Effectiveness of 70% ethanol as a tissue fixation agent in preservation of human tissue morphology

Formaldehyde is the preferred fixative in routine histopathology sample processing. However, the use of formaldehyde is demoted by a variety of drawbacks, including potential health risks, tissue shrinkage, artefactual pigmentation, etc., impacting the quality of the stained sections and affecting certain diagnostic techniques. Alcohol-based fixatives are relatively non-toxic and, therefore, a safer option for tissue fixation. The present study was to assess the effectiveness of 70% ethanol as a tissue fixative in preserving human tissue morphology at different time durations. A total of 40 histology samples including thyroid, breast, appendix, uterus, gallbladder, kidney, etc. of comparable size (thickness; 0.5 – 1 mm) were fixed in 10% formal saline (One part of formaldehyde: nine parts of distilled water: 8.5g of NaCl) and 70% ethanol (6.65 parts of 95% ethanol: 3.35 parts of distilled water) for 24 hours, and two weeks respectively for the assessment of histology on Hematoxylin and Eosin (H & E)-stained tissue sections according to a semi-quantitative scoring system. Ethanol fixation resulted in better preservation of nuclear details, uniformity in H & E staining, and the absence of artefactual pigment depositions. However, preservation of cytoplasmic details and extracellular components was poor in ethanol-fixed sections compared to that of formal saline. The nuclear (24 hour; p=0.174, two-weeks; p=0.000) and cytoplasmic shrinkage (p=0.000 in both 24 hours and two weeks) was more significant in ethanol fixation. Ethanol fixation for two weeks led to worsened nuclear vacuolization, nuclear shrinkage, cytoplasmic cohesiveness, uniformity, texture, cell border preservation, and cytoplasmic shrinkage/swelling in tissues. In conclusion, the results revealed that 70% ethanol fixation was effective in 24 hour fixation of human tissues, however, there were limitations in fixation in 70% ethanol for two weeks. Further studies are warranted for the development of alternative ethanol-based fixatives for applications requiring extended tissue fixation.