Table 2.

Issues related to fixatives in relation to cell-blocking

	Fixatives	Histology	Immunocytochemistry	Molecular testing
1	Formalin[40]	Sections of resultant FFPE would show histomorphology comparable to that with formalin-fixed biopsies and resections	IHC results would be comparable to that with published data predominantly based on FFPE studies	The limiting factor with FFPE is fragmentation of DNA with associated artifacts during sequencing with potential interference RNA-based test (other than miRNA) may be affected due to low yield. However, most of the methodologies are standardized on FFPE
2	Chemical-based fixatives including the fixatives with heavy metal (B5, Zenker’s fixative) or Acidic solutions (Picric acid, Bouin’s fixative)[41,42]	Histomorphology is not affected significantly and is comparable to that with formalin-fixed biopsies and resections Toxicity hazard (e.g., mercury poisoning with Zenker’s fluid)	Morphologically good immunostaining, but results may NOT be comparable to that with FFPE with which the results will be compared. This may lead to aberrant immunoprofile with liability due to potential compromisation of patient care	Little data related to stability of nucleic acids (Some such as picric acid results in DNA damage)
3	Alcohol  Methanol in PreservCyt and CytoLyt used in LBC  Ethanol in SurePath LBC  Cellient™ CB[43,44]	Histomorphology is not affected significantly and is comparable that with formalin-fixed biopsies and resections Shrinkage-related artifacts may interfere	Immunoreactivity may be affected with erroneous immunoprofiles resulting in suboptimal interpretation outcome. This is especially applicable to nuclear immunomarkers including ER/PR, Ki-67, PCNA, p53, S-100 protein, etc.,[9]	Standardized tests/protocols may be required

B5: A fixative; IHC: Immunohistochemistry; LBC: liquid-based cytology; PCNA: Proliferating cell nuclear antigen, ER: Estrogen receptors, PR: Progesterone receptor