Good morning, everybody. It's 10.15, so it's time to get started. Welcome to San Diego and HRS 2025. Thank you for joining us for this session, which is Pulse Field Ablation, Where Every Clinician Should Know, and I really hope this means that by the end of the presentation. So last year, we had a very small room. We asked for something bigger. This is probably way more than we were all expecting for this year, but it's a pleasure to be here. My name is Maria Terricabras. I'm coming from Sunnybrook Health Sciences Center in Toronto, and it's my pleasure to chair this session. Unfortunately, my co-chair couldn't join today, so he apologizes that he's not here. A little bit of housekeeping. I want to remind to our speakers that each talk should only be 12 minutes. We're going to have four excellent speakers, but we're going to keep all the questions for the end of all the presentations. Unfortunately, this room doesn't have a microphone, so try to submit all your questions using the app. It's the HRS 2025 app that you can use the QR code that hopefully we're going to display. You can download it and then submit all the questions. I promise that I'll do my best to review them all and read them. With no further delay, it's my pleasure to introduce Atul Verma from McGill University in Montreal, who is going to tell us how we need to adjust our PFA parameters to optimize lesion depth and minimize the skeletal muscle recruitment. Thanks, Atul. Hello, everyone. Am I able to go back to ... Yeah, I am actually. Thank you. First of all, it's a pleasure being here. I think for those of you who attended the PFA summit yesterday, you probably saw no fewer than 40 technologies for various types of PFA devices. As we're trying to make informed choices about which of these devices to invest in, it's really important that we understand the basic mechanisms of how PFA works and why one device works in a certain way versus another device which works in another way. Hopefully at the end of this session, we'll be able to give you a little bit of insight into how the secret sauce works. These are my disclosures and I want to acknowledge for some of these slides the Abbott R&D team. As you know, pulses are very complex and I'm not going to try and cover each and every one of these aspects because I know that my colleagues here on the DS are going to cover these items, but it is important to understand which of these parameters are key in building the optimal PFA recipe. In terms of what most of the devices are doing today, they're usually using voltages in between about 1,500 and 2,000 volts, which is a peak-to-baseline, so if you're going peak-to-peak, it's actually double that number. They're using biphasic pulses. I think there's only one generator out there right now that is proposing monophasic. The pulse width is somewhere in the low microsecond range or very, very high nanosecond range. Pulse widths usually have about 10 to 30 pulses and you're getting anywhere from about one to eight bursts being delivered. If we move on, how do we actually build the right PFA system? In this particular case, I'm just going to be using a single-point irrigated catheter to illustrate some key points. Here's some data showing the predicted depth of a PFA lesion, so that's what the colors represent, but look at the x-axis. The x-axis goes from one microsecond to two microseconds. By changing the pulse width by only one microsecond, you can have dramatic effects on the results. On the left-hand side, in the blue, you have tissue depths that are around 3.8 to 4 millimeters, but on the extreme right-hand side, you're suddenly getting tissue depths that are close to 6 millimeters. You can achieve this only through the change of one parameter by about one microsecond. This just shows you what an exacting science or idiosyncratic science this can be in terms of coming up with the right recipe. At the same time, however, switching that pulse width from one microsecond to two microseconds can take you from the green zone, which is kind of an acceptable amount of musculoskeletal stimulation, so what we mean here is the patient actually contracting when you deliver the pulses, to the red zone, which is really a completely unacceptable amount of musculoskeletal stimulation, so here we're trying to balance getting the best depth with the least amount of musculoskeletal stimulation, and again, with only a one microsecond change in the pulse width. The pulse width is one of the most important aspects of the recipe. It's not the only aspect, don't get me wrong, but it is one of the most important aspects, and you're going to hear a lot about microsecond delivery and nanosecond delivery. Truthfully, narrowing the duration of the pulse really helps you most with musculoskeletal stimulation. It isn't necessarily going to get you better efficacy, because as you narrow the pulse, if you want to get the same apoptotic result, you basically have to exponentially increase the amount of voltage that you're giving, because at the end of the day, the area under the curve, roughly speaking, has to be the same in order to get the same effect. I'm going to talk a little bit about hemolysis. Devi Nair, who's going to be the final speaker, will talk more about this, but this is such an important concept, and hemolysis is really not so much about the pulse form as it is about the catheter design and how much of the energy you're wasting into the bloodstream, because the more energy you dump into the blood, the more amount of hemolysis you're going to get. So if your catheter is directional or is insulated by a balloon, you're going to waste less of that field in the bloodstream, and therefore, you're going to get hemolysis. And there are big differences in the catheters when it comes to hemolysis. Now you might say, well, you know what? Who cares? Like, why are you making such a big deal of this? If I just give two liters of fluid, I'll get rid of any risk of renal problems and everything will be fine. Well, actually, as it turns out, any kind of minimal damage to the kidney by plasma-free hemoglobin results in a huge inflammatory response. And don't worry, I'm not going to go through this slide step by step, but what I do want you to appreciate is that subclinical damage to the kidneys can result in a chain reaction of events that we don't want. One part of this chain reaction actually happens to be the depletion of nitric oxide. And you might say, oh no, he's going to talk about nitric oxide. Yes, I'm going to talk about nitric oxide. Why? Because first of all, nitric oxide can cause vasospasm. Now I'm not saying that that's necessarily important acutely, but now we are starting to get reports of delayed vasospasm 24 to 72 hours after PFA procedures in certain prone patients. And you have to wonder if hemolysis and depletion of nitric oxide is playing a role here. Furthermore, when you hemolyze, you also activate platelets, and platelet activation causes thrombosis, which your heparin bolus is not going to help you with. So if you think about some of the strokes being caused by some of these devices, you've got to think again about aspects of hemolysis and coagulopathy. Devi will go into this in more detail about the differences between the devices, but there are stark differences. And so you need to be aware of that when you're using one device versus another device. So let's go back to our microsecond delivery, and by optimizing the right amount of microseconds, we can manage to get the right combination between depth and low musculoskeletal stimulation. So the next step is to try and minimize the thermal threshold. So at the bottom, you see waveform D and waveform A, and you can see that waveform D is causing some thermal signal, whereas waveform A doesn't seem to be causing any thermal signal. Waveform A is sacrificing a little bit on tissue depth. It's only 5 millimeters, versus waveform D, which may be a little bit deeper. Irrigation can be used to mitigate some of these thermal effects, however, it should not be the only thing that you are relying on to mitigate thermal effects. You don't need to irrigate with most PFA catheters, but you can irrigate a little bit if it's going to help you with some of that thermal profile. Usually the thermal profile is driven by things like the interphase delay, the inter-train delay, and the post-delivery delay to basically avoid heat stacking. So you may only get a small amount of heat from your PFA device, but if you don't leave enough pauses between the trains or the deliveries, you can get heat stacking. So well-designed devices will avoid that by giving you some cooling time. Repeated bursts will also mitigate thermal effects versus prolonged bursts, and that's why we have gone to these rapid fire bursts as opposed to one long burst. Electrode design is also very important. You need to shape and curve the electrodes in such a way that you avoid edge effect heating, which can also cause significant problems. Finally, I'm not going to spend a lot of time on micro-bubbles because my colleague, Dr. Mokhtarovich, is going to talk about this in more detail, so I'm going to kind of skip over this slide but use it as a teaser. But bubbles are important for a lot of reasons because they can occur due to a variety of mechanisms, hydrolysis, thermal, air. Bubbles can be different sizes, and the larger the bubble you have, the more likely it is to persist and cause cerebral ischemia. So when you have one device that causes bubbles like this, and you have another device which is not causing any bubbles, and yes, the device is delivering, it's pretty clear which device you're going to want to use and which device you're going to want to, I was going to say throw out, but put in the closet. Okay, so these are just, I have had the chance to use a wide variety of PFA devices. I've been very fortunate to be involved in a lot of clinical trials, and there are differences. There are differences in thermal profile, musculoskeletal stimulation, phrenic stimulation, cough, vagal pauses, micro-bubbles. So I've just put a few of these systems up here with my own personal thoughts and observations, and I do put a disclaimer that this does represent my personal thoughts and is not an endorsement of any particular product. So I'd like to conclude by saying that pulse width is a key mediator of tissue depth and minimizing musculoskeletal stimulation. Pulse is mostly catheter-dependent, maybe less so on the pulse parameters. Hemolytic effects extend beyond renal injury. Thermal effects are there with all PFA, don't depend on irrigation as being the only way to eliminate thermal. Use intelligent catheter design and pulse design, and finally, micro-bubbles will be talked about by my colleague, Dr. McClavich. Thank you very much. Thank you, Atul. Just a reminder, to submit any questions that you have for the end of this session, you have to sign in to your HRS 2025 app and then submit the questions there. So now I'm going to introduce Damian McClavich, who's going to talk, sorry, from the University of Ljubljana, who's going to talk about incidental thermal effects of PFA and micro-bubble formation. Thanks, Damian. Thank you very much. I would like to thank, of course, for the organizers who give me the opportunity of being here and sharing this work with us and my views. So I think it's fair to remind ourselves that electroporation is something that has a certain dynamics, that when we expose a cell to the electric field, we actually have an induced transmembrane voltage, which then causes membrane permeabilization. And note the timescale here. Within seconds, we see increased transmembrane transport across the membrane in both directions. And of course, that represents an injury to the cell, which will now try to recover and reestablish the initial conditions. Now within minutes to hours, we will see whether the cell will survive or the cell will die. That will also depend on the amplitude of the electric field to which the cell is exposed. Now quite often, we see a graph like this, and people will say, we are going to choose our parameters to work in this irreversible area. And unfortunately, that does not play exactly like you would want to. Namely, when you move to a, for example, catheter solution, and you try to actually ablate part of the tissue by using irreversible electroporation, there's going to be a rim of reversible electroporation. But there's also going to be an area which will be of thermal damage. And it has been reported. And of course, as we will be driving our lesions deeper and deeper, this will become more and more obvious. Now with the temperature, I'm going to come back. So this comes actually from a colleague of mine, from Dr. Mateusz Jan from Clinical Center in Ljubljana. And you see three different systems. These are all the single-shot devices. And you see different level of bubbles on the eyes. It's the same eyes during the same week that this was taken. So where do the bubbles come from? I think it's a fair question. Now some of you, you may think that I'm obsessed by bubbles. And yes, I am to some extent. You can see the slide, which I actually presented already back in 2020 at the AAF Symposium. And we published these results in a paper in 2020. In Electrochemical Acta, which I think now Vivek and both Atul as well read every day. Yeah, it's a great journal, what Atul says. So what you will see here is monophasic pulses like the defibrillator on the left. And of course, here you see the 100 microsecond. And you see different amount of bubbles on the anode and the cathode. Now here, there's no bubbles. And this is the biphasic high-voltage pulses. So I think if we read the literature, all of these single-shot devices, the producers, the manufacturers disclose that they are using biphasic short electric pulses. Now, where do then the bubbles come from? Because on the previous slide, I showed you there was no bubble. So this was my obsession for a period of time. Bar Kos and Samo Mahnich-Kalamisa from the lab, they set up a very nice test bed using a high-speed camera. And we started to play, they started to play with different waveforms. And I'm going to walk you through quickly. This is the setup. In addition, Peter Lombarger, who is a PhD student of Bar, did put together a model, which allowed us, of course, there's a lot of equations. He coupled the thermal and the electromagnetics together and was able to calculate many things. And that allowed us actually to get a very good spatial, but also temporal resolution of electric field distribution, current density, and temperature distribution. And this is the catheter that we were using. But you only see the distal-most ring. There are two more up there, as I showed at the beginning. Now, I'm going to show you the results, basically, of the... So, yeah. Three examples of the pulses, of the waveforms. One is the monophasic 100 microsecond pulses. Eight, all of them. Then so-called H-fire, the biphasic 2 microsecond positive, negative, and 2 microseconds in between. but also 2 microseconds in between the pulse being repeated. And then a very similar one, now the only thing is that now we have 500 microseconds in between. Now, you see, this is the first one. That's the monophase. You've seen everything. It's going to repeat. It's just nice to see even a small explosion. And this is 8 pulses, 1.6 milliseconds altogether. Now, this is the biphasic. The bubbles start to appear, but then collapse. They start to appear and then collapse. And then this one, there's no bubbles. So what's the difference between the two? Well, it's the duty factor. So how much energy in how much time you actually want to deliver through the catheter. That's the only difference. And of course, we can now match this closely. We've measured the current, the voltage, and of course, visualized the bubbles. And we see that whenever the bubbles start to appear, is actually the temperature hits the 100 Celsius. So the boiling. Remember, this is in the saline. So the conclusion of that paper is, and again, this one's also published in Electrochemica Acta, the great journal that Atul is reading now every day. Well, every fifth year would be good enough. So where do the bubbles come from? The conclusion is that in most of the cases, for the high frequency biphasic pulses, we actually see the bubbles being caused by high temperature. The other two contributions or possible origins are much less likely. Of course, with the bubbles that are of monophasic nature, we see the electrochemistry. And usually that shows if you have two equal geometry electrodes, an asymmetry between the cathode and the anode. Now, I think this now has become a common knowledge. So my obsession will now shift to something else eventually after this talk. Because I think we know, and this is a very nice paper by Brendan Koop, actually, I think last year, where they showed that there are various ways how you can mitigate, actually, the temperature increase, the incidental increase in temperature by number of pulses, the amplitude, and so on. And Atul alluded that there's a lot of parameters in the waveform that we can play. This is nine that you can identify. Nine different variables that you need to tune. And of course, Atul already said that one that is very important is the width, the duration of the pulse, which will also control the lesion depth, the lesion size, and of course the capture, the neuromuscular capture, which all is true. But whatever we change, even if we change the interface delay, that will inevitably also change the effectiveness, so the lesion size. So it's not that you determine the lesion size and you say, this is what I can do with the lesion, and now I'm going to start to mitigate the temperature by making it shorter, making the duration between the two pulses bigger, and so on. So it's much more complex, an interdependent exercise that we need to do. So I think it's fair to say that whoever is trying to develop a new device, it would be good to have this in mind. And again, this is a repetition of the pulse. It's another yet very important part of the engineering of the catheters where you can actually, by brushing off the edges, also mitigate the local heating, the local high current density. Now with this, I would like to conclude that the thermal damage does not, so the increasing temperature is not only present in the cell line that I showed, it's also present in the tissue, like I started off at the beginning. And here's an example from a paper where they clearly delineate between how much is of a thermal damage and how much is of a non-thermal based on the electroporation. Now the other part is, of course, that in addition to the tissue, where maybe this is not so important, we have blood surrounding the catheter. Now blood is also a tissue, and you don't need to actually boil the blood with 100 degrees. You get coagulation, you get protein denaturation, even at lower temperatures. Now of course, we are limited to very small volumes and so on, but the tissue, blood, will respond to the damage. And that is, I think, very important. So with this, I would like to really ask everybody to have this in mind. It's not non-thermal, but let's keep PFA non-thermal as much as possible. Thank you very much. Thank you, Damian. Our next speaker, Debbie Nair, from St. Bernard's Medical Center and Arrhythmia Research Group, who was at another meeting, I think, and she just had to run here, so thanks for coming so quickly. She's going to talk about hemolysis and dependence on systems and pulses. Thank you. Thank you very much, and thank you for having me. So I will speak on hemolysis and dependence on the systems and pulses when it comes to hemolysis. These are my disclosures. So I'm sure you've seen this before, but this is courtesy of Dr. Verma's corporation. The pulses are complex, and there are many parameters that you can optimize. And this has been shown, but the beauty lies in the details, and these parameters can decide how that lesion's going to be delivered, but also what else that lesion can do. And even small changes can make a major effect on these. And there are certain parameters that we typically look at, the voltage, whether it's biphasic or monophasic, the pulse width, the burst, and how many bursts are we delivering. And these are some of the ones that we try to keep up with, but there are definitely more variables in this. In addition, there is the anatomy of the pulses, whether it's a monophasic pulse or an alternating monophasic pulse or a biphasic or maybe an asymmetric pulse design that could also affect how the energy is delivered and what that energy does to the rest of the tissue. Now, we've known that PFA can cause hemolysis for a very long time, but more recently, since pulse field ablation started making its way into AF management, we started seeing case reports of acute renal injury and hemoglobinuria. And what this series of case reports showed was that with the hemoglobinuria, there was an elevation in serum creatinine, but also a significant elevation in the myoglobin. So these were all concerns that we had not initially thought of as we launched PulseField and as we were working with PulseField technology. What happens with pathologic hemolysis in PFA is that you get electroporation of the red blood cells, and that leads to release of free hemoglobin. What happens? Why do we care about free hemoglobin? The problem with free hemoglobin is that the reduced state hemoglobin or the methemoglobin can cause free radical generation and antioxidant dysfunction, which increases plated activation and plated addition. In addition, it consumes nitrous oxide in a one-to-one molar ratio. And what you see in this experiment is that as the plasma hemoglobin goes up, your nitrous oxide consumption goes up as well. And why does that matter? When you have nitrous oxide consumption, now you start having endothelial dysfunction because you have smooth muscle dysfunction as well as vascular constriction. So these are all issues that we have to think about when hemolysis happens. So it's not just the fact that you see hemoglobinuria. There are some other aspects as well, such as coagulopathy, that you have to think about because of platelet activation, because of thromboinflammation, because of immune dysregulation, inflammation, and bacterial clearance. So more things that can be damaging with hemolysis that has to be kept in mind. We know that when free hemoglobin levels are over 200 milligrams per liter, it's associated with hemoglobinuria in healthy individuals. But when it gets to that point, now it starts affecting the haptoglobin levels because haptoglobin is your scavenger and it scavenges the free hemoglobin. So that is one of the ways to assess for hemoglobinuria or hemolysis is to look at, of course, hemoglobinuria, but also drop in hemoglobin as well as your haptoglobin levels that drop below 4 milligrams per deciliter. When it gets to that level, now you're starting to look into acute renal injury, whether any of the stages, and it really depends on what stage the patient starts at. So again, these are all considerations to keep in mind. And what happens with hemolysis is this heme breakdown products, as well as labile iron from this hemolysis, causes iron deficits in the kidney tubules and mitochondrial damage, thereby resulting in tubular injury and renal failure. So it's a very, I would say, a process that is not something that you can just close your eyes to or not worry about because you're sending the patient home the same day and you don't see it. This really affects important organs such as the kidneys. Myoglobin also, as I said, is an important part of the problem and needs to be evaluated. Now, all PFA can cause hemolysis because you are doing electroporation, there's red blood cells around the tissue that you're electroporating, but the degree of hemolysis really is very system specific. And what happens is you have the wasted field that's dumped into the bloodstream and the surrounding tissues that causes hemolysis. And in addition, if you get musculoskeletal recruitment, now you have a double problem and it's a bad combination for your kidneys. Now, the question is, is all PFA the same when it comes to hemolysis? So what you're seeing here is an animal experiment that looked at the variable loop catheter and the pentaspline catheter. And with 50 applications, there was significant amount of haptoglobin reduction as well as hemoglobinuria. And on the right, you're seeing applications with the balloon system as well as with the arc system, which is the pulse select system. And you can see there's significantly lower amount of hemolysis, even with the higher number of applications in that section. And we're seeing more of the hemolysis with the variable loop catheter as well as the pentaspline catheter when it comes to hemolysis. Now, this is just a chart looking at the number of applications. Green is an area where the lower you are on this graph, the better you are. You have less free plasma hemoglobin in your blood. And you can see pulse select usually needs lower applications. So about 32 applications is usually routine for a PVI case. With the volt system, it's about 57 applications. With the sphere 9 catheter, it's about 120 applications. And all of these kind of stay in the lower level with really no free hemoglobin that we worry about. Even if you take the pulse select system and deliver about 120 applications, you still stay in that lower level or maybe mild to moderate level. But when you look at the pentaspline catheter, it becomes concerning with that even with 20 to 29 applications in the porcine model, we were seeing free plasma free hemoglobin levels in the 70 plus or minus 30 milligram per deciliter range, which is concerning, which is in the more moderate hemolysis range. Now, what are the things that affect hemolysis or how the energy is delivered? One of the things is the way the energy is delivered, the waveform is delivered. If you're delivering it as a single field mode, like what you see on the left side versus a dual feed mode, which is where you alternate the electrodes. If you're doing a single field, you're using all the electrodes simultaneously versus if you're doing this dual feed mode, where you're alternating between odd and even electrodes. You get contiguous lesion, but the advantage is that now you are able to deliver a very significant amount of depth without increasing your voltage. And if you don't increase your voltage, that helps with hemolysis and you get durable lesions without having to worry about hemolysis. So these are all things that have to be kept in mind as we think about choosing the technology that we go for. Now, it's important that we understand waveforms. Today we get the catheter, we get, we're told deliver four pulses in the pulmonary vein and that's what you need, but we don't see the waveform. I think it's important for us as clinicians to understand what these waveforms are and how we can maximize depth while minimizing the thermal threshold. And like Damian said, I think it's important that we try to keep PFA non-thermal because it does cause other issues. Now you can have incidental heating from PFA as well. This is an animal experiment looking at the Tacticath catheter using the Galaxy system. And what's seen is that the interface delay, the inter-train delay, the post-delivery delay, these are all essential to avoid heat stacking. So it's not just that it's recharging, but it also is giving the system time to cool down, allowing for less thermal effects and which means less hemolysis. Now, repeated bursts is another thing that can mitigate thermal effect versus a prolonged burst. So again, something that could potentially maybe decrease hemolysis because of the less thermal effect that it has from repeated bursts. Now, Damian just showed the effect of, you know, the edge effect from the electrodes and how the electrodes are. These are things that we don't really pay attention to, but I think it's about time for us to start questioning these things and ask these questions. And when you have this edge effect, you have more heating, which means you worry about hemolysis in that area and you could potentially cause more damage than what you intend to. Now, what are the lesion things? What are things that we could potentially control after the catheter is in our hand? I think there's data looking at the impact of the number of lesions as well as tissue contact. And we definitely see this. This is a swine experiment looking at application with the pentaspline catheter. And when you see that there is contact, there is less hemolysis, whereas when there's no contact and there's more lesions, there's more hemolysis. This is data looking at the pulse-elect system. Again, when there's contact, there's less hemolysis compared to when there's no contact. When it came to the SPHERE-9 system, even with, you know, no contact with or without irrigation, there was really no hemolysis in any of these groups because of the way that probably because the footprint of the catheter. And again, that's different. This is actually a clinical paper that was presented. It's like 145 patients using pentaspline catheter. And you can see that the number of lesions as well as contact assessment is what actually increased the risk of hemolysis in this patient population. The higher the number of lesions, more than 54 PFA deliveries kind of led towards more hemoglobinuria and more hemolysis. This is looking at hemolysis after PFA in a multi-center analysis. And you can see there's the patients that had hemolysis, some of them actually even had acute urinary retention. And again, you have to wonder whether this is because of acute kidney injury or progressing to acute kidney injury. Now, the question is, is this preventable? This was a paper published by Dr. Natalia and his group talking about hydration and how that could prevent probably hemoglobinuria. But I don't truly believe that it can prevent hemolysis. And the question is, is that the solution? Giving, you know, two liters of fluid is a solution. Are we going back in time when we're giving it? Because you have to remember irrigation gives a thermal safety margin, but does not really kind of take the physiology away. It still causes hemolysis. We're probably just diluting it. Having said that, I'd like to summarize saying that hemolysis definitely is one of the collateral injuries that comes along with PFA. The question is, how much hemolysis do we have? Everybody has some. It is mediated mostly by direct effects of PFA on red blood cells. Myolysis also plays an important role in this. And there is hemolytic effects beyond renal injury, such as vasoconstriction, hypertension, coagulopathy, and acute renal injury. And the design and use factors of these devices, as well as how the catheter is used and how the pulses are used is what decides how much hemolysis you have. And you could potentially probably do hydration to minimize hemoglobinuria, but I do think that it is very important to select patients carefully, as well as your tools that you use carefully to mitigate this. Thank you very much. Thank you, Debbie. Our last speaker is Quim Castellví from Universitat Pompeu Fàbrile from Barcelona, and he's going to talk about nanosecond versus microsecond PFA advantages and disadvantages. Thank you. Okay, thank you Dr. Terry Cabras for the introduction and also the organizers for inviting to talk to this advantages and disadvantages of using microsecond and nanosecond for PFA treatments. Okay, as my colleague says, mainly all of you know that we are using electroporation to induce the ablation, and this is using waveforms to deliver the treatments. In this particular case, we can see a very simplistic waveform, but the waveform casts several parameters that everyone plays a key role. However, in doing this talk, I'm going to focus, I'm going to narrow the attention to I'm going to focus the attention mainly on the pulse duration. And the pulse duration plays a key role on electroporation, and if you observe the different applications that have been used and that are currently using electroporation, you can see that the range of pulse durations is huge. For example, probably one of the most extended applications is gene transfection, is gene transfection in cuvettes. In this case, we try to introduce big portions of DNA into the cells. And in those cases, mainly long pulses, in millisecond range pulses, they are used. However, if we go back with smaller and smaller molecules, for example, in cancer treatments, we are trying to introduce some therapeutic agent inside the cell, we are using pulses that range on hundreds of microseconds. And if we just want to kill the cell, mainly with smaller pulses, we have enough. And why this kind of, with big molecules, we need longer pulses, and with small molecules or ions, we need smaller pulses? This is mainly because of electroporation. Electroporation is this phenomenon where we have a cell and we apply mainly an electric field that involves the cell. We are going to start rising up the voltage of the membrane, and if we reach a certain point, the structure of the membrane is going to collapse, and mainly just in this way we create a pore. If we keep the pulse for longer periods of time, we are going to mainly, in a nutshell, enlarge the pore. And that's why for molecules that are large, mainly we are using longer pulses. When we stop the pulse, mainly the pore is going to reseal. This has been shown in molecular dynamics and it's pretty well understood, but what I want the message to came is that the longer the pulsation, the longer the pore size. And why is this important? Why if we want to kill the cells, it's important the pore size? Mainly because with electroporation, we are not only triggering one way to kill the cell, we are triggering multiple ways to kill the cell. And apoptosis and necrosis are the main ones, and actually in one study in our group, we saw that by reducing the pulse duration of the applications that we were performing, actually the necrotic part of the population cells, it was reduced. And this is true. This is something that shortening the pulse, we are going to promote the apoptotic pathway from the cells. And this is something nice. The things that I like to think on the cell, like a boat, if we have small holes, it's easy to put my finger and just to hold the leakage of the boat. However, if I have tons of small holes, I know that I'm not going to be able to repair. I'm going to start packing my things, calling the rescue team and just wait for rescue. However, if in my boat, I have a big hole, no time for call, no time for packing my stuff, I'm going to start mainly a necrotic process as a cell. And this is kind of the concept that I like to think when trying to explain why big pores or longer pulses are going to be more promoting this necrotic pathway. I have to mention that mainly since we are applying the treatments using a minimal invasive applications or catheters, we are going to have not an homogeneous field. And therefore, we can expect that almost in any electroporation application, we are going to have several mechanisms that are taking place. But one of the cool things of nanosecond pulses, going back to the nanosecond versus microsecond pulses, is that nanoseconds actually are targeting to make holes not on the boat, but on the engine. With nanosecond pulses, the key part is that instead of making holes on the boat, we are going to damage the internal organelles of the cell. And this is a very cool stuff because then there's no rush to do necrotic process. I can, as a cell, I have all the time to start packing things, just ordering my papers and just trying to do a less inflammatory reaction cell there. Let me explain why is this happening, why we can, with nanosecond pulses, induce this kind of damage on the internal organelles. And mainly we have an electric field being propagated by the tissue. When we have this pulse, we are going to have a kind of a profile of how we are charging the membrane. If we move farther from the electrode, mainly we are going to have lower electric field. We are going to have something scaled on that, the same profile, but scale of magnitude. The cooler part is what happened with different cell size. When we have different cell size, we have a big cell size, we are going to end up being able to charge that membrane to larger magnitudes with the same electric field, but it's going to be something slow. And we can imagine that it's going to be harder to pull all the ions from the cell and charge the membrane that is big than just if the cell is smaller. If the cell is small, it's going to be very fast. We are going to not charge maybe that much, the cell, but it's going to be really fast. And this key concept is what is behind on why Dr. Verma talked about muscle stimulation, but this is kind of one of the reasons why with shorter pulses we are stimulating less the muscle. And mainly this is some study that we did when we have, there's a theoretical model where if we have the nerve, which is a very big cell, when we are charging at the same time with the other cells, mainly we are going to have the nerve being charged very slow. Therefore, there is a small gap of time at the beginning of the pulse that if we delivered the pulses of that duration, mainly the cell that we are targeting is going to be more sensitive to those pulses than the nerve one. And this is the way that can be achieved, another operation of the cells without the muscle stimulation. These have been evaluated in vitro and also assessed in vivo, and this is kind of the reason why we have this advantage. And this is an advantage of reducing the pulse to nanosecond. We are going to have less muscle stimulation. At the same time, the same principle can be applied for nanosecond pulses. Mainly if we have the internal organelle inside, we are going to have kind of a similar situation. We are going to have a small, very, very small window of opportunity where the pulses that we are delivering are going to be more effective on the organelles than in the membrane itself. However, the problem is that this has been proved that we need very, very short pulses in the terms of few nanosecond pulses and also from huge magnitudes. The main problem of that is that if we are trying to go with those small nanosecond, few nanosecond pulses with large magnitudes and we want to get, for example, a four millimeter lesions, we are going to need those ones in the EP labs. Mainly we are going to need a huge amount of electric voltage being applied on the catheter to get that kind of electric field. This is something not practical from the technological point of view and also from the compatibility of the remaining medical devices. We have to keep in mind that the medical devices are not ready for that magnitudes of pulse and therefore, in order to keep it, it's mainly not feasible to use those kind of pulses along with the other medical humans in the room. This prevents from, if we are picturing this microsecond versus nanosecond, it pictures that we cannot use kind of a clinical practice, those kind of pure nanoseconds. What is most likely that nanoseconds PFA are is more closer values to the microseconds just in the same range of pulse durations and this is something that we are going to keep some features from the nanoseconds but we are going to kind of mitigate or mile down the effectivity of that. With that being said, I can keep trying to see difference between those pulses and this is one interesting concept on lesion homogeneity. It has been shown that on the range of nanosecond pulses that we are now considering, there is kind of a special sensitivity on orientation of the cells and we all know that myocardium is a very anisotropic tissue, therefore, this could have an impact when we are using nanosecond pulses in that range. This is going to affect that maybe on the border, we are going to have more preferential lesions towards one direction than the other or leaving some aisles on the lesion formation. This is something to consider but probably one of the main difference between nanosecond and microsecond pulses is that both technologies came, let's say, from very different points. One is coming from this very, very short nanosecond pulses and at that range, mainly with monophase pulses, there is no muscle stimulation, there is no electrochemical reactions, it's enough. At the same time, from the microsecond point going down, there we're facing some problems on muscle stimulation and they turn the waveform as a biphasic just to prevent the huge muscle stimulation that we have. Now that they are merging, we have using the very similar pulse duration but the main difference that I saw is that one is keep using monophasic pulses and the other is using biphasic pulses and this has an implication. One is that monophasic pulses, it's true, is going to be more effective inducing electroporation. Mainly you can get a cancellation of the negative phase and mainly the drawback is going to be that the muscle stimulation on the nanosecond pulses even being nanosecond just by the fact of being monophasic is going to get increased. At the same time, as Professor McClatchy says, using monophasic sometimes can bring some electrochemical reactions that we want to avoid. That being said, we can summarize a bit on the advantages that I consider that the current technology could have on the PFA. Just showing that probably muscle stimulation, there is not going to be that much difference because this monophasic, biphasic correlation and it's true that it could be a bit more effective on one side, the nanoseconds, but at the same time with the risk of equipment compatibility, which is unknown, depends on the magnitude of the voltage, but also the fact that we are going to have some kind of fiber orientations and also the increase on apoptosis. Those are the conclusions, just summarizing what I just said and just highlighting that mainly now I think that we are merging both worlds in the same area of posturation. It's just that we are going to have to keep in mind those extra specifics of the waveforms and of the catheters and try to see how those kind of play an interdependence between them. Of course, we need additional research. As a researcher, it's something that I always ask and also my team is also asking for more juice for research. Thank you so much. Thank you, Guillermo, and thank you all the speakers for actually finishing on time because we still have seven minutes for questions. Maybe while I'm organizing all the questions, please keep submitting using the app. Maybe I could ask the first question, probably to Damien. We've learned over the last few years that contact is important to achieve good lesion depth and also, as Debbie was mentioning, if our catheter is not in good contact, then we get more hemolysis. My question is, in terms of lesion formation, how important contact is because in our clinical practice and especially when we're using eyes, the moment that the catheter is not in good contact, you see a ton of bubbles. It's probably different depending on what system you use, but even the ones that we know that don't have a lot of thermal effect, we still see those bubbles, which are tiny, but is that a concern or we can just keep going and continually applying? Thanks. I mean, there are several questions in this question, so I'm going to pick the one which I know how to answer. So the lesion depth is affected by the contact. If you lose the contact, even though the blood is conductive, the field will go through. We will, of course, effectively be moving away and we're going to be moving the limit, the depth. So that is, I think, what is clear and I think this is what we understand. I would like, though, to, if you play with one focal catheter, I think it's fairly easy to somehow be sure that you're in contact, either on ice or, you know, any other way. Tactile maybe is less reliant, but nevertheless. But let me ask Davie and Atul, if you operate with single-shot devices, which have from 20, you know, to nine electrodes, how sure can you be that you're going to be at any point delivering the pulses in contact with all the electrodes on the either spline or on the ring? Damien, that's called raw natural talent. I'm kidding. Yeah, I don't think it happens. I don't think you can. I think, I don't believe in every part you can. I think you get away in certain anatomies, maybe over the vein sometimes you could. But in the posterior wall, I don't think you can. I think the topography of the left atrium is not going to let you. But you, I mean, even in the, if you look at the mapping system, I think you can see sometimes if you're assessing contact based off impedance, it'll give you a number of, the number of electrodes that didn't have contact through all of your applications. So I don't think we do, unless Atul gets it, I don't know, we'll see. No, I think it's, it's, you're right, I mean, it's, it's very hard, even like most of the newer catheters are going to come, be developed with some feedback of contact. So whether it's impedance based, force based, whatever, the newer generation catheters are going to tell you, are you in contact or are you not in contact? But with the variation in anatomies, I don't think it's possible to take a large shot device and have 100% contact. Having said that, contact is not binary. It's not, you're fully in contact or you're not at all, you know, some things will be very close and close enough that, yeah, it won't be an ideal lesion, but a lesion will be made. And more importantly, if, if you're seeing a lot of micro bubbles while you're delivering, you probably don't have very good contact. A, you probably don't have a very good system if you're seeing micro bubbles all the time, but B, if you're seeing even more, then you probably don't have very good contact. And so this is part of the problem. I know there are many countries where you cannot use ice for economic reasons, and that's a very valid point because we can't spend an unlimited amount of money on these procedures. But I think that's where ice does become very useful, not only for contact assessment, but that bubble feedback to tell you that, you know what, I really need to do a better job. Either, either the company needs to do a better job or I need to do a better job or both. There's another reason why I would say you can see sometimes bubbles, sometimes not. Some catheters do not have fixed distances, like between the splines. The splines can be sometimes closer to, to each other than the other time because we, in the process of rotating, maybe you didn't go back enough and, you know, you, you bend a little bit one spline towards the other. And that, of course, with the same voltage creates higher current density and that can also produce some bubbles. So I think that also is a sign, if you work with the ice, to actually, you have to correct that somehow. So I think this is, it can be used also as, as a, as a good testing, I would say, a feedback actually, not, not a test, but a feedback to, to the operator. Would you agree? Yeah. We, I know we don't have too much time. I want to know if I could ask one question. Yeah, sure. Kim. So Kim, I really liked your breakdown of the difference between nanosecond, microsecond, and then you also introduced the monophasic aspect. And there is a, there is a commercial system that is trying to get approval for cardiac ablation, which is saying it's in the several hundred nanosecond range and using monophasic pulses. But another aspect of monophasic versus biphasic pulses is arrhythmogenicity and whether you need R-wave gating. So do you maybe want to talk a little bit about that to inform the audience? Yeah, absolutely. I mean, we have to keep in mind that, that just going with monophasic pulses is, is kind of delivering energy without charging, I mean, just balancing the charge that we introduce. And therefore, it's kind of giving some, some energy and letting the system to deal with that energy that you deliver there. You are not getting this, this net balance, and therefore, the charge that is there mainly can do whatever it wants. And if it's triggering some arrhythmogenicity, it's going to trigger some arrhythmogenicity. This is something that, that I thought that, that was clear that we need kind of biphasic pulses just to make less bubbles, more, more, more, less electrochemical reactions and changes of pH, metal release. But it's true that since mainly that company that you mentioned came from the nanosecond, that the true, true, truly nanosecond pulses at that ranges, that things are not that, that required. But now that they are at, at those hundreds of, of nanoseconds, this is something that, that yeah, it should be considered, it should be considered. And we've got a lot of questions, but maybe just one that summarizes, I would say 90% of the questions that were submitted. Given the FDA systems that are currently approved, what are the red flags? How would you choose one or the other? And what would you do to try to minimize the complications that each of these systems can cause? Yeah, maybe I can, I can start with this. And then Debbie, maybe if you want to add, you know, I think if, again, if you're seeing a lot of micro bubbles, every single time you apply, that means you have a thermal, that most likely means that you have a significant thermal signature with your PFA. And so personally, I would choose the system which produces the least amount of thermal and the least amount of bubbles. And, you know, when I showed that table, I was routinely putting esophageal temperature probes in for every single one of the PFA systems that I tried. And there were remarkable differences between the systems. Some didn't cause any change in the esophageal temperature and others caused a lot of change. For those of you who took pictures of the table, you can see for yourself, but, you know, pick the one with the lowest thermal signature and the least amount of bubbles. Debbie? I think that's, it's a great point, but also clinically when you have, you know, more than one commercial system available, some of the clinical things that we look at is, does the patient have renal dysfunction? Does the patient have a history of hypokalemia? Things of that sort that kind of maybe shift you towards using one system over the other to avoid additional problems. Obviously PFA came into the field because of its safety profile, but I think we are seeing newer problems that we have to keep in mind when trying to avoid things like esophageal injury and phrenic nerve injury. Well, I think we're out of time. I want to thank all the speakers for excellent presentations and all the audience also for staying until the end and joining us for this session. Thank you so much.

Pulse Field Ablation

PFA parameters

muscle recruitment

pulse width

thermal effects

micro-bubble formation

hemolysis

nanosecond pulses

apoptosis

device selection