PROCESSING OF TISSUES IN HISTOLOGICAL TECHNIQUES

·                     Definition of tissue processing    

-          any treatment of tissues necessary

-           to impregnate them with solid medium

-          to facilitate the production of sections for microscopy

·                     Aim

-          to embed tissues in solid medium

-          to support the tissues

-          to enable the knife to cut the section with little damage to knife or tissues

Dehydration

·                     A process by which water is removed from fixed tissues

·                     Also removal of aqueous and some of the lipid tissues fluids

·                     Tissue blocks are placed in containers or cassette with tag

                         - lid securely closed

                         - tiny fragments should be wrapped in a single layer of  lens paper

·                     Tissues are passed through a series of increasing concentrations of alcohol

                         - with one change in each concentration

                         - duration of one hour in each

                         - keep covered  to avoid evaporation

·                     Starting alcohol is usually 80%

  - for soft tissues 30%, 50%,70%

                          - it is important that the final bath of alcohol is pure and change 3 times

                          - free from water

                   

Dehydrating agents   

i)          Ethyl alcohol (ethanol) C₂H₅ OH

         -clear colourless inflammable liquid with pleasant odour

         -hydrophillic - miscible with water in all volumes

   - the most common

ii)         Methylated spirit (denatured  alcohol)

         -some physical characteristics as ethanol

         -stronger smell

         -consists of ethanol to which has been added a proportion of methanol

iii)         Methyl alcohol (methanol) CH₃OH    

         -clear ,colourless inflammable and poisonous fluid

         -unpleasant  odour ,miscible with water, ethanol

iv)        Isopropyl  alcohol (2 propanol) CH₃CHOHCH₃

         -miscible with water, ethanol

         -does not harden the tissue

   -99% is the best substitute for ethyl alcohol

v)         Acetone CH₃COCH₃

         -clear ,colourless ,inflammable fluid

         -pungent odours

         -miscible with water, ethanol

         -more volatile 

         -causes brittleness of tissue if treatment is prolonged

         -when speed is essential - acetone is preferred

Clearing

·                     The removal of dehydrating agents from tissue

·                     To make tissue transparent

·                     Selection of a suitable clearing agent must be based on

         -speed of removal of alcohol

         -ease of removal

         -gentleness towards tissue

         -flammability

         -toxicity

         -cost

Clearing agents

i)              xylene

         -for regular size

         -not thicker than 3 -4mm

         -immersion time must not be prolonged

         -tissues become brittle

ii)         Toulene

         -less damaging on prolonged immersion of tissues

         -suitable for automated tissue processing

iii)         Chloroform

        -slower in action causes brittleness

        -thicker tissue up to 1cm thickness can be processed

        -dangerous - releases toxic gas

iv)        Benzene

        -carcinogenic properties

        -similar to xylene

v)         Carbon tetrachloride

        -similar properties to chloroform

        -toxic

vi)        Paraffin

        -variable mixture of hydrocarbon

        -less flammable

        -less dangerous

vii)       Petrol

        -similar properties to xylene

        -various additives

        -not recommended

viii)       Carbon disulphide

        -unpleasant odour

        -danger of fire –not recommended

ix)        Amyl acetate

        -remove alcohol fairly rapidly

        -costly

x)         Methyl benzoate and methyl salicylate

        -fluids moderate in speed action

        -cause minimal distortion of tissue

xi)        Cedarwood oil

        -slow in action

        -causes little hardening or shrinkage of tissues

        -low volatile properties

xii)         Clove oil

              -similar to cedar wood oil

              -more expensive

             -use in humid climates

Infiltration and impregnation

·                     Tissues are transferred to a bath of molten paraffin wax

·                     During this process xylene is eliminated from tissues by diffusion

      -is called infiltration

·                     Wax diffuses into the tissue to replace the clearing agent

        -is called impregnation

·                     To completely remove the clearing agent

·                     Inadequate impregnation lead  to drying and shrinking of tissues

        -crack and crumble develop                 

·                                 High temperature will over - harden the tissues

Embedding

·                     Placing the infiltrated impregnated tissue in warm liquid paraffin wax that

            solidifies into a firm block when cool to room temperature

·                     Known as casting or blocking - use plastic mould

·                     Precaution should be taken

           -wax to be used must contain no trace of clearing agent

           -no dust particles must be present

           -after embedding the wax must be rapidly cooled to reduce the wax crystal

             size

·                     Orientation of tissues

           -tissues of tubular nature cut transversely

     -skin- cut in a plane at right angles

     - muscle biopsies are sectioned both transverse and longitudinal planes

·                     Wax is dispensed into the mould to a depth to cover the thickest tissue block

·                     When a thin film of semi-solid wax has formed on the base of the mould

      -tissue is introduced with warm forceps gently pressing the tissue into the

             semi-solid wax correctly orientated plane

·                     Then fill the mould with wax

           -make sure that there is no air bubbles

·                     Transfer blocks to refrigerator to complete hardening

·                     Identification label must accompany the specimen through all types of  tissues

      processing

Embedding media

i)          Paraffin wax

-most popular

-large number of tissue blocks may be processed in a short time

-sectioning and staining - fewer difficulties

-cheaper

-melting point in range 40-70^oC

-for satisfactory sections melting point 54-58^oC

-additives have been proposed

          -to increase hardness to cut thinner section and to support hard tissues

          -to increase stickiness to produce serial ribbon

          -to reduce crystal size

    eg. bee wax (stickiness) stearic acid (hardness)

ii)         Other waxes

         -ester wax

         -microcrystalline wax

iii)         Resins

          -acrylic

  -epoxy

  -urea - formaldehyde

iv)        Other media

         -agar

         -gelatin

          -celloidin

·                     Different medium is required if

          -medium not sufficiently hard and fail to provide adequate support

          -affected by heat

          -distort the tissue

    -tissue breaking away from the wax during sectioning

          -sections cannot be cut thin enough

Manual tissue processing

·                     Necessary in the following

          -large tissue slices

          -speed processing

          -electrical power failure / machine breakdown

          -small tissue slices - need optimum time

·                     Advantage

         -flexibility tissues are treated for the optimum duration in each fluid

         -use of fluids-not dangerous when using -flammable

                                                                          -volatile   eg. acetone

·                     Rapid technique for thin slices of tissue

i)          10% formalin 60^oC                             20min

      ii)         fresh acetone                                       20min

      iii)        fresh acetone                                       20min

     iv)         fresh acetone                                       20min

     v)          xylene                                                 10min

     vi)         xylene                                                 15min

     vii)        wax                                                     30min

     viii)       wax                                                     60min (185 min)

 -agitate frequently to assist in the transfer of fluids

Automated tissue processing

·                     Most automated tissues processor have 12 containers processing cycle

·                     Some machines apply - heat and vacuum to increase the rate of processing

·                     Care must be taken for type of tissue eg. spleen, muscle, skin, decalcified

       tissue become hardened if heat and vacuum is applied

·                     Vacuum

 -the degree of vacuum should not exceed 40-50mmHg

-normally in a sealed container of molten paraffin wax-applying suction to the 

  container

                  aim   -to remove air bubbles in the tissue

                     -to remove clearing agents

        

           -tissues particularly - lung ,muscle ,spleen, decalcified bone, skin

·                     For increasing speed of processing

                    -use warm (40-50^oC) fixative to ensure fixation is complete

                    -95% alcohol

                    -use a fast - acting clearing agent (eg. xylene)

                    -use of vacuum infiltration at all wax stages

                    -agitation at all stages ,even during fixation

                    -minimum of time at all stages

i.              Routine overnight processing

1                      4% formol saline (1.5 lit.)                                                        2 hrs

                        (40 ml formaldehyde 960 ml water add 9 g. NaCl)

2                      4 % formol saline (1.5 lit.)                                                       2 hrs

3                      70 % alcohol (1.5 lit.)                                                              1 hr     

                        (700 ml alcohol 300 ml water)

4                      90 % alcohol (1.5 lit.)                                                              1 hr

                        (900 ml alcohol 100 ml water)

5                      absolute alcohol (100% alcohol) (1.5 lit.)                                1 hr

6                      absolute alcohol (1.5 lit.)                                                         2 hrs

7                      absolute alcohol (1.5 lit.)                                                         2 hrs

8                      xylene (1.5 lit.)                                                                        1 hr

9                      xylene (1.5 lit.)                                                                        11/2 hrs

10                    xylene (1.5 lit.)                                                                        11/2 hrs

11                    wax                                                                                         2 hrs

12                    wax                                                                                         3 hrs

                                                                                                Total                20 hrs

ii.             Day time processing -for small specimen 

 

1.    formal saline                   15min

2.    formal saline                   15min

3.    70% alcohol                    15min

4.    90%alcohol                     15min

5.    100%alcohol                   15min

6.    100%alcohol                   15min

7.    100%alcohol                   15 min

8.    toluene                             15min

9.    toluene                             15min

10.  toluene                            15min

11.  wax                                 30min

12.  wax                                 30min                                 

                                                 3hrs 30min

Factors influencing the rate of impregnation  

i)    Agitation

    -allow the tissue to sink to the base of container - to make sure interchange of fluids   

      occur

    -mechanical device - vertical agitation

    -average speed of tissue movement is 10-12 inches per minute

    -improved impregnation  by 25-35%

ii)    Heat

      -increases the rate of penetration

      -care must be taken not to overheat tissues and cause shrinkage, brittleness and                                                 

        difficulties  in sectioning

iii)    Viscosity

      -the larger the molecule the higher the viscosity  the slower the rate of penetration

iv)    Vacuum

      -serve to remove air bubbles trapped within the tissue –this will increase the contact

        with fluid to tissue

v)    Ultrasonic - not widely used

·                     Points to note

         -beakers  and wax bath must be filled to the correct fluid level

         -any spillage of fluid should be wiped

         -accumulations of wax must be removed

         -wax bath thermostats set at satisfactory level

         -checked - timing

                         -electrical plugs

         -changing of solution after using them for 2-3 days