The study was financially supported by the South-Eastern Norwegian Health Authority (through grants 2009007 and 2008104).
Address reprint requests to Professor Tor Inge Tønnessen, M.D., Ph.D., Division of Emergencies and Critical Care, Oslo University Hospital, Rikshospitalet, Box 4950 Nydalen, 0424 Oslo, Norway. Telephone: +47-23070000; FAX: +47-23073681; E-mail: firstname.lastname@example.org
The overall complication and retransplantation rates are higher in the pediatric liver transplant population versus the adult liver transplant population. In particular, vascular complications such as hepatic artery thrombosis (HAT; 5%-18%) and portal vein thrombosis (PVT; 5%-10%) occur more frequently in pediatric grafts versus adult grafts and are major contributors to the loss of grafts and patients' lives. Early detection is imperative for enabling interventions before the occurrence of graft necrosis or biliary complications ultimately necessitating retransplantation.[3, 4] Performed by trained radiologists, Doppler ultrasound is a sensitive and specific method for detecting vascular occlusions,[5, 6] but continuous Doppler monitoring with implanted microprobes is currently not available for routine clinical use.[7, 8]
Acute cellular rejection (ACR) is a common complication usually occurring within the first 6 weeks after transplantation with an incidence of 30% to 60%.[1, 2] It is suspected when circulating liver enzymes, bilirubin, or both rise, and although biopsy is not performed routinely at all centers, it is required to confirm the diagnosis. However, particularly during the early stages of rejection, biopsy may be inconclusive and thus may need to be repeated before an antirejection treatment can be initiated. Because children need general anesthesia for the procedure and many patients have a relatively high risk for bleeding,[11, 12] some patients are administered high doses of corticosteroids without a histopathological confirmation of the diagnosis.
Microdialysis catheters are able to sample metabolic substances[13-16] and inflammatory mediators[17, 18] directly from the organ of interest. We have found that ischemia can be detected as increased lactate levels and lactate/pyruvate ratios and that rejection can be detected as increased lactate and pyruvate levels with stable lactate/pyruvate ratios.[19-21] The present study was undertaken because younger patients have more vascular complications and episodes of ACR than older patients.
The aim of this study was to investigate the ability of microdialysis to detect ischemia and rejection in pediatric liver transplants with the optimal cutoff values determined in a previous study. Because the presence of microdialysis catheters theoretically could interfere with the mobilization of patients, we also explored how the catheters were tolerated by children.
PATIENTS AND METHODS
Study Population and Surgical Technique
Between October 2008 and May 2011, 16 patients (11 girls and 5 boys) who underwent 20 liver transplants in all were included. The primary diagnoses for the 16 recipients were biliary atresia (n = 11), cirrhosis of an unknown cause (n = 2), cirrhosis due to an alpha-1-antitrypsin deficiency (n = 1), familial intrahepatic cholestasis (n = 1), and fulminant hepatic failure (n = 1). All grafts were from deceased donors, and 18 of the 20 transplants were performed as split liver transplantation (16 left lateral segments and 2 right lateral segments). Every transplant procedure was performed with the piggyback technique without venovenous bypass, and the portal vein flow was established before perfusion of the hepatic artery. All portal vein anastomoses were performed as end-to-end anastomoses between the donor's and recipient's portal veins. Likewise, 17 of the hepatic artery anastomoses were sutured end to end, whereas anastomoses between the hepatic artery and the aorta were performed in 3 cases. Further recipient, donor, and graft characteristics are listed in Table 1. Eight of the 16 liver transplant recipients in the present study were included in a larger study including 66 liver transplants (58 adults), and 7 were presented in a study of inflammatory parameters, whereas the latter 8 recipients were recruited after the completion of the 2 former studies.
Table 1. Baseline Characteristics of 16 Liver Transplant Recipients and 20 Liver Grafts
For graft recipients who were younger than 12 years.
For graft recipients who were older than 12 years.
Measured intraoperatively with Doppler ultrasound.
The study protocol was approved by the regional ethics committee. Detailed oral and written information was provided, and informed consent forms were signed by relatives before inclusion. Children older than 10 years and adolescents were given individually adjusted oral and written information.
Immunosuppression and Anticoagulant Therapy
Immunosuppression and anticoagulant therapy were administered as part of our routine clinical practice, and these therapies were not part of the study protocol. Immunosuppression was given as a combination of tapering amounts of methylprednisolone/prednisolone (after an initial dose of 10 mg/kg intravenously administered before graft reperfusion), tacrolimus (with a serum level goal of 5-15 μg/L during the first 2 weeks), and mycophenolate mofetil (600 mg/m2 of body surface). Induction therapy was not administered. According to our clinical protocol, low-molecular-weight heparin (LMWH) was administered subcutaneously at a daily dosage of 60 IE/kg until mobilization, and then the patients received acetylsalicylic acid (ASA) at 3 mg/kg (maximum = 75 mg). Antithrombin III concentrations less than 80 IE/L were substituted. However, particularly intraoperatively, individual adjustments, including the administration of unfractionated heparin, were made on the basis of standard blood samples [eg, platelet counts and international normalized ratio (INR)], thromboelastography, and a clinically assessed tendency for bleeding or risk (eg, HAT).
Definition of Clinical Endpoints
With respect to the intrahepatic metabolism as measured by microdialysis, we used the optimal cutoff values determined in our previous study. Ischemia was thus defined as a lactate level > 3.0 mM and a lactate/pyruvate ratio > 20, and rejection was defined as a lactate level > 2.0 mM and a lactate/pyruvate ratio < 20. As in the previous study, the utility of 1, 2, and 3 consecutive hourly measurements for detecting ischemia was explored, and a cutoff of 6 hours was chosen to investigate the utility of repeated measurements for detecting rejection. All episodes of ischemia were confirmed by radiological examinations and/or intraoperative findings, and all episodes of rejection were proven by biopsy. According to our established clinical protocol, which is based on many years of empirical data, indications for performing graft biopsy to diagnose rejection include increases in circulating alanine aminotransferase (ALT) or total bilirubin of 25% or more from one day to the next. All reported bilirubin values are for total bilirubin.
The microdialysis system consisted of a small syringe pump coupled to a double-tubular catheter (CMA 65, M Dialysis AB, Stockholm, Sweden). The catheters had membranes with a 100-kDa pore size, an outer diameter of 0.7 mm, and a length of 30 mm and a secure thread positioned 60 mm from the tip. One catheter was inserted into each liver lobe with a split-needle technique (outer diameter of split introducer = 1 mm). In split transplants, 1 catheter was used. Each catheter entered the abdominal cavity separately through the left side of the abdominal wall to avoid intruding on the access for ultrasound. Postoperatively, the catheters were perfused with a fluid containing dextran and electrolytes (Plasmodex, Meda AB, Stockholm, Sweden) at a velocity of 1 μL/minute via microinjection pumps (CMA 107, M Dialysis AB). Lactate, pyruvate, glucose, and glycerol were analyzed at the bedside every 1 to 2 hours (Iscus, M Dialysis AB) after a patient's arrival at the intensive care unit. The lactate/pyruvate ratio was calculated with the Iscus analyzer. The catheters were kept for as long as they were able to sample the microdialysate and then were pulled out.
Microdialysis parameters are presented as medians and interquartile ranges, and other data are presented as medians and ranges. In 2 cases in which both liver lobes were monitored, values from the catheters that performed satisfactorily for the longest period were chosen for analyses. The sensitivity and the specificity (including the effects of repeated measurements) were calculated with contingency table analyses. For repeated measurements, the Wilcoxon signed-rank test was performed, and for group differences, the Mann-Whitney U test was used. A 2-tailed P value < 0.05 was considered statistically significant. The statistics were performed with PASW 18.0 (IBM, Chicago, IL).
In 6 of the 20 investigated liver grafts, ischemic vascular complications were detected, and biopsy verified ACR in 8 grafts. The characteristics of the recipients and grafts with ischemia and ACR are shown in Table 2. The recipients with ischemic graft complications were significantly younger and had a lower body weight than the recipients who developed ACR, and they had significantly lower intraoperatively measured flow in the hepatic artery. Complications other than ischemia and ACR occurred in 3 grafts: infection (n = 1), biopsy-proven cholestasis (n = 1), and primary nonfunction (PNF; n = 1). One of the 16 recipients died because of ischemic graft failure due to combined HAT and PVT.
Table 2. Characteristics of Liver Graft Recipients and Grafts: Ischemic Complications Versus ACR
In the first samples after the transplant procedure, lactate was elevated, and it decreased to low and stable values after a median of 12 hours (range = 6-22 hours) in circulated and biochemically functioning grafts (n = 15). According to comparisons of samples from grafts before and during rejection with samples collected during ischemia (Fig. 1), lactate was significantly increased during both ischemia and rejection, but it was significantly more increased during ischemia versus rejection. Pyruvate increased only in grafts with ongoing rejection, whereas the lactate/pyruvate ratio increased only in ischemic grafts. Glycerol increased in grafts with severe ischemia (eg, combined HAT and PVT; Fig. 2D), but statistically significant group differences could not be shown. No between-group differences were found for glucose.
Two of the 6 grafts that developed ischemic complications had HAT (Fig. 2A,B), 1 graft had PVT (Fig. 2C), 1 had combined HAT and PVT (Fig. 2D), and 2 had hepatic arteries that were occluded for reasons other than HAT (Fig. 3A,B). When a lactate level > 3.0 mM and a lactate/pyruvate ratio > 20 were used as the criteria, the intrahepatic microdialysis catheters detected ischemia with 100% sensitivity (Table 3). With measurements at only a single time point, the specificity was not more than 57%. With 2 or 3 consecutive positive measurements, the specificity increased to 86%, and the sensitivity remained 100%. When repeated measurements were used as described, only 1 graft with ACR and 1 graft with cholestasis (Fig. 4D) displayed transient episodes of increased lactate levels and lactate/pyruvate ratios that could be misjudged as ischemia; in the graft with ACR, the median value for lactate during a 30-hour period was 3.9 mM (interquartile range = 3.6-4.7 mM), and the respective value for the lactate/pyruvate ratio was 31.4 (interquartile range = 26.6-38.0). The graft with cholestasis displayed a median lactate value of 3.6 mM (interquartile range = 3.5-3.7 mM) and a corresponding lactate/pyruvate ratio of 21.2 (interquartile range = 20.4-21.9) for 16 hours.
Table 3. Contingency Table Analyses Showing How Hourly Repeated Measurements Detected Vascular Occlusion/Ischemia in Liver Transplants
Eight liver graft recipients had histological evidence of ACR (Fig. 4A shows a typical case of rejection). They had rejection activity index (RAI) scores of 4 (n = 5), 5 (n = 2), and 6 (n = 1). The median tacrolimus concentration on the day of biopsy was 5.1 μg/L (range = 2.0-7.2 μg/L). Before biopsy proved ACR, a substantial increase (≥25%) in circulating total bilirubin was observed in 7 cases, and in 5 cases, the activity of circulating ALT increased. In 1 patient, neither ALT nor bilirubin increased; a relatively slow decrease in ALT supported by pathological values in the microdialysate from the liver graft justified the indication for biopsy (RAI = 4) in this particular graft. All rejections were successfully treated with pulses of methylprednisolone.
Rejection was not regarded as a possibility for the graft that was lost because of combined HAT and PVT shortly after graft reperfusion. Thus, 19 grafts were analyzed for possible rejection with the following criteria: a lactate level > 2.0 mM and a lactate/pyruvate ratio < 20 (Table 4). With measurements at only a single time point, the sensitivity was 100%, and the specificity was 0%. With consecutive positive measurements for a period of more than 6 hours, 1 graft that displayed rejection values at just longer than 5 hours was not coded as rejection, and the sensitivity decreased to 88%, but the specificity increased to 45%. In 5 of the 6 grafts in which rejection was incorrectly identified, the clinical picture and context ruled out rejection as a possible diagnosis, and with their exclusion, the specificity was 83%; 2 cases were found during the period of metabolic normalization after an occluded hepatic artery had already been detected, 2 had severe peritonitis and abdominal pain due to leakage from the choledochojejunostomy (Fig. 4B) or a spontaneous perforation of the duodenum, and the last case in which rejection was incorrectly identified involved a graft with PNF (Fig. 4C). In that graft, high values for lactate with normal values for the lactate/pyruvate ratio and high values for glycerol (median = 176 μM) were detected. One episode of histologically proven intracanalicular cholestasis without histological evidence of rejection (RAI = 1) could not be ruled out as possible rejection when the clinical criteria were implemented, and it was thus classified as a true false-positive rejection (Fig. 4D).
Table 4. Contingency Table Analyses Showing How Repeated Measurements of Metabolic Parameters Detected Rejection
Rejection, which was defined as the first episode with lactate values > 2.0 mM and a lactate/pyruvate ratio < 20 lasting for 6 or more hours, occurred at a median of 3.3 days (range = 1.0-6.8 days) after graft reperfusion. This was a median of 4 days (range = 1-7 days) before ALT increased (n = 5, P = 0.11), a median of 4 days (range = 2-9 days) before bilirubin increased (n = 7, P = 0.04), and a median of 6 days (range = 4-11 days) before biopsy was performed (n = 8, P = 0.05).
Rejection was detected by the microdialysis catheters at times when circulating ALT and bilirubin were physiologically declining after high postreperfusion values. In the 5 cases in which intrahepatic lactate increased before ALT, the median peak value for ALT after reperfusion was 365 U/L (range = 55-1005 U/L). When rejection was diagnosed with the microdialysis catheters, ALT had decreased to a median value of 144 U/L (range = 55-273 U/L, P = 0.11). ALT decreased further to a median value of 103 U/L (range = 28-199 U/L, P = 0.11) before it increased to a median value of 159 U/L (range = 116-255 U/L) on the day of biopsy (P = 0.04). During the course of rejection, ALT increased to a median maximum value of 255 U/L (range = 152-459 U/L, P = 0.04 versus the minimum values and P = 0.11 versus the day-of-biopsy values). In the 7 cases in which lactate increased before bilirubin, the median peak value for bilirubin after reperfusion was 77 μM (range = 22-183 μM). When rejection was detected with the microdialysis catheters, bilirubin had decreased to a median value of 47 μM (range = 14-78 μM, P = 0.03). Bilirubin decreased further to a median value of 18 μM (range = 7-72 μM, P = 0.04) before it increased to a median value of 41 μM (range = 10-122 μM) on the day of biopsy (P = 0.03). During the course of rejection, bilirubin increased to a median maximum value of 75 μM (range = 28-219 μM, P = 0.02 versus the minimum values and P = 0.07 versus the day-of-biopsy values).
After the onset of rejection, the microdialysate was sampled for a median of 5 days (range = 2-13 days). In 3 cases, a normalization of the values was observed (ie, lactate level<2.0 mM and lactate/pyruvate ratio < 20). In the other cases, the catheters ceased to sample the microdialysate while the values were still pathologically elevated.
Twenty-two microdialysis catheters were inserted into 20 liver grafts. Two episodes of minor bleeding occurred with the insertion of the splittable needle, and they were easily halted with manual compression and Surgicel. Both these recipients had received unfractionated heparin intraoperatively. They did not receive any platelet inhibitors, but they had relatively low postoperative platelet counts (41 and 48 × 109/L, respectively). Only 2 patients were administered a platelet inhibitor (ASA) intraoperatively, whereas 10 were given unfractionated heparin or LMWH.
At the time of the removal of the catheters, no bleeding was observed. The median platelet count was 129 × 109/L (range = 42-480 × 109/L), and the INR was 1.4 (range = 1.0-2.1) at the time of removal. Eight patients were receiving LMWH, 6 were receiving LMWH and ASA, and 1 was receiving only ASA. No episodes of infection could be related to the catheters.
Microdialysates from livers were sampled for a median of 10 days (range = 1-28 days), and there was no difference in the length of sampling between children who were 2 years or younger and children who were older than 2 years. The parents of 2 patients who were 2 and 7 years old asked for withdrawal of the catheters after 12 and 28 days, respectively, because they perceived the many measurements to be a strain to the children, who were fully mobilized and doing well. All other patients tolerated and kept their catheters until they were removed because of malfunctioning (n = 13), retransplantation (n = 4), or relaparotomy (n = 1). No patients were immobilized because of the catheters.
The present study suggests that through the use of the microdialysis system with predefined criteria for pathological conditions (for ischemia, lactate level > 3.0 mM and lactate/pyruvate ratio > 20; for rejection, lactate level > 2.0 mM and lactate/pyruvate ratio < 20), vascular occlusions in particular may be detected with clinically acceptable levels of sensitivity and specificity earlier in comparison with conventional methods. Especially in children, who constitute a vulnerable patient group, the ability to detect ischemic vascular complications at an early time point is important for improving outcomes.[3, 4] This study also shows that smaller patients are more susceptible to vascular complications than older patients (Table 2). Thus, we think that the microdialysis system may be an important supplemental monitoring tool to use with routine Doppler ultrasound examinations, which are typically performed twice daily.[5, 6] The main advantage would be a close-to-real-time alert for ischemia, and the high sensitivity and specificity with which ischemia was detected suggest that it may also be a qualitatively important supplement to standard Doppler examinations; it has also been shown in this study (Fig. 2B) that a Doppler ultrasound examination performed without a contrast medium can fail to detect a hepatic artery occlusion.[23, 24] The episode of an occluded artery with stable arterial lactate values but a vast elevation of intrahepatic lactate (Fig. 2A) underlines the benefits of intraorgan monitoring in comparison with just measurements in blood.
In contrast to the anaerobic metabolism with increases in the lactate level and lactate/pyruvate ratio detected with ischemia, ACR was detected as simultaneously increased lactate and pyruvate levels with a stable lactate/pyruvate ratio. This phenomenon is physiologically consistent with hypermetabolism due to increased aerobic glycolysis by activated lymphocytes.[20, 25] In comparison with the current standard of care, according to which biopsy is performed after an elevation of liver enzymes and/or bilirubin, microdialysis might represent an opportunity to perform biopsy according to a justified indication and at an earlier time. Whether earlier initiated antirejection therapy would affect the long-term outcome is equivocal.[10, 26] Nevertheless, we consider early antirejection treatment important. It is well known that hepatocytes are able to regenerate. However, hepatocytes may not be the main target in rejection. Sinusoidal endothelial cells, bile ducts, and small intrahepatic arteries are also affected and do not have the same ability as hepatocytes to regenerate. Thus, it is conceivable that the timing of antirejection treatment may have an impact on long-term outcomes.
We consider high sensitivity to be the most important property of a continuous and real-time bedside monitoring tool. Indeed, the sensitivity in detecting rejection would have been 100% (instead of 88%) if we had required consecutive positive measurements for 5 hours (instead of 6 hours or longer). The specificity in detecting rejection only through monitoring metabolism with a detection limit of consecutive positive measurements for more than 6 hours was only 45%. However, when the clinical context was considered, the specificity was 83%. Although it is theoretically possible, we assume rejection occurring simultaneously with peritonitis or severe PNF to be unlikely. Furthermore, according to our experience with intrahepatic metabolism during rejection, we also assume that lactate would display an increasing tendency rather than a decreasing tendency if rejection should occur in the period after revascularization of the hepatic artery. Accordingly, in the presented series of 20 liver transplants, we consider the graft with histological evidence of canalicular cholestasis (Fig. 4D) to be the only case of a true false-positive rejection. Cholestasis with an intrahepatic bile duct pressure exceeding the pressure in the capillaries might explain the metabolic condition. Bilirubin's powerful uncoupling effect on oxidative phosphorylation may be another explanation.[29-32] The patient was administered methylprednisolone intravenously at 10 mg/kg and subsequently recovered, and thus we consider underlying immunological mechanisms to be possible although not revealed by the histopathological examination of the biopsy sample (RAI = 1). All in all, the present study suggests that the specificity with which metabolic mediators detect rejection is restricted. We have recently shown that chemokine (C-X-C motif) ligand 10 (CXCL10) is increased in liver grafts with rejection, and this potentially specific marker will be a subject of investigation in ongoing and future studies.
In the graft with an intrahepatic fungal infection (Fig. 4B), microdialysis revealed an increased metabolism similar to the metabolism observed with rejection. A biological explanation for the hypermetabolism observed with an infection may be an increased demand for energy due to the activation of neutrophil granulocytes (ie, a mechanism similar to that associated with activated lymphocytes during rejection). Additionally, lactate is also released by the oxidative burst mechanism involved in bacterial killing. Accordingly, in addition to cholestasis, infection should be considered a differential diagnosis to rejection in cases in which the microdialysate reveals hypermetabolism. Interestingly, no intrahepatic changes were found in a case of severe peritonitis, and this suggests that the liver graft was not infected in this case. An additional intraperitoneal microdialysis catheter would probably have detected the case of peritonitis,[34-36] and studies aimed at exploring the clinical utility of monitoring vulnerable groups of postoperative immunosuppressed patients with, for example, microdialysis catheters positioned between the loops of the small intestine are ongoing.
When liver biopsy is contraindicated (eg, coexisting coagulopathy or anticoagulant therapy),[11, 12] the additional parameters provided by microdialysis may be of particular value. Heparin and LMWH have previously been demonstrated to be associated with an increased risk of bleeding complications after liver biopsy.[12, 37] Seventy-five percent of the presented recipients were administered LMWH and/or ASA and thus had relative contraindications to graft biopsy. None of these patients had any bleeding complications related to the microdialysis catheters.
One limitation of microdialysis is the limited lifespan of the catheters due to clotting of the pores with cellular debris and proteins.[18, 38] Thus, the method's ability to monitor the effects of antirejection treatment, for example, is restricted. Techniques for percutaneous catheter insertion are not developed, and malfunctioning catheters are not easily replaced by new ones. Another potential limitation of the technique is that the catheters may delay the mobilization of patients and especially young children. Our data indicated, however, that both children who were 2 years and younger and children who were older than 2 years tolerated the catheters, and the feasibility was good. The catheters were not associated with serious complications.
This report is first of all limited by the restricted number of investigated liver transplants and should be considered a preliminary report representing a very small patient population. Our approach needs to be addressed in a larger number of children and at more than 1 center in order to determine whether the method is as valuable as the present data and earlier studies[16, 19, 20] suggest. Unfortunately, we were not able to analyze for CXCL10 and other inflammatory substances in this material. As in our previous reports, we were not able to use the microdialysis method to observe the effects of treatment for all patients, most likely because of the limited lifespan of the catheters. Because the study was observational and, therefore, was not able to show clinical benefits, we chose to terminate the study in its current form despite the very small number of patients. We had clear indications that the open-label approach was increasingly changing our clinical approach without allowing us to evaluate the potential clinical benefits of close intraorgan monitoring. It is unlikely that biopsy would have been performed and rejection diagnosed in the case of a normal course of transaminases and bilirubin if the graft had not been monitored with microdialysis. Because of the very high sensitivity in detecting ischemia, less severe complications such as hepatic artery stenosis and small graft infarctions are likely to be detected more frequently in centers using the microdialysis method in comparison with others. However, this is not likely to be the case in the present study. A new study will be carried out with a decision tree–based study protocol to investigate the clinical impacts of including the microdialysis data in clinical treatment decisions for pediatric liver transplants.
When one considers the implementation of our microdialysis approach in other institutions, it should be noted that in Europe, the catheters are Conformité Européenne–marked for a wide range of organs, including the liver. In the United States, however, brain monitoring is the only indication approved by the Food and Drug Administration. Accordingly, the monitoring of liver transplants in the United States at this stage should be done in a clinical research setting approved by an institutional review board. All equipment used in the present study is commercially available.
In conclusion, ischemic vascular complications and rejection were detected in close to real time with highly satisfactory levels of sensitivity. The specificity for ischemia was acceptable; however, the restricted specificity for rejection emphasizes the need for additional intrahepatic markers. On the basis of the findings from our group, the potential role of CXCL10 will be explored in future studies, and studies aimed at proving potential clinical benefits are under way.