Adrenal Vein Sampling in the Management of Primary Aldosteronism: The Added Value of Intraprocedural Cortisol Assessment

Introduction Primary aldosteronism is the most common cause of secondary hypertension. Adrenal vein sampling is the gold standard for subtyping primary aldosteronism. However, this procedure is technically challenging and often has a low success rate. Our center is one of the very few performing this technique in our country with an increasing experience. Objective The aim of this study was to evaluate the role of the cortisol intraprocedural assay in improving the performance of adrenal vein sampling. Design We enrolled all of the patients with primary aldosteronism that underwent adrenal vein sampling from February 2016 to April 2023. The cortisol intraprocedural assay was introduced in October 2021. Methods We enrolled a total of 50 adrenal vein samplings performed on 43 patients with the diagnosis of primary aldosteronism. In this sample, 19 patients and 24 patients underwent adrenal vein sampling before and after intraprocedural cortisol measurement, respectively. The procedure was repeated in seven patients (one before and six after intraprocedural cortisol measurement), given the unsuccess of the first exam. Selectivity of the adrenal vein sampling was assumed if the serum cortisol concentration from the adrenal vein was at least five times higher than that of the inferior vena cava. Lateralization was assumed if the aldosterone to cortisol ratio of one adrenal vein was at least four times the aldosterone to cortisol ratio of the contralateral side. Results The mean age of the patients that underwent adrenal vein sampling (N = 43) was 55.2 ± 8.9 years, and 53.5% (n = 23) were female. The mean interval between the diagnosis of hypertension and the diagnosis of primary aldosteronism was 9.8 years (±9.9). At diagnosis, 62.8% of the patients (n = 27) had hypokalemia (mean value of 3 mmol/L (±0.34)), 88.4% (n = 38) had adrenal abnormalities on preprocedural CT scan, and 67.4% (n = 29) described as unilateral nodules. There were no statistically significant differences in the patients' baseline characteristics between the two groups (before and after intraprocedural cortisol measurement). Before intraprocedural cortisol measurement, adrenal vein sampling selectivity was achieved in 35% (n = 7) patients. Selectivity increased to 100% (30/30) after intraprocedural cortisol measurement (p < 0.001). With the exception of one patient who refused it, all patients with lateralized disease underwent unilateral adrenalectomy with normalization of the aldosterone to renin ratio postoperatively. Conclusions The lack of effective alternatives in subtyping primary aldosteronism highlights the need to improve the success rate of adrenal vein sampling. In this study, intraprocedural cortisol measurement allowed a selectivity of 100%. Its addition to this procedure protocol should be considered, especially in centers with a low success rate.

Introduction.Primary aldosteronism is the most common cause of secondary hypertension.Adrenal vein sampling is the gold standard for subtyping primary aldosteronism.However, this procedure is technically challenging and often has a low success rate.Our center is one of the very few performing this technique in our country with an increasing experience.Objective.Te aim of this study was to evaluate the role of the cortisol intraprocedural assay in improving the performance of adrenal vein sampling.Design.We enrolled all of the patients with primary aldosteronism that underwent adrenal vein sampling from February 2016 to April 2023.Te cortisol intraprocedural assay was introduced in October 2021.Methods.We enrolled a total of 50 adrenal vein samplings performed on 43 patients with the diagnosis of primary aldosteronism.In this sample, 19 patients and 24 patients underwent adrenal vein sampling before and after intraprocedural cortisol measurement, respectively.Te procedure was repeated in seven patients (one before and six after intraprocedural cortisol measurement), given the unsuccess of the frst exam.Selectivity of the adrenal vein sampling was assumed if the serum cortisol concentration from the adrenal vein was at least fve times higher than that of the inferior vena cava.Lateralization was assumed if the aldosterone to cortisol ratio of one adrenal vein was at least four times the aldosterone to cortisol ratio of the contralateral side.Results.Te mean age of the patients that underwent adrenal vein sampling (N � 43) was 55.2 ± 8.9 years, and 53.5% (n � 23) were female.Te mean interval between the diagnosis of hypertension and the diagnosis of primary aldosteronism was 9.8 years (±9.9).At diagnosis, 62.8% of the patients (n � 27) had hypokalemia (mean value of 3 mmol/L (±0.34)), 88.4% (n � 38) had adrenal abnormalities on preprocedural CT scan, and 67.4% (n � 29) described as unilateral nodules.Tere were no statistically signifcant diferences in the patients' baseline characteristics between the two groups (before and after intraprocedural cortisol measurement).Before intraprocedural cortisol measurement, adrenal vein sampling selectivity was achieved in 35% (n � 7) patients.Selectivity increased to 100% (30/30) after intraprocedural cortisol measurement (p < 0.001).With the exception of one patient who refused it, all patients with lateralized disease underwent unilateral adrenalectomy with normalization of the aldosterone to renin ratio postoperatively.Conclusions.Te lack of efective alternatives in subtyping primary aldosteronism highlights the need to improve the success rate of adrenal vein sampling.In this study, intraprocedural cortisol measurement allowed a selectivity of 100%.Its addition to this procedure protocol should be considered, especially in centers with a low success rate.

Introduction
Primary aldosteronism (PA) afects an estimated 6% of all patients with hypertension and 20% of those with resistant hypertension, being the most common cause of secondary hypertension [1].In PA, there is an autonomous production of aldosterone with low renin levels [1].Aldosterone acts in the principal cells through the activation of the mineralocorticoid receptor of the distal tubule and collecting duct, mediating sodium resorption and secretion of potassium and hydrogen.Reabsorption of sodium is one of the main efects associated with hypertension in PA.Other efects of aldosterone include blood vessel remodeling, fbrosis and endothelial dysfunction, cardiac fbrosis and hypertrophy, and atrial fbrillation [2].PA may be unilateral, usually caused by an aldosterone-producing adenoma, or bilateral caused by bilateral adrenal hyperplasia.Te two main strategies to treat PA include unilateral adrenalectomy or lifelong medical therapy with mineralocorticoid receptor antagonists (MRAs).Most studies show that surgical treatment leads to more favorable results than medical therapy.Patients on MRA maintain residual disease, as suggested by higher blood pressure values, lower serum potassium, higher rates of atrial fbrillation, a decline of renal function, decreased quality of life (QoL), and increased mortality.Tis may be attributable to insufcient dosing of medical treatment in the setting of a lack of adherence to prescribed MRA therapy and/or an insufcient uptitration of MRA therapy.Another explanation for the better outcomes of surgical therapy could be related to the possible concomitant cortisol cosecretion that is corrected by adrenalectomy but not by MRA treatment.In fact, up to 78% of PA patients exhibit at least one pathological test result for excess glucocorticoid [3,4].
To defne the best treatment strategy, subtyping PA is crucial and adrenal vein sampling (AVS) is the gold standard test.However, this procedure is challenging and expensive [5].Success rates for the AVS procedure range from 40 to 94%.Te anatomical complexity and variations lead to frequent AVS failures [6].Cannulation of the right adrenal vein is the most challenging part of AVS, which is also the most common cause of unsuccessful AVS [7].Te confrmation of the technical success of the procedure is often not available until hours or days after its performance, usually after the patient has already been discharged, making resampling cumbersome and bearing higher costs [8].In 2007, the frst proof of concept study was published by an Italian group showing the feasibility of rapid on-site measurement of cortisol during the AVS study in fve subjects.Since then, other studies have been published demonstrating the improvement of successful AVS when using intraprocedural cortisol assays [8,9].
Te purpose of this study is to report and review the role of the cortisol intraprocedural assay in the sampling of adrenal veins in PA in our center.Given the possible interference with AVS results, all patients stopped MRA at least six weeks and diuretics four weeks before the procedure.Alternative antihypertensive drugs used included nifedipine, doxazosin, and diltiazem.

Methods
Tis retrospective study was approved by the Ethics Committee of the Centro Hospitalar Universitário Lisboa Central.All patients had given their written consent for the AVS procedure.

CT Technique.
All patients had adrenal CT before AVS for mapping of the adrenal veins and diagnosis.In addition to axial (3 mm slice thickness), multiplanar sagittal and coronal reformations (3 mm slice thickness) were used.

AVS.
AVS was performed by a team of two interventional radiologists.Before the procedure, patients' blood was collected for analysis and normal serum potassium was ensured before AVS (potassium ≥3.5 mmol/L).
All the patients followed the protocol of our center with ACTH stimulation.ACTH perfusion was started 30 min before the procedure.All procedures were performed in fasting to reduce the efect of food intake in aldosterone levels and in the morning given the circadian fuctuations in aldosterone levels.Tey started at 8 am and took about two hours.Te procedures encompassed the operator learning curves for both interventional radiologists who had no prior experience with these procedures and did not receive proctoring.Both interventional radiologists had experience in vascular and catheter-based procedures for 25 and 10 years before the start of this study.All procedures were performed from a femoral vein access (5Fr).For the left adrenal vein, a 4Fr Berenstein catheter was used (Cordis), and for the right adrenal vein, a 5Fr Cobra 1 or Simmons 1 catheter was used (Cordis).All catheters were used on 0.035inch hydrophilic guidewires (Terumo).Side holes were created near the catheter tip with the use of an intramuscular injection needle.
Blood was collected in EDTA K3E tubes (Sarstedt).Te team started performing the cortisol intraprocedural assay in October 2021.Te radiologist started to collect blood in the right adrenal vein.Te test tubes were immediately transferred to the endocrinology laboratory, centrifuged for fve 2 International Journal of Endocrinology minutes at 3000 g, and the supernatant plasma was used for immediate cortisol analysis.Te time for transport, centrifugation, and measurement was 60-120 min, during which the radiologist attempted sampling of the contralateral adrenal vein.Te sampling catheter was not left in position in the adrenal vein during the measurement.Te patient stayed in the radiology suite until the last sample was analyzed.Following the procedure, the patient remained in the day case ward for four hours.
Tere were no statistically signifcant diferences between the two groups (before and after IPCM), as shown in Table 1.
Globally, 38 patients (88.4%) had adrenal abnormalities on preprocedural CT scan.Te majority (n � 29; 67.4%) were described as unilateral nodules (16 right-sided and 13 leftsided) ranging from 2.9 mm to 35 mm (mean 17.3 ± 7.4 mm).In patients with bilateral nodes (n � 5; 11.6%), the dimension was 18.4 ± 7.1 mm in the right lesions and 15.8 ± 5.8 mm in the left lesions.In three patients, abnormalities were described as unilateral hyperplasia (two left-sided and one right-sided).Both adrenals were considered normal in six (14%) of the patients.

AVS Data.
A total of 50 AVSs were performed in the considered period of time (20 before and 30 after IPCM), with seven of them corresponding to repetitions (one before and six after IPCM introduction) given the unsuccess of the frst procedure.Globally, selectivity was achieved in 37 AVSs (74%).Te rate of bilateral selectivity before IPCM (2016-September 2021) was 35% (7/20) and after the cortisol assay (October 2021-April 2023) was 100% (30/30) (p < 0.001).Te cause of unsuccessful AVS was nonachieving right adrenal selectivity (n � 13).In three of them, selectivity was also not achieved in the left adrenal vein samples. Figure 1 shows the diference in selectivity before and after IPCM.

International Journal of Endocrinology
Lateralization was obtained in 46.7% (n � 14) of the patients who underwent AVS after IPCM, with a median cortisol-corrected aldosterone lateralization index of 20.8 (12.1-25.9).Lateralization was found in 71.4% (n � 5) of the patients who underwent AVS before IPCM, with a median lateralization index of 24.1(12.7-38;p � 0.01 vs. after IPCM).
All AVS procedures occurred without complications.Two patients complained of nausea, sweating, and palpitations after starting the tetracosactide infusion.No major side efects were reported.
Table 3 shows AVS outcomes considering preprocedural imaging and respective outcomes in terms of success of the procedure.
Among all bilaterally selective AVSs (n � 37), the discordance between AVS and CT scan was observed in 51.4% of the patients (n � 19).

Clinical Outcomes.
Except for one patient that refused it, all patients with lateralized AVS (13 in concordance and fve nonconcordant to the preprocedure imaging) underwent unilateral adrenalectomy.After surgery, the patients reduced their medication from an average of 2.93 (±1.1) to 0.8 (±1.1) antihypertensive drugs.All the histopathological exams revealed a cortical adenoma, and a cure of PA was verifed.Postoperatively, there was a normalization of the aldosterone to renin ratio (aldosterone to renin ratio (ARR) <3.7) with a mean ARR of 1.8 (±0.94).
Patients with bilateral disease (with no lateralization in AVS and with assumed bilateral adrenal hyperplasia) were kept under medical therapy.Patients with "grey zone" AVS results were also maintained under medical therapy.All these patients had controlled blood pressure (<140/ 90 mmHg in ambulatory report and in the appointment) with an average of 2.6 (±1.3) antihypertensive drugs.

Discussion
AVS is an essential diagnostic procedure in the workup of PA.Adrenalectomy is the treatment of choice in cases with aldosterone overproduction from one adrenal gland.Rossi et al. and PASO study, a large multicenter study, like other studies, documented the clinical benefts of AVS-guided selection of surgical candidates [11,12].
Te AVS success rate in our center signifcantly improved after incorporating IPCM into our practice.Tis AVS protocol was created by a multidisciplinary team including endocrinology, clinical pathology, and interventional radiology specialties.To the best of our knowledge and investigation, this is the frst published analysis of AVS results in our country.Te Centro Hospitalar Universitário de Lisboa Central (CHULC) is one of the very few centers in Portugal that is performing AVS, and the growing experience is increasing the referral of patients from other hospitals that do not perform this procedure.Te AVS is being performed by the same interventional radiology team.
Te diferentiation between unilateral and bilateral PA should be based on the results of AVS as recommended by the Endocrine Society guidelines concerning the management of PA [10].In fact, the CT/MRI imaging fndings may be misleading [12,13].Discordance rates between imaging and functional studies using AVS are typically 30-40% [14].In our investigation, in 51.4% of the patients, there was no concordance between imaging and AVS results.
Despite being the gold standard, AVS is challenging and frequently unsuccessful, requiring higher experience.Nuclear medicine studies using positron-emitting agents such as [11C]-metomidate or [68 Ga]-pentixafor can detect small tumors, but the high cost and limited availability give little advantage over AVS, at least currently [8,12,15].
Te infusion of cosyntropin may increase the accuracy of AVS, increasing the selective index and thus the confdence of correct catheter placement [14].Its use can also decrease potential ACTH-dependent hormonal fuctuations [12].In this study, all the patients underwent stimulated AVS with cosyntropin according to our protocol.
Adrenal vein cannulation success is determined by a higher cortisol concentration in the adrenal vein sample than in a paired peripheral plasma sample (selectivity ratio).However, this fnding is usually only confrmed after the conclusion of the procedure and after the discharge of the patient [13].In our study, we considered the cutof suggested by the guidelines of the Endocrine Society (2016) for the ACTH-stimulated AVS [10].Some studies such as Mulatero et al. showed that too permissive criteria for successful catheterization led to poor diagnostic reproducibility [16].
Te low success rate of AVS (35%) in our center urged the need to optimize the procedure to achieve better results.In September 2021, we reformulated our AVS protocol to International Journal of Endocrinology include IPCM.During the cortisol assay total time, the radiologist attempted sampling the contralateral adrenal of the same patient.
IPCM has been found to increase the success rate of AVS, allowing a rapid feedback on the selectivity index and, therefore, the success of the procedure.In cases of nonselectivity, this approach allows the resampling while the patient is still in the procedural room.It does not increase discomfort for the patient and adds only the expense of additional procedural time.Furthermore, IPCM had the advantage of not requiring the use of equipment other than that already existing in the hospital laboratory, with no investment costs associated [8].
In Auchus et al.'s study, the success rate increased from 73% to 97% after the use of cortisol assay during AVS.In this study, the AVS was performed during a continuous infusion of cosyntropin and AVS samples were assayed using an immunochemiluminometric assay.Te total turnaround time for serum cortisol assays averaged one to two hours.No adverse efects occurred and the procedure was done by fve diferent interventional radiologists [17].Rossi et al. showed a fnal rate of bilateral selectivity higher than historical series (23/25 vs 16/25) with rapid cortisol measurement during AVS.As in this study, our samples were tested as such and after serial dilution [15].
Betz et al. suggested that the rapid cortisol measurement favors better AVS outcomes, mainly in centers with low success rates, due to an initial efect by resampling of the right adrenal vein and a more delayed training efect [8].Given that, in that study, the success rate previous to the rapid cortisol measurement was low (55%) [8].Similarly, in our study, before IPCM, our AVS success rate was only 35%.
Te increased bilateral selectivity in AVS may also be a consequence of increased experience of interventional radiologist.Te AVS learning curve is estimated to be about 20-30 procedures, with maintenance of profciency of about 15 annual cases [15].In our study, it probably contributed to the diferences in technical success observed before and after the implementation of IPCM during AVS.
Te prevalence of unilateral subtypes of PA is comparable to other published studies.Tere is evidence suggesting a worse clinical prognosis in unilateral disease [7].In our study, there were no statistically signifcant diferences between lateralization and the number of antihypertensive medications, presence of hypokalemia, or complications of PA.However, we must take into account that the small number of patients enrolled in this study, namely, with unilateral disease, may compromise the detection of statistical diferences.
Te prevalence of hypokalemia (62.8%) was higher in our study than typically described in the literature.Nevertheless, there are some comparable studies that had similar percentages as that by Rossi et al. (70% of the patients with hypokalemia) [18].Tis higher prevalence may reveal a selection bias.Hypokalemia with hypertension is a common reason to investigate a possible PA and, therefore, is responsible for some of the referrals for performing AVS [15].
Limitations to this study beyond the ones already discussed include the retrospective design and nonrandomization of the compared cohorts.Te low number of AVS performed can compromise the statistical power of the study.However, the results reinforce the existing evidence, highlighting the importance of the AVS and especially of the IPCM.Tere is still no universal consensus AVS protocol, namely, in terms of the use of ACTH and the selective and lateralization indices.
Ideally, the determination of intraprocedural cortisol assay should be performed using a point of care testing during the AVS in order to minimize the time elapsed between right adrenal vein sampling and confrmation of a successful catheterization.However, this portable measurement system is not available at the time and, if it existed, it would require a study to assess the accuracy and reproducibility of cortisol compared to the one used by the clinical pathology laboratory in order to validate its use.From September 2022, the determination of cortisol performed on Roche equipment Cobas e601 Roche substantially reduced the technical execution time (from 40 minutes to 18 minutes), consequently reducing the total response time (in about 30 minutes) for confrmation of the technical success of the AVS study.

Conclusions
In conclusion, AVS remains the standard for diferentiating aldosterone-producing adenoma from bilateral adrenal hyperplasia.Cannulation of the right adrenal vein remains the major technical challenge in this procedure.IPCM may be an efective tool to improve the AVS success rate, especially in centers with a low success rate.

Table 1 .
[10] August 2022, the determination of cortisol started to be carried out on Roche equipment Cobas e601, an automated eletrochemiluminescence microparticle immunoassay.Cortisol's technical execution time was 40 minutes but with the use of the new referred equipment, it substantially reduced the technical execution time in the Bilateral aldosterone secretion in the context of a bilateral adrenal hyperplasia was assumed if the ratio of aldosterone to cortisol from one adrenal vein was below three times the ratio of the contralateral side.If this ratio was between three and four, a "grey zone" was considered and no conclusions were made[10]. SPSS Statistics 23rd version for Windows (IBM, Armonk, NY, USA). Statitical signifcance was determined with a two-sided p value <0.05.Te mean age was 55.2 ± 8.9 years, and 53.5% (n � 23) were female.At the baseline, patients were taking a mean of 2.8 ± 1.1 antihypertensive drugs.Te mean age at hypertension diagnosis was 42.3 ± 11.5 years ranging between 15 and 67 years.Te mean age at PA diagnosis was 52.1 ± 9.5 years ranging between 31 and 72 years.Te mean interval time between hypertension and PA diagnosis was 9.8 ± 9.9 years.Hypokalemia was observed in 27 patients (62.8%) with a mean of 3 mmol/L (±0.34).
equipment (from 40 minutes to 18 minutes).Te samples with cortisol values above 59.8 μg/dL had serial dilutions (1/10 or 1/20 or 1/40 until the fnal value).Te interval for the measurement of aldosterone samples was 0.97-100 ng/dL.Values above 100 ng/dL had serial dilutions until the fnal value.Cortisol was reported in μg/dl and aldosterone in ng/dl.2.5.Selectivity and Lateralization Criteria.Selective adrenal sampling was assumed if the cortisol concentration in the adrenal vein was at least fve times higher than that in the sample drawn simultaneously from the catheter sheath in the inferior vena cava (IVC) (cortisol in the adrenal vein/cortisol in IVC vein >5.0).Bilateral selectivity was a condition for assuming a successful AVS, as it means that correct cannulation of both the adrenal veins was performed.Te diagnosis of unilateral aldosterone secretion was made if the aldosterone to cortisol ratio from one adrenal vein was at least four times the aldosterone to cortisol ratio of the contralateral side.2.7.Statistical Analysis.Categorical variables are presented as absolute numbers and percentages.Continuous variables with non-normality distribution are presented as the median ± interquartile range.We used the Mann-Whitney test to compare continuous variables.Categorical variables were compared using χ 2 .Data collection and registration were performed with Microsoft Excel and statistical analysis with IBM

Table 1 :
Characteristics of patients submitted to adrenal vein sampling.