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Microwave-stimulated formaldehyde fixation of experimental renal biopsy tissues: computerised morphometric analysis
1Department of Pathology University of Malaya Medical Centre, Kuala Lumpur, Malaysia.
Abstract:
Although microwave irradiation has been used in the histopathology laboratory for several years, there has been minimal published experimental data on its effects on the technical and staining quality of histological sections. Furthermore, it has not been clear whether the advantages gained in reduction of fixation and staining duration has been at the expense of increasing architectural distortion to the tissues. We report here our experience with computerised morphometric analysis to investigate glomerular artifacts caused by microwave-stimulated fixation of renal tissues. 39 rat and 33 human autopsy kidney samples were subjected to (1) fixation in neutral buffered formaldehyde (control), (2) microwave-stimulated fixation followed by neutral buffered formaldehyde, and (3) neutral buffered formaldehyde followed by microwave irradiation. In addition, the effect of post-fixation in 70% ethanol was also investigated. Microwave irradiation was delivered through a dedicated laboratory microwave oven at 80% power and at 55 degrees C for 3 minutes. The different fixation methods were compared with regards to shrinkage (distortion) to glomerular structures (glomeruli and Bowman's spaces) on H and E sections, as determined by morphometric image analysis using a temporary assembled-system consisting of a trinocular microscope, a digital video camera and personal computer. A FlashPoint VGA 3.3 film-grabber card was used to capture images for morphometric analysis by using a Scion Image program. Morphometric analysis of glomerular structures showed that microwaves caused more shrinkage to the area bounded by the Bowman's capsule than the glomerulus proper, but post-fixation with ethanol reduced this shrinkage. These findings have implications on the logistics of tissue preparation of renal biopsies in clinical practice.
Insights
Microwave fixation in histopathology causes glomerular shrinkage, particularly in Bowman's capsule. Post-fixation with ethanol can reduce this distortion in renal tissues.
Area of Science:
- Histopathology
- Renal Pathology
- Biomedical Engineering
Background:
- Microwave irradiation is used in histopathology labs, but its effects on tissue quality and architectural distortion are not well-documented.
- The potential benefits of reduced fixation and staining times need to be weighed against possible tissue artifact.
- Computerized morphometric analysis offers a precise method to quantify tissue changes.
Purpose of the Study:
- To investigate glomerular artifacts caused by microwave-stimulated fixation of renal tissues using morphometric analysis.
- To compare the shrinkage of glomerular structures under different microwave fixation protocols.
- To evaluate the effect of ethanol post-fixation on microwave-induced tissue distortion.
Main Methods:
- 39 rat and 33 human kidney samples were subjected to control fixation, microwave-stimulated fixation, and sequential microwave/formaldehyde fixation.
- Microwave irradiation was performed at 80% power and 55°C for 3 minutes.
- Morphometric image analysis quantified shrinkage in glomeruli and Bowman's spaces on H&E stained sections.
Main Results:
- Microwave irradiation induced greater shrinkage in the area bounded by Bowman's capsule compared to the glomerulus proper.
- The observed shrinkage artifact was reduced when samples underwent post-fixation in 70% ethanol.
- Different microwave fixation protocols showed varying degrees of glomerular distortion.
Conclusions:
- Microwave fixation can lead to architectural distortion in renal tissues, specifically affecting glomerular structures.
- Ethanol post-fixation is a viable method to mitigate microwave-induced shrinkage artifacts.
- These findings are crucial for optimizing tissue preparation protocols in clinical renal biopsy analysis.
