Rapid Tissue Clearing in Pre-warmed Xylene Without Compromising Staining Adequacy and Histoarchitecture

Abstract

Aims: To determine the adequacy of nucleo-cytoplasmic staining characteristics and preservation of nucleo-cytoplasmic morphology of mouse liver tissue cleared in pre-warmed xylene at predetermined temperatures and durations using haematoxylin and eosin staining procedure. Study Design: Tissues for clearing were first divided into 3 broad experimental groups (A, B and C) based on pre-determined tissue clearing temperature. Each broad group were further divided into 4 sub-groups (A1 to A4, B1 to B4 and C1 to C4) based on duration of tissue clearing. Place and Duration of Study: Department of Medical Laboratory Science, Babcock University, Ilishan-Remo, Nigeria and Department of Oral Pathology, College of Medicine, University of Ibadan, Nigeria. January-June, 2015. Methodology: Tissues assigned to sub-groups A1 to A4 were cleared in pre-warmed xylene at 25°C for 30, 45, 60 and 90 minutes respectively, while tissues assigned to sub-groups B1 to B4 were cleared in pre-warmed xylene at 30°C for 30, 45, 60 and 90 minutes respectively and tissues assigned to sub-groups C1 to C4 were cleared in pre-warmed xylene at 35°C for 30, 45, 60 and 90 minutes respectively. As a consequence of this procedure, adequacy of nucleo-cytoplasmic staining characteristics and preservation of nucleo-cytoplasmic morphology were subsequently determined. Results: Adequacy of nuclear and cytoplasmic staining were both assessed using a 2-point grading scale of 0 being inadequate and 1 being adequate, while preservation of both nuclear and cytoplasmic morphology were also assessed using a 2-point grading scale of 0 being poorly preserved and 1 being well preserved. Adequate nuclear and cytoplasmic staining were observed in tissues cleared in pre-warmed xylene at 25°C for 45 and 60 minutes, at 30°C for 30 and 45 minutes, at 35°C for 30, 45, 60 and 90 minutes. Well preserved nuclear morphology were observed in tissues cleared in pre-warmed xylene at 25°C for 30, 45 and 60 minutes, at 30°C for 30 and 45 minutes and at 35°C for 30, 45, 60 and 90 minutes. However, only tissues cleared at 35°C for 30, 45 and 90 minutes exhibited well preserved cytoplasmic morphology.                                                                Conclusion: Adequate nucleo-cytoplasmic staining and well preserved nucleo-cytoplasmic morphology of tissues for histopathologic demonstration may be achieved rapidly by clearing tissues in 35°C pre-warmed xylene for as short as 30 minutes.