Research

Sputum Properties

Sputum and mucus collection

Normal tracheal mucus is collected by the endotracheal tube (ETT) method. We have used this technique to collect mucus from premature infants, older children and adults with and without airway disease. We also use this technique to harvest tracheal mucus from experimental animals.

For the past decade we have collected airway sputum from cystic fibrosis patients during their clinic visits. We also have techniques for collecting nasal secretions from cold, allergy and cystic fibrosis patients.

The analysis of the physical properties of secretions

Viscoelasticity (rheology)

We have developed mathematically validated techniques to measure the dynamic rheology of small specimens of mucus or sputum. We measure rheology using an AR1500ex controlled stress rheometer.Measurements are carried out on 20 μL specimens at 37°C with a humidity shield in place.

Dynamic viscoelasticity is determined from the strain response to an oscillating stress and reported as storage or elastic (G’), and loss or viscous (G’’) moduli, as a function of frequency. When stress in the linear range is used to evaluate the materials, rheology is independent of stress. In order to conduct a frequency sweep from 1 to 100 rad/sec, we calculate viscoelasticity using a creep test at 0.5 Pa for two minutes. The strain response is fitted to a discrete relaxation spectrum, transformed to the retardation spectrum, and then to the storage and loss moduli, as a function of frequency.

King M and Rubin BK. Mucus rheology, Relationship with Transport. Chapter 7 in Airway Secretion: Physiological Bases for the Control of Mucus Hypersecretion. Ed. T. Takishima Marcel Dekker, Inc. New York. 1994; pp. 283-314.

Cohesivity

Cohesivity is the interfacial tension (γ) multiplied by the new area created after a test substance is stretched. For Newtonian fluids this = 2 x γ. For a gel such as sputum, a filancemeter is used to stretch the mucus until broken. The measurement is performed with a 25 µL sample at a velocity of 10 mm/sec. An electric signal conducted through the sample is interrupted when the thread is broken. Using a cone with a base diameter of 1 mm; cohesivity = γ x  π x length in mm/100.

Mucus hydration (% solids) and mucus density

A mucus sample is weighed in a microbalance and completely dried in a lyophilizer. The sample is then reweighed in order to calculate the percent solids composition. The density of the mucus is also calculated by dividing the weight of the sample by the volume.

The analysis of the transport properties of secretions

Sputum mucociliary transportability (MCTR)

A leopard frog (Rana pipiens) is pithed and the palate removed. The palate is placed on gauze saturated with amphibian Ringer’s solution and depleted of mucus over 12 hours. The palate is then placed in a Plexiglas box maintained at 95% humidity and under a microscope so that a 5 mm micrometer scale runs between the optic bulges to the opening of the esophagus. The movement of 4 μL sputum is timed as the trailing edge moves across a 3 mm segment. The mean transport rate is normalized to the transport rate for endogenous frog mucus.

Rubin BK, Ramirez O and King M. The mucus depleted frog palate as a model for the study of mucociliary clearance. J Appl Physiol 1990;69:424-29

In vitro cough transportability

A simulated cough machine is used to measure the airflow-dependent clearability of sputum. A model Plexiglas “trachea,” rectangular in cross section (1.2 x 2 cm) is connected to a 6.4 L tank containing air pressurized to 8 psi giving a flow of about 11 L/s. A solenoid controls air release through a flow-constrictive element used to mimic the airflow pattern of a natural cough. A sinusoidal constriction is used to decrease the airway diameter while minimizing the turbulence (Reynolds number) of the system. A sample, 40 µl in volume and 0.5 mm in depth, is placed in a  line across the base of the Plexiglas trachea. The bulk transport of the sample is measured in mm after a single cough maneuver. Three successive measurements are made and the results averaged

Agarwal M, King M, Rubin BK, Shukla JB. Mucus transport in a miniaturized simulated cough machine: Effect of constriction and serous layer simulant. Biorheology 1989; 26:977-88

Secretion surface properties

Wettability (contact angle )

The wettability test consists of allowing a drop of mucus to fall onto the surface of a glass microscope slide cleaned with chromic sulfuric acid, rinsed with deionized water, then stored in absolute ethanol to maintain dehydration before use. Mucus is transferred to a sharp needle and kept 3 mm above the glass surface until it falls. A stabilization time of one minute is allowed before capturing the image of the sessile drop in the video processing system and calculating the glass-mucus contact angle

Interfacial tension,  γ ,  by the de Noüy ring method

This test measures the surface tension at a liquid-air interface  or the interfacial (adhesion) tension of a sputum-air interface. A platinum-iridium ring is pulled from the sample at a distraction velocity of 10 mm/sec until separation is achieved. The force of separation is measured by a strain gauge connected to the ring. This gives the work of adhesion or the surface tension in dyne/cm. We use a semi-automated tensiometer for these measurements. We have calibrated a custom platinum-iridium ring so that we need only a volume of 12 µl to measure interfacial tension.

DNA and mucin content

Mucin is measured by Western blot using antibodies that we have previously described (5).

DNA is measured by microfluorimetry and compared with calf thymus DNA standard  in the range 0.25 -10.0 µg/mL. Mucin and DNA content can also be determined from confocal microscopy images. Sputum  is placed on concavity slides, fixed in ethanol for five minutes, then stained with 20μL of 10μg/mL L4889 lectin from Ulex europaeus agglutinin (UEA) conjugated with TRITC to identify mucin carbohydrate epitopes.  Sputum is also stained with 20μL of 1 μM YoYo-1. YoYo-1 binds to DNA, excites at 496 nm and emits at 519 nm.

Rubin BK, Tomkiewicz RP, Ramirez OE and King M. Surface properties of respiratory secretions: Relationship with mucus transport. Biorheology 1995; 32:213

Structural analysis by confocal microscopy and polymer discrimination image analysis

We have developed imaging processing techniques that can accurately evaluate mucus and sputum polymer structure including macromolecular length, density and interaction and well as mucin and DNA quantity. Using laser scanning confocal microscopy (LSCM) image sections are acquired as a matrix of pixels at different z-axis by adjusting the stage height. This permits assessment of image integrity and accurate measurement of filament bundle dimensions.

The PolyFiberQuant program is used to measure mucin and DNA co-localization, bundle formation, crosslink density, and branching. Relative differences of specific components are estimated by measuring mean fluorescence intensity. The average point-to-point filament length, branching and interconnectivity, and volume of components is done by morphometric point counting.

 

Figure 1 A representative CF image stained with YoYo-1 is shown above the table. DNA polymers are skeletonized and superimposed on the image. When co-imaged for UEA detected mucous glycoproteins or F-actin, this software can describe the interactions among polymers of different species.

Tomkiewicz RP, Kishioka C, Freeman J, Rubin BK. DNA and actin filament ultrastructure in cystic fibrosis sputum Chapter 34 in Cilia, Mucus, and Mucociliary Interactions. G. Baum ed. 1998. Marcel Dekker, Inc., New York. pp.333-41.