Haematoxylin and eosin (H&E) stain - Principle, Reagents, Procedure, Result, Advantage, Disadvantage

Introduction to Haematoxylin and eosin (H&E) stain

Haematoxylin and eosin (H&E) stain or HE stain is a widely used staining technique in medical diagnosis and is often called the gold standard. It is mostly used in histo-pathology to book biopsy and histological sections.

Haematoxylin and eosin (H&E) stain is a combination of two histological stains – haematoxylin (cationic/positively charged stain) and eosin (anionic/negatively charged stain). Hematoxylin has a deep blue-purple colour and stains nucleic acids while Eosin is pink and stains proteins nonspecifically.

It has been used for more than a century as it displays a broad range of cytoplasmic, nuclear, and extracellular matrix features and morphologic changes that form the basis of contemporary cancer diagnosis.

History of Haematoxylin and eosin (H&E) stain

Haematoxylin and eosin (H&E) stain was first used by chemist N. Wissozky. In the year 1877, he introduced the stain at the Kazan Imperial University in Russia.

Principle of Haematoxylin and eosin (H&E) stain

The Haematoxylin stains the cell nuclei purplish blue while the eosin stains the cytoplasm and xtracellular matric pink. Other cellular structures take on different shades and combinations of both Haematoxylin and eosin stains.

After staining, differentiation can be easily made between the nucleus, cytoplasmic parts, general layout, and distribution of cells. Overall, Haematoxylin and eosin (H&E) stain gives a general overview of a tissue sample's structure. Then the histological information of the tissue can be provided by both expert humans and/or by digital pathology.

Reagents of Haematoxylin and eosin (H&E) stain

1. Harri’s Hematoxylin stain

   A = 1 gm hematoxylin in 10 ml ethanol

   B = 20 gm ammonium alum in hot distilled water

   Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.

2. Eosin solution

   Yellow eosin = 1 gm

   Distilled water = 80 ml

   Ethanol = 320 ml

   Glacial Acetic Acid = 2 drops

3. 0.5% HCl

4. Dilute ammonia water

Procedure of Haematoxylin and eosin (H&E) stain

The procedure for Haematoxylin and eosin (H&E) stain is as follows:

1. The deparaffinization step includes flaming the slide on the burner placing it in xylene and repeating this step to remove the wax.

2. Drain xylene and hydrate the tissue by passing through water and decreasing the concentration of alcohol baths (100%, 90%, 80%, 70%).

3. Add haemoxylin to the tissue section for 3 to 5 minutes to stain the nuclei.

4. Wash gently in running tap water for 5 minutes.

5. Dip in 1% acid alcohol (1% HCl in 70% alcohol) for a few seconds for differentiation (selective removal of excess dye from tissue section).

6. Rinse gently in running tap water for a few seconds.

7. Dip in ammonia water until the section turns blue.

8. Rinse gently in running tap water for a few seconds.

9. Add 1% eosin Y (counterstain) for 10 minutes.

10. Dehydrate the section in increasing concentration of alcohol.

11. Place the slides in two xylene baths for clearing.

12. Mount in DPX or other mounting media.

13. Observe under a compound microscope.

Result, Interpretation of Haematoxylin and eosin (H&E) stain

• Nuclei: blue, dark-purple

• Cytoplasm: Pink/purplish pink

• Muscle fibres: deep red

• RBCs: intense red

• Calcium: Dark blue

• Mucin: Grey-blue

Advantage of of Haematoxylin and eosin (H&E) stain

• Is a quick procedure.

• It is a cheap process so used in routine diagnosis.

• Requires compound microscope.

Disadvantage of of Haematoxylin and eosin (H&E) stain

• Does not always provide enough contrast to differentiate all tissues, cellular structures, or the distribution of chemical substances.