Effectiveness of neutral honey as a tissue fixative in histopathology

Abstract

Background: In routine histopathology, 10% neutral buffered formalin (NBF) is the choice fixative. However, formalin is a human carcinogen, so there is a necessity for a safer alternative. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological samples. This study determined the effectiveness of neutral buffered honey and other types of fixatives to fix histological tissues. Methods: The study was conducted between July 2019 and August 2020 at Sultan Qaboos University, Oman. Sections from three rat livers, kidneys, and stomach tissues were fixed with 10% NBF, neutral buffered Sumer honey, neutral buffered date honey, formalin, Sumer honey, date honey, alcoholic formalin, alcoholic Sumer honey, and alcoholic date honey for 24 hours. Samples were stained with hematoxylin and eosin (H&E), special stains, and vimentin methods. Three expert biomedical scientists then evaluated the fixed and stained samples for the quality of all sections. The fixation ability of the different honey solutions was then compared to 10% NBF and the utility was determined using nuclear and cytoplasmic criteria, specificity, and intensity. Results: H&E showed adequate staining in all groups compared to 10% NBF. The specificity and intensity of all groups for the Periodic acid-Schiff method were identical to 10% NBF except for Sumer honey and alcoholic date honey. Vimentin showed comparable findings with 10% NBF as there were no significant differences. Conclusions: The findings of this study encourage the use of honey, including neutral, as a possible safe substitute fixative for formalin, however, further experiments on larger specimens should be conducted.

Keywords: Histopathology; fixation; formalin; honey; neutral buffered.

Figure 1.. Hematoxylin and eosin-stained liver sections of different fixative groups.

(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).

Figure 2.. Jones’ Methenamine silver-stained kidney sections of different fixative groups.

(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).

Figure 3.. Periodic acid–Schiff-stained kidney sections of different fixative groups.

(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).

Figure 4.. Vimentin-stained kidney sections of different fixative groups.

(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).