The impact of pre-analytic variables on kidney glomerular ultrastructure
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Citation
Abstract
Chronic kidney disease is estimated to affect 37 million Americans, or more than 1 in 7 adults in the United States. Podocytopathies, a cohort of diseases where the primary injury occurs directly to the podocyte, are contributors that lead to progression of chronic kidney disease. The main prototypical feature of podocyte injury is foot process effacement. This is when podocytes, a key supporting cell in the glomeruli, lose their alternating foot process pattern. This may represent a mechanism whereby a foot process will flatten to cover the area where podocyte loss has occurred to maintain integrity of the filtration barrier. Podocyte foot process structure is evident at the sub-micron (ultrastructural) scale. In order to accurately observe podocyte injury and foot process effacement, it is necessary to use transmission electron microscopy (TEM). When analyzing ultrastructural images, optimizing the quality of tissue processing and handling is critical to ensuring a high-quality result. However, in routine clinical and research work, handling errors are inevitable and constitute a source of pre-analytic variation, and therefore it is important to understand the impact of these errors, and how to recover from them. Common handling deviations include placement in or exposure to non-standard fixatives and preservatives, or delays in placing tissue in fixative.
In this research we investigated how pre-analytic variables associated with tissue handling pre-fixation impact the ultrastructure of the glomerulus. First, we investigated the impact of tissue exposure to Zeus preservative, a preservative of tissue for frozen section immunostaining, on glomerular ultrastructure. Additionally, we analyzed the impact of Zeus preservative exposure on foot process width (FPW) measurement in electron micrographs. Finally, we investigated time delay to placement of tissue into fixative. These investigations included both qualitative and quantitative assessment of glomerular ultrastructure.
In the tissue samples exposed to Zeus preservative prior to fixation, we were able to show that it may be possible to extract useful and accurate morphologic information. Tissue may need to be washed according to manufacturer’s protocol prior to being processed for EM depending upon what information is required. We also found that a time delay does have a significant impact in the FPW measurements. Taken together, these findings indicate that tissue handling prior to ultrastructural analysis can have important impacts on qualitative and quantitative assessment, but that it may be possible to mitigate these effects.
Description
2024