PREPARATION OF TISSUES FOR HISTOLOGICAL TECHNIQUES

Fixation

·                    Definition  - is a complex series of chemical events and

       - differs for the different groups of substances found in tissues

·                    Aims - to prevent the process of autolysis (self destruction) and  bacterial attack    

  -   to be as close as possible to their living state

  -   to retain shape or volume

·                  Try as best as possible because eg.- lipids are always lost unless special precaution are       taken                                                           

·                  Most important cellular substance - is the protein component

·                  Fixation is done twice  a)  initial fixation - large amount of tissues or organ involved b) re-  fixing done in histology lab - should be cut thin (3-5mm thick) - to facilitate penetration in a short time 3-5 hours                       

·                Volume of fixative - 10-20 times the volume of tissue block

·                Minute specimens should be wrapped - use cigarette paper or lens paper- to

            avoid losing

                    

 Factors involved in fixation

i)         pH = different fixatives vary - hydrogen ion concentration is adjusted by a suitable buffer - pH suitable for fixation occurs between 6 and 8 - outside this range changes the ultra structure of specimen - for some purposes - fixation at specific pH is chosen eg. gastric mucosa at pH 5.5

  

ii)        Temperature = fixation is carried out at room temperature - suitable temperature - for election microscopy 0 - 4^oC  is chosen  - reason  - autolysis is slowed down

                                                                                             

iii)        Penetration of fixation =>  this process is relatively slow - blocks taken should be small or thin - large blocks of tissue such as uterus should be sliced thinly

iv)       Volume changes => tissues commonly change in volume because-changes in membrane permeability - inhibition of respiration - changes in ion transport through the membrane

The ideal state is not often achieved eg. tissues fixed in paraffin wax shrink by 33%

v)        Duration of fixation =>  common practice allow primary fixation in buffered formalin for 2-6 hours during the day - for electron microscope fixed 3 hours

In formalin - tissues are fixed for 24 hours - prolonged fixation in formaldehyde will cause shrinkage and hardening of tissues                 

                    

Classification of fixatives

i)         Aldehydes => formaldehyde - glutaraldehyde - acrolein

ii)        Oxidizing agents => osmium tetroxide - potassium permanganate - potassium dichromate

iii)         Protein denaturing agents => acetic acid - methyl alcohol - ethyl alcohol

iv)        Unknown mechanism => mercuric chloride - picric acid

Formaldehyde fixatives

 

 a)        Formol saline

                        40% formaldehyde    =           100 cm³    

                                Sodium chloride          =          9g

                        Tap water                  =            900cm³

 

 b)        Neutral buffered formaldehyde

                        40% formaldehyde                  =         100 cm³

                                                Distilled / tap    water               =          900 cm³

                         Add Mg carbonate  in excess

c)         10% formalin

                         40% formaldehyde        =      100 cm³

                         Tap water                       =      900 cm³

Alcoholic fixatives

·                     For nucleic acids study

a)         Carnoy`s fluid

                        Absolute alcohol          =         60 cm³

                        Chloroform                  =         30 cm³

                        Glacial acetic  acid       =        10 cm³

         

3.3.3    Picric acid fixatives

·                     Preserves glycogen

·                     Causes shrinkage of tissues

a)         Bouin`s fluid

                        Saturated aqueous picric acid solution           =         75 cm³

                        40% formaldehyde                                           =        20 cm³

                        Glacial acetic acid                                            =        5 cm³

 Mercuric chloride fixatives

·                     Mercuric chloride penetrates poorly and produces shrinkage tissues - so usually combined with other fixatives

·                     Tissues can be stained effectively

a)         Zenker`s fluid

                         Distilled water                          =        950 cm³

                              Potassium dichromate           =          25 g

                         Mercuric chloride                   =          50 g

                         Glacial acetic acid                  =         50 cm³

·                     The most important reaction are those which stabilize the protein

·                     Fixatives have the property of forming crosslink between proteins - thereby forming a gel - keeping everything in their relation to each other

·                     Glutaraldehyde - causes 30% loss of protein    

·                     Osmium tetroxide -  causes complete denaturation of protein compared  to                                                                                   glutaraldehyde-fixed material

3.4       Secondary fixation (post fixation)

·                     Tissues may be fixed with 2 fixation used in succession

             eg. tissues fixed in buffered formaldehyde then undergo secondary  fixation                                                                                                                                                      

 with mercuric chloride - formaldehyde for a period of several hours - they stain   

 more brilliantly

Fixation for special purposes

·                     Lipids - largely lost during processing

·                     For preservation of lipids

a)    Elftman`s fluid

                  Mercuric chloride            =      5 g

                  Potassium dichromate    =      2.5 g

                  Distilled water                 =      100 cm³

            -keep for over 3 days at room temperature

Decalcification

·                     In order to obtain satisfactory paraffin section of bone and other heavily mineralized tissues - it is necessary to remove the mineral and soften the tissues

·                     It is carried out by treatment with reagents  which react with calcium

·                     Before decalcification

               - cut hard tissues into small pieces (2-6mm)

               - use thin blade or hack saw

               - minimize tearing of surrounding tissues

               - fix the tissue  in buffered or neutral formalin

         - tissues later must be washed

Acid decalcifiers

·                     Strong acid

               

  eg.           hydrochloric acid

                  nitric acid

                

       - if used longer than 24-48 hour deterioration occurs

- strong acid used for urgent biopsy

             

      a)         Aqueous nitric acid

                                          Nitric acid                     =         10 cm³   

                                          Distilled water               =         90 cm³

  

                  b)         Formalin - nitric acid

                                          Formalin                   =             10 cm³

                                          Distilled water          =             80 cm³

                                          Nitric                        =             10 cm³

·                     Weak acid

eg.             formic

                  acetic

                  picric

    - use in non urgent surgical specimen decalcification should be complete 1-10 days

       depending on size

a)         Aqueous formic acid             

                                           90% Formic acid        =          10 cm³

                                           Distilled  water          =           90 cm³

          

                  b)         Formic acid - formalin

                                                  90% Formic acid      =          5 cm³

                                                  Formalin                   =           5 cm ³ 

                                                  Distilled water          =           90 cm³

  

            - excess fixative must be removed

Procedure for decalcification

·                     Suspend the tissue slice in decalcifying  solution - fluid 20  times the volume of

tissue

·                     Change the fluid daily - stirring agitation of the fluid hasten decalcification

·                     The tissue may be mechanically or chemically tested

- bending or piercing with a sharp needle

- generally a day or two will be enough to decalcify

- twice that long for compact bone

·                     Wash specimen for 24 -48 hours in running water before processing the tissue

Treatment of tissues

·                     Special treatment after fixation

             - important to verify the fixation is complete

             - if doubt exists - extra time is necessary

             - if rapid fixation is needed -  use heat

           a)   potassium dichromate fixatives

                   - requires thorough washing

           b)   picric acid - containing fixatives

                   - not to be in contact with water

           c)   Carnoy’s fluid

                   - transferred to 95% absolute alcohol

·                     Treatment of hard tissues with - 4% phenol in 70% alcohol to soften tissues

eg.       tendon

                                    nails

                                    dense fibrous

                                    masses of keratin

                   -labeling of tissues before processing to avoid incorrect reporting or

                    exchange of tissues identity