BASICS IN ANATOMICAL PATHOLOGY

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BASICS IN ANATOMICAL PATHOLOGY
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Patient care is increasingly basedupon
information provided by examination of surgical
specimens biopsies
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Patient report must contain all data necessary
for appropriate patient care
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The ultimate goal is to attain uniformity
consistency ofincluded data found to be
relevant to clinical management of patient
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Quality assurance inanatomical pathology

• Goals
• Accuracy
• Completeness
• Timeliness of all the reports

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Topics

• Specimen collection
• Specimen handling
• Fixation
• Processing
• Tissue embedding
• Staining
• Cover slipping slide mounting
• Reporting

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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

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What should be considered by the surgeon

• Sample handling
• Not slicing the tumor specimen
• Immediately placing the specimen into fixative

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Containers

• Types
• Reusable or Disposable
• Clean uncontaminated
• Adequate size
• Labeled after placing the specimen

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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

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Fixation

• Good preservation of tissue is the most important
  factor in the production of satisfactory
  histology slides

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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

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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

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Essentials to good fixation

• Fresh tissue
• Proper penetration of fixative
• Right choice of a correctly formulated fixative

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No fixative will penetrate a piece of tissue
ticker than 10 mm
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• 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 )

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• All fixative are used once only
• Adequate volume (gt 2/3 of the container volume )
• 10 times volume of fixative to tissue
• Fixation at room temperature ( not be heated )

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Types of fixatives
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Formalin

• Commercially available solutions
• 37 - 40 formaldehyde in water
• Conventional fixation is usually carried out in
  10 neutral buffered formalin ( NBF )

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Formalin

• Suggested fixation time
• gt8 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 )

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Formalin benefits

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

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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

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Notice

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

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Zinc formalin

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

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Zinc sulfate benefits

• Shorter fixation time
• Minimal need for Ag unmasking or retrieval
• Preserve better tissue Ag morphology

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Zinc formalin disadvantages

• Possible quenching of primary fluorescence
• Special handling and disposal requirements

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Alcohol / Acetone

• As 70 - 95 Et OH
• 90 Et OH / 10 acetone
• For
• Histopathology
• Cryostat frozen section
• Cytology smears

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Fixation time

• Variable
• ( often in tissue processing
  secondary
• to formalin fixation )
• 10 - 15 minutes for
• cryostat sections
  
• cytology smears
  

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Alcohol / Acetone benefits

• Shorter fixation time ( 5 mm3 in 4 hrs )
• Better cryostat sections
• Good preservation of cytoplasmic intermediate
  filaments

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Alcohol / Acetone disadvantages

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

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Note

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

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Bouin

• Mixture of formalin and picric acid
• Fixation time 1 -12 hrs

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Bouin

• Is used when enhanced staining for
• connective tissue is required

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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

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Bouin disadvantages

• Preservation of many types of Ags
• ( particularly lipid containing Ags )
• Poor penetration under fixation
• Longer fixation brittle tissue
• 
  nuclear staining

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B 5

• Mixture of formalin mercuric chloride
• sodium acetate
• Fixation time 1 6 hrs

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B 5 benefits

• Primary use in lymph node tissue
• Enhanced cytologic detail and immunoreactivity of
  cytolologic Igs
• intracytoplasmic Ags

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B 5 disadvantages

• Tissue hardening
• Surface Ags not well demonstrated
• Special handling disposal requirements

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Marker dye

• For extremely small biopsies
• 6 drops of 4 aquaous eosin to 1 liter of 10
  buffered formalin

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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 )

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Macroscopic description cutting up the specimens
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Rules

• Qualified and trained staff
• Established and documented macroscopic
• description protocol

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Processing

• Dehydration clearing
• Embedding

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Dehydration clearing

• Removing the water from within the tissue
• cells removing the dehydration agent
  from the tissue prepared for paraffin
  embedding

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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

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Maintenance program for tissue processor

• Clean all wax spills Daily
• Check all solutions wax levels Daily
• Change all solutions Weekly

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Maintenance program for tissue processor

• Clean instruments thoroughly Weekly
• Check of mechanical parts Every six months
• Check of electrical components Annually

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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
  )

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Embedding procedures
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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

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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

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Section cutting procedures

• 5 um sections
• With new disposable blades
• ( degreased and cleaned by xylene )

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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 )

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Drying

• 60 C oven for 30 minutes
• Alternatives
• - Room temperature for 24 hrs
• - Hot air blower
• - Slide plate warmer
• Temperature gt 60 C Ag degradation in ICC

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General comments concerning dyes

• Preparation
• Numbered commercial dye lots
• Careful preparation
• - Specific volume
• - Careful weighing

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General comments concerning dyes

• Contamination
• - Fungal bacterial growth
• - Crystalline precipitation of dye

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General comments concerning dyes

• Times for staining in each reagent
• Documented monitored with
• Internal Quality Control

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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 )