Evaluation of Histomorphometric Changes in Tissue Architecture in Relation to Alteration in Fixation Protocol - An Invitro Study

Abstract

Introduction: Preparation of good tissue specimens for microscopy requires complete fixation. No ideal fixative has been found till date, with every fixative showing advantages and disadvantages. Appropriate fixation is required to maintain clear and consistent morphologic features for histologic examination. Pathologists mostly examine formalin fixed tissue sections and are less used to the morphologic changes induced by other fixatives. Underfixed and overfixed tissue in various fixatives can lead to tissue architectural changes which can affect its diagnostic value.

Aim: To assess sectioning ability, staining intensity and microscopic details of tissues kept in different fixatives at different time intervals.

Materials and methods: Fresh tissue specimen i.e., goat tongue was collected and its middle-third portion was used for the study purpose. The tissue was grossed into 10 equal pieces and kept in various fixatives (10% Buffered formalin, Carnoy's solution, Absolute ethyl alcohol, Bouin's fluid) for five different time intervals (6, 12, 18, 24 and 30 hours) and normal tissue processing steps were carried out followed by sectioning and staining. During sectioning, sectioning parameter was assessed. Following sectioning, sections were observed under light microscope and were histologically evaluated for staining and microscopic details. To calculate the sectioning parameter Fisher's exact test was used and to assess parameters for staining and microscopic details Mann-Whitney U test was used.

Results: According to the study, 10% buffered formaldehyde is considered as a superior fixative under all parameters followed by Bouin's fluid, Carnoy's solution and Absolute alcohol.

Conclusion: In our study, it was concluded that 10% buffered formaldehyde should be continued as a routine fixative however, other fixatives can be used depending upon the non-availability of required fixative or in case of emergencies. Pathologist should be accustomed to histologic and morphologic changes of underfixed and overfixed tissue which can affect its diagnostic value.

Keywords: Absolute ethyl alcohol; Bouin’s fluid; Buffered formalin; Carnoy’s solution; Incomplete fixation; Over-fixation; Under-fixation.

[Table/Fig-1]:

Collection of specimen (goat tongue).

[Table/Fig-2]:

Immediate transfer of specimen.

[Table/Fig-5]:

Comparison of sectioning criteria. Graphs showing hard to cut (a) and crumbling (b) frequency during sectioning.

[Table/Fig-8e-h]:

Photomicrographs (10X) of tissue kept in Carnoy’s fluid for 6 hours (a) and 30 hours (b), in Absolute alcohol for 6 hours (c) and 18 hours (d). Photomicrographs (10X) of tissue kept in 10% buffered formaldehyde for 24 hours (a) and 6 hours (b), in Bouin’s solution for 24 hours (c) and 12 hours (d).