intra‐pulmonary arteriovenous anastomoses and pulmonary ... · intra-pulmonary arteriovenous...

J Physiol 597.22 (2019) pp 5365–5384 5365

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Intra-pulmonary arteriovenous anastomoses andpulmonary gas exchange: evaluation by microspheres,contrast echocardiography and inert gas elimination

Michael K. Stickland1,2 , Vincent Tedjasaputra1,3, Cameron Seaman4 , Desi P. Fuhr1,Sophie E. Collins1,5 , Harrieth Wagner6, Sean van Diepen7, Bradley W. Byers1,3, Peter D. Wagner6

and Susan R. Hopkins6

1Division of Pulmonary Medicine, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada2G.F. MacDonald Centre for Lung Health, Covenant Health, Edmonton, Alberta, Canada3Faculty of Kinesiology, Sport and Recreation, University of Alberta, Edmonton, Alberta, Canada4Division of Pediatric Cardiology, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada5Faculty of Rehabilitation Medicine, University of Alberta, Alberta, Canada6Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, University of California San Diego, San Diego, USA7Department of Critical Care and Division of Cardiology, Faculty of Medicine and Dentistry, University of Alberta, Alberta, Canada

Edited by: Harold Schultz & Philip Ainslie

Linked articles: This article is highlighted in a Perspectives article by Foster. To read this article, visithttps://doi.org/10.1113/JP278820.

Key points

� Imaging techniques such as contrast echocardiography suggest that anatomicalintra-pulmonary arteriovenous anastomoses (IPAVAs) are present at rest and are recruitedto a greater extent in conditions such as exercise. IPAVAs have the potential to act as a shunt,although gas exchange methods have not demonstrated significant shunt in the normal lung.

� To evaluate this discrepancy, we compared anatomical shunt with 25-µm microspheres tocontrast echocardiography, and gas exchange shunt measured by the multiple inert gaselimination technique (MIGET).

� Intra-pulmonary shunt measured by 25-µm microspheres was not significantly different fromgas exchange shunt determined by MIGET, suggesting that MIGET does not underestimate thegas exchange consequences of anatomical shunt.

� A positive agitated saline contrast echocardiography score was associated with anatomical shuntmeasured by microspheres. Agitated saline contrast echocardiography had high sensitivitybut low specificity to detect a �1% anatomical shunt, frequently detecting small shuntsinconsequential for gas exchange.

Michael Stickland received his Doctorate in 2004 from the University of Alberta, and completedpost-doctoral training at the University of Wisconsin – Madison in 2006. He is currently a Professorin the Pulmonary Division within the Faculty of Medicine and Dentistry at the University of Alberta.Clinically, Dr Stickland is the Director of the G. F. MacDonald Centre for Lung Health, which delivers theprimary pulmonary rehabilitation programme in Edmonton. His research interests in physiology includeexamining the cardiovascular consequences of chronic lung disease, the determinants of pulmonary gasexchange, and autonomic control of cardiovascular function in health and disease. Sue Hopkins is aProfessor of Medicine and Radiology and Director of the Pulmonary Imaging Laboratory at UC-SanDiego. Dr Hopkins obtained her Medical degree from Memorial University of Newfoundland (1980),and completed a fellowship in Sports Medicine (1997) and a PhD (Pulmonary and Exercise Physiology,1993) at the University of British Columbia, before completing a postdoctoral research fellowship with John West and Peter Wagner at the UC-San Diego.Dr Hopkins’ research focuses on the lung response to stress, such as hypoxia and exercise. In addition to classic gas exchange techniques such as themultiple inert gas elimination technique (MIGET), she uses novel functional MRI techniques to quantify pulmonary blood flow, ventilation, density andventilation–perfusion ratio distributions and offer insights into mechanisms of disease.

C© 2019 The Authors. The Journal of Physiology C© 2019 The Physiological Society DOI: 10.1113/JP277695

https://orcid.org/0000-0001-8234-4760

https://orcid.org/0000-0002-6367-0752

https://orcid.org/0000-0002-6323-1127

https://doi.org/10.1113/JP278820

5366 M. K. Stickland and others J Physiol 597.22

Abstract The echocardiographic visualization of transpulmonary agitated saline microbubblessuggests that anatomical intra-pulmonary arteriovenous anastomoses are recruited duringexercise, in hypoxia, and when cardiac output is increased pharmacologically. However, themultiple inert gas elimination technique (MIGET) shows insignificant right-to-left gas exchangeshunt in normal humans and canines. To evaluate this discrepancy, we measured anatomicalshunt with 25-µm microspheres and compared the results to contrast echocardiography andMIGET-determined gas exchange shunt in nine anaesthetized, ventilated canines. Data wereacquired under the following conditions: (1) at baseline, (2) 2 µg kg−1 min−1 I.V. dopamine, (3)10 µg kg−1 min−1 I.V. dobutamine, and (4) following creation of an intra-atrial shunt (in fouranimals). Right to left anatomical shunt was quantified by the number of 25-µm microspheresrecovered in systemic arterial blood. Ventilation–perfusion mismatch and gas exchange shuntwere quantified by MIGET and cardiac output by direct Fick. Left ventricular contrast scores wereassessed by agitated saline bubble counts, and separately by appearance of 25-µm microspheres.Across all conditions, anatomical shunt measured by 25-µm microspheres was not different fromgas exchange shunt measured by MIGET (microspheres: 2.3 ± 7.4%; MIGET: 2.6 ± 6.1%,P = 0.64). Saline contrast bubble score was associated with microsphere shunt (ρ = 0.60,P < 0.001). Agitated saline contrast score had high sensitivity (100%) to detect a �1% shunt,but low specificity (22–48%). Gas exchange shunt by MIGET does not underestimate anatomicalshunt measured using 25-µm microspheres. Contrast echocardiography is extremely sensitive, butnot specific, often detecting small anatomical shunts which are inconsequential for gas exchange.

(Received 16 January 2019; accepted after revision 12 August 2019; first published online 20 August 2019)Corresponding author M. Stickland: 3-135 Clinical Sciences Building, Edmonton, Alberta, Canada, T6G 2J3.Email: [email protected]

Introduction

The human red blood cell is approximately 8 µm indiameter, and barely fits through pulmonary capillaries,which are typically 7–10 µm in diameter. Studies usingimaging techniques suggest that there are anatomicarteriovenous pathways (IPAVAs) in the human lung thatare potentially large enough to allow red blood cells tobypass the gas-exchanging regions. These anastomosesmay be present in some subjects at rest (Elliott et al.2013) and show increased recruitment during exercise,or when cardiac output and/or the pulmonary circulationis physiologically or pharmacologically altered (Eldridgeet al. 2004; Stickland et al. 2004, 2006; Lovering et al.2008a; Bryan et al. 2012; Laurie et al. 2012; Elliottet al. 2014). For example, during exercise there is trans-pulmonary passage of saline contrast bubbles as imagedby echocardiography (Eldridge et al. 2004; Stickland et al.2004; Lovering et al. 2008a). Similarly, single-photonemission computed tomography (SPECT) imaging showsdecreased retention of Technetium-99m-labelled albuminmicrospheres (Whyte et al. 1992), and macroaggregatedalbumin (Lovering et al. 2009b; Duke et al. 2017b) withexercise, suggesting that exercise recruits arteriovenousanatomical pathways that are large enough to permitpassage of these particles.

Transpulmonary passage of bubbles has been shown tobe correlated with cardiac output, mean pulmonary arterypressure, and the alveolar–arterial difference for oxygen

(AaDO2) in exercising humans (Stickland et al. 2004).Inotrope infusion increases transpulmonary passage ofagitated saline contrast and estimated shunt fractionin healthy resting humans (Bryan et al. 2012; Elliottet al. 2014). Consistent with the human imaging studies,intra-pulmonary arteriovenous anatomical pathwaysalso have been documented by microspheres in dogsduring exercise (Stickland et al. 2007). This supportsprevious anatomical work in humans and animalsdemonstrating arteriovenous anatomical pathways in thelung (Prinzmetal et al. 1948; Tobin, 1966; Wilkinson &Fagan, 1990). Collectively, this information suggests thatarteriovenous anastomoses have the potential to impactthe arterial partial pressure of oxygen (PaO2 ) (Sticklandet al. 2004, 2013; Stickland & Lovering, 2006; Loveringet al. 2009a).

A shunt, defined as the admixture of pulmonary mixedvenous blood into the systemic arterial circulation, of1–2% of cardiac output would explain all of the increasedAaDO2 observed during exercise (Dempsey & Wagner,1999). The multiple inert gas elimination technique(MIGET) allows for evaluation of ventilation–perfusionrelationships including shunt from either cardiac and/orintra-pulmonary sources. Importantly, none of the studiesusing MIGET has documented shunts of 1–2% at rest orduring exercise in normal human subjects (Hammondet al. 1986; Wagner et al. 1986; Hopkins et al. 1994, 1998;Podolsky et al. 1996; Rice et al. 1999; Olfert et al. 2004; Jonket al. 2007). Studies using the 100% oxygen technique

C© 2019 The Authors. The Journal of Physiology C© 2019 The Physiological Society

J Physiol 597.22 Arteriovenous anastomoses and pulmonary gas exchange 5367

to quantify shunt have similarly confirmed a venousadmixture/shunt fraction of<1% of cardiac output duringexercise (Dempsey et al. 1984; Torre-Bueno et al. 1985;Hammond et al. 1986; Wagner et al. 1986; Vogiatziset al. 2008). Results obtained from MIGET studies inresting and exercising animals including dogs (Hsia et al.1990), horses (Wagner et al. 1989) and pigs (Hopkinset al. 1999) have also not demonstrated a shunt >1%.Despite the observation of intra-pulmonary arteriovenousanatomical pathways in dogs during exercise (Sticklandet al. 2007), these animals had no evidence of a gasexchange impairment. Thus, the contribution of shuntto the gas exchange impairment during exercise has beenargued to be minimal (Dempsey & Wagner, 1999; Vogiatziset al. 2008; Hopkins et al. 2009a,b).

There are several possible reasons for the discrepancybetween imaging studies suggesting the presence ofanatomical shunt pathways in the lung with the potentialto allow deoxygenated blood to bypass the pulmonarycapillaries, and gas exchange studies showing the absenceof significant shunt. First, agitated saline contrastechocardiography, as currently implemented, does notallow for a quantitative determination of anatomic arterio-venous shunt magnitude. Consequently, while contrastbubbles in the left ventricle may detect arteriovenouspathways, this may represent a very small amount ofshunting (Hopkins et al. 2009b), consistent with gasexchange measurements. Second, the appearance of salinecontrast bubbles in the left ventricle could be due toother factors including the presence of small-diameterbubbles (<10 µm) which are able to pass through normalcapillaries, the deformation of larger bubbles which areable to fit through the pulmonary capillaries, and/orcapillary distention (Warrell et al. 1972). Finally, it hasbeen suggested that MIGET may underestimate the truemagnitude of arteriovenous shunt because of gas exchangethat may be occurring proximal to the capillary, orwithin arteriovenous pathways (Stickland et al. 2004, 2013;Stickland & Lovering, 2006; Lovering et al. 2009a).

Rigid 25 µm radiolabelled microspheres are largerthan a pulmonary capillary, and when injected into thepulmonary circulation should not traverse the intactcapillary bed. Thus, the appearance of microspheres inthe systemic circulation allows for the quantificationof blood flow through extra-capillary anatomic arterio-venous pathways. Accordingly, the purpose of thisstudy was to compare anatomic shunt (intra-pulmonaryand/or intra-cardiac) using 25 µm microspheres, contrastechocardiography and gas exchange shunt quantifiedby MIGET in the dog under manipulations previouslyshown by imaging studies to recruit IPAVAs secondaryto pulmonary vasodilatation or increased cardiac output.Based on previous imaging studies, we hypothesized thatMIGET would underestimate shunt as measured by trans-pulmonary passage of 25 µm microspheres. A companion

paper (Stickland et al. 2019) reports a comparison of pre-capillary gas exchange between inert gases and oxygen.

Methods

Ethical approval

All surgical and experimental procedures were approvedby the University of Alberta Research Ethics Office(Protocol #AUP00001296). Studies were conducted inaccordance with the American Physiological Society’s‘Guiding Principles in the Care and Use of Animals’.

Study overview

This work was part of a larger project that also examinedprecapillary pulmonary gas exchange of O2, CO2 andinert gas (Stickland et al. 2019). Nine mixed-breed hounddogs weighing between 19 and 25 kg were studied(Marshall BioResources, North Rose, NY, USA). Forlogistical reasons, the first four animal investigations wereconducted several months earlier than the last five.

The experiments were conducted in atemperature-controlled (21–23°C), ventilated room.Once anaesthetized, ventilated and instrumented, allanimals were studied under the following conditions:(1) baseline, (2) 2 µg kg−1 min−1 dopamine, and (3)10 µg kg−1 min−1 dobutamine, with the order of the druginfusions randomized. At low doses (i.e. 2µg kg−1 min−1),dopamine stimulates the pulmonary dopaminergicreceptors, without alpha and beta stimulation, resultingin pulmonary vascular vasodilatation (Hoshino et al.1986; Ricci et al. 2006), and has previously been shownto result in IPAVA recruitment as evaluated by contrastechocardiography, as well as an increase in fractionalshunt (Qs/Qt) in humans (Bryan et al. 2012). At a highdose (i.e. 10 µg�kg−1�min−1), dobutamine acts as analpha and beta agonist resulting in a large increase incardiac output. Dobutamine has also been shown torecruit IPAVAs as evaluated by contrast echocardiographyand to increase Qs/Qt in humans (Bryan et al. 2012).

Data were obtained 15 min after the start of each 30 minpharmacological infusion. Steady-state was confirmedwithin each condition by stable oxygen consumption andheart rate for at least 5 min before data collection. Eachdata set included: pulmonary arterial, pulmonary arterialwedge and systemic arterial pressures; inert gas, O2 andCO2 levels in mixed venous blood, systemic arterialblood and mixed expired gas; metabolic rate, ventilationand cardiac output; shunt determined by microspheres;and transpulmonary bubble and microsphere trans-mission by contrast echocardiography. Following eachpharmacological condition, a 30 min recovery periodensured drug wash out, and additional cardiopulmonarydata (blood pressure, blood gases, inert gas sampling,



oxygen consumption, cardiac output and ventilation)were obtained.

Given that the magnitude of any anatomicintra-pulmonary shunt was expected to be relatively small,a positive control condition was also created to evaluatesensitivity during a condition in which a large anatomicshunt was known to be present. This control was created byatrial trans-septal puncture in the final five animals; it wassuccessfully completed in four. In these four animals, datawere collected following atrial communication creation innormoxia and hypoxia (see below).

Anaesthesia

All animals were premedicated with I.M. acepromazine(0.05 mg�kg−1) and hydromorphone (0.1 mg�kg−1). Inthe first four animals, surgical plane anaesthesia wasmaintained by I.V. propofol (1 mg�kg−1 I.V. bolus, thenmaintenance doses of 0.2 mg�kg−1�min−1). In the lastfive animals, surgical plane anaesthesia was maintainedwith pentobarbital (20–30 mg�kg−1 I.V. bolus, thenmaintenance doses of 1–5 mg�kg−1�h−1). Veterinarianstaff continuously monitored the level of anaesthesia.Following completion of data collection, animals werekilled with 120 mg�kg−1 I.V. pentobarbital.

Instrumentation

Following induction of anaesthesia, an endotracheal tubewas inserted, and animals were ventilated on room air(target PaCO2 �35 mmHg). A positive end-expiratorypressure of 5 cmH2O was used throughout, and frequentsighs were conducted between experimental conditions inan attempt to prevent atelectasis. Four venous catheterswere inserted, one in each hind limb for (1) infusionof anaesthetic, (2) infusion of saline containing inertgases, (3) infusion of dobutamine/dopamine and (4)back-up catheter. The femoral artery was cannulatedand a catheter was advanced into the abdominal aortafor peripheral arterial blood sampling. Two Swan-Ganzcatheters were introduced, one through each jugularvein. The first Swan-Ganz catheter (7fr, 2.3 mm) waspositioned approximately 1–2 cm distal to the rightventricular/pulmonary artery interface as confirmed bydirect pressure monitoring, and was used for mixed venoussampling and pressure monitoring. Another Swan-Ganzcatheter (5fr, 1.7 mm) was positioned approximately 1 cmproximal to the wedge position, also confirmed by directpressure monitoring, and was part of a separate study notreported here (Stickland et al. 2019).

Cardiopulmonary data

Expired gases were passed through heated tubing to amixing chamber set to maintain temperature >37°C inorder to avoid condensation of water vapour. Expired

O2 and CO2 were measured (Analysers 17625/17630;Vacumed, Ventura, CA, USA), ventilation was determinedby pneumotachometer (Series 3700A, Hans Rudolph Inc.,Shawnee, KS, USA), and O2 consumption and CO2

production were calculated (Powerlab, AD Instruments,Colorado Springs, CO, USA). The pressure transducers(Surgivet Advisor, Smiths Medical, Dublin, OH, USA)were zeroed to the level of the right atrium. Meanarterial, pulmonary arterial and pulmonary arterialwedge pressures were recorded immediately before eachset of inert-gas measurements. Cardiac output wascalculated from the systemic arterial–pulmonary arterialO2 concentration difference, and oxygen consumptionusing the Fick equation.

Multiple inert gas elimination technique

MIGET was applied as previously described (Wagner et al.1974a,b, 1975; Dueck et al. 1978). The inert-gas solutionwas prepared in normal saline and infused for 20 minbefore collection of baseline samples. The infusion rate ofMIGET solution was set to 0.25 ml�min−1 per l�min−1 ofventilation for an average infusion rate of �1 ml�min−1

in these animals. The total volume of fluid infused duringthe study from all sources was 1 litre over a period of3–4 h.

Quadruplicate 15 ml samples of mixed expiredgas and duplicate 6 ml samples of pulmonary andsystemic arterial blood were obtained in gas-tight glasssyringes at each condition for measurement of thesteady-state concentrations of the six inert gases by gaschromatography (model 5890A; Hewlett-Packard,Wilmington, DE, USA). Ventilation–perfusiondistributions as well as right-to-left shunt weredetermined by MIGET as described (Hopkins & Wagner,2017). LogSDQ, and LogSDV, which are the secondmoments about the mean on a log scale of the perfusionand ventilation distributions, respectively, were used toquantify the extent of ventilation–perfusion mismatch, acalculation that excludes both shunt (VA/Q = 0) anddead space (VA/Q = infinity). The value of PaO2 expectedfrom the observed VA/Q mismatch and shunt was alsocalculated (predicted PaO2 ). This should agree with themeasured value unless there is diffusion limitation ofO2 uptake across the blood–gas barrier, in which casethe measured value will be systematically less than thatpredicted (Hopkins & Wagner, 2017). Of note, MIGET isunable to distinguish a right-to-left shunt that arises fromintra-pulmonary sources (i.e. unventilated lung regionscaused by alveolar filling or atelectasis, or, potentially,from intra-pulmonary arteriovenous anastomoses) fromthat arising from intra-cardiac sources. Rather, MIGETaggregates shunt from these different sources into asingle value. However, the small amount of venous bloodfrom the bronchial circulation and the Thebesian veins,



which is returned to the left side of the circulation, canreduce PaO2 , but will not affect inert gas exchange, and istherefore not detected by MIGET (or by intra-venouslyinjected microspheres) (Hopkins & Wagner, 2017). Theresidual sum of squares (RSS) was used as an indicatorof the adequacy of fit of the data to the 50-compartmentmodel of the lung. RSS follows the chi-square distributionfor six degrees of freedom (because six inert gases areused) and is never zero because of random experimentalerror. The RSS indicates the level of such error; thechi-square distribution predicts that 50% of the time,the RSS should be less than 5.3, that 90% of the timeit should be less than 10.6, and that 99% of the time itshould be less than 16.8 (Hopkins & Wagner, 2017). Dataare reported as the average of the two duplicate measuresunder each condition.

Blood gas measurements

Systemic arterial and mixed venous blood gas samples(3 ml each) were collected simultaneously at identicalsampling rates, immediately after each arterial andmixed venous blood sample for MIGET. Samples wereimmediately cleared of any bubbles and were maintainedon ice until analysed for PO2 , PCO2 , pH, and haemoglobinconcentration using a blood-gas analyser (ABL80 FLEX,Radiometer, Copenhagen, Denmark). O2 saturation(SaO2 ) was calculated based on PO2 , PCO2 , pH and rectaltemperature (Kelman, 1966,1967,1968), and an assumedpartial pressure of O2 at 50% saturation of haemoglobin of30 mmHg, as previously reported for dog blood (Cambieret al. 2004; Zaldivar-Lopez et al. 2011).

Contrast echocardiography

Agitated saline contrast echocardiography was used todetect intra-pulmonary arteriovenous anastomoses andintra-cardiac shunt, according to previously publishedmethodologies (Stickland et al. 2004; Lovering et al. 2008a;Bryan et al. 2012). Briefly, 10 ml of saline was combinedwith 1.0 ml of air, and the solution was forcefully agitatedbetween two syringes through a three-way stopcock toform fine suspended bubbles, which were then injectedthrough the right atrial port of the Swan-Ganz catheter. Afour-chamber view of the heart was recorded before andduring contrast injection with a minimum of 20 cardiaccycles recorded. Saline contrast injections were alwaysconducted before microsphere injections, and followingcompletion of the agitated saline injection, the catheterwas flushed with normal saline to ensure that no furtherbubbles entered the right ventricle.

All echocardiograms were performed by the samesonographer using a Vivid Q (GE, Boston, MA, USA)ultrasound, and were later analysed by a cardiologistwho was blinded to the experimental condition. The

appearance of contrast in the left ventricle within fewerthan five cardiac cycles from appearance of contrast in theright ventricle suggests intra-cardiac shunt, whereas theappearance of contrast in the left ventricle after five cardiaccycles suggests an IPAVA (Stickland et al. 2004; Loveringet al. 2008a; Bryan et al. 2012). Contrast intensity for bothagitated saline and microspheres was scored based on apreviously described scoring system used in our laboratoryand others: no contrast bubbles in the left ventricle in anysingle frame received a score of 0; �3 bubbles a scoreof 1; 4–12 bubbles a score of 2; >12 bubbles a score of3; many bubbles heterogeneously distributed within theleft ventricle a score of 4; many bubbles homogeneouslydistributed within the left ventricle a score of 5 (Loveringet al. 2008b; Bryan et al. 2012). A contrast score of 1 wasconsidered positive for shunt (either intra-pulmonary orintra-cardiac).

Microspheres

During each condition, all dogs received a bolus injectionof 25-µm stable isotope-labelled neutron activated micro-spheres suspended in 10 ml of normal saline containing0.01% Tween 80 and 0.01% Thimerosal (STERIspheres,BioPAL Inc., Worcester, MA, USA). A unique iso-tope was used for each condition (gold, samarium,lanthanum, ytterbium, lutetium), and the isotope labelwas randomized across conditions and animals. In thefirst four animals, one half-vial (approximately 2.7 millionmicrospheres) was injected at baseline and dopamineinfusion. A full vial (5.4 million microspheres) was injectedduring the dobutamine infusion to maximize sensitivitywhen cardiac output was expected to be elevated. Duringthe second phase of experiments, 5.4 million microsphereswere injected in each condition to increase sensitivity todetect shunt. Before injection, a small sample (20 µl)from the microsphere vial was obtained and evaluatedto determine total counts injected.

Microspheres were injected directly into the rightatrial port of the large Swan-Ganz catheter. Immediatelyfollowing injection, the 10-ml syringe was refilled withsaline and the catheter was rapidly flushed three times.Successful injection of microspheres into the right atriawas confirmed by ultrasound. To evaluate the trans-pulmonary passage of microspheres by echocardiography,the intensity of microsphere contrast observed in the leftventricle was evaluated using the agitated saline contrastscoring system as described above.

Beginning at the time of microsphere injection, and forthe next 60 s, systemic arterial blood was withdrawn fromthe catheter in the abdominal aorta at a rate of 0.5 ml�s−1

to provide a 30 ml sample of blood. Samples wereprepared as per the manufacturer’s guidelines and lateranalysed for the presence of microspheres (commercialanalysis by BioPAL).



When microspheres were detected in arterial bloodsamples, shunt as a fraction of cardiac output wascalculated based upon the following data: total countsinjected, cardiac output, arterial blood sample volumeand counts detected in arterial sample (Stickland et al.2007). The manufacturer’s specifications indicate that themethodology is sensitive enough to detect even one micro-sphere in an arterial blood sample. Based on cardiacoutput, a 30 ml arterial blood sample and the goal of200 microspheres in each arterial sample, this quantity ofmicrospheres had the sensitivity to quantify an anatomicalshunt fraction greater than 0.4% of cardiac output. Duringthe second phase of experiments, 5.4 million microsphereswere injected in each condition, which allowed for thequantification of an anatomical shunt larger than 0.2%.

Trans-septal puncture and inter-atrial communicationcreation

In the second phase of the study, creation of aninter-atrial communication was successfully performed infour animals to create a large anatomic shunt condition.Following removal of the proximal pulmonary arterialcatheters, a 4fr Coe 2 catheter (Terumo Medical Corp,Somerset, NJ, USA) was used to advance a 0.64 mm,260 cm Fixed Core J-curve guide wire to the super-ior vena cava. A 59 cm Mullins Transseptal sheath(Medtronic, Minneapolis, MN, USA) was introduced, andsubsequently a 71 cm Brockenbrough needle (Medtronic)was used to puncture the atrial septum under fluoro-scopic guidance. A guide-wire was placed across theseptum and a 3.5 mm × 10 mm Flextome CuttingBalloon (Boston Scientific, Marlborough, MA, USA) waspositioned across the atrial septum and inflated to 12atmospheres using a pressure inflation device (Encore 26,Boston Scientific). A 4 mm × 26 mm stent (Medtronic)was then positioned across the septum and inflated to 15atmospheres. The stent was then post-dilatated using a6 mm × 2 cm Sterling Over-the-Wire Balloon (BostonScientific) at 14 atmospheres. Injected contrast (Isovue370, Bracco Diagnostics, Monroe Township, NJ, USA)was used to confirm the right-to-left cardiac shunt.The second pulmonary arterial catheter was kept in theanimal, and inflated to provide partial occlusion of thepulmonary artery in order to maximize intra-cardiacshunt. Following data collection in normoxia, the animalsalso breathed hypoxic gas (F IO2 =0.125) in order to furtherincrease pulmonary artery pressure, and potentially theintra-cardiac shunt.

Statistical analysis

All statistical analyses were performed using SPSSStatistical software v. 24.0 (IBM Corp, Armonk, NY, USA).

For all inferential analyses, the probability of Type I errorwas set at 0.05. Continuous variables were evaluatedacross conditions by repeated measures ANOVA. Shuntdetermined by the recovery of microspheres in arterialblood and by MIGET was compared across experimentalconditions using two-way repeated measures ANOVA,with shunt type as the fixed factor (microspheres vs.MIGET). Saline contrast bubble score and microspherecontrast score were evaluated separately across conditionsby Friedman’s ANOVA. Saline contrast bubble scores werecompared to microsphere contrast scores by combiningdata from all conditions and comparing saline vs. micro-sphere contrast score using a Wilcoxon signed-rank test.Sensitivity and specificity of the saline and microspherecontrast scores to detect a 1% shunt measured by micro-spheres were calculated with all data as:

Sensitivity = number of true positives/(number of true positives + false negatives

).

Specificity = number of true negatives/(number of true negatives + false positives

)

The intra-cardiac procedure was successful in fouranimals, and these animals were evaluated under twopost-intra-cardiac conditions (normoxia and hypoxia). Inan attempt to maximize the power to detect a differencebetween MIGET and microsphere shunt, these eightpost intra-cardiac shunt (IC) conditions were consideredindependent conditions, and were compared to thecorresponding baseline data. Pearson product momentcorrelation was used to evaluate the relationships betweencontinuous variables. Spearman rank order correlationcoefficients (ρ) were used to evaluate bubble/microsphereshunt score vs. continuous variables. As the intra-cardiacshunt procedure resulted in a high degree of shuntthat could inflate the results of the correlation analysis,comparisons were evaluated with and without these data.

Results

Haemodynamic, blood gas and MIGET data acrossconditions are reported in Tables 1 and 2, while shuntand contrast data are shown in Figs 1 and 2. AppendixTable A1 provides individual data for select variables. Dataare reported as mean ± SD unless otherwise indicated.

Baseline

The ventilation–perfusion distribution was normal atbaseline (mean LogSDQ = 0.55 ± 0.11, mean LogSDV =1.69 ± 0.41), aside from a high ventilation–perfusionmode increasing the LogSDV that is consistent with theuse of positive end-expiratory pressure (PEEP) in these



Table 1. Mean (±SD) haemodynamic and ventilation data during all conditions

Baseline(n = 9)

Dopamine(n = 8)

Post-dopamine(n = 8)

Dobutamine(n = 9)

Post-dobutamine(n = 9)

Cardiac shunt(n = 8)

Heart rate (bpm) 103 116∗ 128 193∗ 139 172∗

25 16 19 11 16 12Stroke volume (ml) 14 18 16 15 14 13

3 7 4 6 4 5Cardiac output (litres) 1.4 2.0 2.1 2.9∗ 2.0 2.3∗

0.4 0.7 0.5 1.2 0.4 0.8Mean arterial pressure (mmHg) 83 85 92 74 90 54

17 22 16 20 16 30Pulmonary arterial pressure (mmHg) 13 14 14 19∗ 14 23

4 3 4 4 3 8Pulmonary wedge pressure (mmHg) 9 9 9 10 9 11

2.8 3 2.1 3.4 2.4 3.5Pulmonary vascular resistance

(mmHg l–1 min–1)3.5 3.1 3.2 3.4 3.0 5.02.0 1.4 2.4 1.3 2.1 2.1

VO2 (ml min–1) 67 78 80 98 83 6011 15 15 14 11 23

VCO2 (ml min–1) 54 55 55 69 59 5311 13 12 12 8 14

VE (l min–1) 4.0 3.8 3.7 3.9 4.0 4.00.9 1.0 1.0 1.1 1.2 1.2

Tidal volume (litres) 0.26 0.26 0.25 0.25 0.25 0.240.04 0.04 0.04 0.03 0.02 0.03

Breathing frequency (breaths min–1) 15 15 15 16 16 173 3 3 4 4 4

∗P < 0.05 vs. baseline.

animals (Hedenstierna et al. 1979). Right-to-left shuntcalculated by MIGET (0.4 ± 0.4% of the cardiac output),and anatomic shunt calculated by microsphere passage(0.2 ± 0.3% of the cardiac output) were both small andnot statistically different from each other (P = 0.10). Fiveof the nine animals had evidence of IPAVAs at baseline asevaluated by saline contrast, with a score of 1 in one animaland a score of 2 in four. Three of these five animals hadevidence of IPAVAs at baseline as evaluated by microspherecontrast echocardiography – two with a score of 1, andone with a score of 2. Of note, no contrast was visualizedin the left ventricle in fewer than five cardiac cycles atbaseline, with dopamine or dobutamine, indicating nointra-cardiac shunt in these conditions.

Dopamine

Compared to baseline, dopamine resulted in an increasein heart rate (from 103 ± 25 to 116 ± 16 bpm,P = 0.02), with no other differences in cardiovascular data.There was a small improvement in ventilation–perfusionmatching with dopamine as LogSDQ was reduced from0.55 ± 0.11 to 0.49 ± 0.10, compared to baseline(P = 0.048). Right-to-left shunt calculated by MIGET was

small (0.6 ± 0.2%), and was not significantly increasedfrom baseline (0.4 ± 0.4%, P = 0.07). Likewise, anatomicalshunt as determined by microsphere passage was also small(0.2 ± 0.3%) and was not significantly increased frombaseline (0.2 ± 0.3%, P = 0.46). Seven of eight animals hadevidence of IPAVAs during dopamine infusion as evaluatedby saline contrast – four with a score of 1, two with a scoreof 2, and one with a score of 3. For technical reasons,a microsphere contrast echo score was not obtained inone animal. Six of seven animals had evidence of IPAVAsduring dopamine infusion as evaluated by microspherecontrast, all with a score of 1.

Dobutamine

As expected, dobutamine increased heart rate, cardiacoutput and pulmonary artery pressure compared tobaseline (all P < 0.01; Table 1). Ventilation–perfusionmatching, as evaluated by LogSDQ and LogSDV, wasnot significantly changed with dobutamine (0.55 ± 0.11vs. 0.55 ± 0.09, P = 0.87, and from 1.69 ± 0.41 to1.92 ± 0.32, P = 0.07, respectively). Deadspace ventilationwas decreased (from 47 ± 5 to 40 ± 4%, P < 0.01) withdobutamine. Relative to baseline, shunt as determined by



Table 2. Mean (±SD) blood gas data during all conditions

Baseline(n = 9)

Dopamine(n = 8)

Post-dopamine(n = 8)

Dobutamine(n = 9)

Post-dobutamine(n = 9)

Cardiac shunt(n = 8)

PaO2 (mmHg) 95 93 88 83∗ 86 51∗

11 8 7 7 7 22PaCO2 (mmHg) 31.4 30.9 31.9 35.4∗ 33.5 33.2

6.2 5.0 3.3 3.0 2.6 5.5AaDO2 (mmHg) 5.6 7.1 7.1 8.3 11.2 23.5∗

9.5 6.4 7.1 5.7 6.4 14.0SaO2 (%) 95.8 95.8 95.0 93.4∗ 94.2 70.2∗

2.4 1.1 1.3 2.0 1.5 22.6Low VA/Q (0.005–0.1) (%) 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0High VA/Q (10–100) (%) 14 15 19 19∗ 16 22

5 6 8 6 4 10Predicted PaO2 (mmHg) 88 89 85 81 84 56

10 8 7 8 6 25Deadspace (%) 47 46 44 40∗ 45 42∗

5 3 4 4 5 12LogSDQ 0.55 0.49∗ 0.51 0.55 0.51 0.75∗

0.11 0.10 0.11 0.09 0.08 0.22LogSDV 1.69 1.78 1.91 1.92 1.92 1.76

0.41 0.43 0.30 0.35 0.27 0.40PvO2 (mmHg) 41.9 44.4 43.4 48.5 44.4 32.5

6.3 6.8 6.2 6.6 5.5 12.3

∗P < 0.05 vs. baseline. Low VA/Q, proportion of lung units with a ventilation/perfusion ratio between 0.005 and 0.01; high VA/Q,proportion of lung units with a ventilation/perfusion ratio between 10 and 100; LogSDV, SD of the long-normal ventilation distribution;LogSDQ, SD of the log-normal perfusion distribution.

MIGET and shunt as determined by microsphere passagewere increased with dobutamine (from 0.4 ± 0.4 to1.1 ± 0.6%, and from 0.2 ± 0.3 to 1.0 ± 1.3%, respectively,P = 0.02 for both). Eight of nine animals had evidence ofIPAVAs with dobutamine as evaluated by saline contrast,with scores of 1 in three animals, 2 in two animals and 3 inthree animals. A microsphere contrast echo score was notobtained in one animal. Six of eight animals had evidenceof IPAVAs with dobutamine as evaluated by microspherecontrast echocardiography with scores of 1 in five animalsand 2 in one animal.

Intra-cardiac shunt

The trans-septal puncture and creation of aninter-atrial communication was successful in providingan intra-cardiac anatomic shunt for a positive controlcondition in four animals, each studied in normoxiaand hypoxia. Intra-cardiac shunt was confirmed by theappearance of saline (and microsphere) contrast in the leftventricle within five cardiac cycles after the appearanceof contrast in the right ventricle. Compared to base-line, intra-cardiac shunt resulted in a significant increase

in heart rate (from 103 ± 25 to 172 ± 12 bpm,P < 0.01) and cardiac output (from 1.44 ± 0.38 to2.29 ± 0.80 l�min−1, P = 0.02). AaDO2 was alsosignificantly increased from baseline (from 5.6 ± 9.5 to23.5 ± 14.0 mmHg, P < 0.01). Ventilation–perfusionmatching was impaired with LogSDQ being increasedrelative to baseline (from 0.55 ± 0.11 to 0.75 ± 0.22,P = 0.01). This increase in LogSDQ could be explainedby partial occlusion of the pulmonary circulation frominflation of the Swan-Ganz catheter, which would havecaused a reduction in pulmonary blood flow in selectregions, and/or some oedema formation secondary tothe large increase in pulmonary artery pressure, and thusmean capillary pressure in this condition. Right-to-leftshunt as evaluated by MIGET was significantly increased(from 0.4 ± 0.4 to 9.1 ± 10.7%, P = 0.05). Similarly,anatomical shunt as determined by microsphere passagewas elevated, but did not reach statistical significance(from 0.2 ± 0.3 to 8.8 ± 14.3%, P = 0.16), probablybecause of marked measurement variability betweenanimals. The associated agitated saline contrast scoreswere 2 in two animals, 3 in four animals and 4 in twoanimals, while the microsphere contrast scores were 1 intwo animals and 3 in the remainder.



Comparison of shunt determined by MIGET to shuntquantified by transpulmonary passage of 25 µmmicrospheres

The two-way repeated measures ANOVA showed thatshunt varied across the conditions (P < 0.01), with

no significant interaction (P = 0.80) observed betweencondition and shunt measurement type (i.e. MIGET vs.microspheres). There was no significant difference acrossall conditions in the amount of shunt determined byMIGET vs. shunt measured by passage of microspheresusing pairwise comparisons (Fig. 1, mean MIGET shunt:

*

#

*

Baseline Dobutamine IC Shunt

0

00 3 6 9 12 15

Microsphere Shunt (%) Microsphere Shunt (%)

3

6

9

12

15 1.5

0.5

0.00.0 0.5 1.0 1.5

1.0

10

20

30

40

503.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

MIGET

IC SHUNT

IDENTITY IDENTITY

DOBUTAMINE

DOPAMINE

BASELINE

Shunt(%)

MIG

ET

Shu

nt (

%)

MIG

ET

Shu

nt (

%)

Shunt(%)

Microsphere

Dopamine

Figure 1. TOP: Box and whisker plot ofmicrosphere and MIGET shunt data atbaseline (n = 9), dopamine (n = 8),dobutamine (n = 9), and followingintra-cardiac (IC) shunt procedure (n = 4,2 observations each). BOTTOM: Mean(±SE) Microsphere vs. MIGET shunt atbaseline, dopamine, dobutamine, andfollowing IC shunt procedure (left) andwith IC shunt removed (right)Note: individual data are included with adifferent colour used for each animalthroughout all the figures. Dashed line =mean, continuous line = median. The boxrepresents observations between the 25thand 75th percentiles. The whiskers representthe 10–90th percentile. #P < 0.05 vs. baselinemicrosphere shunt, ∗P < 0.05 vs. baselineMIGET shunt. Across all conditions, there wasno difference between shunt measured bymicrospheres vs. MIGET (P = 0.64).

4

3

2

Saline ContrastMicrosphere Contrast

1

0

Baseline Dopamine Dobutamine IC Shunt

EchocardiographicContrast

Score

Figure 2. Box and whisker plot ofagitated saline contrast andmicrosphere contrast data at baseline(n = 9), dopamine (n = 8), dobutamine(n = 9) and following intra-cardiac (IC)shunt procedure (n = 4, twoobservations each)Note: individual data are included with adifferent colour used for each animalthroughout all the figures. Dashed line =mean, continuous line = median. The boxrepresents observations between the 25thand 75th percentiles. The whiskersrepresent the 10–90th percentile.



2.6 ± 6.1% vs. mean microsphere shunt: 2.3 ± 7.4%,P = 0.64). Shunt evaluated by MIGET vs. shunt evaluatedby microspheres was significantly correlated across allconditions (R2 = 0.78, P < 0.001; Fig. 3), whenintra-cardiac shunt data were removed (R2 = 0.35,P = 0.002; Fig. 3) and when only the intra-cardiac shuntdata were evaluated (R2 = 0.72, P < 0.001). Figure 4shows Bland–Altman plots comparing shunt evaluated byMIGET vs. shunt evaluated by transpulmonary passage ofmicrospheres. The mean bias of MIGET was+0.28% when

50

40

30MIGETShunt(%)

MIGETShunt(%)

20

10

0

0

1

2

3

4

0

0 1 2 3 4

10

Baseline

Baseline

R2 = 0.78

R2 = 0.35

P < 0.001

P = 0.002

Dopamine

Dopamine

Dobutamine

Dobutamine

Intra-Cardiac Shunt

20 30 40 50

Microsphere Shunt (%)

Microsphere Shunt (%)

Figure 3. Pearson correlation coefficient of all individual datacomparing shunt as determined by microspheres and shunt asdetermined by MIGET across all conditions (top, n = 34), andwith intra-cardiac shunt condition removed (bottom, n = 26)Dashed line = line of identity. Different coloured symbols denoteindividual animals.

all data were included and +0.27% when intra-cardiacshunt data were removed.

Comparison of saline contrast echocardiographyscores to microsphere contrast scores

Comparison of scores from the agitated saline contrastto the scores obtained from imaged microspheres allowsfor evaluation of potential factors related to bubbledeformation and bubble survival. Friedman’s ANOVAfound that there was no significant difference in salinecontrast score across the various conditions, i.e. baselineor intervention (P = 0.15); and similarly, there was nosignificant difference in microsphere contrast score acrossconditions (P = 0.19; Fig. 2). Pooled comparisons ofsaline contrast score as compared to microsphere contrastscore across all conditions found a significant differencein pooled median score, indicating that the median salinecontrast score was higher than the median score formicrosphere contrast (median saline contrast score: 2 vs.median microsphere contrast score: 1, P < 0.01). Withall the individual data combined, saline contrast bubblescore showed a significant rank order compared to micro-sphere contrast score (ρ = 0.635, P < 0.001; Fig. 5) onSpearman rank order correlation; however, with the ICdata removed, saline contrast bubble score did not show asignificant rank order compared to microsphere contrastscore (ρ = 0.374, P = 0.07). Note that a Spearman rankorder correlation evaluates the relative position betweentwo sets of variables, and differs from the Pearson productmoment correlation, which evaluates the strength of alinear association. Spearman’s rho is relatively insensitiveto extremes of data because an outlier in the Spearman’srho analysis is limited to its rank in the overall data.

Comparison of saline contrast scores and microspherecontrast scores to microsphere shuntSaline contrast bubble score showed a significant rankorder compared to shunt as evaluated by recovery ofmicrospheres in arterial blood with all data combined(ρ = 0.601, P < 0.001; Fig. 6), and with the intra-cardiacshunt condition removed (ρ=0.429, P=0.029). Similarly,microsphere contrast score showed a significant rankorder compared to shunt as evaluated by recovery ofmicrospheres in arterial blood with all data combined(ρ = 0.665, P < 0.001; Fig. 6) and with the intra-cardiacshunt condition removed (ρ = 0.555, P = 0.005).

Sensitivity/specificity of contrast echocardiography todetect anatomic shunt

Sensitivity and specificity to detect a 1% shunt forsaline contrast echocardiography and microsphere scores



are given in Table 3. An agitated saline score of �1demonstrated very high sensitivity (100%) to detect aphysiologically meaningful (�1%) shunt as measured byarterial sampling of microspheres, but very low specificity(22%); a score of �1 was associated with a micro-sphere shunt of <1% in 21 of 27 observations. Witha threshold saline contrast score of �2, sensitivity wasreduced to 86%, but specificity was only 48%. In otherwords, in roughly half of the observations, a bubble scoreof 2 was associated with an intra-pulmonary anatomicalshunt measured by microspheres recovered in the systemicarterial blood of <1% of cardiac output. Similarly, amicrosphere contrast score of �1 also demonstrated veryhigh sensitivity (100%), but very low specificity (36%) todetect a >1% shunt measured by microspheres. However,when the threshold for a significant microsphere contrast

score was raised to �2, sensitivity was reduced to 71% andspecificity improved to 88%.

Sensitivity/specificity of MIGET to detect anatomicshunt

Sensitivity and specificity to detect a 1% shunt forMIGET is also presented in Table 3. A MIGET shuntof 1% demonstrated high sensitivity (86%) to detect aphysiologically meaningful (�1%) shunt as measured byarterial sampling of microspheres, and high specificity(78%). Specificity was affected by the fact that MIGET,as expected, detected shunt even when there was noevidence of anatomical shunt. This is because MIGETdetects shunt as the inability to excrete inert gases, andthus has contributions from additional sources such as

Figure 4. Bland–Altman figures demonstrating the relative difference in shunt (MIGET, microspherecalculated shunt) relative to mean shunt calculated by the two methodsNote: left = all conditions, right = excluding intra-cardiac shunt conditions. Dotted line = 95% confidence interval.Mean bias was 0.28% shunt when all data were included and 0.27% with intra-cardiac shunt data excluded.Different coloured symbols denote individual animals.

MicrosphereContrast

Score

0

0 1

BaselineDobutamineDopamineIntra-Cardiac Shunt

ρ = 0.635P < 0.001

2

Saline Contrast Score

4

1

2

3

4

Figure 5. Comparison of agitated salinecontrast echocardiography score andmicrosphere contrast score across allconditionsNote: 32 observations are reported. All contrastscores were always reported and analysed inwhole numbers; however, to allow visualizationwithin this figure, saline contrast scores aredisplayed with variance around each wholenumber. Spearman rank correlationdemonstrated a rank order association betweensaline contrast score and microsphere contrastscore (ρ = 0.635, P < 0.001). Different colouredsymbols denote individual animals.



atelectasis or alveolar flooding and not from anatomicshunting alone.

Discussion

The purpose of the present study was to betterunderstand the physiological significance of trans-pulmonary microbubble transmission for pulmonarygas exchange. We approached this by comparing

anatomical intra-pulmonary/intra-cardiac arteriovenousconnections evaluated using 25-µm microspheres to(1) right-to-left shunt gas exchange shunt quantifiedby MIGET, and (2) to the anatomical connections asassessed by contrast echocardiography. Under a varietyof conditions, anatomical shunt, as determined by micro-spheres, largely paralleled the right-to-left gas exchangeshunt calculated by MIGET, suggesting that contrary toour hypothesis, shunt determined by MIGET does not

Figure 6. Agitated saline contrast bubble score (Left) and microsphere contrast score (Right) vs. shuntas determined by recovery of 25 µm microspheres in systemic arterial blood for all data (Top) and withintra-cardiac shunt condition removed (Bottom)Note: all contrast scores were always reported and analysed in whole numbers; however, to allow visualizationwithin each figure, contrast scores are displayed with variance around each whole number. Different colouredsymbols denote individual animals.



Table 3. Sensitivity and specificity of agitated saline contrast,microsphere contrast scoring and MIGET to detect shuntdetermined by microspheres of �1%

Microsphere-quantifiedshunt

<1.0% �1.0% Sensitivity Specificity

Saline contrastscore

(n = 27) (n = 7)

0 6 (22%) 0 (0%) 100 22�1 21 (78%) 7 (100%)0-1 13 (48%) 1 (14%) 86 48�2 14 (52%) 6 (86%)

Microspherescore

(n = 25) (n = 7)

0 9 (36%) 0 (0%) 100 36�1 16 (62%) 7 (100%)0-1 22 (88%) 2 (29%) 71 88�2 3 (12%) 5 (71%)

MIGET (n = 27) (n = 7)<1% 21 (78%) 1 (14%) 86 78�1% 6 (22%) 6 (86%)

Comparison of agitated saline, microsphere scores derived fromultrasound scores and MIGET shunt (percentage of observations)to shunt quantified by microspheres recovered in arterial blood.No shunts were detected with microspheres in 14 cases, <1% in14 cases and �1% in seven cases. A shunt of �1% was acceptedas being physiologically significant. Agitated saline and micro-sphere scores, and MIGET all showed high sensitivity to detectphysiologically significant shunt. A bubble or microsphere scoreof �1 was always observed when there was a �1% shunt present.In one case, MIGET reported a shunt of <1% (0.9%) when therewas a �1% shunt present as quantified by microspheres. Bothagitated saline and microsphere scores showed low specificity,and in 62% (microspheres) to 78% (agitated saline) of cases ashunt score of �1 was associated with a quantified shunt of<1%. Specificity was improved for the microsphere score whena score of 2 was used and an indicator of significant shunt, but foragitated saline contrast the specificity was still very poor. MIGETshowed a shunt >1% in 22% of cases with a shunt quantifiedby microspheres of <1%. However, specificity is affected by thefact that MIGET does not just measure shunt through arterio-venous anastomoses, but also measures other sources of shuntthat contribute to gas exchange impairment.

significantly underestimate the gas exchange contributionfrom intra-pulmonary or intra-cardiac connections.Agitated saline contrast score had a rank order relationshipwith microsphere contrast score (ρ = 0.635, P < 0.001),suggesting that agitated saline contrast bubble appearancein the left ventricle is the result of passage of bubblesthrough anatomical connections that are �25 µm.However, while agitated saline contrast score was foundto be very sensitive (i.e. 100%) to detect a >1% anatomicshunt, the technique demonstrated low specificity (22%),and contrast bubbles were frequently visualized in the left

ventricle when only a very small (or absent) anatomicshunt was measured using microspheres. Furthermore,considerable variability was seen in contrast score for agiven shunt. These results suggest that saline contrastechocardiography, as currently applied, is useful toconfirm the absence of physiologically meaningful arterio-venous connections �25 µm, but is not able to accuratelyquantify or assess the magnitude of anatomic shuntwhen present. Furthermore, the gas exchange abnormalityassociated with intra-pulmonary connections detectedby contrast echocardiography is often small and largelyinsignificant for gas exchange.

Gas exchange

Previous work using agitated saline contrastechocardiography (Eldridge et al. 2004; Stickland et al.2004, 2006; Lovering et al. 2008a), technetium-labelledmicrospheres (Whyte et al. 1992), macroaggregatedalbumin (Lovering et al. 2009b; Duke et al. 2017b)and microspheres (Stickland et al. 2007) suggests thatincreased cardiac output and/or pulmonary vascular pre-ssures during exercise recruits arteriovenous anatomicalpathways in humans and animals. However, these findingshave not been reconciled with the many reports of MIGETdata that have not demonstrated significant (�1% ofcardiac output) gas exchange shunt during exercise(Hammond et al. 1986; Wagner et al. 1986; Hopkins et al.1994, 1998; Podolsky et al. 1996; Rice et al. 1999; Olfertet al. 2004; Jonk et al. 2007).

It has been hypothesized that MIGET may under-estimate the gas exchange consequences of right-to-leftanatomic shunt, possibly because of precapillary gasexchange of respiratory gases, such that inert gases areeliminated to some extent before reaching the capillarybed, while the respiratory gases are not (Stickland et al.2004, 2007, 2013; Lovering et al. 2009a). If so, thismight potentially explain why there can be apparentevidence of anatomical shunt, but no right-to-left shuntas quantified by MIGET. However, when shunt calculatedby MIGET was directly compared to anatomical shuntquantified by 25 µm microspheres in the current study,it is evident that MIGET has the sensitivity to detecta gas exchange shunt, even when the magnitude of theright-to-left shunt via anatomical pathways is extremelysmall (see Fig. 1). Furthermore, when individual data arecompared, shunt determined by microspheres is stronglyrelated to shunt determined by MIGET (see Fig. 3).Finally, Bland–Altman figures demonstrate that there isminimal bias (equal to � +0.3% shunt) between shuntdetermined by MIGET vs. microspheres (see Fig. 4),with limits of agreement of �1% in the intra-pulmonaryshunt conditions. Importantly, this bias is towards MIGETmeasuring greater gas exchange shunt, which is contrary tothe original hypothesis that MIGET would underestimate



shunt when compared to microspheres. It has beenpreviously suggested that because of precapillary gasexchange, IPAVAs may be detected as low VA/Q regionsof the lung, and not as pure shunt. However, as notedin Table 2, there was virtually no low VA/Q regions(i.e. regions where VA/Q = 0.005–0.1) with any of theinterventions, suggesting that when recruited, IPAVAsdo not appear to function as regions of low VA/Qratio. Alternately, as discussed in our companion paper(Stickland et al. 2019) these may be so low that MIGETcannot distinguish these regions from shunt.

Whenever a study returns a negative result, it isreasonable to question whether this represents an issueof statistical power and a failure to reject a false nullhypothesis. We used a repeated measures design in thisstudy, whereby each animal acts as their own control.We assumed that a fractional shunt of 0.01 (i.e. 1%of cardiac output) was a biologically meaningful shunt.We also assumed: (1) that a 10% difference (i.e. 0.1%shunt) between methods was a physiologically importantdifference between the two measurement techniques, (2)a modest correlation between MIGET and microspheremeasures of r = 0.5, and (3) a standard deviation ofmeasurement in healthy normal animals of 10% (0.1%shunt). Our a priori power calculations showed that n = 6animals gave a power of >0.95 to detect a 10% differenceat P < 0.05, two-tailed (Faul et al. 2007). We studiedthree additional animals, and thus our sample size isadequate to test our hypothesis. In order for the observeddifference between MIGET vs. microsphere shunt to bestatistically significant if sustained in a larger study sample,360 animals would be required to give a power of 0.8 todetect a difference at P < 0.05. Furthermore, the apparentdifference between MIGET and microsphere shunt was inthe opposite direction of our hypothesis. Given this, we canbe reasonably certain that our failure to find a significantdifference between MIGET and microspheres is not dueto an inadequate sample size, and the difference, if any, isbiologically unimportant.

In order to study multiple conditions in the sameanimal, we were limited in the total number of micro-spheres (and associated blood samples) that could beinjected (discussed below). The number of microspheresinjected had the sensitivity to quantify a 0.2–0.4%anatomical shunt, based on the number of microspheresinjected, observed cardiac output and the volume ofarterial blood sampled. Consequently, very small shuntsof less than this magnitude may have been missed withthe microsphere technique. In keeping with this, MIGEToften showed the presence of a small gas exchange shuntin cases when anatomic pathways were not detectedby microspheres. While shunt determined by MIGETvs. microspheres were highly related (R2 = 0.78), it isworth emphasizing that MIGET and microspheres do notmeasure the identical shunt. Microsphere transmission

measures an anatomic shunt as perfusion through large(i.e. 25 µm or larger in our study) intra-pulmonary andintra-cardiac vascular channels, whether or not thesechannels impact O2 exchange and arterial oxygenation.MIGET detects a gas exchange shunt defined by theinability to excrete inert gases, which can reflect severalparts of the respiratory system: (1) unventilated butperfused alveoli (atelectasis, fluid or debris-filled alveoli,i.e. with VA/Q ratio of 0, which would be invisible tomicrospheres if the capillaries are of normal size), (2)enlarged vascular channels (but only when they do notallow gas exchange) and (3) intra-cardiac right-to-leftshunts. Thus, some degree of disagreement betweenshunt determined by microspheres and shunt determinedby MIGET is expected. In keeping with this idea, as seenin Fig. 4, MIGET measured a mean shunt �0.3% greaterthan that measured by microspheres. This difference inthe type of shunt measured would also potentially affectthe sensitivity and specificity calculations for MIGETcompared to microsphere transmission, reducing thecalculated specificity of MIGET for an anatomic shunt>1%. In spite of this, MIGET had a high degree ofsensitivity (86%) to detect a significant anatomic shunt.In trying to reconcile the observations of apparentarteriovenous pathways being recruited during exercisevs. a lack of significant right-to-left gas exchange shuntcalculated with MIGET during exercise, our data do notsupport the hypothesis that MIGET has low sensitivity todetect a small shunt.

Confirmation of IPAVAs in the intact lung

In the present study, contrast echocardiography wasconducted during the 25 µm microsphere injections.Previous morphological data indicate that pulmonarycapillaries are �13 µm in diameter, even under conditionsof high perfusion pressure (Glazier et al. 1969). As 25 µmmicrospheres were observed in the left ventricle of at leastsome of the animals under each condition, this wouldsuggest that arteriovenous connections greater than 25µmare present in the intact lung and are thus able to bedetected with contrast echocardiography.

The appearance of microsphere contrast in the leftventricle was correlated with the appearance of salinecontrast in the left ventricle. It has been suggestedthat IPAVAs may be functionally open at all times, andthat the increase in cardiac output under conditions ofphysiological stress (exercise, hypoxia, pharmacologicalinterventions) may reduce transit time, thus allowingfor greater bubble survival for visualization in the leftventricle (Hackett et al. 2016). Transpulmonary passageof microspheres was not typically observed under base-line conditions but was observed with the administrationof dobutamine and following intra-cardiac shunt.Microspheres are incompressible, with an infinite survival



time. Combined with the association between salineand microsphere contrast scores, this suggests that theincreased visualization of contrast agents in conditions ofphysiological stress is unlikely to be due entirely to reducedtransit time and greater bubble survival.

However, agitated saline contrast score was typicallyhigher than the contrast score with injected microspheres.This may reflect increased sensitivity of echocardiographyto saline contrast such that saline bubbles are more easilyvisualized (i.e. more echogenic) than 25µm microspheres.Alternatively, this may reflect a lack of uniformity ofthe saline bubbles being injected, and it is possible thatsome larger bubbles may be compressed and travellingthrough capillaries or that smaller bubbles (i.e. smaller indiameter than a pulmonary capillary) may be traversingthe pulmonary capillaries and subsequently imaged in theleft ventricle (Hopkins et al. 2009b). While the specificity ofthe scoring system to detect meaningful anatomic shuntwas improved when microspheres were imaged, it wasstill variable, suggesting that some of the issues relatedto specificity of the contrast echocardiography techniquerelate to echocardiography itself, rather than the choice ofcontrast agent.

Agitated saline contrast and scoring system

Sensitivity. When agitated saline contrastechocardiography is used to evaluate IPAVAs, manyhave used a scoring system in an attempt to assess theirmagnitude (Lovering et al. 2008b; Bryan et al. 2012;Duke et al. 2017a,b). With this system, a score of 1,i.e. up to three bubbles appearing in the left ventricleafter five cardiac cycles, has been considered evidenceof a significant IPAVA (Lovering et al. 2008b; Bryanet al. 2012). On a gross level, the agitated saline contrastscore, as well as the microsphere contrast score, showsome relationship to the magnitude of the anatomicshunt across the various physiological conditions (seeFigs 1 and 2), with a key caveat. Our data suggestthat the contrast echocardiography technique hashigh sensitivity to detect small anatomic shunts (i.e.<1% of cardiac output), and when a small number ofbubbles or microspheres are visualized within the leftventricle with echocardiography, that these may in factreflect a very small arteriovenous connection. However,the magnitude of shunt fraction is extremely small,and not physiologically important for gas exchange.For example, a median score of 1 on agitated salinecontrast echocardiography at baseline corresponded toa gas exchange shunt of 0.4 ± 0.4% of cardiac outputdetermined by MIGET, and a 0.2 ± 3% anatomic shuntmeasured with microspheres. Importantly, an anatomicshunt of this magnitude would not be sufficient to explainthe gas exchange impairment observed during exercise(Dempsey & Wagner, 1999). Conversely, there were no

cases where an anatomic shunt �1% of cardiac outputwas measured with microspheres without an associatedsaline contrast score or microsphere score greater than1. Indeed, the same observation held true for a shunt�0.5% of cardiac output. Thus, when saline contrast ormicrospheres are not observed in the left ventricle, onecan be confident that an appreciable anatomic shunt isnot present.

Specificity. While contrast echocardiography appears tobe highly sensitive, the scoring system lacks specificity,because there were many observations where either salineor microsphere contrast was detected in the left ventriclewith ultrasound, despite minimal shunt detected witheither MIGET or arterial microsphere sampling. Theintra-cardiac shunt condition resulted in an averageshunt increase of roughly 20- to 40-fold from base-line (MIGET: 0.4–9.1%, microsphere: 0.2–8.8%). Incomparison, contrast saline bubble score increased onlyby about two units even with a significant intra-cardiacshunt. Further evidence for the lack of specificity withcontrast echocardiography is apparent in the regressionplots displayed in Fig. 6. A bubble score of 3 (defined as>12 bubbles in any frame) corresponded to a wide range ofshunt as determined by microspheres. With all conditionscombined, the eight conditions with a bubble score of 3corresponded to microsphere shunts of 0, 0.16, 0.31, 0.38,0.75, 2.4, 5.3 and 42.3% (see Fig. 6, top left panel). Inaddition, five of the eight observations where the bubblescore was 3 corresponded to a microsphere shunt of lessthan 1%. This suggests that simply increasing the scorethat is accepted as representing a significant shunt to 3 isnot sufficient. Although there were two instances wherethe bubble score was 4, and in both of these instancesthis corresponded to a large intra-cardiac shunt of around10%, the small number of measurements and the lack ofscores of this magnitude in the absence of an intra-cardiacshunt limit potential conclusions. Together, these resultssuggest that contrast echocardiography as currently usedis highly sensitive in detecting small, but often physio-logically insignificant, arteriovenous channels; however,the methodology lacks the ability to provide a reasonableestimate of arteriovenous flow. Thus, if agitated salinecontrast is used in a research setting in order to accuratelyevaluate IPAVA magnitude, further improvement in theshunt scoring system is needed. The observations in ourstudy support suggestions by Duke et al. (2017b) thatbubble scores, as currently used, are not linear, and thus abubble score should not be assigned to a given shunt (orIPAVA) magnitude.

Study limitations

The requirement to use a precise incompressible micro-sphere necessitated the use of an animal model. Canines



were used as the experimental model as they have pre-viously been shown to demonstrate IPAVAs during exercise(Stickland et al. 2007), and their relatively large bloodvolume allowed for repeated blood sampling. Duringpositive pressure ventilation, PEEP (5 cmH2O) was usedto reduce the chance of atelectasis. It is possible thata larger anatomic shunt might have been observedwith microspheres in the absence of PEEP or with theanimal breathing spontaneously; however, it is likelythat atelectasis would have developed, thus confoundingshunt comparisons between MIGET and microspheres, asexplained above.

As the recruitment of IPAVAs has been described pre-dominantly during exercise, it would have been ideal tocharacterize gas exchange, shunt and IPAVAs in an exercisemodel. However, it was simply not feasible to acquirehigh-quality echocardiography images during exercisein a canine. It is possible that higher contrast scores(suggesting more IPAVAs) would have been observedduring exercise. As mentioned previously, a right-to-leftshunt of 1–2% would explain the entire AaDO2 duringexercise (Dempsey & Wagner, 1999). It has been arguedthat IPAVAs may not act like a pure shunt, but rathercontribute to diffusion limitation. Previous MIGETwork has shown that VA/Q mismatch explains �50%of the AaDO2 during near-maximal exercise, leavingthe remainder to be explained by diffusion limitation(Hopkins et al. 1994). In this case, the amount of IPAVAflow would need to be �1% to explain the AaDO2 notaccounted for by VA/Q mismatch. In the current study,microsphere data demonstrated an anatomical shunt of1.0 ± 1.3% with dobutamine and an 8.8 ± 14.3%shunt following the surgical creation of an intra-cardiacshunt. While the animal model does not entirely replicateexercise, dobutamine resulted in an anatomical shuntwhich would be similar in magnitude to that whichwould explain all the AaDO2 observed during exercise.Furthermore, the intra-cardiac shunt condition allowedfor comparisons of MIGET, microsphere shunt andcontrast echocardiography during a physiological shuntthat is more extreme than would be observed duringexercise in health. While the metabolic rate, cardiacoutput and mixed venous O2 values were not altered tothe same level as would be observed with exercise, ourexperimental approach allowed for the precise evaluationof shunt within a shunt range expected during exercise.It may be argued that the intra-cardiac shunt condition isvery different from the other conditions studied whichresulted in IPAVAs; however, the intra-cardiac shuntcondition resulted in increases in cardiac output andpulmonary artery pressure, both of which have beenpreviously associated with IPAVA recruitment (Sticklandet al. 2004, 2006; Bryan et al. 2012; Elliott et al. 2014).Thus, it is likely that the intra-cardiac shunt conditionalso resulted in greater perfusion of IPAVAs; however,

there is no way to separate IPAVA perfusion fromintra-cardiac shunt.

The microsphere methodology we used was based onthe goal of injecting a substantial number of microspheresto allow for sensitivity in detecting an anatomical shunt,without over-embolizing the lung. This is confirmed bypulmonary vascular resistance data in Table 1, showingthat pulmonary vascular resistance did not increase withrepeated injections, suggesting that over-embolization wasunlikely. The selection of microsphere size to be injectedwas based on maximizing the sensitivity to detect a shunt,while ensuring that microspheres would not pass throughdistended capillaries (Warrell et al. 1972). Had 15 µmmicrospheres been used, it is likely that shunt magnitudewould have been larger. However, there would have beenconcern that some of these 15 µm microspheres couldhave passed through pulmonary capillaries dilatated bythe dopamine and dobutamine interventions, and it wasfor this reason that 25 µm microspheres were used.

Microspheres were injected directly into the rightatrium to limit microsphere loss, while a relatively largevolume of arterial blood (30 ml) was drawn over 1 minimmediately following injection to maximize sensitivity indetecting microspheres in the systemic blood. The basictechnique of injecting a known quantity of microspheresinto the left atria (or in the present study the right atria)and sampling at a fixed rate from the femoral artery hasbeen shown to be very accurate in determining bloodflow in canines (Archie et al. 1973). Our methodologywas based on collecting a portion of left ventricularoutflow, and it is possible that collecting all of theoutflow would have increased sensitivity to detect a shunt,particularly when the shunt was very small. However,this would have necessitated the use of one animal perinjection, which is impractical. As mentioned above,manufacturer specifications indicate that even one micro-sphere should have been detected in the arterial blood, andthe number of microspheres injected had the sensitivityto accurately quantify a 0.2–0.4% anatomical shunt, ifpresent. Furthermore, a small sample from each micro-sphere vial was obtained before injection to determinetotal counts injected, and counts were reported on all ofthese samples, confirming that microspheres present couldbe activated. Thus, the failure to detect anatomical shuntwith microspheres under some conditions is unlikely tobe explained by inadequate sensitivity of the microspheretechnique.

Summary

The goal of this study was to help resolve the ongoingdiscrepancy between saline contrast data suggestingarteriovenous connections under a variety of conditions,despite no evidence of significant right-to-left gasexchange shunt as quantified by MIGET. There were



three main findings. First, anatomic shunt (whetherintra-pulmonary or intra-cardiac) determined by micro-spheres recovered in the arterial circulation paralleledright-to-left shunt calculated by MIGET, suggestingthat MIGET does not underestimate the gas exchangeconsequences of an anatomic shunt. Second, positivecontrast echocardiography probably detects arterio-venous pathways >25 µm in diameter. Third, contrastechocardiography appears to be extremely sensitive atdetecting small arteriovenous connections, but lacksspecificity at providing an estimate of shunt, oftenidentifying very small anatomic shunts that have no

measurable gas exchange consequence. Based on theseobservations, we suggest that the discrepancy betweencontrast echocardiographic data and MIGET data in pre-vious work can be explained as follows: (1) conditionswhere cardiac output is increased (such as exercise,hypoxia and pharmacological interventions) probablyrecruit small arteriovenous connections that are detectedas positive contrast echocardiography scores; and (2)these arteriovenous connections are probably verysmall (i.e. <0.5% of cardiac output), and thus areunlikely to substantially contribute to gas exchangeimpairment.

Appendix

Table A1. Individual data for select variables

Condition AnimalPaO2 measured

(mmHg)PaO2 predicted

(mmHg) SaO2 (%) PvO2 (mmHg)Cardiac Output

(L min−1)

Baseline 1 89 81 97 35 1.03Baseline 2 114 102 98 32 1.07Baseline 3 88 78 95 45 1.92Baseline 4 98 81 97 38 1.09Baseline 5 105 99 97 46 1.45Baseline 6 102 92 97 47 1.69Baseline 7 100 96 96 52 2.04Baseline 8 87 88 95 40 1.19Baseline 9 76 75 90 42 1.50

Dobutamine 1 91 82 96 37 1.62Dobutamine 2 79 77 94 45 2.20Dobutamine 3 87 82 94 55 4.90Dobutamine 4 80 70 93 43 1.92Dobutamine 5 87 88 94 56 3.94Dobutamine 6 85 82 93 54 2.72Dobutamine 7 91 97 95 54 2.70Dobutamine 8 74 75 91 49 2.20Dobutamine 9 74 76 90 45 2.01

Dopamine 1 87 81 96 36 1.36Dopamine 2 107 100 98 35 1.27Dopamine 3 89 85 95 49 3.12Dopamine 4 88 78 96 43 1.54Dopamine 5 98 96 96 49 2.21Dopamine 6 99 92 96 50 2.38Dopamine 7 93 92 95 52 2.86Dopamine 8 82 85 94 41 1.34Dopamine 9

Cardiac S 6 70 89 92 41 2.25Cardiac S 7 82 87 93 50 2.36Cardiac S 8 78 79 93 41 1.91Cardiac S 9 48 52 69 29 1.64Cardiac S HYP 6 38 40 69 32 3.21Cardiac S HYP 7 34 36 52 28 4.64Cardiac S HYP 8 37 39 64 29 2.05Cardiac S HYP 9 26 30 29 9 0.85



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Additional information

Competing interests

The author have no conflicts of interest.

Author contributions

Experimental studies were performed in the Health SciencesLaboratory Animal Services at the University of Alberta. Allauthors approved the final manuscript and contributed to theconception of the work, the acquisition and analysis of data anddrafting and revising intellectual content.

Funding

Natural Sciences and Engineering Research Council of Canada(NSERC), NIH HL119201, NIH HL129990.



Disclaimer

The views expressed in the submitted article are our own andnot an official position of the institutions.

Acknowledgements

This work was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada (NSERC) and the

National Institute of Health (NIH HL119201, NIH HL129990,USA).

Keywords

arteriovenous anastomoses, pulmonary circulation, pulmonarygas exchange, shunt

Translational perspective

Imaging techniques such as agitated saline contrast echocardiography are often used clinically todetect intra-pulmonary or intra-cardiac shunt connections and, more recently, are used in researchevaluating anatomic intra-pulmonary arteriovenous connections. This technique has shown that theseintra-pulmonary arteriovenous connections can be recruited in conditions such as exercise, or whencardiac output is increased pharmacologically. If present, arteriovenous connections potentially allowblood to bypass the lungs (i.e. cause a shunt) and therefore reduce systemic arterial oxygen content.The purpose of this study was to quantify the amount of flow through arteriovenous connectionswith 25 µm microspheres and compare these results to contrast echocardiography and pulmonarygas exchange shunt as determined by the multiple inert gas elimination technique under a variety ofconditions in canines. Based on previous imaging studies, we hypothesized that the multiple inert gaselimination technique would underestimate shunt as measured by transpulmonary passage of 25 µmmicrospheres. The results indicate that flow through arteriovenous connections measured by 25 µmmicrospheres was not significantly different from gas exchange shunt determined by the multipleinert gas elimination technique. A positive agitated saline contrast echocardiogram was associatedwith anatomic shunt measured by microspheres. However, clinicians and scientists should be awarethat contrast echocardiography often detects very small anatomic intrapulmonary shunts which areinconsequential for gas exchange.


intra‐pulmonary arteriovenous anastomoses and pulmonary ... · intra-pulmonary arteriovenous...

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