Muco-obstructive diseases are characterized by abnormal viscosity of the airway mucus. The protocol shows how to use rheology to quantify mucus viscosity and analyze data to test mucus-targeting treatments. Mucus rheology provides specific quantitative biomarkers.
Using a benchtop device greatly simplifies the logistics of measuring these biomarkers in clinical specimens like human sputum samples. Using mucus rheology as a diagnosis tool for obstructive diseases is still challenge. An increasing body of evidence shows a strong correlation between rheology properties, inflammation, and bacterial infection.
Mucus is always present at the body interface where it lubricates and plays a protective role. Mucus rheology can be applied to other systems like the gastrointestinal and genital tracks. To acquire accurate measurements, correct handling and placement of the sample is critical.
A visual demonstration can enhance the precision of this assay. Demonstrating this procedure will be Kendall Shaffer, a research technician from the air laboratory. Begin by sorting the collected airway sputum or mucus in sterile specimen cups.
In the case of sputum, remove excess saliva from the sample immediately upon collection. Place the samples on ice for transport. Limit the transport time to less than four hours.
Analyze the samples at the time of collection. Homogenize the mucus by gently pipetting up and down three to five times with a positive displacement pipette. Or pipette directly into the microcentrifuge tubes.
Store the samples at 80 degrees Celsius until processed. Aliquot the samples for storage in volumes greater than or equal to 500 microliters to ensure sufficient volume for experiments. Before aliquoting, pipette fresh and frozen sputum or mucus directly or homogenize specimens.
Repair the required aliquots for repeat measurements and treatments. Incubate the aliquots to be tested at 37 degrees Celsius for at least five minutes prior to measurement. For testing pharmacological agents, use high concentrations of stock solutions to prevent sample dilution.
Add between 1%to 10%volume of the desired reagent directly onto the sample. Make sure no drop of the compound stays on the side of the tube. Incubate the samples at 37 degrees Celsius for the required length of time, to allow a chemical reaction.
Mix the mucus sample and reagent by flicking the bottom of the microcentrifuge tube every two minutes to allow progressive penetration of the reagent into the mucus sample without compromising the mucin network. When comparing multiple drug reagents, ensure that the incubation time is similar. Turn on the machine and initialize the software.
Select new measurement. Enter the sample identification number under Measure ID and the name of the operator under Operator to continue. Enter additional information or comments under Comments.
Select a geometry set and inspect large and small plates carefully, to ensure that plates are clean and in perfect condition. Insert the large plate firmly on the bottom pulpit. Insert the small plate gently on the upper pulpit and lock the plate by slightly rotating until hearing a click, which indicates that the plate is properly clamped.
Note that free oscillation of the upper plate is normal. Wait until the temperature reaches 37 degrees Celsius target value. Then initiate automatic calibration, as prompted by the software.
Using a positive displacement pipette, slowly pipette between 250 and 500 microliters of the sample on the center of the large bottom plate. Once deposited on the plate, viscous samples will adopt a dome shape, whereas, highly elastic samples may require physical severing. Lower the measuring head carrying the small plate via the software and observe the sample.
If properly loaded on the bottom plate the sample will make contact and be centered between the two plates. To ensure that the sample fills the gap, use the reduced gap function until the sample is no longer in the biconcave shape or aligned with the plate's edge. The reduced gap function lowers the measuring edge in 0.1 millimeter increments and is limited to 7 increments.
If a gap remains after the 7 increments, click on Redo Installation to return to the initial position and adjust the sample's position and volume. If the gap is exceedingly reduced, remove the excess sample with a spatula by a circular motion along the edge of the upper plate. Trim the excess sample gently to avoid shear stress.
Lower the protective cover to avoid any accidental projection of contaminated fluids during oscillation. To initiate measurement, click on Start Analysis. An entire cycle will take four to seven minutes.
Avoid talking loudly and touching the device or the bench during the entire length of the cycle. A quiet environment is crucial for the first two minutes. Once the cycle is complete, click next to raise the measuring head and generate the sample analysis report.
Once the measuring head is fully retracted, raise the protective cover. Discard the sample and carefully remove the plates. Clean and disinfect the plates using warm water and soap.
The viscoelastic characteristics of eight megadalton PEO were measured at five concentrations and directly compared between the evaluated benchtop rheometer and a traditional bulk rheometer. In contrast with PEO solutions, status asthmaticus mucus represents a solid-like elastic dominated behavior at low strain and features a crossover at high strain. In addition, triplicate measurements performed in 1.5%PEO solution and clinical assay mucus sample, confirmed that linear viscoelastic characteristics were highly repeatable for the values obtained from the biological sample.
Changes were measured in the viscoelastic properties of mucus following treatment with a mucolytic agent. TCEP effects on assay mucus viscoelasticity were tested in a clinical setting using the benchtop rheometer. Mucolytic treatment resulted in a more fluid-like sample with a decrease in the complex modulus by 4.6 fold, elastic modulus by 5.1 fold, viscous modulus by 1.9 fold, crossover strain by 3.3 fold, and crossover yield stress by 5.7 fold, and an increase in the damping ratio by 2.8 fold.
Measuring airway mucus viscoelasticity in a fast paced clinical setting can help tailor patients'treatment on a case-by-case basis, as well as provide a standardized protocol for testing novel pharmacological compounds. This technique is currently used to examine the effects of highly effective CFTR modulators on cystic fibrosis sputum and to test the potency of various drugs aimed at altering mucus properties.
