Conventional Papanicolaou staining technique and its modification

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Conventional Papanicolaou staining technique and
its modification

• Ganesh Prasad Acharya
• M.Sc.M.L.T. (Path with Cytology)

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ModbGan
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• The screening for cervical cancer with
  Papanicolau(Pap) testing is recommended for all
  women who are or have been sexually active and
  who have a cervix. Pap smears should begin with
  the onset of sexual activity and should be
  repeated at least every 3 years.

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• Staining of the smear is the key part among
  other essential parts of the job like sample
  collection, sample processing, labeling, record
  keeping etc.
• There are some important staining objectives
  which need to be fulfilled to meet the diagnostic
  criteria so as to reach the final diagnosis
• Well stained nuclear chromatin.
• Differential counter staining, i.e., staining
  the cytoplasm of different cell types into
  different colors and intensity.
• Retaining cytoplasmic transparency.

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• Most of the time we can not meet the above
  mentioned staining objectives due to various
  reasons

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•  We have to compromise with impure dyes,
  chemicals, reagents and Test kits
• (Lack of quality assurance system)
• Lack of knowledge of quality assurance and
  passing the low price quoted tender without
  giving preference to the quality of goods.
• Most of the time it is difficult to make
  them available in our laboratory at the right
  time
• (Error in purchasing system)

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• Considering above all facts and to meet the
  diagnostic criteria, following modification was
  brought about during a period of one and half
  years (2003-2005) on conventional Papanicolaou
  methodology.

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• This modified "ModbGan" technique was applied at
  the Department of Clinical Pathology, Maternity
  Hospital, Thapathali, Kathmandu to stain the
  received Cervical smears and Body fluid samples
  smear.

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• Materials and Methods 
• The number of samples were more than one
  thousand.
• Types of samples were
• Wet fixed cervical smears in fixative
  container/coplin jars.
• Pre-fixed and dried cervical smears.
• Anticoagulated (1 ammonium oxalate in 1
  9 ratio) body fluid specimens i.e. pleural
  ascitic, Tumor or cystic and
• peritoneal fluids etc.

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• Characteristics of this Modification
• ModbGan technique is mainly based on the
  preparation of the staining solution, especially
  the EA modified staining solution.
• Other variations of EA solutions like EA 36, EA
  50, EA 65 are omitted in this technique, only
  timing of the stain varies in different types of
  sample.

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• CHEMICALS AND REAGENTS (Conventional
  Papanicolaou stain)
•     Harris hematoxylin
• 1. Hematoxylin dye (colour index) CI no.75290.
• 2. Aluminium ammonium sulphate (alum) or
  Potassium ammonium sulphate (AR/GR).
• 3. Mercuric oxide (AR/GR).
• 4. Absolute methanol (LR).
• 5. Glacial acetic acid (LR).
• 6. Ethylene glycol (for Gills hematoxylin)
  (AR/GR.).
• 7. Sodium iodate (for Gills hematoxylin )
  (AR/GR).
• 8. Aluminium sulphate (for Gills hematoxylin )(
  AR/GR).
• 9.  Citric acid (for Gills hematoxylin, if
  glacial acetic acid is not available) (AR/GR)

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•   OG-modified
• 1. Orange G CI no. 16230 (AR).
• 2. Phosphotungstic acid (LR/GR).
• 3. Ethanol (AR/GR).
• 4. Glacial Acetic Acid (AR/GR)

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Preparation of original Modbgan EA Solution

• Preparation of EA- ModbGan staining solution
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• ModbGan Solution (TDC-A)
• Eosin Y 4.0 g
• Phosphotungstic acid 2.0 g
• Ethanol 480 ml
• Glacial acetic acid 20 ml
• Dist. Water 500 ml
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• ModbGan solution (TDC-B)
• Light green 10 g
• Absolute Methanol 1000 ml
• Store in daark bottle
• For working solution
• Add 65 parts of solution (A) to 35 parts of
  solution (B). Mix well and filter before use.
• Note Do not prepare the solution B in
  iso-propanol or Ethanol.

• Preparation of EA modified staining solution
  (original method)
• Light green 0.3 g
• Eosin 4.0 g
• Phosphotungstic acid 2.0 g
• Dist. water 480 ml (Dissolve
  all above).
• 95 Ethanol 500 ml (Add)
• Glacial acetic acid 20 ml.
• Mix well, store in a dark bottle and filter
  before use.

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• The Staining time required are as follows-
• Cervical smears 1-5 minutes.
• FNAC smears 1-2 minutes.
• Body fluid smear 45 sec-1 minutes.

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• Conventional Papanicolaou Stained Smears

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• ModbGan Stained Smears

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Comparison of conventional and ModbGan technique
 
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Namaste