Hrev_master Veins and Lymphatics 2014; volume 3:4037 [Veins and Lymphatics 2014; 3:4037] [page 47] Scintigraphy-based analysis of possible pulmonary lesions after foam sclerotherapy: a pilot study Lorenzo Tessari,1 Marcello Izzo,2 Attilio Cavezzi,3 Francesco Zini,4 Mirko Tessari,5 Daniela Grigolato6 1Glauco Bassi Foundation, Trieste; 2Mathematics for Technology, Medicine & Biosciences Research Center, University of Ferrara; 3Eurocenter Venalinfa, San Benedetto del Tronto (AP); 4Casa di Cura Città di Parma, Parma; 5Vascular Disease Center, University of Ferrara; 6UOC of Nuclear Medicine, University Hospital of Verona, Italy Abstract The aims of this study were to assess extempo- raneous in vivo binding between 99mTcO4- and two sclerosant detergents in foam sclerotherapy, and subsequently to control any possible damage in lungs and other organs related to sclerosant foam passage. A prospective comparative pilot study was performed on two male patients (62 and 56 years old) affected by varicose veins; each of them underwent scintigraphy investigations with free radiotracer and a scintigraphy investi- gation after each of the four sessions of scle- rotherapy of varicose tributaries of the lower limbs with labeled sclerosant foam. One of the two patients underwent two further scintigraphic investigations, with free radiotracer and with labeled sclerosant foam, at a later stage. Four mL of 2% polidocanol (POL) foam, or four mL of 1% sodiumtetradecylsulfate (STS) foam for session were injected. The sclerosant foam was labeled with the radioactive tracer technetium pertech- netate, 99mTcO4- (120 MBq per exam). Two scintigraphy assessments for free tracer (basal) and five scintigraphy investigations of bound-to- sclerosant tracer uptake/transit were obtained. No relevant variations in time/activity curves of the lungs and other organs were documented between the basal and post-sclerotherapy find- ings, also at the later stage. Free radiotracer mean region-of-interest data were: 336 counts (heart), 208 counts (lungs) and 371 counts (thy- roid). Mean values extrapolated from each curve at each step for labeled CO2O2-based sclerosant foam were respectively: 351 counts (POL) and 328 counts (STS) for heart, 202 counts (POL) and 188 counts (STS) for lungs, 335 (POL) and 263 (STS) for thyroid. No pulmonary damage by scle- rosant foam was caused. Neither immediately after treatments, nor at short-term follow-up. Introduction Foam sclerotherapy was introduced more than 60 years ago, and Cabrera and Monfreux’s innovative methods in the late 1990s gave a new impulse to using sclerosant foam (SF).1 The extended use of this treat- ment was achieved after the introduction of the Tessari method.2,3 Foam sclerotherapy has proved to be a vali- dated, effective and safe method to treat vari- cose veins of the lower limbs,4 and possibili- ties and limitations for this technique have been elucidated in several publications.5,6 More recently, issues have been raised and discussed as to potential risks, side effects and complications of SF.7 Distal embolization of SF bubbles to lungs and possible lung cap- illary/parenchyma inflammation has been postulated in the past8 and recent9 literature. Furthermore, especially in the presence of a right-to left shunt such as a patent foramen ovale, isolated cases of neurological complica- tions have been reported.4 The authors aimed to assess any possible change in the tissues of various organs, par- ticularly the lungs, which may be affected by possible passage/impact of SF bubbles. Furthermore, any difference between air- based SF and CO2O2-based SF was investigat- ed in order to assess a possible higher safety profile of biocompatible gases vs room air. Finally, the clinical and experimental trial aimed at assessing any difference between the two commonly used detergent sclerosant drugs [sodiumtetradecysulfate (STS) and polidocanol (POL)] as to the above-men- tioned issues. In order to get a reproducible and quantifiable method for detecting possi- ble tissue changes in the target/transit organs after sclerosant foam injections, scintigraphy was employed in a qualitative and semi-quantitative way. On the basis of the chemical/physical properties of pertech- netate ion (99mTcO4-), a protocol to assess extemporaneous in vivo binding between 99mTcO4- and POL/STS foam during various steps was designed. P 99mTcO4- shows a stable chemical structure not easily suited to bind other molecules. The study aimed at assessing the possibility of linkage between this radiotracer and the SF to check the presumed possibility of tracing the pathway and accumulation of SF bubbles by means of 99mTcO4-, as reported in a previous publication.8 In fact, in the vascular stream Pertechnetate ions remain in equilibrium, partially in free form and partially bound to serum proteins. More specifically, free ions leave the vascular compartment and migrate to interstitial fluids as a result of their small size, so as to decrease the pertechnetate blood concentration This leads to an equiva- lent release of 99mTcO4- that is bound to the proteins as an unavoidable result of its elimi- nation.10 The Pertechnetate ion is removed by various organs and systems into the intersti- tial liquid, as seen in the stomach, thyroid, salivary glands, bowel, choroid plexus, kid- neys, vascular structures and mucosae.11 Interestingly, the pertechnetate ion is strong- ly taken up by the thyroid gland only in its free form and not bound to other molecules.12 Noticeably, lungs represent a transit organ for 99mTcO4- and its temporal concentration peak declines along the 30 min of the scinti- graphic observation and this pattern is very similar also for other various organs.13 Finally and more importantly, the increased capillary permeability leads to an increased 99mTcO4- uptake in inflamed tissues. Therefore, in the case of possible drug-induced lung damage following foam sclerotherapy, there would be an increased 99mTcO4- persistence into the lungs after its injection during the post-treat- ment period. Materials and Methods A prospective comparative pilot study was performed on two male patients (56 and 62 years old) affected by primary varicose veins (C2 of CEAP - Clinical-Etiology-Anatomic- Pathophysiologic - classification) related to Correspondence: Lorenzo Tessari, via Giovanni Falcone 24/B, Peschiera del Garda (VR), 37019 Italy. Tel.: +39.0456401681. E-mail: lorenzo@tessaristudi.it Key words: foam sclerotherapy, scintigraphy. Contributions: LT, study design, data collection, data analysis, critical review, final approval; MI, data analysis, critical review, final approval; AC, data collection, data analysis, writing, critical review, final approval; FZ, data collection, data analysis, final approval; MT, data collection, final approval; DG, data analysis, critical review, writ- ing, final approval. Conflict of interests: the authors declare no potential conflict of interests. Received for publication: 24 April 2014. Revision received: 23 September 2014. Accepted for publication: 24 September 2014. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright L. Tessari et al., 2014 Licensee PAGEPress, Italy Veins and Lymphatics 2014; 3:4037 doi:10.4081/vl.2014.4037 No n c om me rci al us e o nly Article [page 48] [Veins and Lymphatics 2014; 3:4037] great saphenous vein (GSV) reflux. The patients were treated with SF which was labeled with a radionuclide (99mTcO4-) and they gave informed written consent to the procedures; no ethical committee approval was required, as per any observational study in Italy. Basal scintigraphy was performed immediately after injection of 4 mL of saline solution with the radiotracer (RT) 99mTcO4- in a varicose tributary of the lower leg. Subsequently, scintigraphy was performed after each of the four sessions of foam scle- rotherapy which were performed on each patient. An overall imaging of the outcomes is reported in Figure 1. Injections were per- formed into the tributaries and the GSV stems, tailoring each session according to the outcomes achieved, using 4 mL of 2% POL foam or 4 mL of 1% STS foam per session with the patient in the supine position. Sclerosant foam was obtained through Tessari’s method2 with one part of sclerosant drug (POL or STS) and four parts of gas (air or CO270%+O230%). Prior to foam formation, the sclerosant drug was mixed with 99mTcO4- (120 MBq per exam): more specifically 1 cc of POL 2%, or 1 cc of STS 1% were mixed with 0.2 cc of RT. In more detail, the flow chart of the proto- col for each patient was as follows: - Step 1, basal scintigraphy examination after injection of free 99mTcO4- (Figure 2). - Step 2 at day 7, scintigraphy examination 10 min after POL 2% + air radiolabelled sclerosant foam injection (Figure 3A). - Step 3 at day 14, scintigraphy examination 10 min after POL 2% + CO2O2 radiolabeled sclerosant foam injection (Figure 3B). - Step 4 at day 21, scintigraphy examination 10 min after STS 1% + CO2O2 radiolabeled sclerosant foam injection (Figure 3C). - Step 5 at day 28, scintigraphy examination 10 min after STS 1% + air radiolabeled sclerosant foam injection (Figure 3D). - Step 6 at day 42, scintigraphy examination for one of the two patients during the 90 min following free RT injection and 90 min after STS 1% + air radiolabeled sclerosant foam injection (Figure 4). This final step was undertaken to assess any possible late lung (and thyroid) tissue change due to several/repeated foam sclerotherapy ses- sions, as in the previous steps. Radiation exposition was controlled in each patient after every step and no limit- exceeding figure was recorded. Uptake and transit of free RT and of scle- rosant-bound RT were assessed in transit organs (lungs and heart) and in target organs (thyroid, salivary glands, stomach) with semi- quantitative scintigraphy by means of a dou- ble-head gamma camera and region-of-inter- est (ROI) investigation. All scans lasted 30 min except for the step 6 scan (90 min). All dynamic evaluations were focused on the chest and neck, in anterior and posterior pro- jections. Ninety photograms (1800 s in total) were taken at intervals of 20 s every frame (matrix 128¥128 pixel). The count per second in ROIs were plotted against time and the curves were fitted (counting out the initial transient phase). Activity in the site of sclerosant injection was measured at different interval times (see above) and the total count was divided by the background activity registered in the con- tralateral leg. Results At step 1 after injection of pertechnetate ion, the two patients showed a typical pattern of RT distribution in all transit and target organs and more specifically the semi-quanti- tative calculations resulted in a mean half time of 57.26 s in the lung, 57.35 s in the heart and -138.68 s in the thyroid. The integrated areas between 9th and 30th min were 336 (heart), 208 (lung) and 371 (thyroid). For each patient at step 1, step 2, step 3 and step 4, step 5 the var- ious ROI images (same size) and relative Figure 1. Curves and images of various regions-of-interest of different organ uptake after- free radiotracer injection (step 1). Figure 2. Organ uptake at step 1 for one patient. No n c om me rci al us e o nly Article [Veins and Lymphatics 2014; 3:4037] [page 49] curves of uptake have been summarized in Table 1 where mean figures for each organ and each session are summarized. The mean fig- ures have been calculated for each organ after summation of all resulting values for each step and from both patients. Comparison between step 1 and steps 2, 3, 4 and 5 showed overlapping time/activity curves related to lung transit. Assessment of overall amplitude of the curves showed a min- imal difference in comparison to free RT curve related to the radioactivity which has remained trapped in the vessels of the leg which was the site of the sclerosant foam injection. The only relevant difference between the basal step 1 and all other foam sclerotherapy steps (2 to 5) concerned the delayed transit time and a small reduction in amplitude in all samples, which is a consequence of peripheral vein uptake of the RT mixed with sclerosant foam. In fact, spasm of the treated vein and sclerosant foam stagnation in the treated veins (varicose tributary and consequently GSV trunk) contributed to the delayed arrival of RT to the lungs. Imaging of the varicose tributary and saphenous veins (Figure 5A) clearly depicted RT stagnation in these peripheral vein territories and gradual 99mTcO4- release over time (Figure 5). The activity/time curves related to the main up taking organs (thyroid and stomach in step 2,3,4 and 5 showed a pattern distribution which was overlapping the free RT curve of step 1. Data on these dose uptakes are summa- rized in Figure 1 and lung/thyroid/heart values are reported in Table 1. As thyroid normally takes up 99mTcO4- in its free form, the findings above highlight how RT in thyroid circulation is neither bound to the sclerosant bubbles nor to the sclerosant drug. Examination of any possible difference in the patterns and curves for the two sclerosant drugs STS and POL at the four different scle- rotherapy steps (2 to 5) in comparison to step 1 (free RT) showed overall similar results. Conversely, air-based SF showed a more pro- longed delay in time overall to reach the maxi- mum radioactivity in the curves (time to max) in comparison to CO2+O2-based SF (Table 2). We can speculate that bubbles of CO2+O2 scle- rosant foam tend to break up earlier than air- based SF so that RT release is accelerated. Data analysis of the procedures of step 6 in one of the two investigated patients showed free 99mTcO4- curves at the pulmonary and thy- roid levels which overlap the step 1 activity/time curves (Figure 4). As no signifi- Table 1. Figures deriving from total counts in the different regions-of-interest between the 9th and the 30th min in each curve. Heart Lung Difference between Thyroid basal (step 1) and sclerosant foam in lungs at step 2, 3, 4, 5 99mTcO4–(BASAL) 336.02 208.49 - 371.64 POL air 293.00 191.74 −9% 292.42 POL CO2 O2 351.36 202.04 −4% 335.08 STS CO2 O2 328.51 188.29 −10% 263.23 STS air 265.61 191.29 −9% 324.43 POL, polidocanol; STS, sodiumtetradecylsulfate. Figure 3. A) Organ uptake at step 2 for one patient; B) organ uptake at step 3 for one patient; C) organ uptake at step 4 for one patient; D) Organ uptake at step 5 for one patient. Figure 4. A) Step 6 images and curves of lungs after free radiotracer (RT) injection (left) and after last labeled sodiumtetradecylsulfate (STS) injection (right); B) step 6 images and curves of thyroid after free RT (left) and last labeled STS foam. No n c om me rci al us e o nly Article [page 50] [Veins and Lymphatics 2014; 3:4037] cant difference in uptake was highlighted, these data confirm that at 42 days after four sclerotherapy sessions, no relevant changes in lung and thyroid circulation/tissues occurred. Furthermore, the second part of step 6 con- sisted in one last foam sclerotherapy session with STS 1% sclerosant foam and subsequent scintigraphic assessment of RT uptake in the lungs and thyroid again showed no relevant changes in the curves in comparison to curves originating from free RT injection performed at the first part of step 6 (Figure 4). As a consequence of these findings, no lung circulation changes due to sclerosant foam can be postulated. The lack of important variations in the time/activity curves of the lungs between the first and the last foam sclerotherapy session indicates that sclerosant foam does not reach or does not induce any lung lesion. Discussion This observational experimental study was carried out to assess sclerosant foam effect on a few organs that may potentially receive a tis- sue lesion following sclerotherapy sessions. After a preliminary investigation on tech- netium pertechnetate chemical-physical pro- prieties and on the possible binding interac- tions with SF, the authors finalized a clinical and scintigraphy protocol. The aim was to assess the pathway of SF bubbles/drug through the blood stream and the binding/effect of SF on target and transit organ tissues. In a previous study,8 99mTcO4- was used to assess pulmonary patterns and bubble accu- mulation during foam sclerotherapy. In our study, the use of 99mTcO4- proved to be unreli- able to demonstrate the sclerosant drug path- way along the blood stream and within the transit organs (e.g. lungs), as the radiotracer flows independently. In fact, to bind the scle- rosant drug to the 99mTc we would need an ade- quate procedure of reduction 99mTcO4-+ Sn2+ + STS or POL and hence the extemporaneous way to combine sclerosant drug with RT proved to be of no utility. From the chemical and scintigraphy point of view, it is recognized that radionuclide 99mTc is obtained in physiologic solution as pertechne- tate ion, and it has a coordination compound among technetium and oxygen. The metal atom is bound to four binding oxygen (0-2) thus forming a very compact tethraedron-like structure, with four identical faces of triangu- lar shape. Technetium oxidation state in ion is +7. This constitutes the most stable oxidation state and represents one of the most stable pertechnetate chemical species in watery solu- tions. In order to prepare a radiolabelled drug from the 99mTcO4-, with coordinated bindings to give particular biological properties to the complex, it would be necessary to remove, partially or totally, the oxygen atoms that are bound to the metal. Finally substitution of these O2 atoms with the coordinated atoms of new binders cannot happen with a simple extemporaneous mixing. Hence tracing SF pathway by means of this radiolabelled ion would require a more complex process to overcome the chemical/physical difficulties as to above. Contrasting evidence from a few studies high- lighted the possible alterations induced by the sclerosant foam on the lung capillaries/tissues. After a preliminary non-conclusive scinti- graphic study from Milleret,8 on one side Grandi9 and collaborators showed lung parenchyma lesions in rats after foam scle- rotherapy, on the other side Moro14 and co- workers recently showed no significant changes in pulmonary ventilation parameters after sclerosant foam injection. One of the objective limitation of Grandi’s study9 subsides in the injected dose of SF in the rats which was by far much higher than the maximum permitted dose in humans; sim- ilarly the very short time/pathway between ears (the injected site) and lungs may have influenced their conclusions as well. Notwithstanding the very small sample size, the variability of the methodology (gas, Table 2. Time to maximal radioactivity in each curve. Lungs Heart Thyroid Background (vs contralateral limb) 99mTcO4- 60 s 60 s 27 min 15 min CO2O2-based SF 120 s 120 s 17 min 19 min Air-based SF 5 min 4 min 26 min 26 min SF, sclerosant foam. Figure 5. A) Lung and great saphenous vein imaging and lung uptake curves (for the first five steps); B) heart imaging and relative uptake curves (for the five steps); C) thyroid imaging and uptake curves (for the five steps); D) stomach imaging and uptake curves (for the five steps). No n c om me rci al us e o nly Article [Veins and Lymphatics 2014; 3:4037] [page 51] agents) and the limited pathology treated, this preliminary study confirms the overall safety of foam sclerotherapy as to the possible chemical lesion to transit/target organs. The invariable and total neutralization of STS or POL drug by blood components (proteins firstly) was demonstrated by Parsi15-17 and Watkins.18 Similarly Tessari and collaborators showed no free STS in the blood of common femoral vein (at short/mid/long term time intervals) after injecting STS-based sclerosant foam in the great saphenous vein/tributaries of the lower leg.19 Due to the very small sample size no sta- tistical analysis was performed, though all data seem to be coherent with the conclusions on the safety of foam sclerotherapy as to the pos- sible chemical lesion in distal transit/target organs. Similarly no specific speculation on the pos- sible foam bubbles accumulation in the lungs is possibly drawn from this study, as the mechanical passage of the SF bubbles was not quantified with this methodology; conversely the clinical/experimental study was addressed to investigate any possible residual lesion on the pulmonary capillaries/tissues after SF pas- sage. Though our investigations were aimed mainly at pulmonary circulation and no specif- ic scintigraphic examination was carried out for brain/ocular circulation, the final total body scintigraphy of the second patient (step 6) did not show any residual lesion in any organs, including cerebral parenchyma. The data from this observational study rein- force the concept of the absence of any direct chemical activity of SF within lungs and other central organs, which is coherent with the results of our scintigraphic study and with the results of other studies.15-19 Conversely all scintigraphic investigations showed a clear increase of RT accumulation in the injected areas of the lower limbs, where inflammation and sclerothrombosis obviously occurred. Conclusions A few conclusions may be drawn from the data collected through this scintigraphy-based preliminary study: i) definitely no damage of the sclerosant foam on the pulmonary tissues was demonstrated; ii) extemoraneous mixing of a sclerosant drug with pertechnetate ion is not a correct procedure to highlight the drug pathways in foam sclerotherapy, differently from what was shown in the past; new, alterna- tive procedures for a stable binding between STS/POL and RT would be required if the scle- rosant drug tracing within the blood stream is targeted; iii) little delayed uptake differences were shown as to the time/curves between free RT and SF, due to the sclerosant foam transient persistence in the peripheral vein where it was injected; iv) air-based sclerosant foam top radioactivity was reached later than CO2+O2 based SF, due to the early release of the RT from the bubbles in the second case; v) no sig- nificant differences were highlighted between POL and STS curves. 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