Microwave fixation

30. It operates with a frequency of 2.45 GHz and 0.915 GHz . The electromagnetic field is created by the microwave, and Dipolar molecules ( water) rapidly oscillate in this electromagnetic field. This rapid kinetic motion of these molecules generates uniform heat. Homogeneous increase of temperature within the tissue every part of the tissue is heated. The generated heat accelerates the fixation. How Scientific and medical Microwave works?

31. Factors controlling the temperature rise : The rise of temperature in the microwave heated media depends mainly on: The dielectric property of the media Thermal properties of the material Radiation level Orientation and shape of the object

32. Advantages : Rapid processing. No change in volume of tissue. Preservation of the tissue antigen and good for immunohistochemistry. Other fixation methods may prevent antibody bindings. It facilitates the staining reaction without any bad effect.

33. Disadvantages : Safety problems The tissue immersed in formalin during microwave fixation may generate large amount of toxic gas. Solution: Overhead hood  for the removal of this toxic substance from the microwave. Heat injury . Overhead hood

34. Applications : Surgical pathology laboratory Electron microscopy after osmium tetroxide fixation Urgent processing of biopsy (e.g. kidney biopsy)

35. The simplest form of fixation. boiling tissue in normal saline. Boiling an egg precipitates the proteins Cutting an egg  the yolk and egg white can be identified separately . Each component is less soluble in water after heat fixation than the same component of a fresh egg. (7) Heat fixation

36. The tissue should be free from excessive blood before putting it into fixative . Essential Precautions for Proper Fixation in General

37. Tissue must be very thin. Tissue should be thinly cut in 3–5 mm thickness.

38. The amount of fixative must be more than the tissue. The amount of fixative fluid should be 20 times more than the volume of the tissue. So that it can penetrate all parts.

39. The tissue with fixative should be in a tightly screw-capped bottle.

40. " Basic and Advanced Laboratory Techniques in Histopathology and Cytology " by  Pranab Dey Bancroft’s Theory and Practice of Histological Techniques Books