Enumeration of islets by nuclei counting and light microscopic analysis

Abstract

Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with: (1) visual counting in a hemacytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg DNA/cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about 160 or more islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IE). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared to LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from light microscopy is a novel method for achieving accurate islet enumeration.

Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

“…2 and Table 4) and were comparable to expected values, 16,18 thereby verifying the quantitative reliability of both nuclei counting and DNA measurement with tissue encapsulated in alginate. Data suggested that 10% (w/v) Triton X-100 in the lysis buffer resulted in effective liberation of nuclei from cells in barium alginate.…”

“…15,16 Examination of viability by assessment of membrane integrity with vital stains such as fluorescein diacetate/propidium iodide does not reflect true viability because it may not account for cells undergoing early cell death processes, during which cells have not yet developed damage to their cell membrane. 8,9,17,18 Measures of cellular energetic state, including ATP content [19][20][21][22][23][24] and ATP/ADP and related ratios, [25][26][27] have been investigated with modest success.…”

“…Combination of this measurement with purity data from light microscopy provided accurate measurement of the number or volume of islet cells. 16 Cell nuclei were conserved during lysis of islet tissue, 16 which provided an advantage over dissociation into cells that invariably entails cell loss. Last, combination of nuclei counts and DNA measurements provided insight into the extent of recent cell damage.…”

“…16 For cell suspensions in dissolved alginate with or without PFC, a 100-mL sample was added to an equal volume of lysis solution (0.1 M citric acid and 1%, 2%, or 10% [v/v] Triton X-100; Sigma Aldrich) and incubated for 5 min with vortex mixing three times. For islets, the dissolved sample was resuspended in 100 mL PBS before adding 100 mL of lysis buffer (0.1 M citric acid and 10% [v/v] Triton X-100) to remove EDTA, which interfered with nuclei liberation, and incubated for 15 min with vortex mixing three times.…”

Classification of microscopy images of Langerhans islets

“…However, these techniques turn out to perform poorly in predicting the transplantation success, because the content of the mentioned substances varies from donor to donor. Pisania et al, 11 2010, evaluated the number of islet nuclei taking advantage of their homogeneity. Other methods are based on estimation of the amount of tissue from the extracted DNA (Stegenmann et al, 1998) or the total protein contents.…”