BASICS IN ANATOMICAL PATHOLOGY

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					BASICS IN ANATOMICAL
    PATHOLOGY
Patient care is increasingly based
  upon information provided by
examination of surgical specimens
           & biopsies
Patient report must contain all
data necessary for appropriate
         patient care
The ultimate goal is to attain
uniformity & consistency of
 included data found to be
     relevant to clinical
   management of patient
       Quality assurance in
      anatomical pathology :
• Goals :


            Accuracy
            Completeness
            Timeliness of all the reports
    Topics :
•   Specimen collection
•   Specimen handling
•   Fixation
•   Processing
•   Tissue embedding
•   Staining
•   Cover slipping & slide mounting
•   Reporting
   What should be considered by
   the surgeon :
• Sample collection :
   • Preoperative consultation with pathology
     staff about :
     • Requirements for selection , handling ,
       transporting and processing of tissues
     • Size of biopsy
     • Number of biopsies
     • Surgical margins
    What should be considered by
     the surgeon :
• Sample handling :

• Not slicing the tumor specimen

• Immediately placing the specimen into
  fixative
  Containers :
• Types :
       Reusable or Disposable
• Clean & uncontaminated
• Adequate size
• Labeled after placing the specimen
                 Labeling :
Two patient identifiers are required through
 the whole processing of the specimen :
  •   Patient , s name
  •   Patient , s date of birth
  •   Laboratory number
  •   Hospital number
  Fixation :


Good preservation of tissue is the most
 important factor in the production of
 satisfactory histology slides
     Aims of fixation :
• To prevent autolysis or decomposition ( due to bacterial
  or osmotic change )


• To preserve tissue as near to its original form as possible


• To protect tissue against subsequent changes during
  processing & embedding
  Aims of fixation :
• To give tissue a texture which permits easy
  sectioning


• To render the various constituents of the
  tissue reactive to the proposed stains
  Essentials to good fixation:

• Fresh tissue
• Proper penetration of fixative
• Right choice of a correctly formulated
  fixative
No fixative will penetrate a piece
   of tissue ticker than 10 mm
• Solid organs : cut slices not ticker than 5
  mm
• Hollow organs : open out or fill with
  fixative
• Large specimens : inject fixative along
  vessels (or bronchi in case of lungs )
• All fixative are used once only
• Adequate volume (> 2/3 of the container
  volume )
• 10 times volume of fixative to tissue
• Fixation at room temperature ( not be
  heated )
Types of fixatives
   Formalin :

• Commercially available solutions :
   37 - 40 % formaldehyde in water


• Conventional fixation is usually carried out
  in 10% neutral buffered formalin ( NBF )
   Formalin :
• Suggested fixation time :
              >8 hrs
              24 - 48 hrs for complete fixation
             (1/10 specimen to fixation ratio)
• Formalin in containers should be replaced
  weekly and a standard PH should be
  adopted.( either neutral or slightly acidic )
    Formalin benefits :

• Readily available
• Penetrates tissue quickly
• Long term storage in formalin is possible
Formalin disadvantages :

 • penetrates quickly but fixation is slow
 • ( may not be complete with shorter times )
 • May not be suited to long term storage of
   tissue for ICC
 • Hardens specimens
 • Antigen cross-linking
 • Partial Ag disappearance
 • Special handling & disposal requirements
  Notice :

• HCL and formalin should be avoided in
  combination
• Formalin has respiratory and carcinogenic
  effects .
   Zinc formalin :


• Mixture of zinc sulfate and formalin
• Fixation time :
               4 – 48 hrs
              ( 4 - 6 hrs for complete fixation )
   Zinc sulfate benefits :


• Shorter fixation time
• Minimal need for Ag unmasking or retrieval
• Preserve better tissue Ag morphology
 Zinc formalin disadvantages :


• Possible quenching of primary fluorescence

• Special handling and disposal requirements
  Alcohol / Acetone :
• As : 70 - 95 % Et OH
       90 % Et OH / 10 % acetone
• For :
       Histopathology
       Cryostat frozen section
       Cytology smears
    Fixation time :
• Variable
          ( often in tissue processing secondary
            to formalin fixation )
• 10 - 15 minutes for
                    cryostat sections
                    cytology smears
  Alcohol / Acetone benefits :

• Shorter fixation time ( 5 mm in 4 hrs )
                              3


• Better cryostat sections
• Good preservation of cytoplasmic
  intermediate filaments
   Alcohol / Acetone disadvantages :

• quality and integrity of ICC staining
        ( especially after long term storage )
• Ethanol has shrinking & hardening effects
   on tissues
 Note :

Tissue incompletely fixed by NBF or Zinc
 formalin will be subjected to alcohol
 fixation in the tissue processor
   Bouin :


• Mixture of formalin and picric acid

• Fixation time : 1 -12 hrs
Bouin :


Is used when enhanced staining for
    connective tissue is required
   Bouin benefits :

• Fixes tissues rapidly
• An excellent premordant fixative for chromatin to
  be demonstrated ( if using iron – haematoxylin
  methods )


• Useful particularly for endocrine tissues and
  tumors
  Bouin disadvantages :
• Preservation of many types of Ags
    ( particularly lipid containing Ags )
• Poor penetration       under fixation
• Longer fixation        brittle tissue
                          nuclear staining
   B–5:

• Mixture of formalin &mercuric chloride &
  sodium acetate

• Fixation time : 1 – 6 hrs
  B – 5 benefits :

• Primary use in lymph node tissue

• Enhanced cytologic detail and
  immunoreactivity of cytolologic Igs &
  intracytoplasmic Ags
  B – 5 disadvantages :

• Tissue hardening
• Surface Ags not well demonstrated
• Special handling & disposal requirements
  Marker dye :

• For extremely small biopsies

• 6 drops of 4 % aquaous eosin to 1 liter of
  10 % buffered formalin
   Decalcification :
• Tissue disruption and removal of minerals ,
  particularly hemosiderin
• The procedure :
        - Adequately fixed tissue ( 48 hrs )
        - Draining off fixative
       - Replacing decalcifier ( 10 X to 20 X
                                  specimen volume )
      - Everyday checking for presence of Ca ++
         ( with dipstick or ammonium oxalate )
Macroscopic description &
 cutting up the specimens
   Rules :

• Qualified and trained staff

• Established and documented macroscopic
  description protocol
Processing :

• Dehydration & clearing

• Embedding
    Dehydration & clearing :

Removing the water from within the tissue &
cells     removing the dehydration agent
 from the tissue    prepared for paraffin
 embedding
  Typical schedule for sample
   preparation :

• Fixation ( room temperature)    8 – 24 hrs

• Dehydration ( room temperature ) 25 hrs

• Clearing ( room temperature )      2 hrs

• Impregnation (58 – 60 C)           5 hrs
      Maintenance program for tissue
      processor :

• Clean all wax spills :   Daily

• Check all solutions & wax levels : Daily

• Change all solutions : Weekly
     Maintenance program for tissue
     processor :

• Clean instruments thoroughly : Weekly

• Check of mechanical parts : Every six months

• Check of electrical components : Annually
         Xylene :
• Most commonly used clearing agent
• Tissue storage for prolonged period :
                    Over hardening the specimen
• Background staining
• Alternatives :
                - Toluene (no tissue hardening )
                - Chloroform ( slower penetration )
                - Limonene ( no hazards )
Embedding procedures
    Notes :
• The size of mould allow 1- 2 mm margins
  around the specimen
• Not to be too hot : Charring of tissue
• Minimal pressure on the forceps
                       ( tissues are delicate )
• Correct orientation of tissue
   Notes :
• Melting point of paraffin : 50 – 80 C
   ( usually 65 C )
   - for ICC : lower degrees ( 55 - 58 C )

• Temperature of paraffin should be monitored
  and recorded daily
  Section cutting procedures :


• 5 um sections

• With new disposable blades
      ( degreased and cleaned by xylene )
   Water ( Floatation ) bath :
• Monitoring its temperature constantly
  5 – 10 C below the melting point of wax
   ( 47 – 49 C )

• Cleaning the water surface between cases
  ( preventing cross – contamination )
    Drying :
• 60 C oven for 30 minutes
• Alternatives :
           - Room temperature for 24 hrs
           - Hot air blower
           - Slide plate warmer
• Temperature > 60 C         Ag degradation in ICC
       General comments
       concerning dyes :
• Preparation :

   Numbered commercial dye lots
   Careful preparation
          - Specific volume
          - Careful weighing
       General comments
       concerning dyes :
• Contamination :

   - Fungal & bacterial growth

  - Crystalline precipitation of dye
        General comments
        concerning dyes :


• Times for staining in each reagent :

     Documented & monitored with
        Internal Quality Control
          Turnaround time in
            pathology lab :
              Verbal report      Written report
• Rushes             1                  2
• Biopsies           2                  3
• Surgicals          2                  3

  ( For each of the additional necessary
    procedures the extra time must be
    regarded )

				
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posted:10/21/2011
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