The Subtyping Of Primary Aldosteronism by Adrenal Vein Sampling:
Sequential Blood Sampling Causes Factitious Lateralization
Giacomo Rossitto, MD1,3; Michele Battistel, MD2; Giulio Barbiero, MD2; Valeria Bisogni, MD1; Giuseppe Maiolino, MD1; Diego Miotto, MD2, Teresa Maria Seccia, MD, PhD1
and Gian Paolo Rossi, MD, FACC, FAHA1
1Clinica dell’Ipertensione Arteriosa, DIMED, University of Padua, Italy
2Institute of Radiology, Department of Medicine, DIMED, University of Padua, Italy
3Institute of Cardiovascular and Medical Sciences, University of Glasgow, UK
DATA SUPPLEMENT
Corresponding author (Address for correspondence):
Prof. Gian Paolo Rossi, MD. FACC, FAHA DIMED –Clinica dell’Ipertensione Arteriosa University Hospital
via Giustiniani, 2; 35126 Padova, Italy Phone: 39-(0)49-821-7821 or 2279 Fax: 39-049-821-7873
E-mail: [email protected]
SUPPLEMENTAL METHODS Study Population
We prospectively recruited consecutive patients referred to our Specialized Centre for Hypertension for suspected PA from 2007 to 2015 who met the current guidelines indications for AVS.[1,2[ They were asked to have a nor - mal sodium intake [3] and to withdraw mineralocorticoid receptor antagonists at least 6 weeks before the study. To minimize the risks of uncontrolled HT, agents that affect the renin-angiotensin-aldosterone system, including di- uretics, beta-blockers, angiotensin-converting enzyme inhibitors, and angiotensin II type 1 receptor antagonists, were withdrawn for at least 2 weeks, and a long-acting calcium channel blocker and/or doxazosin were prescribed in patients without severe drug-resistant hypertension. Hypokalemia, if present, was systematically corrected with K+ supplementation before AVS. Diagnosis of APA was made according to the following “four-corners’ criteria”:
i) biochemical diagnosis of PA; ii) evidence of lateralized aldosterone secretion at bilaterally selective AVS; iii) identification of a CYP11B2-immunostained adrenocortical adenoma at pathology, which was performed starting in 2015 when the human monoclonal antibody for CYP11B2 became available [4]; iv) cure or improvement of hy- pertension, and correction of the biochemical picture of PA at follow-up.[3-5]
Adrenalectomy was offered on clinical ground and regardless of the AVS results, considering the likelihood of an APA [6] and the need to achieve BP control with pharmacologic therapy [1], in only few cases with non-bilater- ally selective AVS. Even in such cases the diagnosis of APA had to be confirmed by the aforementioned follow- up criteria. All procedures followed the principles of the Declaration of Helsinki and the institutional guidelines and a written consent was obtained from all participants.
Exclusion criteria
Patients were excluded from the analysis if: i) refused to undergo adrenalectomy ii) 1 and 6 months post-surgical follow-up data were not available for APA diagnosis confirmation; iii) AVS was non-bilaterally selective and they were not treated with adrenalectomy, iv) AVS was performed under potential confounding drugs owing to resistant hypertension [1], without evidence of PRA suppression, or history of allergic reactions to the contrast medium.
AVS Procedure
AVS was performed by highly experienced interventional radiologists (D.M., M.B., G.B.) after a 3-hour rest in the supine position, between 08:00 and 12:00 AM to minimize effect of circadian variations of cortisol and aldos- terone. The procedure was performed with local anesthesia (lidocaine 2%). Two sheath 5F and 6F (Glidesheath, Terumo, Tokyo, Japan), respectively, were inserted into the right common femoral vein: the first introducer was in- serted by puncture of the right common femoral vein, under US-guide or not; the second introducer was inserted by slightly more cranial puncture of the right common femoral vein under fluoroscopic guide of the first introducer (Supplemental Figure 1).
To cannulate the right adrenal vein we used a Simmons 1 (5- to 6-F) catheter (Cordis), or, less frequently, a Cobra 2 (C2) or a Cobra 1 (C1) catheter (Cordis), all with a side hole at 1-2 mm from the tip to facilitate blood collection;
for left adrenal vein we used a Simmons 2 or 3 (5- to 6-F) catheter or, less often, a C2 catheter (Figure 1). To eval- uate the adequacy of the catheterization, a small amount, usually < 3 mL, of ionic contrast material (37 g/dL of Iopamidolo, Iopamiro 370, Bracco) was used; injection was performed very gently manually, under fluoroscopic control, to minimize the risk of rupture of small intra-glandular veins (video 1 and 2, supplemental material, http://links.lww.com/HJH/A849, http://links.lww.com/HJH/A850). Blood samples for the measurements of plasma aldosterone concentration (PAC) and cortisol concentration (PCC) were obtained immediately after confirmation of adequate bilateral cannulation (t-15) and again after 15 minutes (t0). They were collected simultaneously from the right and left adrenal veins and from the infra-renal IVC by gravity. To avoid the risk of thrombosis the catheter was withdrawn from the right adrenal vein after blood sampling at t-15; it was then repositioned just be- fore collecting the samples at t0; the left catheter was left in place after t-15 cannulation.
Baseline and t-15’ Index definitions
The selectivity index (SI) and the lateralization Index (LI) were calculated as described for bilateral simultaneous AVS at each time, but only values derived at t0 were used for diagnostic purposes.
A cut-off of 2.00 was used for the SI and the LI to define selectivity and also lateralization, and therefore the indi- cation to adrenalectomy.
The Relative Aldosterone Secretion Index (RASI) for each side was determined as the ratio of plasma aldosterone concentration (PAC) in the adrenal vein over PAC in the infra-renal inferior vena cava (IVC) blood (taken as a sur- rogate of PAC in arterial blood entering the adrenal), divided by the ipsilateral SI as follows:
RASI = (PACside/PACivc)/(PCCside/PCCivc).
This index estimates the secretion of aldosterone relative to cortisol, from each adrenal cortex [5]. For the purpose of data analysis, a side was identified as “dominant” by the higher RASI value compared to the contralateral side.
Simulation of Sequential Techniques Conditions.
To simulate conditions occurring with the sequential AVS technique hormonal values derived from different times and sides were combined: we used hormonal values from the right adrenal vein at t-15, from the left adrenal vein at t0 and from IVC at both times (for calculation of the selectivity index and correction for the degree of selectivity achieved). This approach simulates a sequential “first Right, second Left” Technique (R⇒L) sequence, which is how sequential sampling is usually performed.
A further analysis was conducted, to investigate potential systematic errors involved in diagnostic discordance be- tween simultaneous and sequential approaches: using hormonal values from the left adrenal vein at t-15, from the right adrenal vein at t0 and from IVC at both times, thus simulating a sequential “first Left, second Right” tech - nique (L⇒R) was simulated. Online Tab 1 provides detailed description of all the indexes calculated.
Biochemical Measurements
Serum, K+ levels, PAC, PCC, and plasma renin activity (PRA) were measured as described [3]. Normal ranges, in- tra- and inter-assay coefficient of variation and antibody cross-reactivity for the measurements of PAC, PCC, and PRA have already been reported [3].
Statistical analysis
As AVS is based on the premise that bilaterally selective samples are obtained, the main analysis aimed at evaluat- ing the impact of timing of blood sampling on aldosterone secretion and lateralization was limited to the patients with bilaterally selective AVS at both time points, e.g. t-15 and t0.
Results are expressed as mean ± SD, or median and interquartile range, as appropriate. Continuous variables were tested for normal distribution with Kolmogorov-Smirnov test. Log-transformed data were used in case of a skewed distribution before parametric statistical analysis was performed; data that remained skewed were analyzed with non parametric tests. Paired or Mann-Whitney test were for within-patient comparisons; Pearson’s χ2 test was used for categorical variables, as appropriate.
The concordance between log-transformed indexes derived with the compared strategies (simultaneous sampling at t-15 vs t0; simulated sequential sampling vs simultaneous at t0) was examined by calculating the concordance cor- relation coefficient (ρc = ρ Cb), which estimates the degree to which pairs of observations fall on the identity line (the 45° line through the origin); ρ is the Pearson correlation coefficient, and Cb a bias correction factor that mea - sures to what extent the best-fit line deviates from the identity line, and estimates accuracy [7]. Similarly, a Bland- Altman plot was used to detect systematic biases [8,9].
The value of Cohen’s Kappa (K) was used to evaluate the diagnostic agreement between compared strategies and interpreted as follows: K < 0.20 poor; K = 0.21 - 0.40 fair; K = 0.41 - 0.60 moderate; K = 0.61 - 0.80 good; K = 0.81 - 1.00 very good strength of agreement [10].
The area under (AUC) the Receiver operating characteristic (ROC) analysis was calculated to assess the accuracy of LI derived with the simultaneous or the sequential techniques, using APA as diagnosis as status. Significance was set at p < 0.05. MedCalc (MedCalc Software Ostend Belgium, vers. 15.8) and SPSS (vers. 24 for Mac, SPSS Bologna, Italy) were used for data analysis.
Supplemental Table 1. Calculation of Indexes (RASI and LI) for simulated sequential technique
7 Main analysis R⇒L(sequential first RIght, second Left technique)
Relative Aldosterone Secretion Index
RASIDOMINANT_R⇒L
= RASIRIGHT_t-15’ if RASIRIGHT_t-15’ > RASILEFT_t0
= RASILEFT_t0 if RASIRIGHT_t-15’ < RASILEFT_t0
RASINON-DOMINANT_R⇒L
= RASILEFT_t0 if RASIRIGHT_t-15’ > RASILEFT_t0
= RASIRIGHT_t-15’ if RASIRIGHT_t-15’ < RASILEFT_t0
Lateralization Index LI_R⇒L RASIDOMINANT_R⇒L / RASINON-DOMINANT_R⇒L
Secondary analysis L⇒Requential first Left, second Right technique)
Relative Aldosterone Secretion Index
RASIDOMINANT_L⇒R
= RASILEFT_t-15’ if RASILEFT_t-15’ > RASIRIGHT_t0
= RASIRIGHT_t0 if RASILEFT_t-15’ < RASIRIGHT_t0
RASINON-DOMINANT_L⇒R
= RASIRIGHT_t0 if RASILEFT_t-15’ > RASIRIGHT_t0
= RASILEFT_t-15’ if RASILEFT_t-15’ < RASIRIGHT_t0
Lateralization Index LI_L⇒R RASIDOMINANT_L⇒R / RASINON-DOMINANT_L⇒R
IVC = inferior vena cava; PAC = plasma aldosterone concentration, PCC = plasma cortisol concentration; L⇒R = sequential first Left, second Right technique; R⇒L = sequential first RIght, second Left technique; RASI = Relative Aldosterone Secretion Index = (PACside_time/PACIVC_time)/
(PCCside_time/PCCIVC_time); RASI was calculated at each time and side with simultaneous IVC PAC and PCC values and only upon demonstration of Selec- tivity on that side at each specific time.
Supplemental Table 2: Rate of selective AVS studies at t-15 and t0. n = 138
% (n) SI cut-off = 2.00 t-15’ t 0 p (McNemar)
Bilaterally selective 70.3 (97) 60.9 (84) 0.037
Left selective 84.1 (116) 73.2 (101) 0.007
Right selective* 76.8 (106) 74.6 (103) NS
*right catheter was withdrawn from the adrenal vein after blood sampling at t-15 and was thereafter replaced just before collecting the samples at t0 to avoid the risk of vein thrombosis; the left catheter was left in place after t-15 cannulation.
Supplemental Figure 1. Impact of stress reaction and time delay on the selectivity index (SI) values. The SI fell from t-15 to t0 on both sides. Median and IQ range. Dashed line = SI cut-off (2.00).
Supplemental Figure 2. The relative changes of LI between t-15 and t0 (normalized for LIt0) show no correlations whatsoever with the selectivity index (SI) values at t-15 and t0, indicating no relationship with side and time of AVS.
Supplemental Figure 3. Comparison of LI values between the simultaneous and the sequential AVS technique according to the final diagnosis. For APA patients (upper panels), LI was similar (left), although the sequential approach showed a much larger variability, despite no systematic bias (Cb, right). For non-APA patients (lower panels), LI was systematically overestimated with the sequential technique as compared to the simultaneous technique.
* p < 0.005; R⇒L = Sequential first Right -> second Left Technique; SIMt0 = simultaneous at t0; left panels:
dashed line = LI cut-off for lateralization. Right panels: the solid lines correspond to the best-fit line; the dotted lines corresponding to the identity line is shown as reference. Please note the narrower range in the non APA patients.
SUPPLEMENTAL REFERENCES
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VIDEO FILES LEGENDS
Video AVS1. Left adrenal vein cannulation: a gentle injection of less than 3 mL of ionic contrast material is used, under fluoroscopic control, to confirm correct placement in the phrenic trunk.
Video AVS2. Right adrenal vein catheterization: the tip of the catheter is positioned at the orifice of the adrenal vein. An accessory hepatic vein draining nearby is stained by the injection of the contrast just cranially to the adrenal venous network.