HEMATOXYLIN AND EOSIN STAIN(H AND E STAIN)

Prepared by

Kanchan ShresthaCMLT-3rd Year2068 batch

Roll no. 13

Hospital: Kathmandu Model Hospital

Hospital type: 125 beded Community Referral HospitalDepartment: Histopathololy and CytopathologyDuration: 30 daysTotal case performed: 291Cancerous cells: 15Benign cells: 276Repeated case: 18

Objective

• To differentiate the tumor/cancer or benign cells from autopsy and biopsy.

• To identify cell constituents demonstrated with H and E stain.

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.

Principle

The acidic component of the cells has theaffinity to basic dye and basic component ofthe cells has the affinity to acidic dye.

In hematoxylin and eosin stain, hematoxylinstains the acidic part of the cell, i.e. Nucleus.So hematoxylin is called as nuclear stain .While eosin acts as a acidic stain and bindwith the basic part of the cell, i.e cytoplasm ofthe cells staining pink.

Requirements

• Xylene (I & II)

• 10% formalin

• Alcohol (70%, 80%, 90%, 95%, absolute I & absolute II)

• 1% lithium bi-carbonate

• Histokinate

• Microtome

• Microtome knife

• DPX ( mounting xylene)

• H&E stain

• Microscope(light or electron microscope).etc

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:

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.

Result

Total performed specimens = 291Cancerous cell = 15Benign cell = 276Repeated sample = 18Out of 100% specimens 5.2 of sample was detected asa cancer cell remaining 94.8% was benign cells.

Expected result from stains on normal cells.Nuclei : blueRBCs : orange to pink

Cytoplasm : pink

Fig: Performed result

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Performedspecimen

Cancerous cell Benign cell

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Fig: Cancerous Cell of brain (Diagnosed as Gliobastomamultiforme (WHO – Grade IV)

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 MallaThakuri 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.