ABH-Glycan Microarray Characterizes ABO Subtype Antibodies: Fine Specificity of Immune Tolerance After ABO-Incompatible Transplantation.

Organ transplantation from ABO blood group-incompatible (ABOi) donors requires accurate detection, effective removal and subsequent surveillance of antidonor antibodies. Because ABH antigen subtypes are expressed differently in various cells and organs, measurement of antibodies specific for the antigen subtypes in the graft is essential. Erythrocyte agglutination, the century-old assay used clinically, does not discriminate subtype-specific ABO antibodies and provides limited information on antibody isotypes. We designed and created an ABO-glycan microarray and demonstrated the precise assessment of both the presence and, importantly, the absence of donor-specific antibodies in an international study of pediatric heart transplant patients. Specific IgM, IgG, and IgA isotype antibodies to nonself ABH subtypes were detected in control participants and recipients of ABO-compatible transplants. Conversely, in children who received ABOi transplants, antibodies specific for A subtype II and/or B subtype II antigens-the only ABH antigen subtypes expressed in heart tissue-were absent, demonstrating the fine specificity of B cell tolerance to donor/graft blood group antigens. In contrast to the hemagglutination assay, the ABO-glycan microarray allows detailed characterization of donor-specific antibodies necessary for effective transplant management, representing a major step forward in precise ABO antibody detection.

Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells.

Regulatory T cell (Treg)-based therapy is a promising approach to treat many immune-mediated disorders such as autoimmune diseases, organ transplant rejection, and graft-versus-host disease (GVHD). Challenges to successful clinical implementation of adoptive Treg therapy include difficulties isolating homogeneous cell populations and developing expansion protocols that result in adequate numbers of cells that remain stable, even under inflammatory conditions. We investigated the potential of discarded human thymuses, routinely removed during pediatric cardiac surgery, to be used as a novel source of therapeutic Tregs. Here, we show that large numbers of FOXP3(+) Tregs can be isolated and expanded from a single thymus. Expanded thymic Tregs had stable FOXP3 expression and long telomeres, and suppressed proliferation and cytokine production of activated allogeneic T cells in vitro. Moreover, expanded thymic Tregs delayed development of xenogeneic GVHD in vivo more effectively than expanded Tregs isolated based on CD25 expression from peripheral blood. Importantly, in contrast to expanded blood Tregs, expanded thymic Tregs remained stable under inflammatory conditions. Our results demonstrate that discarded pediatric thymuses are an excellent source of therapeutic Tregs, having the potential to overcome limitations currently hindering the use of Tregs derived from peripheral or cord blood.

Successful Semi-Ambulatory Veno-Arterial Extracorporeal Membrane Oxygenation Bridge to Heart-Lung Transplantation in a Very Small Child.

Lung transplantation (LTx) may be denied for children on extracorporeal membrane oxygenation (ECMO) due to high risk of cerebral hemorrhage. Rarely has successful LTx been reported in children over 10 years of age receiving awake or ambulatory veno-venous ECMO. LTx following support with ambulatory veno-arterial ECMO (VA ECMO) in children has never been reported to our knowledge. We present the case of a 4-year-old, 12-kg child with heritable pulmonary artery hypertension and refractory right ventricular failure. She was successfully bridged to heart-lung transplantation (HLTx) using ambulatory VA ECMO. Initial resuscitation with standard VA ECMO was converted to an ambulatory circuit using Berlin heart cannulae. She was extubated and ambulating around her bed while on VA ECMO for 40 days. She received an HLTx from an oversized marginal lung donor. Despite a cardiac arrest and Grade 3 primary graft dysfunction, she made a full recovery without neurological deficits. She achieved 104% force expiratory volume in 1 s 33 months post-HLTx. Ambulatory VA ECMO may be a useful strategy to bridge very young children to LTx or HLTx. Patient tailored ECMO cannulation, minimization of hemorrhage, and thrombosis risks while on ECMO contributed to a successful HLTx in our patient.

Real-time 3-dimensional echocardiography provides new insight into mechanisms of tricuspid valve regurgitation in patients with hypoplastic left heart syndrome.

BACKGROUND: Tricuspid regurgitation in hypoplastic left heart syndrome has an impact on outcome, but its mechanisms remain unclear. METHODS AND RESULTS: Real-time 3-dimensional echocardiography was performed in 35 patients with hypoplastic left heart syndrome (age, 1 month to 10 years; 10 after first-stage Norwood, 12 after superior cavopulmonary shunt, 13 after Fontan). From the 3-dimensional data set, we marked the annulus in systole and diastole. At mid systole, we marked the location of the papillary muscle tip and point of chordal attachment to the leaflet. We traced the surfaces of the tricuspid valve leaflets and measured the volume of leaflet prolapse, tethering, annular and septal leaflet areas, and papillary muscle position. Seventeen patients had moderate tricuspid regurgitation (prolapse, 7; tethered leaflets, 7) and 18 mild (prolapse, 0; tethered leaflets, 7). Multiple linear regression analysis revealed that moderate tricuspid regurgitation is associated with leaflet tethering and prolapse; that in hypoplastic left heart syndrome with tethered leaflets, the papillary muscle is displaced laterally and the tricuspid annulus is more planar; and that enlargement of the annulus at mid systole, small septal leaflet area, and age affect the degree of prolapse. CONCLUSIONS: In hypoplastic left heart syndrome, moderate tricuspid regurgitation may be associated with increasing age, geometrical changes of the annulus, leaflet prolapse, lateral papillary muscle displacement, and subsequent leaflet tethering, as well as a smaller septal leaflet.

Management of infants with idiopathic dilatation of the right atrium and atrial tachycardia.

Idiopathic dilatation of the right atrium (IDRA) is a rare anomaly defined as isolated enlargement of the right atrium in the absence of other cardiac lesions known to cause right atrial dilatation. IDRA is a congenital anomaly with unknown pathogenesis and highly variable clinical presentation. Optimal management of severe IDRA is controversial and individualized. Literature reports of long-term follow-up have been limited. We describe a child with IDRA with rapid atrial tachycardia (AT) refractory to both medical and surgical management, and we provide long-term follow-up on our two previously reported cases, both of whom had documented AT. For infants with AT, the clinical course is unpredictable, and medical therapy is the first line of treatment. The decision to proceed with surgical resection of a giant right atrium should be made on an individual basis. Atrial resection along with a modified right atrial MAZE procedure could be considered in infants with life-threatening atrial tachyarrhythmia refractory to medical treatment. Surgical scarring of the right atrium may produce substrate for atrial arrhythmia, which may also be refractory to medical therapy. Histological examination of excised atrial tissue remains inconsistent and not contributory to the determination of the etiology of IDRA. Our three infants with IDRA illustrate unique features of their variable clinical courses, as well as continued difficulties with establishing clear guidelines with regard to surgical management of this unusual disorder.

Fetal cardiac intervention and surgery.

Fetal echocardiography has changed our understanding of congenital heart disease by allowing us to diagnose and observe the malformed human heart within weeks of primary morphogenesis. Serial echocardiographic studies have shown that some complex heart malformations result from relatively simple primary lesions that occur early during heart development and may be amenable to an in utero intervention aimed at altering abnormal growth patterns. Although fetal cardiac intervention or surgery does not presently exist as a realistic therapeutic option in the management of critical congenital heart disease, progress in several areas of investigation give merit to the concept and future potential of in utero cardiac repair.

pH paradox and neonatal heart.

BACKGROUND: To evaluate the functional status of the Na+/H+ exchanger in the neonatal heart. METHODS: On the Langendorff system, isolated neonatal rabbit hearts were arrested by using cardioplegia with or without a specific Na+/H+ exchanger blocker, 5-(N,N dimethyl) amiloride (DMA) (20 microM). Ischemic period was 40 minutes at 37 degrees C or 120 minutes at 20 degrees C before 30 minutes of reperfusion at 37 degrees C. When DMA was added to the cardioplegia solution, it was also added to the reperfusate for the first 5 minutes of reperfusion (20 microM). RESULTS: Postischemic developed pressure was 50.3+/-7.1 mmHg in the DMA group versus 25.9+/-6 mmHg in the control group (p<0.05) at 37 degrees C and 74.8+/-14.6 mmHg in the DMA group versus 60.6+/-11.5 mmHg in the control group (p<0.05) at 20 degrees C. Postischeimic diastolic pressure was 40.4+/-3.3 mmHg in the DMA group versus 28.4+/-7 mmHg in the control group (p<0.05) at 37 degrees C and 9.6+/-3.1 mmHg in the DMA group versus 15+/-3.7 in the control group (p<0.05) at 20 degrees C. Creatine kinase washout was 296+/-97 IU/L in the DMA group versus 1253+/-537 IU/L in the control group (p<0.05) at 37 degrees C and 370+/-156 IU/L in the DMA group versus 524+/-104 IU/L in the control group (p<0.05) at 20 degrees C. CONCLUSIONS: 1) The Na+/H+ exchanger is active in the neonatal heart. 2) The Na+/H+ exchanger plays a key-role in the pathogenesis of reperfusion injury of the neonatal myocardium. 3) This exchanger is sensitive even for low H+ transmembrane gradients and even under hypothermic conditions.

ABO-incompatible heart transplantation in infants.

BACKGROUND: Transplantation of hearts from ABO-incompatible donors is contraindicated because of the risk of hyperacute rejection mediated by preformed antibodies in the recipient to blood-group antigens of the donor. This contraindication may not apply to newborn infants, who do not yet produce antibodies to T-cell-independent antigens, including the major blood-group antigens. METHODS: We studied 10 infants 4 hours to 14 months old (median, 2 months) who had congenital heart disease or cardiomyopathy and who received heart transplants from donors of incompatible blood type between 1996 and 2000. Serum isohemagglutinin titers were measured before and after transplantation. Plasma exchange was performed during cardiopulmonary bypass; no other procedures for the removal of antibodies were used. Standard immunosuppressive therapy was given, and rejection was monitored by means of endomyocardial biopsy. The results were compared with those in 10 infants who received heart transplants from ABO-compatible donors. RESULTS: The overall survival rate among the 10 recipients with ABO-incompatible donors was 80 percent, with 2 early deaths due to causes presumed to be unrelated to ABO incompatibility. The duration of follow-up ranged from 11 months to 4.6 years. Two infants had serum antibodies to antigens of the donor's blood group before transplantation. No hyperacute rejection occurred; mild humoral rejection was noted at autopsy in one of the infants with antibodies. No morbidity attributable to ABO incompatibility has been observed. Despite the eventual development of antibodies to antigens of the donor's blood group in two infants, no damage to the graft has occurred. Because of the use of ABO-incompatible donors, the mortality rate among infants on the waiting list declined from 58 percent to 7 percent. CONCLUSIONS: ABO-incompatible heart transplantation can be performed safely during infancy before the onset of isohemagglutinin production; this technique thus contributes to a marked reduction in mortality among infants on the waiting list.

Voltage-gated potassium channels in human ductus arteriosus.

We studied tone in the human ductus arteriosus and show that the constriction to oxygen is due to inhibition of voltage-gated potassium channels and, in the acute phase, is independent of endothelin-1.

[Role of the Na+/H+ pump in heart ischemia-reperfusion lesions in the neonatal period]. / Implications de l'échangeur Na+/H+ dans les lésions d'ischémie reperfusion du coeur en période néonatale.

OBJECTIVE: Amiloride 5-(N,N-dimethyl)(AMD), an Na+/H+ pump inhibitor, was used to evaluate the maturity and sensitivity of the Na+/H+ pump in neonatal myocardial cells. METHOD: Using the isolated contraction isometric heart model, the hearts of neonatal rabbits (10-12 days) were stopped by cardioplegia with and without AMD (20 mumoles/l), then submitted to 40 minutes of ischaemia at 37 degrees C or 120 minutes of ischaemia at 20 degrees C before being reperfused for 30 minutes at 37 degrees C. When AMD was added to the cardioplegic solution, it was also added (20 mumoles/l) to the Krebs solution for the first five minutes of reperfusion. Functional assessment of myocardial contractility and relaxation was performed before aortic clamping and after 30 minutes of reperfusion. Coronary venous drainage during reperfusion was collected to evaluate CPK levels. RESULTS: To 37 degrees C or 20 degrees C, Na+/H+ pump inhibition was marked by improvement of all myocardial contractility and relaxation parameters at the end of the reperfusion period. A significantly reduced CPK release wase also observed during reperfusion associated with Na+/H+ pump inhibition. CONCLUSION: This study demonstrates maturation of the Na+/H+ pump in neonatal myocardial cells and confirms the role of this ion exchange pump in the pathogenesis of ischaemia-reperfusion lesions. The study at 20 degrees C demonstrated the sensitivity of the Na+/H+ pump to a low transmembrane proton gradient.

Pyruvate augments mechanical function via activation of the pyruvate dehydrogenase complex in reperfused ischemic immature rabbit hearts.

BACKGROUND: Reperfusion of ischemic adult hearts is associated with increased fatty acid oxidation, reduced pyruvate oxidation, and reduced pyruvate dehydrogenase (PDH) activity, leading to a decrease in cardiac efficiency. These effects may be amplified in newborn hearts because of the immaturity of their PDH pathway. We hypothesize that pyruvate can augment mechanical function in the immature heart by activating the PDH complex (PDC) during reperfusion in severely ischemic hearts. MATERIALS AND METHODS: Seven-day old isolated working rabbit hearts (n = 12) were perfused with modified Krebs solution containing 0.4 mM palmitate. Pyruvate (5 mM) was added for a 10-min period either before or after a 30-min period of normothermic global ischemia. Cardiac functional indices before global ischemia and during reperfusion were correlated with active and total PDC activity measured in 28 additional hearts frozen at the various time points throughout the perfusion protocol. RESULTS: Addition of pyruvate before ischemia increased the proportion of active PDC but did not affect any measured functional indices. During early reperfusion, aortic flow, cardiac output, and cardiac work were all significantly depressed compared to preischemic values. Addition of pyruvate significantly increased the proportion of active PDC and was also associated with a significant increase in aortic flow, cardiac work, and developed pressure. Removal of pyruvate from the perfusate resulted in a subsequent significant decrease in PDC activity and these functional parameters. CONCLUSION: During reperfusion of neonatal rabbit hearts, addition of pyruvate improves cardiac performance in association with activation of PDC.

Role of nucleoside transport and purine release in a rabbit model of myocardial stunning.

Previously, we have demonstrated the role of nucleoside transport and purine release in post-ischemic reperfusion injury (myocardial stunning) in several canine models of ischemia. Since rabbits are deficient of xanthine oxidase, it is not known whether selective blockade of purine release is beneficial in a rabbit model of coronary artery occlusion and reperfusion (stunning). Therefore, we determined the hemodynamic and metabolic correlates in response to myocardial stunning in the presence or absence of selective nucleoside transport blocker (p-nitrobenzylthioinosine, NBMPR) and adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA). Sixty adult anaesthetized rabbits were surgically prepared for hemodynamic measurements. After stabilization period, the left anterior descending coronary artery was occluded for 15 min and reperfused for 30 min. Transmural myocardial biopsies were obtained from the ischemic LAD area and from the non-ischemic posterior (circumflex, CFX) segment of the myocardium. Rabbits (n = 60) were randomly assigned to either the control or the EHNA/NBMPR-treated group (n = 30 each). Each group was further divided to either functional or metabolic groups (n = 15 each subgroup). Each animal received intravenously 30 ml of either a vehicle solution or 100 M EHNA and 25 M NBMPR 10 min before ischemia. Although administration of EHNA/NBMPR did not affect the heart rate, it did cause mild hypotension (about 20-30%). Fifteen minutes of LAD occlusion resulted in significant ATP depletion and concomitant accumulation of nucleosides in both groups (p < 0.05 vs. baseline and non-ischemic CFX segment). AMP was higher in the LAD compared to the CFX segment. Significant accumulation of adenosine was observed in the treated group compared to the control group. It is concluded that EHNA/NBMPR induced site specific entrapment of adenosine of nucleoside transport in the rabbit heart, in vivo.

Complete atrioventricular septal defects: results of repair, risk factors, and freedom from reoperation.

BACKGROUND: Several perioperative risk factors influence outcomes after repair of complete atrioventricular septal defects. This study was conducted to determine their association with perioperative mortality and need for reoperation. METHOD AND RESULTS: Between July 1982 and February 1995, 363 children underwent defect repair (median age, 8.3 months; mean, 17.4+/-1.3). Tetralogy of Fallot was present in 21 patients, double-outlet right ventricle in 4, subaortic stenosis in 8, ventricular hypoplasia in 8, coarctation in 5, atrial isomerism in 2, and other congenital anomalies in 9. Down's syndrome was present in 235 (65%). One-patch technique was applied in 99, two-patch in 243. During repair, the anterior bridging leaflet was divided in 12, the posterior bridging leaflet in 31, both leaflets in 71, and neither in 249. Left atrioventricular valve (LAVV) cleft was closed partially in 181 and completely in 112. Early mortality was 10.5%; 10-year survival, 83% (95% confidence interval, 0.79 to 0.87). At 10 years, freedom from reoperation for LAVV repair was 86%; LAVV replacement, 90%; subaortic stenosis, 95%; residual ventricular septal defect, 97%; permanent pacemaker insertion, 98%; and other types of reoperation, 95%. At the time of operation, greater age, shorter ischemic time, absence of a double-orifice LAVV, and cleft closure were found to be significant independent predictors of survival. CONCLUSIONS: Repair of atrioventricular septal defects has acceptable early and late mortality and a low incidence of reoperation. Double-orifice LAVV remains a risk factor. Repairs that include complete cleft closure may confer better survival.

Pulmonary valve replacement late after repair of tetralogy of Fallot.

BACKGROUND: Pulmonary valve incompetence is usually well tolerated after tetralogy of Fallot repair but may result in late progressive right heart failure as manifested by increasing fatigue, dyspnea, and frequently arrhythmias. METHODS: All patients who underwent pulmonary valve replacement in our center late after repair of tetralogy of Fallot were reviewed. RESULTS: Eighty-five patients had elective pulmonary valve replacement late (median, 9.3 years) after repair. Operative risk was low (1.1%). Ninety percent of survivors are in New York Heart Association class I. Survival 10 years after pulmonary valve replacement is 95%, with 86% of the patients free of reoperation for valve failure. CONCLUSIONS: Pulmonary valve replacement is infrequently required after repair of tetralogy of Fallot. Pulmonary valve replacement may be performed electively with little risk; it improves symptoms of right heart failure and provides satisfactory long-term survival with low risk of early valve failure. As the population of patients who have had repair of tetralogy of Fallot ages, pulmonary valve replacement will become a more frequent consideration.

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes.

Cardiac surgery is usually performed under conditions of cardioplegic ischemic arrest. To protect the heart during the ischemic period, the myocardium is exposed to varying degrees of hypothermia. Although hyperthermia is known to induce the heat shock response, the molecular effects of hypothermia on the myocardium have not been investigated. We have studied the effect of hypothermia on the induction of heat shock proteins in primary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytes induced a 6 fold increase in the heat shock protein HSP70 as compared to control. Increased HSP70 protein levels correlated with induction of HSP70 mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recovery from cold shock, indicating the transient nature of the response. Induction of HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even though increased HSP25 protein levels were not detected. Our results indicate that hypothermia is capable of inducing the heat shock response in neonatal cardiomyocytes.

Hemodynamic alteration by fetal surgery accelerates myocyte proliferation in fetal guinea pig hearts.

BACKGROUND: Fetal heart development occurs by hyperplasia as myocytes lose the capacity to proliferate at birth. This potential for cell division may have application in altering fetal growth patterns in congenital cardiac malformations, but it is not known whether the proliferative activity can be modified by intrauterine surgical manipulation. The purpose of this study was to determine whether hemodynamic alteration by fetal surgery influences myocyte proliferation and myocardial development. METHODS: Six pregnant guinea pigs of 50 to 52 days of gestation (term, 65 days) underwent hysterotomy, and the fetal ascending aorta was banded and narrowed by 50% (AoB). Cesarean section was performed near term, and the heart was assessed for myocyte proliferative activity (Ki-67 monoclonal antibody), apoptosis, and morphologic features. RESULTS: The heart to body weight ratio (1.02% +/- 0.12% versus 0.42% +/- 0.02%, p < 0.01) and left ventricular posterior wall thickness (1.89 +/- 0.25 mm versus 1.31 +/- 0.19 mm, p < 0.01) were significantly higher in the AoB group. The percentage of Ki-67 positive cells was increased in AoB group (29.5% +/- 4.4% versus 15.3% +/- 1.3% in right ventricle, 35.8% +/- 5.1% versus 13.1% +/- 1.7% in interventricular septum, and 39.8% +/- 3.2% versus 12.0% +/- 2.0% in left ventricle (p < 0.01). The apoptotic cell to myocyte ratio was less than 1/1000 in both groups. CONCLUSIONS: Fetal hemodynamic alteration by aortic banding accelerates myocardial cellular proliferation without affecting apoptosis, suggesting that in utero cardiac interventions have a greater influence on myocardial development compared with postnatal intervention.

Reducing the deleterious effects of intrauterine CO2 during fetoscopic surgery.

The fetoscopic approach to fetal intervention is a promising minimally invasive technique for correcting congenital anomalies in utero. However, expansion of the amniotic cavity with CO2 to visualize the fetus causes fetal hypercarbia and acidosis. We assessed whether maternal hyperventilation during intrauterine CO2 insufflation could attenuate the fetal hypercarbic acidosis. Seven fetal lambs of 105 +/- 2 days (mean +/- SEM) gestation (term = 145 days) were instrumented with a carotid arterial catheter in utero. After 7 +/- 1 days of recovery, fetoscopic exposure was obtained with intrauterine insufflation of CO2 at 10 mmHg of intraamniotic pressure. After 30 min, the ewe was hyperventilated at a mean respiratory rate of 23/min for 30 min under continuous insufflation. The uterus was then deflated and following 1 hr of stabilization, and the same protocol of CO2 pneumometrium was repeated. Fetal and maternal arterial blood was sampled at baseline and at 15 min intervals. Fetal PaCO2 increased during 30 min of CO2 insufflation (50.8 +/- 2.8 vs. 72.3 +/- 5.0 mmHg, P < 0.01); however, this change was reversed (to 51.5 +/- 3.0 mmHg, P < 0.01) by 30 min of maternal hyperventilation. The fetus developed acidosis after 30 min of CO2 pneumometrium (pH 7.350 +/- 0.012 vs. 7.236 +/- 0.026, P < 0.01); this was also reversed (to 7.366 +/- 0.019, P < 0.01) by maternal hyperventilation. These results were reproducible during the second CO2 insufflation challenge. Fetal hypercarbic acidosis during fetoscopy with CO2 insufflation is reduced by maternal hyperventilation.

Pulmonary atresia with intact ventricular septum: results of the Fontan procedure.

BACKGROUND: Children with pulmonary atresia and an intact ventricular septum show a heterogeneous spectrum of cardiac anomalies. A biventricular repair is attainable in some; a Fontan procedure or a one-and-a-half ventricle is the only possible repair for others. Children with right ventricle-to-coronary artery connections, with or without right ventricle-dependent coronary artery blood flow, are a high-risk group. METHODS: Between May 1980 and December 1994, 22 children underwent a Fontan operation for the treatment of pulmonary atresia with an intact ventricular septum at The Hospital for Sick Children, Toronto. The mean age was 5.8 years (median, 4.9 years). All children had had at least one pre-Fontan palliative procedure; 19 had two, and 7 of these had three or more. Right ventricle-to-coronary artery connections were present in 15 children, including 5 with right ventricle-dependent coronary artery blood flow. Thromboexclusion of the right ventricle was done in 10 children, with 7 undergoing it before and 3 at the time of the Fontan procedure. RESULTS: There were three early deaths (13.6%) and one late death. The actuarial survival at 10 years after the Fontan operation was 80%. Early postoperative complications occurred in 4 children. Follow-up was completed in all children at a mean of 4 years (range, 1 to 12.5 years) after the Fontan operation. Atrial arrhythmia occurred in 3 children, and permanent pacemakers were required in 4. CONCLUSIONS: Results of the Fontan operation for the treatment of pulmonary atresia with an intact ventricular septum are satisfactory. Thromboexclusion of the right ventricle is indicated in the presence of right ventricle-to-coronary artery connections without right ventricle-dependent coronary artery blood flow. The right ventricle should not be decompressed or thromboexcluded in children with right ventricle-dependent coronary artery blood flow, and at the Fontan operation, saturated blood must enter the right ventricle.