Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

Abstract

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden.

A novel fluorescein-bisphosphonate based diagnostic tool for the detection of hydroxyapatite in both cell and tissue models

“…The ready availability of the FDA-approved probe components makes the alendronate-based Fluorescein-BP conjugate 1 particularly attractive for development and human translation as a method for the detection of calcification. Recently the Leong group reported the use of Fluorescein-BP 1 for the study of urinary calculi 15 ; although the fluorescence intensity of the probe in the presence of macroscopic calcium oxalate (CaOx) was found to be relatively moderate.…”

“…Fluorescein-BP was synthesised following the procedure reported by the Leong group 15 . Specificity of the probe towards HAP was shown by incubating Fluorescein-BP with a range of calcium minerals including HAP, calcium pyrophosphate (CaPy), CaOx and calcium phosphate (CaPi).…”

“…In this study, the fluorescein-bisphosphonate conjugate 1 (Fluorescein-BP, Fig. 1 ) 15 was repurposed to allow for a rapid and inexpensive method of studying calcification and its use was investigated in a range of cell based models as well as mouse and human vascular tissue.

Figure 1 Selectivity towards calcium minerals of Fluorescein-BP ( 1 ) in comparison to Alizarin S stain ( 2 ).

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Drosophila melanogaster as a function-based high-throughput screening model for antinephrolithiasis agents in kidney stone patients

“…We previously demonstrated that alendronate-fluorescein isothiocyanate (FITC), a fluorescently labeled bisphosphonate, binds to oxalate calculi via petrographic thin sections of calcium oxalate kidney stones and to nanocrystals in patient urine samples ( Gavin et al, 2016 ). Pulverized calcium oxalate kidney stones were stained with alendronate-FITC and hydroxyapatite nanoparticles were used as a positive control, revealing a high binding affinity of the alendronate-FITC bisphosphonate probe to calcium oxalate calculi ( Fig.…”

“…Stone samples were collected using either duckbill forceps or an endoscopic grasping basket and deposited directly into a sterile container and stored at −20°C. Patient data, stone and urine analysis data, including oxalate crystal data, were previously presented in an independent report ( Gavin et al, 2016 ).…”