Measurements

Experimental Conditions

• Measurements of respiratory mechanics are highly dependent on the experimental conditions under which they are measured. For example, resistance readings can vary dramatically with frequency even when there are no changes to the underlying lung mechanics.


• When working with spontaneously breathing subjects, it is impossible to maintain the same experimental conditions in different subjects, particularly when a subject is undergoing a challenge. Breathing frequency, tidal volume and/or volume history will inevitably be altered, introducing significant measurement uncertainty.


• Because the flexiVent fully controls both the ventilation between measurements and the perturbation waveform that is applied during a manoeuvre, it ensures that frequency and tidal volume are tightly controlled and independent of the subject's condition, and that volume history is standardized.

Reproducibility & Accuracy

• The combination of these controlled experimental conditions and the flexiVent's advanced indirect measurement technique results in highly reproducible measurements with outstanding accuracy.


• Using the flexiVent, it is possible to resolve small group differences of only 10% with excellent statistical significance. In mechanical test loads, differences as small as 2% can be measured without difficulty.

Efficient Experimentation

• For many applications, the flexiVent's outstanding measurement accuracy directly translates into fewer subjects per group for a desired degree of statistical separation, therefore shortening experimental protocols and contributing to more efficient experimentation.


• Ultimately, the most inefficient experiments are those that provide inconclusive, ambiguous or even misleading results. For scientists striving to minimize the risk of misleading data, using a highly sensitive and specific tool such as the flexiVent is not only the most efficient, but the only truly efficient preclinical pulmonary function measurement solution.

Forced Manoeuvres vs. Passive Observation

The measurements taken by the flexiVent can be categorized as "Forced Manoeuvres". Because these types of measurements actively "perturb" the system to extract information, they offer more accurate and detailed results than passive measurement techniques. The Forced Manoeuvres performed by the flexiVent differ from passive measurements in a number of ways:

About Standard Perturbations

When a manoeuvre is triggered, the flexiVent briefly pauses mechanical ventilation and applies a test signal, also called a "Perturbation". Several families of pre-defined standard perturbations are available to serve a range of different applications. All measurements listed are automatically calculated by the flexiVent software.

List of Standard Perturbations

TLC: Deep inflation of the subject's lungs to a pressure of 30 cmH20 (or other user-specified value) followed by a breath hold of typically a few seconds.
Purpose: open up airspaces, standardize volume history.

SnapShots: Sinusoidal (single-frequency) forced oscillation waveform. The oscillation frequency is typically matched to the subject's respiratory rate, e.g. "SnapShot-150" for mice breathing at 150 br/min.
Purpose: obtain accurate, reproducible resistance & compliance data.

Primewaves: Broadband (multi-frequency) forced oscillation waveforms, typically denoted by duration (e.g. "Prime-8") which also reflects frequency content.
Purpose: measure input impedance, distinguish between airways and tissues.

PV: Slow stepwise or continuous (ramp) inflation to TLC and deflation back to FRC, controlling either volume or, preferably, pressure.
Purpose: assess nonlinearities in P-V loops, measure quasi-static compliance.

NPFE: Inflation to TLC followed by rapid switch to negative pressure reservoir (hardware add-on required).
Purpose: assess F-V loops, expiratory flow limitation & forced expired volumes.

Imaging Perturbations: pressure-controlled breath-hold, with or without preceding deep inflation to TLC, with synchronized trigger signals for imaging modalities such as Micro-CT or microscopes.
Purpose: provide steady, reproducible configuration for multi-frame image acquisition.

Parameters Measured

The flexiVent software calculates the following parameters from the data collected from different types of manoeuvres:

• Resistance, Compliance & Elastance from single-frequency FOT.
• Respiratory System Input Impedance from broadband FOT.
• Constant-Phase Model parameters from broadband FOT.
• Quasi-static Compliance & Elastance from P-V loops.
• Salazar-Knowles Equation coefficients from P-V loops.
• Hysteresis/enclosed area from P-V loops.
• Forced Expired Volumes & Vital Capacity from Forced Expirations.
• Various characteristic flows from Forced Expirations.
• Inspiratory capacity and plateau values from various manoeuvres.
• Descriptive Waveform Parameters for quality control from all manoeuvres.
• Goodness of fit indicators for quality control from all model fitting procedures.

The FEV Extension

With the new FEV extension, flexiVent systems can measure Flow-Volume loops and parameters such as FEVx and FVC, uniquely permitting Forced Expiration and Forced Oscillation measurements to be obtained simultaneously in the same subject. The FEV extension adds the following components to standard flexiVent systems:

• A modular negative pressure reservoir that is easily adapted to subject size.
• An electronic negative pressure controller that automatically maintains the negative pressure.
• A large, fast-acting shutter valve, plus a second valve to isolate the flexiVent piston from large negative pressures.
• An optional Aeroneb nebulizer
• A whole body plethysmograph with built-in pneumotachograph, required to measure flow during the Forced Expiration.
• The FEV Extension readily connects to most existing flexiVent systems. It is currently available for mice only, with solutions for rats and larger rodents in preparation for the near future.

Negative pressure Forced Expirations

• As opposed to clinical spirometry, the Forced Expirations produced by the flexiVent are driven by negative pressure at the airway opening. To emphasize this difference, we refer to them as Negative Pressure Forced Expirations (NPFEs).


• For each NPFE manoeuvre, the subject is inflated to total lung capacity and then rapidly switched to the negative pressure reservoir, which is automatically held at a user adjustable negative pressure.


• Because the expiratory flow is not generated by the flexiVent piston, a body plethysmograph is used for independent measurement of the forced expired flow. The plethysmograph can be easily opened to access the subject between NPFE manoeuvres.


• Upon completion of the NPFE, the flexiVent software immediately displays flow-volume loops and automatically calculates all pertinent volume and flow parameters.

Advantages

• Forced Expirations have previously required dedicated systems. The flexiVent is the first platform that permits NPFEs concurrently with a wide variety of other respiratory mechanics measurements in the same cohort of animals.


• The assessment of challenge responses with Forced Expirations alone poses timing difficulties that often result in substantial data variability. With the flexiVent, the timing of NPFE measurements can be optimized in order to improve reproducibility and separation between groups.


• The addition of Forced Expirations extends the range of flexiVent measurements and offers a means to more directly assess expiratory flow limitation, which is a key element of diseases such as Emphysema and Chronic Bronchitis.


• Although NPFE manoeuvres are obtained under conditions that differ substantially from clinical spirometry, they may provide a useful cross-species correlate for drug development studies.

Technical Challenges

Conventional approaches to measuring respiratory mechanics typically rely on pneumotachographs and body plethysmographs to measure or estimate airflow at the airway opening. When scaled down to sizes suitable for rodents, these approaches often suffer from the following problems:

• Insufficient measurement accuracy
• Poor common mode rejection
• Poor frequency response
• Susceptibility to thermodynamic disturbances
• Excessive dead space

The flexiVent Solution

• The flexiVent does not directly measure the airflow in and out of the subject's lungs. Instead, it uses an indirect measuring technique that has several advantages over direct flow measurements using pneumotachographs or plethysmographs.


• During a Forced Oscillation manoeuvre, the flexiVent measures the volume displaced by its piston and the pressure in the cylinder. From these data, the mechanical load presented by the total of the subject's lungs and the ventilator tubing can be calculated.


• Using sophisticated signal processing along with calibration data established in a prior dynamic tube calibration manoeuvre, the load presented by the subject can be separated from the ventilator compartment.


• This technique has been thoroughly validated and has proven to be more accurate and sensitive than approaches using a direct flow measurement.