Stefano Di Marco°

Beatrice Boldrini #

Umberto Conti

Gabriella Marcucci°

Cecilia Morgantini #

Ele Ferrannini #

Andrea Natali
email: anatali@ifc.cnr.it

Prof Andrea Natali

Effects of GIK (glucose-insulin-potassium) on stressinduced myocardial ischaemia

Keywords: insulin, myocardial ischaemia, GIK Clinical Science Immediate

Despite the evidence in experimental animal models that insulin -or GIK (glucose-insulinpotassium) -improves left ventricular function and perfusion during both acute and chronic ischaemia, clinical studies have generated conflicting results. We tested the hypothesis that pretreatment with GIK attenuates the vascular and functional effects of stress-induced myocardial ischaemia in man. Twenty-two patients with evidence of inducible myocardial ischaemia were enrolled; 11 patients with normal ventricular function underwent two dypiridamole echocardiography tests and 11 with regional contractility defects from previous myocardial infarction were submitted to two ECG exercise tests combined with 201 Tl myocardial perfusion scintigraphy; the tests were preceded by 60 minutes of either normal saline or an isoglycaemic GIK infusion. On stress echo, a 30% reduction in the severity of ischaemia was observed. On ECG ergometry, GIK infusion slightly increased the time to ischaemia (+0.6 min, p=0.07); however, the higher workload (+8%, p=0.07) was achieved at a similar rate-pressure plateau. On scintigraphy, an increase in ischaemic segments (+48%, p<0.001) was imaged mainly at the expense of viable (but non-ischaemic) and non-viable segments, which were reduced by 60%.

GIK affected stress-induced left ventricular underperfusion only marginally (GIK: 39.7±2.5 vs saline: 35.4±2.2 units, p<0.05), but significantly improved its acute reversibility (-42±4 vs -25±4%, p<0.001).

We conclude that GIK pretreatment attenuates the effect of ischaemia on myocardial contractility, slightly improves exercise tolerance and causes a more rapid and diffuse recovery of post ischaemic reperfusion.

Introduction

Insulin's direct and indirect effects on myocardial metabolism [START_REF] Ferrannini | Metabolic and hemodynamic effects of insulin on human hearts[END_REF], vascular reactivity [START_REF] Taddei | Effect of insulin on acetylcholine-induced vasodilation in normal subjects and in patients with essential hypertension[END_REF], microcirculation [START_REF] Bertuglia | Glucose-insulin-potassium treatment in combination with dipyridamole inhibits ischaemia-reperfusion-induced damage[END_REF] and cell membrane potential [4; 5] are all potentially beneficial for the heart when a mismatch between blood flow and energy demand occurs. Both in isolated heart and in experimental animals -especially in the model of low-flow ischaemia [START_REF] Apstein | Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Effects on contractile function, diastolic compliance, metabolism, and ultrastructure during ischemia and reperfusion[END_REF], where residual tissue washout prevents intracellular lactate and proton accumulation -, there is evidence that insulin attenuates the mechanical consequences of ischaemia [START_REF] Ahmed | Sustained effect of glucoseinsulin-potassium on myocardial performance during regional ischemia. Role of free fatty acid and osmolality[END_REF] and accelerates function recovery during reperfusion [START_REF] Apstein | Determinants of a protective effect of glucose and insulin on the ischemic myocardium. Effects on contractile function, diastolic compliance, metabolism, and ultrastructure during ischemia and reperfusion[END_REF]. In addition to the lower oxygen cost of ATP (-13%) due to the shift from lipid to carbohydrate oxidation, insulin enhances anaerobic glycolysis, thereby providing the energy to fuel membrane ion exchange and to prevent intracellular Ca ++ overload during hypoxia [START_REF] Hendrickson | Free fatty acid metabolism during myocardial ischemia and reperfusion[END_REF]. By directly stimulating the ATPase-dependent sodium-potassium pump [START_REF] Ferrannini | Independent stimulation of glucose metabolism and Na+/K+ exchange by insulin in the human forearm[END_REF], insulin could further stabilise myocardial membrane potential and attenuate the impaired repolarisation induced by ischaemia. In addition, insulin has been shown to favour endothelium-dependent vasodilatation of resistance arteries and the microcirculation by enhancing nitric oxide (NO) bioavailability and hyperpolarisation [START_REF] Taddei | Effect of insulin on acetylcholine-induced vasodilation in normal subjects and in patients with essential hypertension[END_REF], to amplify ß-adrenergic vasodilatation and to counteract α-adrenergic vascoconstriction [START_REF] Lembo | Insulin modulation of beta-adrenergic vasodilator pathway in human forearm[END_REF]. Collectively, these actions, if extended to the coronary vasculature, would be beneficial in the setting of acute ischaemia. Despite this physiological rationale, a favourable effect of insulin in acute coronary syndromes has not emerged as clinically significant. In fact, the CREATE-ECLA study failed to show any acute positive effect of glucoseinsulin-potassium infusions (GIK) in a large cohort of patients admitted for acute myocardial infarction [START_REF] Mehta | Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial[END_REF]; likewise, the DIGAMI 2 study in patients with type 2 diabetes could not demonstrate a beneficial effect of insulin infusion on cardiovascular outcomes beyond plasma glucose control [START_REF] Malmberg | Intense metabolic control by means of insulin in patients with diabetes mellitus and acute myocardial infarction (DIGAMI 2): effects on mortality and morbidity[END_REF]. Possible explanations for these partially unexpected results are that insulin was always administered after the occurrence of the acute event and that the model of myocardial infarction is not the most likely to show a protective effect of insulin-induced myocardial metabolic conditioning. As elegantly demonstrated by studies in experimental animals [START_REF] Zhu | Glucose-insulin-potassium preserves systolic and diastolic function in ischemia and reperfusion in pigs[END_REF], the optimal condition to show benefit from for metabolic support is when GIK precedes the event. In this regard, it is interesting that in the CREATE-ECLA study the only patients to benefit from GIK (i.e., an odds ratio of 0.75 for 30-day mortality) were those undergoing primary A c c e p t e d M a n u s c r i p t percutaneous angioplasty (PTCA), which was in every case performed after the start of GIK infusion (due to the small size of this subgroup, the effect did not reach full statistical significance) [START_REF] Mehta | Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial[END_REF]. A recent study has also observed a smaller (-44%) troponine rise after elective PTCA if this was preceded by GIK [START_REF] Yazici | Effect of glucoseinsulin-potassium infusion on myocardial damage due to percutaneous coronary revascularization[END_REF]. Presumably, insulin pre-treatment protects against reperfusion damage and transient ischaemia because these are models of ischemia in which residual tissue washout is present and the changes in cell metabolism induced by insulin are more likely to be protective.

Despite the abundant experimental evidence and the long time since the seminal clinical observation of Sodi-Pallares et al. [START_REF] Sodi-Pallares | Effects of an intravenous infusion of a potassium-glucose-insulin solution on the electrocardiographic signs of myocardial infarction. A preliminary clinical report[END_REF], only two studies [16; 17] have directly addressed the question, whether GIK modifies the threshold and/or the clinical severity of stress-induced ischaemia in humans; their results were not conclusive possibly because the techniques employed (ECG) did not allow the evaluation of the effect of GIK on stress-induced changes in regional myocardial perfusion and contractility.

More consistent, if preliminary, is the evidence on the effect of GIK in the model of chronic ischaemia (viability). Here, insulin infusion has been demonstrated to improve the contractility of the hypokinetic regions to a similar extent as low-dose dobutamine infusion [18; 19]. Whether this effect is entirely metabolic or also involves blood flow redistribution is unclear.

In the present study, we tested whether in patients with ischaemia on effort GIK pretreatment alters the ischaemic threshold, the extension and/or the severity of stress-induced myocardial dysfunction in the normal myocardium and/or of the exercise-induced myocardial perfusion defects in the damaged myocardium.

Methods

Patients We recruited a total of 22 patients of either gender with a positive or suggestive exercise test (ST-segment depression ≥1.5 mm or ≥1.0 mm with symptoms of angina) (Table 1). A c c e p t e d M a n u s c r i p t HbA 1c value above the upper limit of normal (6.3%); chronic hepatic or kidney failure; hyper-or hypokalaemia; ongoing treatment with digitalis. ACE inhibitors or angiotensin receptor blockers (all the patients with previous myocardial infarction and 6 patients with myocardial ischaemia), aspirin and statins (all patients) were maintained; calcium antagonists and ß-blockers were suspended on the day of the tests or 24 hours before at the discretion of the cardiologist (adopting an equal strategy on both study occasions); nitrates were withdrawn on the day of testing. After the study, all patients underwent coronary angiography. Two additional patients in whom angiography did not confirm the presence of haemodynamically significant stenoses consistent with the scintigraphic data were excluded from the study. The study was approved by the Institutional Ethics Committee and all patients signed an informed consent before participating.

Eleven patients without previous myocardial infarction

Study design Each patient received two stress tests within 8-14 days. All tests were preceded by 60 min of either normal saline or GIK infusion: the latter consisted of 500 ml of a 10% glucose solution containing 10 UI of human rapid-acting insulin and 10 mEq of potassium.

The infusion rate was initially set at 3 ml . kg -1. hour -1 ; if plasma glucose rose >20% above baseline additional insulin was added to the infusate (in three subjects in Echo/NoPMI) to increase its concentration ~1.5 fold.

Dyiridamole echocardiograpy test Patients were invited not to eat after the dinner of the day prior to the study and reached the echo lab between 8 and 10 am the next morning. Two indwelling catheters were placed into the right arm, one for insulin and glucose (or saline) infusion in an antecubital vein and another in a hand vein for blood sampling. Another catheter was placed in the left arm for dypiridamole (Persantin, Boehringer-Ingelheim) and atropine (Atropine, Farmigea, Italia) administration. After a 15-min rest, the saline/GIK infusion was started; 2 hours later the sonographer initiated the basal acquisition, which was followed by drug infusions according to a standard format (dypiridamole 0.56 mg/kg in 4 min, followed by no infusion for 4 min after which further 0.28 mg/kg were given in 2 min followed by 2 min of no infusion; then, at min 12 and 13 atropine (0.5 mg) was infused in 30 s). Long-and short-axis images acquired through a 3.5 MHz probe (Acuson Sequoia C256 Ultrasound Instrument, Siemens, Mountain wiew, CA) were continuously recorded on an optic disk. The test was interrupted when a clear change in segmental left-ventricular function occurred; aminophylline (80-240 mg i.v.) and/or atenolol (5 mg i.v.) were immediately administered to restore normal wall kinetics. Segmental left-ventricular kinetics were quantitated using a score from 1 to 5 Myocardial perfusion scintigraphy test Bicycle ergometry started with an initial load of 25 watt followed by increments of 25 watt every 3 min. Six-lead continuous ECG was recorded throughout the test. A standard dose (3 mCi) of 201 Tl was injected at the onset of ischaemic ECG signs and/or at the onset of chest pain; the patient then exercised for further 30-60 s and images were acquired, according to current guidelines [START_REF] Hesse | Clinical Science Immediate Publication[END_REF], trough a SPECT apparatus in three consecutive occasions: a) immediately after interrupting the test (STRESS), b) after 3-4 hours (REST), c) and 20-30 min after a second dose (3 mCi) of Tl 201 was injected (REINJ). Leftventricle walls were subdivided into 20 segments and a score from 0 to 4 (0 corresponding to a normal tracer uptake and 4 to no tracer uptake) was assigned to each segment; the score was estimated by a nuclear medicine specialist (G.M.) who was blind to the presence or absence of GIK infusion. Segments with a post-stress score ≥2 were considered abnormal. A perfusion defect score was calculated as the sum of all the segment scores both in each patient (individual score) and in the whole group (global score). The changes between STRESS, REST and REINJ were used to quantitatively estimate the reversibility of the defects. To avoid volume overload, the GIK infusion rate was set at 40 ml/h after the first SPECT acquisition (STRESS), returned to 3 ml . kg -1. hour -1 one hour before the second acquisition (REST) and not changed until the end of the test.

Assayes Plasma samples were collected before the GIK/saline infusion and immediately before the first two scintigrafic acquisitions (STRESS and REST) and before and at the end of the DET. Plasma glucose concentrations were monitored throughout the tests, while potassium concentrations were measured (Beckman Analyser 2) at the end of the test; plasma insulin concentration was measured within 3 months on stored (-20 C°) plasma aliquots (INSKIT, Linco USA).

Statistical analysis Differences between two tests were evaluated with the use of the χ 2 or the Wilcoxon Signed Rank test, as appropriate.

Results

Both stress tests, echo-dypiridamole and exercise scintigraphy, were well tolerated; neither local nor systemic undesirable effects were observed during the GIK infusion. As shown in Table 2, during GIK plasma insulin rose to high physiological levels; in 3 Echo/NoPMI patients additional insulin was required to clamp plasma glucose levels. Plasma glucose and potassium levels were not different throughout tests, with the only exception of the STRESS phase in the MS/PMI group, during which plasma glucose tended to be higher during GIK than saline (p<0.05).

Echo/NoPMI -Blood pressure and heart rate during STRESS were similar in GIK and saline (Table 3). The test was positive for ischaemia in all saline studies and became negative in 3 GIK studies; the threshold, expressed as time since the beginning of the pharmacologic stress, rose in 7 patients, remained stable in 3 patients and worsened in one patient, giving an average increase of 22% (p=0.06). The frequency of ST depression and typical pain were similar in the two tests (Table 3). Despite the slightly higher pharmacologic load, both the severity (as indexed by the variation in wall motion score -∂WMS) and the extension of ischaemia (as estimated by the number of left ventricular segments showing a worsening of the kinetics) tended to be attenuated by GIK, resulting in a 30-35% lower (p<0.04) left ventricular dysfunction as measured by the LV-WMS or the Ischemic Index (Table 3 and Fig. 1). 4), blood pressure and heart rate were similar with GIK and saline; however, the same maximal rate-pressure product was achieved at a slightly higher workload (+8%, p=0.07) during GIK. No test became negative during GIK.

MS/PMI -During maximal exercise (Table

As shown in Fig. 2, GIK markedly changed the distribution of the tracer uptake defects detected by the myocardial perfusion scintigraphy (p<0.001). The increase in ischaemic segments occurred mainly at the expense of viable and/or non-viable segments. All (n=3) patients without viable myocardium became positive for viability in at least 2 left ventricular segments. Most (95%) of the myocardial segments that the saline test read as ischaemic were also detected as ischaemic by the GIK test (p=ns, Fig. 2). The between-test agreement was good for normal segments (84%) but poor for both viable (29%) and non-viable (32%) segments (p<0.001). We used the Global Perfusion Defect score to estimate the overall effect of GIK (on both extension and severity) independently of the segment localisation, also including minor changes in perfusion (a change in score of 1 unit). Between STRESS and REST the perfusion defect score decreased -i.e., myocardial tracer uptake increased -by 24% during saline infusion and by 41% during GIK infusion (p<0.001), corresponding to a 70% increment in the acutely reversible defects (that is, overall ischaemia) (Fig. 3). Following reinjection, the tracer uptake further improved by 13% and 11% with saline and GIK respectively (p=ns), indicating a quantitatively similar estimate of overall viability.

The severity of ischaemia was further assessed by considering the 76 ischaemic segments on which there was agreement between the two tests. During GIK the segmental perfusion defect score at STRESS was 1 unit higher than with saline in 22 segments (from 2 to 3 in 10 and from 3 to 4 in 11) and 1 unit lower in 4 segments (from 3 to 2 in 2 and from 4 to 3 in 2 segments), yielding an 8% greater global score (239 vs 222 units, p=ns). In contrast, the number of segments showing a 2-unit improvement at REST was greater (38 vs 10), while the number of 1-unit gradients was lower (38 vs 65) for GIK and saline, respectively (p<0.001).

No regional heterogeneity was observed in the effect of GIK, all the 7 main regions of the left ventricle (anterior, antero-septal, inferior, infero-septal, lateral, infero-lateral and apical) showing a similar spatial distribution of the perfusion changes.

Discussion

GIK improved the ischaemic threshold and exercise tolerance only marginally but significantly reduced (by ~30%) the stress-induced left ventricular contractile dysfunction and accelerated the acute perfusion recovery in ischaemic regions.

During GIK, the maximal workload and maximal pharmacologic burden tolerated were 8% and 22% higher, respectively, than with saline. This result should be interpreted cautiously not only because of borderline statistical significance but also because the differences were small and the ECG signs of ischaemia were observed at similar double-product rates. The effect on exercise tolerance could be due to improved systemic haemodynamics (secondary to peripheral A c c e p t e d M a n u s c r i p t vasodilatation, as seen in patients with chronic heart failure [START_REF] Parsonage | Beneficial haemodynamic effects of insulin in chronic heart failure[END_REF]) and/or to a slower rise in heart rate; this, in turn, could be the consequence of an improved skeletal muscle efficiency (i.e., ATP production per unit of oxygen consumption). Thadani et al [START_REF] Thadani | Effects of low and high glucose in a glucoseinsulin-potassium infusion on hemodynamics and exercise tolerance in patients with angina pectoris[END_REF] also reported some increase in the time to angina despite the heterogeneity of the individual responses, whereas Bogaty et al [START_REF] Bogaty | Effects of exogenous metabolic substrate modulation on exercise-induced myocardial ischemia[END_REF] found no difference; in the latter study, however, plasma glucose was not controlled and rose from 5.7 to 9.4 mM before the exercise. Our patients also showed variability, with a change in exercise duration that was positive in 6 patients, nil in 4 and negative in 1. In any event, this modest improvement is unlikely to be of clinical relevance.

The data of the Echo/NoPMI study also suggest that GIK only marginally affects the ischemic threshold although it attenuates the mechanical consequences of ischemia. A 13% increase in the ischaemic threshold would be the expected if the only underlying mechanism was the improved metabolic efficiency (i.e., ATP/oxygen); and such a difference, however, might have been missed given the limited sample size and the study design. In their elegant study, Bogaty et al [START_REF] Bogaty | Effects of exogenous metabolic substrate modulation on exercise-induced myocardial ischemia[END_REF] induced marked metabolic changes in circulating substrates, but saw no effects on the ischaemic threshold. In contrast, in perfused isolated hearts [START_REF] Korvald | Myocardial substrate metabolism influences left ventricular energetics in vivo[END_REF], complete substitution of glucose for free fatty acids (FFA) as a fuel causes a 30% improvement of cardiac efficiency, suggesting that more extreme metabolic manipulations may be more effective, possibly through additional mechanisms (e.g., inhibition of futile cycles); however, the clinical relevance of these manipulations remains questionable. The large inter individual variability of the response to GIK (Figure 1), which was unexplained by the available clinical variables, however, suggest that the effect of GIK pre treatment on ischemia might be more relevant in some specific patients.

With regard to perfusion, the scintigraphic data showed that, for a similar myocardial stress (double product), extension and severity of perfusion defects were if any accentuated during GIK.

However, the comparison with the control situation (saline) shows that most (76 out of 80) of the ischaemic segments (including only ischaemic and ischaemic-and-viable) on the saline test were confirmed ischaemic during GIK; the majority of the "new", i.e., GIK-induced ischaemic segments (30 out of 42) were either non-viable (n=15) or viable but not ischaemic (n=15) segments on saline. Thus, the worsening of ischaemia by GIK was mostly apparent because the infusion "upgraded" chronically underperfused segments while causing a true perfusion deterioration (mostly of 1 unit) in only 12 out of 91 normally perfused segments. This interpretation is supported by the perfusion defect score. As recapitulated in Fig. 3, under stress conditions GIK increased the perfusion defect score by 14% relative to saline, but the drop in score from stress to resting conditions was almost double with GIK. This was true regardless of whether the score was individual or global, absolute or relative, and also applied to the subset of myocardial segments that were ischaemic on both tests. That the overall ischaemic burden was not significantly increased by GIK is also confirmed by the clinical indices: mean ST-T segment depression averaged 1.5 mm during saline and 1.3 mm during GIK and angina was present in 5 patients during saline and in 3 during GIK. Provided that the two stresses induce ischaemia through different mechanisms, our data suggest that the effect of GIK on stress ischaemia is mostly metabolic. Although unlikely, we cannot exclude that GIK exerts different vascular and metabolic effect on the working myocardium depending on whether a portion of it has been damaged by a previous infarction.

The MS/PMI study indicates that post-stress tissue perfusion (i.e., reperfusion) is improved by GIK; with regard to the mechanism(s) and consequences of this improvement we can only speculate. In the Hamster cheek pouch model, insulin has been shown to improve post-ischaemic tissue microcirculation through arteriolar vasodilatation and reduction in platelet adhesion [START_REF] Bertuglia | Glucose-insulin-potassium treatment in combination with dipyridamole inhibits ischaemia-reperfusion-induced damage[END_REF].

Whether this phenomenon impacts on post-ischaemic recovery of myocardial function remains to be tested by techniques that measure absolute tissue perfusion and contractility simultaneously (e.g., contrast echocardiography, magnetic resonance imaging). With regard to this, it is interesting to note that pretreatment with GIK has been shown to reduce the troponine-I rise produced by the coronary angioplasty procedure [START_REF] Yazici | Effect of glucoseinsulin-potassium infusion on myocardial damage due to percutaneous coronary revascularization[END_REF]. Another possible explanation, suggested by some studies [START_REF] Tartagni | Detecting hibernated myocardium with SPECT and thallium-glucose-insulin infusion[END_REF], is that insulin might simply enhance myocardial avidity for thallium (which is handled like potassium). We think that this is unlikely for the following reasons: a) in vivo potassium and thallium are handled with different kinetics by the human myocardium [START_REF] L'abbate | Myocardial kinetics of thallium and potassium in man[END_REF], b) post-injection (stress and reinjection) thallium uptake has been shown not to be affected by insulin and to be dependent only on blood flow [START_REF] Weich | The extraction of thallium-201 by the myocardium[END_REF], c) delayed post-stress imaging depends on thallium redistribution (i.e., loss of normal tissue and enrichment in ischaemic tissue); during this phase, GIK enhances tissue thallium wash-out from both ischaemic and normal regions [START_REF] Wilson | Effect of glucose-insulinpotassium infusion on thallium myocardial clearance[END_REF], thereby exerting, if any, a negative effect on the sensitivity of the test to detect ischaemia [START_REF] Wilson | The effect of eating on thallium myocardial imaging[END_REF].

The conclusion, that GIK favours postischaemic reperfusion, is strengthened by the data on myocardial viability. The global perfusion defect score on reinjection during saline (243 units) was similar to the score measured under resting conditions with GIK (258 units) (Fig. 2). On A c c e p t e d M a n u s c r i p t tracer reinjection, GIK decreased the score further (to 212 units), i.e., it increased myocardial perfusion. In fact, non-viable segments decreased from 31 to 13 (p<0.05), being "upgraded" either to ischaemic (n=15) or viable (n=5) (Fig. 2). That GIK can recruit non-viable tissue to viability has been confirmed in a recent study comparing a modified GIK scintigraphy (coinfusion of thallium and GIK) with the gold standard technique, namely, 18 F-deoxyglucose uptake [START_REF] Toyama | Evaluation of myocardial viability following acute myocardial infarction using 201Tl SPECT after thallium-glucose-insulin infusion-comparison with 18F-FDG positron emission tomography[END_REF]. Again the mechanism appears to be related to myocardial energy metabolism, rather than tracer handling, since in patients with previous myocardial infarction GIK was able to improve the contractility of the myocardial segments with stable kinetic defects to an extent similar to low-dose dobutamine infusion [START_REF] Yetkin | Comparison of low-dose dobutamine stress echocardiography and echocardiography during glucose-insulin-potassium infusion for detection of myocardial viability after anterior myocardial infarction[END_REF].

It is important to note that our GIK protocol was essentially euglycaemic and hyperinsulinaemia was within the physiological postprandial range. In previous studies, different GIK mixtures gave rise to variable rates of both hypoglycaemia and hyperglycaemia. In the CREATE-ECLA trial [START_REF] Mehta | Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial[END_REF], for example, the group randomised to GIK had significantly higher plasma glucose levels and more hypoglycaemia during the 24 hours of infusion as compared to the usual care group. Both hypoglycaemia -through sympathetic activation [START_REF] Rowe | Effects of insulin and glucose infusions on sympathetic nervous system activity in normal man[END_REF] -and hyperglycaemia -through oxidative stress [START_REF] Yetkin | Comparison of low-dose dobutamine stress echocardiography and echocardiography during glucose-insulin-potassium infusion for detection of myocardial viability after anterior myocardial infarction[END_REF] -can offset any beneficial effects of insulin.

In conclusion, pretreatment with GIK slightly enhances the myocardial ischaemic threshold, reduces the severity of the stress-induced contractile dysfunction and improves postischaemic reperfusion (reflow); in addition, it also enhances tissue viability identification.

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

  underwent echo-dypiridamole testing for ischaemia (Echo/NoPMI study). The other 11 patients with previous myocardial infarction (>3 months) underwent exercise myocardial perfusion scintigraphy for the detection of ischaemia and viability (MS/PMI study). Exclusion criteria were: previous diagnosis of diabetes mellitus or a Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438 A c c e p t e d M a n u s c r i p t (1=normal, 2=hypokinetic, 3=akinetic, 4=dyskinetic, 5=aneurismatic) for each of the 16 standardised left-ventricular wall segments; a global wall motion score (LV-WMS) was then calculated as the mean of these 16 values. We also calculated an index of ischaemia severity (Ischaemic Index) by summing all the changes in the wall motion score of each segment and normalising this value by the pharmacological stress expressed as the time elapsed between the start of drug administration and the end of the test (aminophylline/atenolol administration).

  Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

Clinical

  Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 -

 1 Figure 1 -Dypiridamole echocardiography test: individual, and mean, ischemic index value (see methods) in the Saline and GIK tests.

Figure 2 -

 2 Figure 2 -Myocardial segment status distribution as defined by the results of myocardial stress scintigraphy in the Saline and GIK test. The arrows, whose thickness is proportional to the number of segments, illustrate the fluxes of the segments that changed status.

Figure 3 -

 3 Figure 3 -Distribution of the perfusion defect scores (histogram and left Y axis) in all the left ventricle myocardial segments of the 11 patients and Global perfusion defect score values (dotted line and right Y axis) during the three SPECT acquisitions Stress, Rest and Reinjection.

Table 1 -Demographic and clinical parameters Echo/NoPMI MS/PMI

 1 Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438	Gender (m/f) Age (years) Hypertension (y/n) Exercise ECG positive/suggestive Effort angina (y/n) Anterior Infero-lateral Inferior Previuos MI Apical One vessel Two vessels Angiography Tree vessels Ejection Fraction (%) A c c e p t e d M a n u s c r i p t 8/3 10/1 67 ± 3 64 ± 2 6/5 4/10 8/3 9/2 10/1 5/6 -6 -4 -2 -2 1 5 4 2 6 4 59 ± 4 43 ± 2

Table 2 -Plasma concentrations of glucose, insulin and potassium

 2 Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

			Echo/NoPMI	MS/PMI
			Saline	GIK	Saline	GIK
	Glucose (mmol/l) Baseline	6.0 ± 0.5	6.1 ± 0.6	5.4 ± 0.2	5.9 ± 0.3
		Stress	6.3 ± 0.3	6.8 ± 0.8	6.3 ± 0.3	7.7 ± 0.1*
		Rest	5.9 ± 0.2	6.1 ± 0.5	5.6 ± 0.2	5.7 ± 0.4
	Insulin (pmol/l)	Baseline	98 ± 16	105 ± 28	78 ± 6	72 ± 6
		Stress	108 ± 22	1,130 ± 282*	48 ± 6	754 ± 54*
		Rest	98 ± 17	1,060 ± 275*	48 ± 6	652 ± 60*
	Potassium (mEq/l) Baseline	3.9 ± 0.3	3.8 ± 0.2	3.9 ± 0.3	4.1 ± 0.2
		Stress	3.8 ± 0.3	3.6 ± 0.4	3.9 ± 0.3	4.0 ± 0.2
		Rest	3.9 ± 0.3	3.7 ± 0.4	4.0 ± 0.3	4.1± 0.4

* p<0.05 vs Baseline by Wilcoxon Signed Rank test

Table 3 -Echocardiographic parameters during echo dipyridamole stress test. Saline GIK

 3 Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

	Statistics°

SBP: systolic blood pressure; DBP: diastolic blood pressure; HR: Heart rate; ∂WMS: wall motion score change; LV-WMS stress: left ventricular mean wall motion score in stress condition

° Wilcoxon Signed Rank Test

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Table 4 -Ergometry Saline GIK

 4 Clinical Science Immediate Publication. Published on 15 Dec 2009 as manuscript CS20090438 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090438

	p

SBP: systolic blood pressure; DBP: diastolic blood pressure; RP: rate pressure product;

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited


MD Alexander ; Zipprich
email: alexander.zipprich@medizin.uni-halle.de

Matthias Winkler

Thomas Seufferlein

Matthias M Dollinger

Martin

Comparison of balloon versus straight catheter for measurement of portal hypertension

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Introduction

In patients with cirrhosis increased intrahepatic resistance and vasodilatation of the splanchnic circulation will eventually lead to a rise in portal pressure [START_REF] Wiest | The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough[END_REF]. Portal hypertension, however, directly correlates with the development of cirrhotic complications and the mortality thereof. To assess portal pressure quantitatively, a catheter has to be introduced into the hepatic vein to determine free (FHVP) and wedge (WHVP) hepatic venous pressure. The difference between the two measurements, i.e. the hepatic venous pressure gradient (HVPG), is then commonly used as an estimation of the portal pressure [START_REF] Groszmann | The hepatic venous pressure gradient: anything worth doing should be done right[END_REF].

Traditionally regarded as a tool for research, HVPG has first been suggested to be of clinical value in the early 1950s [START_REF] Sherlock | Hepatic vein catheterization in clinical research[END_REF][START_REF] Browne | Hepatic catheterization and upper gastrointestinal hemorrhage in portal hypertension[END_REF]. Since then, its clinical potential has been repeatedly re-evaluated when studies demonstrated that the degree of portal hypertension quantified by HVPG may predict decompensation of liver disease, outcome after variceal bleeding or surgery for hepatocellular carcinoma, and ultimately long-term survival of patients with cirrhosis [START_REF] Sherlock | Hepatic vein catheterization in clinical research[END_REF][START_REF] Browne | Hepatic catheterization and upper gastrointestinal hemorrhage in portal hypertension[END_REF][START_REF] Boyer | Wedged hepatic vein pressure (WHVP): ready for prime time[END_REF][START_REF] Leevy | Practical and research value of hepatic-vein catheterization[END_REF][START_REF] Groszmann | The hepatic venous pressure gradient: has the time arrived for its application in clinical practice?[END_REF][START_REF] Mukherjee | Beta-blockers to prevent esophageal varices--an unfulfilled promise[END_REF][START_REF] Abraldes | Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis[END_REF][START_REF] Ripoll | Influence of hepatic venous pressure gradient on the prediction of survival of patients with cirrhosis in the MELD Era[END_REF](11). A 20% reduction of HVPG is usually considered a good hemodynamic response associated with a lower risk to develop complications related to portal hypertension, mainly variceal bleeding [START_REF] D'amico | Hepatic vein pressure gradient reduction and prevention of variceal bleeding in cirrhosis: a systematic review[END_REF]. However, reproducibility and reliability of HVPG measurements are crucial before a method to determine HVPG can be implemented in everyday clinical practice used by a multitude of different investigators.

Several authors investigated the reliability of HVPG measurements as a surrogate marker for portal pressure. In one study, correlation between a single measurement of HVPG and portal pressure was high with a correlation coefficient of at least 0.93 independently of the etiology of the cirrhosis [START_REF] Perello | Wedged hepatic venous pressure adequately reflects portal pressure in hepatitis C virus-related cirrhosis[END_REF]. In contrast, measurements in three different hepatic veins varied by as much as 60% in another study, questioning reproducibility and hence the clinical validity of the procedure [START_REF] Keiding | Intrahepatic heterogeneity of hepatic venous pressure gradient in human cirrhosis[END_REF]. The contrasting results, however, were obtained by two different methods of measurement using a balloon catheter [START_REF] Groszmann | Wedged and free hepatic venous pressure measured with a balloon catheter[END_REF] or a straight catheter to wedge the hepatic venous flow.

Although the balloon catheter is considered to be more reproducible and reliable, a direct comparison of both methods has not been performed so far [START_REF] Groszmann | The hepatic venous pressure gradient: anything worth doing should be done right[END_REF][START_REF] Groszmann | Wedged and free hepatic venous pressure measured with a balloon catheter[END_REF].

The aim of this study was to compare the two available methods to measure HVPGballoon and straight catheter -head-to-head and to assess reproducibility and reliability of both. the position of the each catheter controlled radiologically. To measure the WHVP, the straight catheter was advanced into the hepatic vein until a wedged position was achieved. Alternatively, the balloon catheter was inflated achieving likewise a wedge position of the catheter. In both measurements, the appropriate wedge position was documented radiologically and by the characteristic pattern of the wedged curve (figure 1) as suggested by Groszmann et al. [START_REF] Groszmann | The hepatic venous pressure gradient: anything worth doing should be done right[END_REF]. Before the recording of the tracings, both free and wedge hepatic venous pressure were allowed to stabilize for 45s to 60s. The HVPG was calculated as the difference of the two measurements (HVPG = WHVP -FHVP).

Measurement of the WHVP and portal pressure

The direct intravascular portal pressure was assessed together with the WHVP during the procedure of implanting a transjugular intrahepatic portosystemic shunt.

The WHVP was measured either with a straight or a balloon catheter as described above. Thereafter, the portal vein was punctured using a transjugular biopsy needle.

A guide wire was introduced into the portal vein and advanced to the confluence of the vessel followed by a 7 French catheter for measuring the portal pressure. The guide wire was removed and the right position of the catheter in the portal vein documented radiologically. Before the measurements the portal pressure tracings were allowed to stabilize for 45s to 60s.

Statistical examination

Data were expressed as the means ± standard deviation (SD). The variation coefficient for each catheter was calculated by the standard deviation divided by the means. T-test for paired samples was used to compare data of repeated Measurements with balloon and straight catheter in comparison 47 patients were assessed hemodynamically using straight and balloon catheter in sequence. Hepatic pressures determined with the balloon catheter (figure 1) were 14.7±7.2 mmHg (HVPG), 13.0±4.4 mmHg (FHVP) and 27.9±8.6 mmHg (WHVP), respectively. Calculation of the variation coefficients for the triplicate measurements revealed only minor variations of the absolute values with a coefficient of 0.07 for the HVPG, 0.02 for the WHVP and 0.06 for the FVHP (figure 2). Hepatic pressures determined using the straight catheter (figure 1) were 13.1±6.5 mm Hg (HVPG), 12.8±4.6 mm Hg (FHVP) and 26.1±8.4 mm Hg (WHVP). Compared with the values obtained using the balloon catheter, variation coefficients for the triplicate measurements using the straight catheter demonstrated a significantly wider variation with a coefficient of 0.17 for the HVPG (p<0.001; figure 3) and 0.06 for the WHVP (p<0.001), respectively. Only the measurements of the FHVP showed the same consistency with the straight as compared with the balloon catheter with no statistically significant difference between the two coefficients (p=0.57). Thus, the In addition to the variation coefficient of each method, the intraclass correlation coefficient was calculated to assess the concordance between measurements obtained with both catheters. With high intraclass correlation coefficients between all three hepatic pressures, i.e. HVPG 0.87 (95%CI 0.74-0.93, p<0.001), FHVP 0.88 (95%CI 0.80-0.93, p<0.001) and WHVP 0.92 (95%CI 0.79-0.96, p<0.001), both methods showed good agreement on absolute values.

Comparison of WHVP and portal pressure

In 29 patients, the WHVP obtained by either balloon (n=13) or straight (n=16) catheter was correlated with a direct intravascular assessment of the portal pressure.

Using the balloon catheter, the WHVP was 26.8±5.4 mmHg (HVPG 24.3±5.4 mmHg) compared with a pressure of 26.8±4.6 mmHg as determined directly within the portal vein. The median of the difference between the two measurements was 0.5 mmHg (interquartile range: -3.25 -2.5; figure 4) with an intraclass correlation coefficient of 0.72 (p=0.004).

Using the straight catheter, the WHVP was 32.8±8.8 mmHg (HVPG 24.1±5.6 mmHg) compared with a direct intravascular portal pressure of 33.4 ±7.4 mmHg. The median difference between the two measurements was 1 mmHg (interquartile range: -9.00 -3.00; figure 4) and the intraclass correlation coefficient only 0.58 (p=0.011). Thus, measurements obtained with the balloon catheter showed a higher correlation with

Discussion

Within recent years, assessment of the HVPG has become a clinical tool with prognostic relevance recommended by an increasing number of experts in the management of patients with liver cirrhosis [START_REF] Groszmann | The hepatic venous pressure gradient: anything worth doing should be done right[END_REF][START_REF] Garcia-Tsao | Portal hypertension and variceal bleeding--unresolved issues. Summary of an American Association for the study of liver diseases and European Association for the study of the liver single-topic conference[END_REF]. Although qualitative evaluation of portal hypertension is possible by indirect means such as endoscopy or ultrasound, only the quantitative HVPG offers the potential for rapid and subtle therapeutic adjustments in affected patients [START_REF] Abraldes | Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis[END_REF][START_REF] Groszmann | Beta-blockers to prevent gastroesophageal varices in patients with cirrhosis[END_REF][START_REF] Ripoll | Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis[END_REF][START_REF] Bosch | The clinical use of HVPG measurements in chronic liver disease[END_REF]. The hemodynamic response to treatment with beta-blockers, for instance, has long been advocated to prevent variceal bleeding [START_REF] Abraldes | Hemodynamic response to pharmacological treatment of portal hypertension and long-term prognosis of cirrhosis[END_REF][START_REF] D'amico | Pharmacological treatment of portal hypertension: an evidence-based approach[END_REF]. A good response can be determined by an absolute value or by a relative decrease in HVPG. Traditionally, a 20% drop in HVPG is the threshold value considered an adequate response. However, recent studies have suggested that a 10% reduction may have a protective effect, too [START_REF] Groszmann | Beta-blockers to prevent gastroesophageal varices in patients with cirrhosis[END_REF][START_REF] Villanueva | Acute hemodynamic response to betablockers and prediction of long-term outcome in primary prophylaxis of variceal bleeding[END_REF][START_REF] Mura | Prognostic value of acute hemodynamic response to i.v. propranolol in patients with cirrhosis and portal hypertension[END_REF]. Reproducibility and reliability of HVPG measurements are therefore crucial taking into account that even minor changes in HVPG have been proven relevant and may be used to support clinical decisions.

In response to conflicting reports on the accuracy of available methods, a recent systematic review attempted to evaluate HVPG measurements as an estimation of portal pressure [START_REF] Keiding | Intrahepatic heterogeneity of hepatic venous pressure gradient in human cirrhosis[END_REF][START_REF] Thalheimer | Assessment of the agreement between wedge hepatic vein pressure and portal vein pressure in cirrhotic patients[END_REF]. The two techniques -balloon and straight cathetercorrelated well with direct portal pressure measurements, however, a comparison between the two methods failed on account of the available data. Our study is the first to compare balloon and straight catheter head-to-head, with the balloon catheter producing the more consistent and reliable results. Assessment of the WHVP, in particular, appears to be the weakness of the straight catheter, affecting the calculation of the HVPG as a consequence. One possible explanation could be hemodynamic differences between hepatic veins as a result of heterogenic liver damage proven in a number of studies assessing the sampling variability of liver biopsies [START_REF] Ratziu | Sampling variability of liver biopsy in nonalcoholic fatty liver disease[END_REF]. The peripheral positioning of the straight catheter, too, might cause an underestimation of the portal pressure in case of small intrahepatic shunts, present in up to 50% of patients [START_REF] Butzow | Clinical value of hepatic vein catheterization. Improved pracability by balloon catheter technique[END_REF]. In fact, the only study evaluating HVPG measurements (straight catheter) in different hepatic veins describes considerable hemodynamic heterogeneity, in part due to the presence of intrahepatic shunts [START_REF] Keiding | Intrahepatic heterogeneity of hepatic venous pressure gradient in human cirrhosis[END_REF].

The technical properties of the balloon catheter appear to eliminate such hemodynamic variations by covering a greater territory. Its clinical application has even been suggested to complement liver biopsy [START_REF] Burroughs | Assessment of therapeutic benefit of antiviral therapy in chronic hepatitis C: is hepatic venous pressure gradient a better end point[END_REF][START_REF] Rincon | Antiviral therapy decreases hepatic venous pressure gradient in patients with chronic hepatitis C and advanced fibrosis[END_REF][START_REF] Blasco | Hepatic venous pressure gradient identifies patients at risk of severe hepatitis C recurrence after liver transplantation[END_REF]. In our study too, the balloon catheter produced more consistent wedged pressure measurements which also correlated better with the direct intravascular portal pressure. The correlation between WHVP determined by the straight catheter and the direct portal pressure, in contrast, remained poor supporting the concept of heterogeneous hemodynamics within different hepatic veins.

One limitation of our study is the comparably low correlation between WHVP and portal pressure. Previous reports demonstrated more conform results for the two pressures, although with the lowest intraclass correlation coefficient in alcoholic cirrhosis [START_REF] Perello | Wedged hepatic venous pressure adequately reflects portal pressure in hepatitis C virus-related cirrhosis[END_REF]. In patients with sinusoidal portal hypertension, WHVP equals portal pressure. However, a number of studies suggest a pre-sinusoidal component to the portal hypertension in non-alcoholic cirrhosis such as primary biliary cirrhosis or viral hepatitis [START_REF] Navasa | Portal hypertension in primary biliary cirrhosis. Relationship with histological features[END_REF][START_REF] Pomier-Layrargues | Presinusoidal portal hypertension in non-alcoholic cirrhosis[END_REF]. In our study with nearly 16% non-alcoholic liver disease, presinusoidal changes might well be more prevalent than previously reported.

  in 76 patients with histologically proven cirrhosis prior to treating portal hypertension by medication or TIPS implantation. In 47 of the patients, a straight and a balloon catheter were used in sequence to measure HVPG during the same procedure. In another 29 patients, the WHVP was measured using either the straight or the balloon catheter followed by direct intraportal assessment of the pressure. All patients gave written informed consent to the above described procedures; the study was carried out in agreement with the declaration of Helsinki.Measurement of the hepatic venous pressure gradient using two different methods All measurements were performed in fasting patients in a supine position after stopping any portal pressure reducing agents or anti-hypertensive medication for a minimum of 24 hours. The hemodynamic assessment included right heart catheterization and measurement of the HVPG. Right heart catheterization was conducted using a quadruple-lumen thermodilution catheter (Abbott Lab., USA) inserted into the internal jugular vein under local anaesthesia with continuous assessment of mean arterial pressure (MAP) and heart rate using automated noninvasive monitoring (Sirecust, Siemens AG, Germany). Standard measurements included central venous pressure (CVP), cardiac output (CO), and systemic vascular resistance (SVR).Each FHVP and WHVP measurement was repeated three times using a 7 French straight catheter (MP A1, Cordis Corporation, USA) and a 7 French balloon catheter (Standard Occlusion Balloon Catheter, Boston Scientific, USA) in sequence. The FHVP was assessed 2 to 3 cm proximal to the confluence of the hepatic veins with

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 7 Zipprich et al.: Reproducibility of HVPG measurement measurements. To compare the two different catheters to each other, the mean of the three single measurements were calculated. The intraclass correlation coefficient was calculated to compare data of different measurements. Bland-Altman plots were constructed to compare the different measurements.

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 8 Zipprich et al.: Reproducibility of HVPG measurement R2 Results Patients description Mean age of patients was 55±12 years, the mean Child-Pugh score 9.2 ± 2.4 (Child-Pugh class: A: n=9; B: n=30; C: n=37). The etiology of cirrhosis was in the majority alcoholic liver disease (n=62). Remaining patients suffered from cryptogenic cirrhosis (n=4), NASH (n=3), hepatitis C (n=2), hemochromatosis (n=2), PSC (n=1), PBC (n=1) or autoimmune hepatitis (n=1). Refractory ascites was the indication for TIPS implantation in the respectively treated 29 patients.

  Zipprich et al.: Reproducibility of HVPG measurement balloon catheter was more precise in determining the HVPG than the straight catheter, a result of the smaller variation of the WHVP when using the balloon to assess the wedged pressure.

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 10 Zipprich et al.: Reproducibility of HVPG measurement R2 the directly determined intraportal pressure than measurements obtained with the straight catheter. Zipprich et al.: Reproducibility of HVPG measurement

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 13 Zipprich et al.: Reproducibility of HVPG measurementIn conclusion, the balloon catheter currently represents the most reliable and reproducible method to assess HVPG. The consistency of the results obtained with the balloon catheter is particularly important, if repeated measurements are required for therapeutic adjustments in the management of cirrhotic patients.

Figure 1

 1 Figure 1

Figure 2 Bland-

 2 Figure 2

Figure 3 Bland-

 3 Figure 3

Figure 4 Bland-

 4 Figure 4

  reporting of studies of diagnostic accuracy (version January 2003)

  participants satisfying the criteria for inclusion who did or did not undergo the index tests and/or the reference standard; describe why participants failed to undergo either test (a flow diagram is strongly recommended). , missing data and outliers of the index tests were handled.[START_REF] Thalheimer | Assessment of the agreement between wedge hepatic vein pressure and portal vein pressure in cirrhotic patients[END_REF] Estimates of variability of diagnostic accuracy between subgroups of participants, readers or centers, if done. 24 Estimates of test reproducibility, if done. DISCUSSION 25 Discuss the clinical applicability of the study findings. 11

	Section and Topic	Item		On page #
		#
	TITLE/ABSTRACT/	1	Identify the article as a study of diagnostic accuracy (recommend MeSH	1
	KEYWORDS		heading 'sensitivity and specificity').
	INTRODUCTION	2	State the research questions or study aims, such as estimating diagnostic	3
			accuracy or comparing accuracy between tests or across participant
			groups.
	METHODS
	Participants	3	The study population: The inclusion and exclusion criteria, setting and	5
			locations where data were collected.
		4	Participant recruitment: Was recruitment based on presenting symptoms,	5
			results from previous tests, or the fact that the participants had received
			the index tests or the reference standard?
		5	Participant sampling: Was the study population a consecutive series of	Yes
			participants defined by the selection criteria in item 3 and 4? If not,
			specify how participants were further selected.
		6	Data collection: Was data collection planned before the index test and	5
			reference standard were performed (prospective study) or after
			(retrospective study)?
	Test methods	7	The reference standard and its rationale.	N/A
		8	Technical specifications of material and methods involved including how	5
			and when measurements were taken, and/or cite references for index
			tests and reference standard.
		9	Definition of and rationale for the units, cut-offs and/or categories of the	5
			results of the index tests and the reference standard.
		10	The number, training and expertise of the persons executing and reading	N/A
			the index tests and the reference standard.
		11	Whether or not the readers of the index tests and reference standard	N/A
			were blind (masked) to the results of the other test and describe any
			other clinical information available to the readers.
	Statistical methods	12	Methods for calculating or comparing measures of diagnostic accuracy,	6
			and the statistical methods used to quantify uncertainty (e.g. 95%
			confidence intervals).
		13	Methods for calculating test reproducibility, if done.	6
	RESULTS
	Participants	14	When study was performed, including beginning and end dates of
			recruitment.
		15	Clinical and demographic characteristics of the study population (at least	8
			information on age, gender, spectrum of presenting symptoms).
		16	The number of N/A
	Test results	17	Time-interval between the index tests and the reference standard, and	5
			any treatment administered in between.
		18	Distribution of severity of disease (define criteria) in those with the target	5
			condition; other diagnoses in participants without the target condition.
		19	A cross tabulation of the results of the index tests (including
			indeterminate and missing results) by the results of the reference
			standard; for continuous results, the distribution of the test results by the
			results of the reference standard.
		20	Any adverse events from performing the index tests or the reference
			standard.
	Estimates	21	Estimates of diagnostic accuracy and measures of statistical uncertainty	8
			(e.g. 95% confidence intervals).
		22	How indeterminate results