1. HEMATOXYLIN AND EOSIN STAIN
(H AND E STAIN)
Prepared by
Kanchan Shrestha
CMLT-3rd Year
2068 batch
Roll no. 13
2. Hospital: Kathmandu Model Hospital
Hospital type: 125 beded Community Referral Hospital
Department: Histopathololy and Cytopathology
Duration: 30 days
Total case performed: 291
Cancerous cells: 15
Benign cells: 276
Repeated case: 18
3. Objective
• To differentiate the tumor/cancer or benign cells from
autopsy and biopsy.
• To identify cell constituents demonstrated with H and E stain.
4. Theory
• The hematoxylin and eosin stain (H&E) is the most widely used stain in
histology and histopathology laboratories.
• When it is properly performed it has the ability to demonstrate a wide
range of normal and abnormal cell and tissue components.
• The hematoxylin component stains the cell nuclei blue-black, showing
good intra-nuclear detail.
• While the eosin stains cell cytoplasm and most connective tissue fibers in
varying shades and intensities of pink, orange, and red.
• Hematoxylin is extracted from the heartwood of the central American
logwood Haematoxylon campechianum Linnaeus. Heamatoxylon is
derived from Greek, haimatodec (blood - like) and xylon (wood).
• Hematoxylin by itself cannot stain. It must first be oxidized to hematein.
• Eosin can be used to stain cytoplasm, collagen & muscle fibers for
examination under the microscope. Structure that stain readily with eosin
are termed eosinophilic.
5. Principle
The acidic component of the cells has the
affinity to basic dye and basic component of
the cells has the affinity to acidic dye.
In hematoxylin and eosin stain, hematoxylin
stains the acidic part of the cell, i.e. Nucleus.
So hematoxylin is called as nuclear stain .
While eosin acts as a acidic stain and bind
with the basic part of the cell, i.e cytoplasm of
the cells staining pink.
7. Procedure
After the formalin fixation, tissue processing and trimming
process:
The thin ribbon like structure was made by fine cutting, this
was taken in new grease free albuminated slide in a centre.
And after dried and this was taken in H&E staining by
following procedure:
8. continued…
I. Left Xylene I,II&III for 8 minutes.
II. Absolute alcohol---------10 dips.
III. 90% alcohol --------10 dips.
IV. 80% alcohol ---------10 dips.
V. Washed in tap water.
VI. Hematoxylin stain for 20 minutes.
VII. Washed in tap water.
VIII. 90% alcohol ------------10 dips.
IX. 1% acid alcohol-----------1-2 dips.
X. Washed in water.
XI. 95% alcohol ---------10 dips.
XII. 1% lithium carbonate ------1-2 dips.
XIII. Washed in tap water.
XIV. Absolute alcohol---------10 dips.
XV. Eosin stain for 4 minutes.
XVI. Washed in tap water.
XVII. Finally dried and mounting by DPX. Now it is ready for microscopic examination.
9. Result
Total performed specimens = 291
Cancerous cell = 15
Benign cell = 276
Repeated sample = 18
Out of 100% specimens 5.2 of sample was detected as
a cancer cell remaining 94.8% was benign cells.
Expected result from stains on normal cells.
Nuclei : blue
RBCs : orange to pink
Cytoplasm : pink
10. 100
90
80
70
60
50
40
30
20
10
0
Performed
specimen
Cancerous cell Benign cell
Fig: Performed result
Series 1
Column2
Column1
11. Fig: Cancerous Cell of brain (Diagnosed as Gliobastoma
multiforme (WHO – Grade IV)
12. ACKNOWLEDGEMENT
I am very grateful to Ojesbi Academy for provided me an
opportunity to completed all laboratory practical directly in
the hospital. Also I would appreciate Kathmandu Model
Hospital Administration and general staff members for gave
me a chance to completed all my practical (On the job
training – OJT). I would like to thank pathologist Dr.
Shrestha Yagya because she gave me a chance to find out
cancer cell in brain. I would like to thank Rajan Malla
Thakuri because he gave me an opportunity of presentation
in “HEMATOXYLIN AND EOSIN STAIN”.I’m obliged with all
my friends for their kind support and discussion about
laboratory rule, procedure, and safety measures.
