INSTRUCTIONS FOR USE FOR IVD DIRECTIVE 9879EC ISSUE NUMBER by mikelbyington

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									INSTRUCTIONS FOR USE FOR IVD DIRECTIVE 98/79/EC ISSUE NUMBER 1 31/07/03

MANUFACTURER: Surgipath Europe Ltd., Venture Park, Stirling Way, Bretton, Peterborough, UK
                        PE3 8YD. Telephone: +044-(0)1733 333100. Fax: +44-(0)1733 331111.
PRODUCT NAME: Powdered Stain Haematoxylin Dye. Haematoxylin Colour Index Number
75290.
INTENDED PURPOSE: Used as a constituent in the preparation of nuclear stain.
 This product should only be used for its intended purpose.
HANDLING AND TREATMENT BEFOREUSE:
Haematein must be produced from haematoxylin, as it is this that stains the tissue. This may be done by
oxidation either naturally or by the use of chemicals. Haematein has a poor affinity for tissue and
should therefore be combined with a mordant for best results. Many formulations are available for the
production of nuclear stains. Protocols for their production are many and varied but easily available in
textbooks and publications. The following are suggested but by no means exhaustive. Harris, Gills,
Mayer’s, Ehrlich’s, Delafield’s, Carazzi’s, Cole’s, Weigerts, Heidenheims, PTAH, Loyez, Solcia.

Please refer to the Material Safety Data Sheet or product label for information on handling, disposal,
chemical warnings, precautions and product composition.

STORAGE INSTRUCTIONS: This product should be stored upright in a tightly closed container, at
room temperature away from direct sunlight. Check that powder is dry prior to use.
TYPE OF HISTOLOGICAL MATERIAL TO BE USED: Fresh frozen cryostat, paraffin or plastic
sections of various types of tissue.
HANDLING AND TREATMENT BEFORE STAINING: Suggested fixatives include 10%
formaldehyde buffered with phosphate to a pH of 7. Routine dehydration, clearing and paraffin
infiltration and embedding and routine preparation of microtome sections. Poor fixation, processing,
rehydration and sectioning will adversely affect the staining quality. Sections of 2-5 micron thickness
should be used.

TYPE OF CYTOLOGICAL MATERIAL TO BE USED: Smear, tissue imprint, cell culture,
monolayer.
HANDLING AND TREATMENT BEFORE STAINING: Wet fixed in ethanol or routine cytology
fixative. Ensure that any Polyethylene Glycol has been fully removed in ethanol before rehydration.

EXAMPLE OF PROCEDURE TO BE USED FOR SURGIPATH HARRIS HAEMATOXYLIN
HISTOLOGICAL SPECIMENS:
1. De-wax and rehydrate section as appropriate using routine procedure.
2. Immerse in Haematoxylin for 3 minutes if hand staining or until desired intensity has been reached
    if using a staining machine. (Times will vary according to the type of machine used, and the
    manufacturer’s manual should be consulted and times adjusted accordingly.)
3. Wash in running tap water until excess stain is removed. (The pH of the tap water should be above
    7 to ensure correct colour of nuclei.)
4. Differentiate in 1% Acid Alcohol (4 dips) if hand staining or until stain is removed from all
    background components if using a staining machine. The stain should now only remain in the
    nuclei. This step should be controlled microscopically for optimum results. Under staining or over
    staining can be corrected at this stage by repeating the above steps as appropriate.
5. Wash in running tap water.
6. Blue if necessary in alkaline tap water.
7. Wash well in tap water.
8. Counterstain as required. Dehydrate, clear and mount in synthetic resin.
Results: The nuclei and their components should now be stained in varying shades of blue/purple.

EXAMPLE OF PROCEDURE TO BE USED FOR SURGIPATH HARRIS HAEMATOXYLIN
CYTOLOGICAL SPECIMENS:
1. Rehydrate smears as necessary using routine procedure. Immerse in Harris Haematoxylin for 2
   minutes if hand staining or until desired intensity is reached if using a staining machine. (Times
   will vary according to the type of machine used, and the manufacturer’s manual should be
   consulted and times adjusted accordingly.)
2. Wash in tap water. (The pH of the tap water should be above 7 to ensure correct colour of nuclei.)
3.  Differentiate in 1% Acid Alcohol (1 dip) if hand staining or until stain is removed from all
    background components if using a staining machine. The stain should now only remain in the
4. nuclei. This step should be controlled microscopically for optimum results. Under staining or over
    staining can be corrected at this stage by repeating the above steps as appropriate.
5. Wash well in water.
6. Blue if necessary in alkaline tap water.
7. Wash well in water.
8. Counterstain as required. Dehydrate, clear and mount in synthetic resin.
Results: The nuclei and their components should now be stained in varying shades of blue/purple.

EXAMPLE OF PROCEDURE TO BE USED IF USING SURGIPATH HARRIS
HAEMATOXYLIN AS A COUNTERSTAIN:
1. After staining with the chosen technique, wash with water.
2. Immerse in Harris Haematoxylin for 30 seconds or until desired intensity has been reached.
3. Wash in water.
4. Blue if necessary in alkaline tap water.
5. Wash in water.
Results: Nuclei should now be stained light blue to provide contrast to the main staining technique.

PRINCIPLE OF THE METHOD: Haematoxylin is converted to Haematein by the use of an
oxidising agent. The oxidising agent may be natural or chemical. Haematein is the actual dye which
when combined with a mordant stains nuclei red. When the nuclei are washed with an alkaline solution
they will convert to a blue/black colour.

PERFORMANCE CHARACTERISTICS AND METHOD LIMITATIONS:
Haematoxylin is used in the preparation of nuclear stains whose recipes are many and varied. The
chosen protocol is employed as part of a routine staining procedure, which varies between users and
can be adapted to give an intensity and specificity required by the diagnosing pathologist. It is both
repeatable and reproducible within the bounds of the subjective nature of the procedure.
Under staining or over staining that is only noticed at the end of the procedure requires that a repeat
section should be stained, paying attention to control the times as indicated in the protocol. Due to the
subjective nature of the stain, precise times for each step are impossible to predict.

RECOMMENDATIONS:
1. Whichever formulation the user chooses to adopt it is recommended that a control section be tested
   daily before work commences to ensure the best possible results are obtained.
2. It is recommended that an adequate volume of solution be used in the staining dishes to ensure
   complete coverage of the section during staining.
3. It is recommended that any solutions made are filtered daily to avoid carryover of cells or tissue,
   and to remove any surface precipitate.
4. It is recommended that any solutions made are replaced with new solutions at least weekly.
5. Large throughput of slides may cause any solutions made to become diluted and it is
   recommended that the staining dishes be rotated to avoid over dilution.


Post-Market Surveillance: In the unlikely event that you should experience any problems with this
product, please contact Surgipath Europe Ltd. directly or your local supplier, and your complaint will
be dealt with promptly.

								
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