1.0 Purpose
To establish a standardized procedure for Hematoxylin &
Eosin (H&E) staining of tissue sections.
2.0 Scope
This procedure applies to all tissues received for
processing, blocks received for review, or as a preliminary step before immunohistochemistry
(IHC) staining.
3.0 Introduction
Hematoxylin and Eosin (H&E) staining is the most widely
used staining technique in histology. Hematoxylin is used for nuclear staining,
while eosin is used for cytoplasmic staining. The procedure provides an
overview of the tissue, allowing differentiation between normal, inflamed, or degeneratively changed structures, and is often sufficient for diagnosis. It is
applicable to paraffin sections, frozen sections, and clinico-cytological
specimens.
4.0 Principle
H&E staining involves two main steps:
1. Nuclear Staining: Hematoxylin stains the nuclei blue to
dark violet/black.
2. Counterstaining: A xanthene dye (eosin Y, eosin B, or
erythrosin B) stains the cytoplasm, collagen, keratin, and erythrocytes red.
This standard staining method enables differentiation of tissue structures, which is critical for determining if further specialized staining techniques are necessary.
5.0 Analytical Measuring Range and Limit of Detection
Not applicable.
6.0 Responsibility
- Technical Staff
- Pathologist
7.0 Abbreviations
- H&E: Hematoxylin & Eosin
- DPX: Distrene Dibutylphthalate Xylene
8.0 Requirements
- Hematoxylin
- Eosin
- Alcohol
- Xylene
- DPX
- Coplin jars/Staining troughs/Slide rack/Slides
Disposables:
- Not applicable
- Not applicable
- Handle all specimens with gloves, mask, and goggles.
- Handle all instruments with care to prevent injury.
Not applicable.
11.0 Documentation
Maintain the following records:
- Daily Record Sheet: H&E Staining
- Record of Change of Stain: H&E
12.0 Instructions
- Rinse a clean bottle with distilled water.
- Take 80 ml of ethanol in the reagent bottle.
- Add 20 ml of distilled water.
- Discard 2 ml of the solution.
- Add 2 ml of concentrated hydrochloric acid.
- Mix well by shaking. The acid-alcohol solution is ready
for use.
- Dissolve 2.5 gm of hematoxylin in 25 ml of absolute
alcohol (Solution A).
- Rinse a 2-liter flask with distilled water.
- Take 500 ml of distilled water in the flask.
- Add 50 gm of potassium alum and mix well using heat
(Solution B).
- Mix Solution A with Solution B.
- Boil carefully and gently.
- Remove from heat and add 1.25 gm of mercuric oxide.
- Boil for 5 minutes.
- Cool rapidly by placing the flask in cold water or on
chipped ice.
- Once cold, add 20 ml of glacial acetic acid.
- The stain is ready for use. Filter before use.
- Rinse the reagent bottle with distilled water.
- Take 20 ml of distilled water.
- Add 1 gram of eosin powder and mix well.
- Add 20 ml of ethanol and mix well.
- Add 1 ml of glacial acetic acid and mix well.
- Let the stain stand for 24 hours before use.
- Prepare a working solution by mixing 20 ml of stock
solution with 20 ml of ethanol. Filter before use.
- Rinse the reagent bottle with distilled water.
- Take 100 ml of distilled water.
- Add 1 gram of eosin powder and mix well.
- Add a few drops of glacial acetic acid and mix well.
- The stain is ready for use. Filter before use.
12.2.2 Wash in water.
12.2.3 Dip slides in hematoxylin for 5-10 minutes.
12.2.4 Wash in running tap water.
12.2.5 Give one dip in acid-alcohol.
12.2.6 Wash in running tap water for 3-5 minutes for
blueing.
12.2.7 Check under a microscope.
12.2.8 Dip in eosin for 1 minute.
12.2.9 Dip in 90% alcohol for 3 minutes.
12.2.10 Dip in absolute alcohol for 3 minutes.
12.2.11 Dip in xylene I for 1-3 minutes.
12.2.12 Dip in xylene II for 3 minutes.
12.2.13 Mount in DPX.
12.2.14 Label the slides.
- At the beginning of each daily run, one tissue section is
reviewed for staining quality. Additional sections are stained only if the
initial results are acceptable or after necessary corrective actions.
- Not applicable.
- Slides and blocks are stored for 10 years at room
temperature.
13.0 Reference Range
Not applicable.
14.0 Critical Value
Results are reported as per the Work Instruction (WI) for
reporting results.
15.0 Reporting of Results and Interpretation
Results are reported based on the morphology of tissues in
sections and are documented in an approved format.
16.0 Contingency Plan
In case of reagent shortages, manpower issues, or instrument
breakdowns, multiple trained staff members are available as backups to ensure
continuity.
17.0 References
- Bancroft, J.D. *Theory and Practice of Histological
Techniques*, Fourth Edition.
END OF THE DOCUMENT
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