 My talk today will be on therapeutic plasma exchange for pediatric open heart transplantation. I have no disclosures. So the overview for today, I'm gonna review the basics of humoral immunity and the sensitization process responsible for organ rejection. I'll touch on some TPE objectives, some TPE circuitry and mechanics. And then we'll take a look at our transplant, cardiac transplant population at Cincinnati Children's Hospital. And then look at some of our process guidelines for pre-peri and post-TPE with special attention to the anticoagulation and calcium supplementation. And then I'll finish up the presentation with a case presentation of an ABO compatible heart transplant that I did last December. So pediatric orthotopic heart transplantation has been an accepted therapy for children with end-stage heart disease since the 80s. Since that time, advancements in perioperative management and transplant immunosuppression have led to significant improved outcomes for this class of patients. A 16-year median survival rate has been reported in the literature with up to 22-year survival rates for patients that were transplanted during their infancy. Improved management of congenital heart disease and advanced technologies for managing critical patients, awaiting transplant means that there's more children are surviving to the point where transplant can actually become an option. Children who are immunologically sensitized to the human leukocyte antigen remain at increased risk for morbidity and mortality while waiting for end-after transplantation. And 18% of the heart transplant recipients with anti-ABO or acquired anti-HLA antibodies go on to develop antibody mediated rejection. Antibodies are protein molecules that are produced by B cells in response to an initial antigen exposure. The lag period or the amount of time it takes to develop the full antibody production takes about 14 days. And the antibody production continues until the antigen has been destroyed. The follow-up or secondary exposure also known as the anomestic response is a re-exposure to the same antigen where the memory B cells from the primary exposure produce the antibody much quicker in five days instead of the 14 days on the initial activation and they remain active forever. The IgG antibodies are the most potent and effective antibody in the immune system. They bind and destroy antigens by antibody dependent cell mediated cytotoxicity which is cell lysis. And the IgG is produced in the greatest amounts during the subsequent exposures. It makes up 80% of the antibody in the serum and it can also diffuse into the tissue to bind the donor antigen. So the body's humoral response to donor antigen during and after organ transplantation can be very quick and very destructive to the transplanted organ. So the repeat exposure is more rapid than the initial sensitization exposure. The antibody responds to lower concentrations of the antigen. It has a higher affinity for the antigen. It's produced faster and the antibody is produced in larger quantities. Sensitization results from a non-self immunologic exposure such as a prior organ transplant. An important risk factor in children with congenital heart disease is exposure to human homograph tissue during their initial surgical palliation for the congenital heart disease. Blood transfusions and mechanical circulatory support patients are triggered either due to increased blood transfusions while they're on the device or possibly an immunologic response to the textured surfaces of some devices. And the IgG is the only antibody that can cross the placenta and is responsible for hemolytic disease of a newborn from an RH mismatch between the mother and the fetus. I wanted to point out that I'm a member of the AMSEC, Peds and Congenital Committee and we just finished a project where we're developing pediatric based protocols. And I was on the task force that just completed a therapeutic plasma exchange protocol. And I looked last night, it's been approved by AMSEC and approved by the membership and by AMSEC legal but it isn't yet on the website. So a lot of the information that I'm speaking about today is actually in this protocol if anybody's interested. TPE is an accepted treatment for antibody mediated disorders. The most established indications for perioperative TPE are usually related to immunomodulation during organ transplantation by removing the HLA antibodies or to prevent or to treat humeral rejection. Using immunosuppressive meds and TPE for patients all sensitized pre-transplant increases the likelihood of a successful match. TPE is also useful in the management of intentional and inadvertent ABO incompatible recipients. And it's an essential treatment for hyper acute rejection. The type of phoresis is based on the type of cell or pathologic solutes that are being removed. Cytophoresis refers to selective removal of various formed elements that are listed here on this slide. And although the terms of therapeutic plasma exchange and plasma phoresis are used interchangeably the procedures are clinically different. Plasma phoresis refers to a broad range of procedures resulting in the removal of plasma only. Therapeutic plasma exchange refers to plasma phoresis or the removal of donor plasma and the associated HLA antibodies. And the re-infusion of the remaining ontologous components followed by transfusion of a replacement product in equal volumes back to the donor. For the purposes of this presentation I'll be referring to TPE. So this graph shows that the continual plasma replacement is necessary and results in exponentially decreasing removal efficiency. Because if you think about it during the TPE process you're removing the patient's donor plasma that has the HLA antibodies in it as you remove the concentration, the concentration drops and then towards the end of the procedure you're removing some of the FFP that you've actually re-infused back into the patient. So the HLA antibody levels are lower and you need to process more of the patient's blood volume in order to continue removing the antibodies. So with a 1.0 blood volume exchange you can remove approximately 63% of the antibodies. A 1.5 exchange removes about 78% and the highest rate of removal that I was able to find in the literature that I reviewed talked about removing up to 90% of the HLA antibodies. At Cincinnati Children's these are the exchange volumes that we use for these different classes of patients. Highly sensitized patients get a 1.5 volume exchange, liver freezes at 2.0 and ABO and compatible patients get a 2.5 blood volume exchange. So this is the device that we use at our institution. I'm not promoting the use of this device just due to the time limitations for this talk. I'm gonna just talk about what we do at Cincinnati Children's. The aphoresis circuit is set up in parallel to the bypass circuit and the aphoresis technology uses a computerized process to retrieve whole blood from the patient fractionated into cellular and plasma layers via centrifugation, separate and remove the target layer or component and return the remaining fractions to the patient along with a volume replacement fluid and in this case that replacement fluid is FFP. Aphoresis devices also detect the Buffy code and automatically re-infuse the Buffy code and the contents of the Buffy code which is platelets and white blood cells automatically determined intervals during the process. So intraoperative TPE can be completed in half the time because there's a lower resistance when the aphoresis machine is withdrawing the volume from the venous line on the heart lung machine. And TPE can be, the objective is to complete TPE prior to donor organ reperfusion. And the typical aphoresis inflow blood flow rates to the aphoresis device without bypass are about 30 to 50 mLs per minute for infants and 80 to 100 mLs per minute for adults. And on bypass we can get flow rates and the aphoresis device up to 150 to 200 mLs per minute. So pre-vipass considerations. We place the aphoresis device very close to the heart lung machine. Close enough obviously that you can connect the aphoresis circuit to the heart lung machine. The aphoresis circuit is blood-primed when the circuit volume is greater than 10% of the patient's total blood volume. With the optia the circuit volume is about 185 CCs if they do not use a warmer. If the heat exchanger is needed, it adds an extra 42 mLs for a total prime volume of about 227 mLs. The inlet connection for the aphoresis device is indicated by the blue arrow there. It's a stopcock on a lured connector on the venus line. And then the outlet from the aphoresis device is indicated by the red arrow, which returns it to the top of a lured port on the top of our heart shell venus reservoir. So the TPE technicians may not always have the same sense of urgency as the perfusion team does in the middle of the night for a transplant. And it's easier to coordinate the placement of their device and connecting it to our heart lung machine before we actually go on bypass. They have a lot of stuff that they need to do. They need to check a lot of units of FFP. They need to have their system primed. I like to have them in the OR and set up behind our heart lung machine and actually connected by the time the loading dose of Heparin is given so that there's no delaying once or twice when we were ready to go on bypass, they were still checking blood. And the objective, as I stated earlier, is you wanna get the TPE completed before the cross clamp comes off. And we like to start TPE flow into their aphoresis device within the first five to 10 minutes of initiating bypass. So going on bypass, we initiate in the usual manner. Post initiation, we do blood gases, ACTs, and we also monitor the anticoagulation status by using the HAPCON HMS system. We do that within five to 10 minutes after full stable flow of bypass. We notify the aphoresis team as soon as we know what our post-dilutional hematocrit value is from the first blood gas. And then we open the inlet stopcock on the venous line and the outlet stopcock to the top of our hardshell venous reservoir and they can start their aphoresis flow. And it's important to check for kinks in the aphoresis tubing. It usually happens when on the outflow line from the TPE device coming up to the top of our hardshell and simply just draping it up over something on the mass to support it helps. So post-TPE, we flush the aphoresis prime volume into the pump circuit post-TPE to conserve the RBCs. We didn't realize they were wasting the prime volume of the circuit during the first few cases that we did. And the perfusion documents all the aphoresis prime volume in the CPD INOs. And they also give any unused units of FFP to the perfusionist rather than discarding that as well. So during the three and a half year timeframe that's listed here, we've done 61 total transplants at Cincinnati Children's, 41 of those 61 patients or 67% were sensitized pre-transplant and required TPE. And if you go to the literature, you can see that the estimated incidence of positive PRA titers in pre-transplant is 11 to 15%. And I was initially a little surprised to see that our incidence of TPE was as high as it was, but it matches up very nicely with the MCS patients who are at increased risk for PRA positive titers. And you can tell that the largest percentage of our patients that we've done are VAD X-plant patients at the time of transplant. So anticoagulation is required when you do TPE and citrate anticoagulation is generally used except for liver patients because to prevent citrate toxicity since citrate is metabolized in the liver. And we also do not use citrate anticoagulation for bypass patients. Cause as we all know, they're completely heparinized, anticoagulated with heparin. And the large volume of FFP that's used during the TPE process contains about 15 to 18% citrate per unit and utilizing a citrate anticoagulation in addition to the citrate that's in the FFP would further decrease our ionized calcium levels. Well, we run serial ACTs and HMS heparin concentrations during the entire TPE process. As Jim mentioned on our first day that the new heparin that we use has a higher seeding coefficient and it's actively removed from the circuit. And I've got some examples of that during the case presentation on the final slides. We maintain our ACTs at greater than 480 seconds and our HPT values are at our projected HPT value from the HDR cartridge. So we verify an accurate hematocrit value to the aphorosis team every 10 to 15 minutes during TPE. An accurate hematocrit is important to establish an interface for how the plasma is separated from the whole blood. And if the hematocrit becomes inaccurate for any reason during the course of TPE, we risk spilling the patient's RBCs over into the waste bag. So serial APGs are used to identify any changes in hematocrit from hemidilution, hemoconcentration, washed PRBC, transfusion, et cetera, during bypass. Adequate ionized calcium levels are very important as well. We use calcium chloride as to supplement the chelation of the removal of calcium from the citrate in the FFP. Calcium chloride is set up at a concentration by the aphorosis team at eight milligrams per ML. Calcium gluconate is used, when it is used, is set up at a concentration of 50 milligrams per ML. And calcium gluconate can exacerbate acidosis. So like I said, we use calcium chloride on all of our patients. I instruct the aphorosis team to maintain a calcium, an ionized calcium level between 1.0 and 1.1 during the TPE period. And depending on how fast they're flowing, you can get big fluctuations in the calcium levels. So I wanna be in more control, I guess, is the right word to use to supplement calcium PRN as needed to keep the calcium levels where they need to be. And if you get behind, it's just hard to catch up. So this is an off-label use of calcium chloride, but it's how we calculate it at Cincinnati Children's. And a lot of you may be doing the same thing, but it works very well for pediatric patients up to about 35 kilos or so. And then you can use variations on the same theme. If you take 10 milligrams of calcium chloride per kilogram body weight, that will increase your ionized calcium levels by 0.1 millimoles per liter. So on a five kilogram patient, if you wanna increase the calcium from 1.0 to 1.3, you would give three doses of 50 milligrams each to bump it from 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3. So this is an ABO incompatible heart transplant that I did on December 29th, 2021. The patient was a 72 day old male with pulmonary atresia and intact ventricular septum status post-ASFAD, which was a PDMAG inserted on day of life 14. Patient was 4.81 kilos of BSA of 0.26 meters squared. And the procedure was a PDMAG ASFAD X-plant, a PDA stent removal, PIA reconstruction, ABO incompatible heart transplant with therapeutic plasma exchange. The circuit I used was a 1.25, 3.16 AV loop. The prime volume was 260 mLs. And the HMS data for this patient was skewed because while he was on the PDMAG, he was on bivalve and the HDR information came back with showing a very, very sensitive slope. So I chose to maintain this patient at HPT of 3.4 during the case. Something else that we do on every single, one of our open heart patients is to do continuous delusional ultrafiltration and continuous ultrafiltration. I removed 2.6 liters from this patient. The total volume in was 3,200. The total out was almost 3,400. The fluid balance was negative 175 or negative 36 and a half mLs per kilo. We try to have a negative fluid balance at the end of every procedure, but not more negative than negative 40 per kilo because the literature shows that that can be detrimental to the patients. And then the TPE time was 54.2 minutes. So looking at the data from this case, the heparin doses are the blue columns. The ACT values are the orange columns and they're graduated against the left vertical axis. The heparin protamine titration results values are shown by the gray line and graduated against the right vertical axis. The TPE time is shown by the shaded box, which was 54.2 total minutes. And the cross-plamp removal was indicated by the green arrow. You can see that TPE was completed 98 minutes prior to reperfusion of the donor heart. Because the timeline on this horizontal axis is not linear, I put 30 minute intervals by these dashed red lines so that you could see what samples were run during each timeframe. And typically on bypass, we would run a blood gas and an ACT every 30 minutes. On these TPE cases, I ran an ABG and ACT HPT every eight minutes. And what we typically do is as soon as we go on bypass we'll run a series of samples, get the post delusional hematocrit to the aphoresis team and then run an HPT, ACT and a blood gas, the blood gas to keep track of what your calcium value is and your hematocrit value. And the HPT value is gonna come back in a matter of seconds. And as soon as the ACT reaches 480 seconds, we stop the test and put in new cartridges and run another one. So it takes about eight minutes to get to 480 seconds. And that's how we keep up with things during the course of the case like this. So if you look at the heparin administration on this case specifically, the patient's heparin loading dose was 416 units per kilogram or 2000 units based on the HMS slope. The patient was given an additional 5,600 units of heparin during the TPE period or 2.8 times the heparin loading dose. During the entire period of bypass, the patient was given 10,800 units of heparin including the loading dose or 5.4 times the loading dose which equaled very close to 2,100 units of heparin per kilogram on a 4.8 kilogram child. So that's a whopping dose of heparin. So now looking at the calcium information, the calcium chloride doses are indicated by the orange columns and graduated against the left vertical access and the resulting ionized calcium values are indicated by the blue line and graduated against the right vertical access. TPE time is again shown by the shaded box. Clamp time is shown by the green arrow. 30 minute intervals by the red dotted lines. And our protocol is to keep the ionized calcium levels higher than 0.8 millimoles per liter. The aphoresis team is running their calcium chloride delivery at four times their blood flow. So if it's at 50 mLs per minute, they'll run it at 200 mLs the calcium supplements at 200 mLs per hour, not per minute per hour. So they're delivering approximately 27 milligrams per minute of calcium chloride during the entire course of TPE. In addition to the calcium administration by the aphoresis team to maintain those levels, I gave four boluses of 20 milligrams and one 40 milligram calcium bolus for a total of 120 milligrams of additional calcium to maintain the level at 0.8 millimoles per liter. You can see the ionized calcium level was dropping as TPE continued, even though the calcium supplementation was being done by both the aphoresis team and myself. Then as TPE was completed, three additional boluses of calcium chloride indicated by the yellow circle were given to maintain the level at 1.0. And prior to weaning from bypass, I supplemented 180 milligrams of calcium chloride over four doses to achieve an ionized calcium level of 1.3. We like to get the calcium value up to 1.3 before we wean from bypass because we muff at the end of every case and we continue to remove calcium through the muff process. So by getting the ionized calcium to at least 1.3 before we come off, it usually is always at 1.2 after the muff procedure, which you can tell from this final value. That's where we ended up. So in closing, there seems to be a little agreement between the institutions and how to effectively manage these patients. And the number of sensitized pediatric heart transplant candidates is increasing as our bad technology and care for these patients improves. Patients with preformed antibodies and elevated PRAs have long-term higher incidence of rejection, graph loss, vascular disease, and lower survival rates. TPE during bypass allows for high volume plasma exchanges in a hemodynamically unstable patient that otherwise would not be able to tolerate this procedure. An intraoperative TPE is safe and effective for patients who would otherwise not be transplanted or suffer from high rates of morbidity and mortality post-transplant due to their elevated PRA levels. These are my references. Thank you very much.