Enumeration of islets by nuclei counting and light microscopic analysis

Area of Science:

• Endocrinology
• Cell Biology
• Transplantation Research

Background:

• Islet enumeration is critical for pancreatic islet transplantation and research.
• Conventional dithizone staining and visual counting methods are prone to inaccuracies and operator variability.

Purpose of the Study:

• To develop and validate a novel, accurate method for enumerating islet equivalents (IEs) in impure islet preparations.
• To compare the accuracy of the new method against the standard dithizone staining technique.

Main Methods:

• Disruption of islet preparations into single cells and nuclei using lysis solution and shear.
• Accurate nuclei counting using visual counting, aperture electrical resistance, and flow cytometry.
• Morphological analysis via light microscopy (LM) and stereological point counting to determine islet volume fraction.
• Calculation of islet equivalents (IEs) by combining nuclei counts and islet volume fraction.

Main Results:

• Nuclei counting methods demonstrated accuracy and precision, with DNA content per cell aligning with literature values.
• The novel method combining LM and nuclei counting provided more accurate islet enumeration compared to dithizone staining.
• The standard dithizone staining method overestimated islet equivalents by approximately 90% in human islet preparations.

Conclusions:

• Nuclei counting combined with light microscopy-based islet volume fraction analysis is a reliable and accurate method for islet enumeration.
• This novel approach significantly improves the precision of islet quantification, essential for research and clinical applications.
• The findings highlight the limitations of traditional dithizone staining and advocate for the adoption of more accurate enumeration techniques.