Problems and Solutions in Histological Technique

 

Problems in Histopathological Technique

 

Prepared by

ROY ELLIS

IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

Email: roy.ellis@imvs.sa.gov.au

 

 

 

PROBLEM NUMBER 32

Cells in stained tissue sections appear crenated

 

This is a section of kidney stained Haematoxylin and Eosin showing crenation in cells and overstaining with eosin. The eosin definitely lacks differentiation.

 

Cause?

 

This effect is caused by hypertonic solutions resulting in water flow from the cell into the fixative solution during fixation. Its a difference in osmolarity between the tissue and the fixative. Osmolarity of fixatives needs to be balanced to that of the tissue being preserved. Its best to use an isotonic fixative solution which exerts the same osmotic pressure as the cells in tissue, in other words a fixative made up in physiological saline such as a buffered formol saline or neutral buffered formalin.

Solution

 

The best average is achieved with aqueous fixatives, not alcoholic, buffered to a neutral pH. In fact the effect of crenation is seen more markedly with fixatives which contain alcohols, like Carnoy’s and is sometimes observed in cytology preparations when absolute alcohol, absolute isopropanol or methanol are used as the fixative. Better osmolarity is achieved by using 80 to 95% ethanol, 60% isopropanol or 95% methanol. There will still be some shrinkage of cellular material, but the crenated effect is avoided.

 

This is also a section of kidney stained H&E but showing well rounded, well preserved nuclei and with differential eosin staining.

Just as it should be after fixation in neutral buffered formalin.

 

 

 

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© Roy C. Ellis 2002