Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

This protocol can specifically detect calcium-containing nanocrystals in human urine. This technique could potentially be useful to predict early stages of kidney stone formation as well as other diseases associated with crystal urea. Demonstrating the procedure will be Dr.Parveen Kumar, a researcher IV in my laboratory.

Have participants consume a low-oxalate diet for three days and fast overnight. Then collect a 24-hour urine sample from the participants before having them consume an oxalate load. Feed the participants with a smoothie containing fruits and vegetables having an oxalate load of approximately eight millimoles.

Have participants collect their urine for 24 hours post-oxalate consumption and return it on the following day. Maintain all urine samples at room temperature prior to processing. Measure and record urine pH and volume of the urine samples.

Mix thoroughly and add 50 milliliters of urine into a labeled sterile 50 milliliter conical tube. Centrifuge the sample at 1, 200 times G for 10 minutes at room temperature using a bench-top centrifuge. Discard the supernatant, then wash and resuspend the pellet again with five milliliters of 100%ethanol.

Centrifuge the sample at 1, 200 times G for 10 minutes at room temperature using a bench-top centrifuge. Discard the supernatant and resuspend the pellet in one milliliter of 100%ethanol. Store the sample at minus 20 degrees Celsius for later processing or immediately proceed with staining the samples.

Dilute 100 nanometer-sized gold nanoparticles in ultrapure water at a ratio of 1:1, 000 and use them for optimizing the settings on the instrument. Dilute the urine samples 20 times in water prior to staining with five millimolar Fluo-4 AM for 30 minutes in the dark and analyze the samples using NTA. Prepare calcium oxalate and calcium phosphate crystals as described in the text manuscript prior to NTA analysis.

Turn on the computer and the instrument, then open the software and turn on the camera. Click the capture icon in the top left corner of the window to start the capture mode. Clean the platform by first pumping air into it using a one milliliter syringe until the platform appears clean.

Gently add water to the apparatus two to three times and use another one milliliter syringe to remove any air bubbles. Once the platform is clean, add water and check for any contamination on the surface by viewing the camera, then add gold nanoparticles as a control to the sample loading pump injector to set up the instrument. Adjust the camera level on the screen or on the knob to the right side of the instrument until the image starts to display colored pixels, then reduce the camera level.

Next, adjust the screen to optimize the image. Left click the mouse button on the video image. Hold the left mouse button and drag the image up and down to get the entire view.

Set up the infusion speed and focus the camera so that the gold nanoparticles are visible on the camera screen. Set the infusion speed to high for initial setup to ensure the gold nanoparticles are detected. Once detected, reduce the speed to 50 microliters per minute to visualize gold nanoparticles.

Adjust the camera level to visualize the particles. For unstained samples, adjust the screen gain at level five to achieve the camera focus and set the camera level at eight. Once the focus is set, record the sample.

After optimization, clean the apparatus again with water before assessing other samples to ensure that the tubing is clean and particles are not present. To analyze stained samples, adjust the camera to the filter position containing the suitable fluorescent filter and load diluted and stained samples onto the sample loading pump injector, reducing the speed to 20 microliters per minute for analysis. Adjust the screen gain to five and camera level to 13.

Save the data after each measurement. Calculate the average number of nanoparticles for all five readings for each individual sample and analyze the data using standard deviation or standard error of the mean. Use T-tests for paired analysis.

Fluo-4 AM was able to bind to both calcium oxalate crystals, which were between 50 and 270 nanometers in size and had a mean concentration of 1, 260 million particles per milliliter, and calcium phosphate crystals, which were between 30 and 225 nanometers and had a mean concentration of 2, 220 million particles per milliliter. 24-hour urine samples collected before the oxalate load contained some urinary nanocrystals between 110 to 300 nanometers in size. In contrast, there was a significant increase in urinary nanocrystals present in post-oxalate samples.

To confirm the reproducibility of the method, samples were measured three times and there was no significant variation in technical replicates. When attempting this protocol, there may be some difficulty with adding samples without bubbles to the machine. To prevent this, add air and water to the machine, and then slowly add samples.

Also, make sure to continually monitor your samples and that there are no bubbles present. This new method will be tested in individuals with calcium oxalate kidney stone disease to monitor disease progression and/or to predict kidney stone risk.