Domino liver transplants for metabolic disorders: experience with familial amyloidotic polyneuropathy

Background: Shortage of liver donors means that new methods of liver procurement must be explored. In domino transplantation, organs explanted during transplantation in one patient are transplanted into a second patient. Domino procedures can be performed with livers from patients having transplantion for hepatic metabolic disorders that cause systemic disease without affecting other liver functions. Familial amyloidotic polyneuropathy (FAP) type I is one of these.

Study Design: We reviewed the Paul Brousse experience with a domino liver transplant program for FAP, hoping to extend the approach to other metabolic disorders.

Results: Livers from 10 patients transplanted for FAP type 1 were used for domino transplants to patients with unresectable primary or metastatic liver cancers. There was no perioperative mortality. Neuropathy or cardiomyopathy did not increase the morbidity of the domino liver explant and transplant procedures. Morbidity for the domino recipients did not appear to be increased. Variant transthyretin was detected in the serum in FAP liver recipients, with no immediate clinical consequences.

Conclusions: The domino approach is feasible and requires careful planning of the surgical procedures for liver explantation, particularly for the nature and site of vascular anastomoses. Domino transplantation of metabolically dysfunctional livers creates new categories of potential donors and potential recipients. It raises new ethical, technical, and societal issues. The domino approach could be used in several genetic or biochemical disorders now treated by liver transplantation. It has the potential to increase the number of liver grafts available for transplantation.     

Domino liver transplantation in living donors

Domino liver transplantation (DLT) has been developed as a method to expand the donor pool. In living donors DLT, the prime concern is to avoid any disadvantage to the donor and the first recipient. Seven DLTs were performed among 211 patients who underwent living donor liver transplantation. The domino recipients included six with hepatocellular carcinoma and one with citrullinemia. The domino grafts were obtained from patients with familial amyloid polyneuropathy (FAP) including the left liver in three cases and the right liver in four. Among the seven domino recipients, a 64-year-old woman with advanced hepatocellular carcinoma died of lung metastasis. The other six domino recipients are alive without FAP symptoms. In living donor liver transplantation, because the vessels of the graft from the first donor are not long enough for anastomosis, the hepatic vessels must be left as long as possible when removing the liver from the FAP patients in order to ensure sufficient safety for vascular reconstruction. With careful decision making during the procedure, such as where to divide the vessels in the FAP patients, DLT may help address the shortage of liver grafts.     

Operative Risks of Domino Liver Transplantation for the Familial Amyloid Polyneuropathy Liver Donor and Recipient: A Double Analysis

Although domino liver transplantation (LT) is an established procedure, data about the operative risks are limited. This study aimed at evaluating the operative risks of domino LT. Two retrospective analyses were conducted (comparison of familial amyloid polyneuropathy [FAP] liver donors [61 patients] vs. FAP nondonors [39 patients] and FAP liver recipients [61 patients] vs. deceased donor liver recipients [61 patients]). First analysis showed a 60‐day mortality of 6.6% for FAP donors and 7.7% for FAP nondonors (p = 1.0). No patient developed primary graft nonfunction. Acute rejection was higher in FAP nondonors compared to FAP donors (38.5% vs. 13.1%). Both groups had similar vascular and biliary complication rates. ICU stay was similar, whereas total hospitalization was longer for FAP nondonors. Both groups had similar 1‐ and 5‐year patient and graft survival rates (83.4% vs. 87.2%, and 79.8% vs. 71.8%, p = 0.7) and (83.3% vs. 87.2%, and 79.1% vs.71.8%, p = 0.7). The second analysis showed a 1.6% mortality for FAP liver recipients vs. 3.2% of the control group (p = 1). Both groups had similar morbidity and technical complication rates (18.0% vs. 13.1%, p = 0.45) and (0.18 vs. 0.15, p = 0.65). The domino procedure does not add any risk to FAP donor or recipient. It increases the organ pool allowing transplantation of marginal recipients who otherwise are denied deceased donor liver transplantation.     

Domino liver transplantation: risks and benefits

Domino liver transplantation, wherein a patient who himself undergoes liver transplantation in turn donates his liver to another recipient, has been performed since the mid-1990 s. Although livers from a handful of metabolic disorders cured by liver transplantation have been used for domino transplantation, familial amyloidotic polyneuropathy (FAP) livers are by far the most common source. FAP is an inherited disorder never presenting its clinical manifestation before the age of 15. In many carriers, the genetic disorder never manifests during lifetime. Thus, only a proportion of patients with FAP develop disease symptoms, which has been the rationale for using such livers for other patients on the waiting list for liver transplantation. According to the Familial Amyloidotic Polyneuropathy World Transplant Registry (FAPWTR), only 2 out of more than 500 patients so far have developed symptoms after domino liver transplantation using an FAP liver. Domino recipients with nonmalignant indications for liver transplantation show excellent long-term survivals. With careful selection of recipients, the procedure helps to reduce the organ shortage and the time on the waiting list for patients with malignant disorders.     

No ocular involvement in familial amyloidotic polyneuropathy ATTR V30M domino liver recipients

In many transplantation centers domino liver transplantation is an established procedure, increasing the number of available liver grafts. Increasingly, grafts from familial amyloidotic polyneuropathy (FAP) patients are used. Ocular involvement is a well known manifestation of FAP, and can be vision-threatening. The aim of this study was to evaluate the risk of development of familial amyloidotic polyneuropathy ocular manifestations in domino liver recipients. Forty-four cirrhotic patients submitted to liver transplantation were studied, with an average of 6 years of follow up after the procedure. Twenty two patients had received a liver from a FAP donor (Group 1) and 22 had received a liver from a non-FAP cadaveric donor (Group 2). Both groups were similar for mean age and gender. Routine ophthalmological examinations with particular attention to amyloid deposition in the anterior segment and vitreous, peripheral retina state, lacrimal functions tests (Schirmer and tear break-up time) and pupillometry (dynamic and static) were performed. No statistically significant differences were observed in all studied ophthalmic parameters between the two groups. No FAP related ophthalmic manifestations were detected after 6 years of domino liver transplantation, but further prospective regular ophthalmological examinations are necessary to detect the eventual development of late ocular manifestations.     

Transthyretin-derived amyloid deposition on the gastric mucosa in domino recipients of familial amyloid polyneuropathy liver.

Familial amyloid polyneuropathy (FAP) is a form of hereditary generalized amyloidosis. Liver tissue explanted from FAP patients has normal structure and function, except for the production of amyloidogenic variant transthyretin (TTR), and domino liver transplantation (DLT) using grafts from FAP patients was first performed in 1995. FAP symptoms usually develop in genetically determined individuals after the age of 20, but it is difficult to estimate when FAP symptoms will appear in domino recipients. Concerning this problem, histological findings showing amyloid deposition have recently been obtained in a few domino recipients of FAP livers. This study investigated the presence of de novo amyloid deposition in the gastroduodenal mucosa of domino recipients transplanted at our institution. Biopsy of gastroduodenal mucosa was carried out in 5 recipients of FAP livers and TTR-derived amyloid deposits were detected in 2 patients, both of whom had undergone DLT 47 months previously. In FAP liver recipients, de novo systemic amyloid deposition may begin much sooner than previously supposed. Therefore, careful follow-up of domino recipients of FAP livers is required.     

Living donor liver transplantation for biliary atresia: An analysis of 2085 cases in the registry of the Japanese Liver Transplantation Society

Biliary atresia (BA) is the most common indication for liver transplantation (LT) in pediatric population. This study analyzed the comprehensive factors that might influence the outcomes of patients with BA who undergo living donor LT by evaluating the largest cohort with the longest follow‐up in the world. Between November 1989 and December 2015, 2,085 BA patients underwent LDLT in Japan. There were 763 male and 1,322 female recipients with a mean age of 5.9 years and body weight of 18.6 kg. The 1‐, 5‐, 10‐, 15‐, and 20‐year graft survival rates for the BA patients undergoing LDLT were 90.5%, 90.4%, 84.6%, 82.0%, and 79.9%, respectively. The donor body mass index, ABO incompatibility, graft type, recipient age, center experience, and transplant era were found to be significant predictors of the overall graft survival. Adolescent age (12 to <18 years) was associated with a significantly worse long‐term graft survival rate than younger or older ages. We conclude that LDLT for BA is a safe and effective treatment modality that does not compromise living donors. The optimum timing for LT is crucial for a successful outcome, and early referral to transplantation center can improve the short‐term outcomes of LT for BA. Further investigation of the major cause of death in liver transplanted recipients with BA in the long‐term is essential, especially among adolescents     

Temporary Auxiliary Partial Orthotopic Liver Transplantation Using a Small Graft for Familial Amyloid Polyneuropathy

Donor shortage is a major issue in liver transplantation. We have successfully performed temporary auxiliary partial orthotopic liver transplantation (APOLT) using a small volume graft procured from a living donor for recipients with familial amyloid polyneuropathy (FAP). The aim of this study was to evaluate this procedure by comparing it with standard living donor liver transplantation (LDLT). We compared 13 recipients undergoing this procedure with 23 recipients undergoing a standard LDLT for the treatment of FAP. The estimated donor graft volume and the graft volume/recipient's standard liver volume ratio were significantly smaller in the temporary APOLT group than in the standard LDLT group. Postoperative complications were comparable, although the hospital stay was longer in the temporary APOLT group. All the patients safely underwent a remnant native liver resection about 2 months after their first operation in the temporary APOLT group. No symptoms related to FAP developed before the remnant liver resection, and no significant differences in graft and patient survival were observed between the two groups. We successfully performed temporary APOLT using a small volume liver graft without postoperative liver failure for FAP. Temporary APOLT for FAP might be a useful alternative procedure for expanding the donor pool for LDLT.     

成人间双供体活体肝脏移植成功2例报告

目的供肝短缺是影响肝脏移植发展的主要因素之一，活体供肝是解决这一矛盾的重要措施，供者提供足够的肝脏是影响活体肝脏移植的重要因素。方法施行成人间双供体活体肝移植2例，1例由受者的两位姐姐分别提供左半肝作为供肝，另1例由受者母亲提供右半肝，由无心跳供者提供左半肝(采用劈裂方式，其另一部分肝脏同时为另一成人受者实施肝脏移植)作为供肝。结果术后供、受者肝功能均恢复良好。结论成人问双供肝活体肝脏移植可以为受者提供更大重量的肝脏，又可减少供者提供较多肝脏所带来的风险；双供肝一受者肝脏移植手术操作复杂。     

Outcomes of Domino Liver Transplantation: A Single Institution's Experience

Aims

Domino liver transplantation (DLT) is a strategy to increase the donor pool. Explanted liver from patients with familial amyloidotic polyneuropathy (FAP) are often used as domino grafts, because the liver is normal apart from the production of the mutated transthyretin variant. We present the outcomes for both donors and recipients of DLT.

Materials and Methods

Retrospective analysis of initial DLT for 16 consecutive adult patients performed between July 2004 and July 2009. All cases of FAP donor to grafts were removed preserving the cava vein with reconstruction of the hepatic veins, except the first and seventh cases, where in we removed the retrohepatic vena cava with the liver without venovenous bypass. The postoperative follow-up period for surviving DLT recipients at the end of September 2009 was 2-62 months (mean, 26).

Results

Two patients out of 8 FAP donors died due to pulmonary thromboembolism on the 31st postransplant day, or sepsis at 35 days namely, an overall survival of 75%. One patient out of 8 recipients died namely, an early portal thrombosis on the 22nd postransplant day) with a crude survival of 87.5% in the recipient group (P = no significant [NS]). Four grafts from 8 FAP donors were lost—2 deaths and 2 retransplants due to thrombotic events on the first and second postransplant day—with a crude survival of 50%. Two of 8 recipients lost their grafts: 1 death and 1 retransplantation for an acute Budd-Chiari syndrome on the first postransplant day with a crude survival of 75% in the recipient group (P = not significant [NS]).

Conclusion

We believe that the FAP liver graft is an excellent option for selected patients. Special care must be taken with thrombotic events.     

Domino liver transplantation

Orthotopic liver transplantation is today an established treatment for end stage liver diseases. However, the ongoing shortage of suitable livers together with progressively longer waiting lists prevents many patients from being transplanted, and many patients die while being on the waiting list. Using livers from living donors is one way to increase the supply of liver grafts. Another group of potential living liver donors are some selected liver recipients, whose native explanted liver in turn can be considered for transplantation into another patient. This unorthodox procedure have been named domino liver transplantation (DLT). The domino approach can be considered in patients with some genetic or biochemical disorders that today are treated by liver transplantation. The underlying rationale is that such livers ultimately cause severe systemic disease but are otherwise normal. In this review we present the current world status of DLT as well as updated results from the Domino Liver World Transplant Register (DLTR) and our own experience at the Karolinska University Hospital Huddinge with the DLT procedure.     

Living Donor Liver Transplantation for Neonates Using Segment 2 Monosubsegment Graft

The prognosis of liver transplantation for neonates with fulminant hepatic failure (FHF) continues to be extremely poor, especially in patients whose body weight is less than 3 kg. To address this problem, we have developed a safe living donor liver transplantation (LDLT) modality for neonates. We performed LDLTs with segment 2 monosubsegment (S2) grafts for three neonatal FHF. The recipient age and body weight at LDLT were 13–27 days, 2.59–2.84 kg, respectively. S2 or reduced S2 grafts (93–98 g) obtained from their fathers were implanted using temporary portacaval shunt. The recipient portal vein was reconstructed at a more distal site, such as the umbilical portion, to have the graft liver move freely during hepatic artery (HA) reconstruction. The recipient operation time and bleeding were 11 h 58 min–15 h 27 min and 200–395 mL, respectively. The graft‐to‐recipient weight ratio was 3.3–3.8% and primary abdominal wall closure was possible in all cases. Although hepatic artery thrombosis occurred in one case, all cases survived with normal growth. Emergency LDLT with S2 grafts weighing less than 100 g can save neonates with FHF whose body weight is less than 3 kg. This LDLT modality using S2 grafts could become a new option for neonates and very small infants requiring LT.     

Liver transplantation from living donors with Gilbert's syndrome is a safe procedure for both donors and recipients

Liver transplantation (LT) has become a favorable therapeutic option for patients with end‐stage liver diseases. Gilbert's syndrome (GS) is a benign condition characterized by intermittent mild jaundice due to unconjugated hyperbilirubinemia. It is not obvious whether living‐donor liver transplantation (LDLT) from a donor with GS could result in a normal outcome for both the recipient and the donor. We aimed to determine whether right lobe hepatectomy is a safe procedure for living donors with GS and LT recipients. Between September 2011 and March 2015, 305 LDLT procedures using right lobe grafts were performed at Atasehir Memorial Hospital, Istanbul, Turkey. Nineteen of 305 LT candidates who had been diagnosed with GS were included in the current study. After a 12‐h overnight fast, total and indirect bilirubin levels of donors and recipients were measured. The median follow‐up after transplant was 16 months (range 3–36 months). The median age of donors was 25 (range 20–55 yr). Four donors (21%) were female, and 15 donors (89%) were male. The median age of donors was 51 (range 23–68 yr). Eleven recipients (57%) were female, and 8 (43%) were male. The median preoperative total bilirubin level of donors was 1.69 mg/dL (range 1.26–2.43 mg/dL) (normal range <1.2 mg/dL). The median total bilirubin level of donors on postoperative day 7 was 1.04 mg/dL (range 0.71–3.23 mg/dL). As our study has included a large number of donors with GS, it produced reliable evidence that right lobe hepatectomy is a safe procedure for living donors with GS and LT recipients.     

肝移植术后胆道并发症病因及预防

目的分析原位肝移植术后胆1道并发症的病因。方法回顾性分析307例尸体供肝和40例活体供肝原位肝移植的临床资料,总结术后胆道并发症的病因。结果40例活体肝移植受体术后胆道并发症的发生率5.0%,307例尸体供肝肝移植受体术后胆道并发症的发生率为18.9%;肝内胆道狭窄和胆道铸型结石形成等严重胆道并发症在活体肝移植和放置“T”管的尸肝移植未发生。结论缺血时间尤其热缺血时间是导致严重胆道并发症的最主要的原因,放置“T”管引流能降低胆道并发症的发生率。     

Clinical analysis of emergency liver transplantation: the role of living donor liver transplantation

The current liver allocation system requires reevaluation because of the advancements in peri‐transplantation care and surgical techniques. And, the role of living donor liver transplantation (LDLT) in an emergency has not been determined yet. Retrospective review of all patients undergoing emergency liver transplantation (LT) from January 2000 to June 2010 was conducted, and clinical data were analyzed. Of the total 505 LTs, 69 patients (13.7%) underwent an emergency LT. Of these, 54 patients (78.3%) underwent LDLT using a right liver, and 15 patients (21.7%) underwent deceased donor liver transplantation (DDLT). The overall hospital mortality was 21.7% (15/69). The leading cause of death after transplantation was sepsis (60.0%). Multivariate analysis demonstrated that a model for end‐stage liver disease (MELD) >33 [hazard ratio (HR), 16.6; 95% confidence interval (CI), 1.443–191.632; p = 0.024] and existence of pre‐transplantation intubation (HR, 18.2; 95% CI, 1.463–225.483; p = 0.024) were independent factors associated with poor survival after emergency LT. LDLT group and DDLT group showed no difference in hospital mortality (p = 0.854) and graft survival (p = 0.861). Thus, MELD score and respiratory insufficiency could be parameters predicting post‐transplant survival. And, LDLT using the right liver could be an appropriate alternative to DDLT in an emergency.     

Emergent right lobe adult-to-adult living-donor liver transplantation for high model for end-stage liver disease score severe hepatitis

The aim of this study was to explore the feasibility of emergency right lobe adult-to-adult living-donor liver transplantation (LDLT) for high model for end-stage liver disease (MELD) score severe hepatitis. Consecutive 10 high MELD score severe hepatitis patients underwent emergency right lobe adult-to-adult LDLT in our hospital from April to December 2007. The MELD score was 34.50 ± 2.088. The outcomes of these recipients were retrospectively analyzed. Among them, eight cases of ABO blood group were identical and two cases compatible, one case was Rh negative. Two recipients died and the rest of the recipients and all donors are safe; perioperative and 2-year survival rate was 80%. The mean graft-recipient weight ratio (GRWR) was 1.27% ± 0.25%, and graft volume to recipient standard liver volume ratio (GV/ESLVR) was 56.7% ± 6.75%. Of the 10 patients, three received right lobe grafts with middle hepatic vein (MHV), four without MHV, three without MHV but followed by V and VIII hepatic vein outflow reconstruction. An encouraging outcome was achieved in this group: elevated serum creatinine, serum endotoxin, decreased serum prothrombin activity, and Tbil returned to normal on postoperative days 3, 7, 14, and 28, respectively. One-year survival rate was 80%. Outcomes of emergency right lobe adult-to-adult LDLT for high MELD score severe hepatitis were fairly encouraging and acceptable. Emergency right lobe adult-to-adult LDLT is an effective and life-saving modality for high MELD score acute liver failure patients following severe hepatitis.     

Critical graft size and functional recovery in living donor liver transplantation

With a high prevalence of chronic hepatitis B and a low cadaveric organ donation rate, living donor liver transplantation (LDLT) remains the only option for many patients in Hong Kong. In such cases, the liver graft volume is smaller owing to a partial liver graft; therefore, a problem of small-for-size grafts often occurs. Between September 1999 and April 2003, 25 cadaveric, 16 living related, and 1 auto-LTs were performed at our center. The outcomes of LDLT were analyzed to assess the critical graft size and functional recovery. Among the 16 LDLT recipients (mean age, 44.4 +/- 14.4 years; mean weight, 61.9 +/- 11.4 kg), 1 patient received a graft from a donor left lobe (weight, 400 g) in an auxillary partial orthotopic LT (APOLT), 12 received right lobes, and 3 received left lobes. Besides the APOLT case, the overall graft/recipient weight ratio (GRWR) for the 15 LDLTs was 1.11 (0.76 to 1.75). The GRWR in the 25 cadaveric LTs was 1.92 (1.05 to 3.69) (P < .001). Among the 12 successful LDLTs, there were 5 (41.7%) cases of small-for-size graft syndrome: 3 of 3 (100%) in GRWR < or = 0.8%; 5 of 6 (83.3%) in GRWR < 1%; and 0 of 6 with GRWR > 1%. The initial post-LT graft function parameters were significantly higher among the LDLT group: International normalized ratio (INR), 1.42 vs 1.24, P = .03; alanine aminotransferase (ALT, 387 vs 201 IU/L, P = .005, and bilirubin, 170 vs 48 micromol/L, P < .001 as compared to the cadaveric transplant group). Small-for-size graft syndrome can be avoided if GRWR > 1%, but often occurs when GRWR < 0.8%. Graft function in LDLT recovers more slowly than in cadaveric liver transplant.     

The Risk of End‐Stage Renal Disease Among Living Donor Liver Transplant Recipients in the United States

Since initiation of model for end‐stage liver disease (MELD)‐based allocation for liver transplantation, the risk of posttransplant end‐stage renal disease (ESRD) has increased. Recent US data have demonstrated comparable, if not superior survival, among recipients of living donor liver transplants (LDLT) when compared to deceased donor liver transplant (DDLT) recipients. However, little is known about the incidence of ESRD post‐LDLT. We analyzed linked Scientific Registry of Transplant Recipients (SRTR) and US Renal Data System (USRDS) data of first‐time liver‐alone transplant recipients from February 27, 2002 to March 1, 2011, and restricted the cohort to recipients with a laboratory MELD score ≤25 not on dialysis prior to transplantation, in order to evaluate the incidence of ESRD post‐LDLT, and to compare the incidence among LDLT versus DDLT recipients. There were 28 707 DDLT and 1917 LDLT recipients included in the analyses. The 1‐, 3‐ and 5‐year unadjusted risk of ESRD was 1.7%, 2.9% and 3.4% in LDLT recipients, compared with 1.5%, 3.0% and 4.8% in DDLT recipients (p > 0.05), respectively. In multivariable competing risk Cox regression models, there was no association between receiving an LDLT and risk of ESRD (sub‐hazard ratio: 0.99, 95% CI: 0.77–1.26, p = 0.92). In conclusion, the incidence of ESRD post‐LDLT in the United States is low, and there are no significant differences among LDLT and DDLT recipients with MELD scores ≤25 at transplantation.     

Severe Course of Primary Hyperoxaluria and Renal Failure After Domino Hepatic Transplantation

We report herein a domino orthotopic liver transplantation (LT), from a 38-year-old woman undergoing liver-kidney transplantation (LKT) for primary hyperoxaluria type I (PH1) to a recipient with cirrhosis and hepatocellular carcinoma. Delayed onset of PH1 and renal failure and 10% residual alanine-glyoxylate aminotransferase (AGT) activity in domino liver justified its use for domino procedure. The clinical course after LKT was similar to that described in other series, including ours. Renal function started promptly and maintained despite sustained hyperoxaluria from dissolution of oxalotic deposits. Conversely, the domino recipient manifested severe hyperoxaluria and developed nephrolithiasis and renal insufficiency with rapid progression over 2 months. A new LT resulted in slow decrease of oxaluria and improvement of renal function. Therefore, PH1 behaved quite differently in these two patients, leading us to conclude that domino LT using livers from PH1 patients should be considered very carefully, only as a bridge to definitive LT in recipients with critical clinical conditions.     

Prevention of de novo hepatitis B virus infection in living donor liver transplantation using hepatitis B core antibody positive donors

Exclusion of liver grafts from hepatitis B core antibody (anti-HBc) positive donors to prevent de novo hepatitis B virus (HBV) infection after liver transplantation is not feasible in areas highly endemic for HBV virus like Taiwan, where approximately 80% of adults are anti-HBc(+). The efficacy of lamivudine monotherapy to prevent de novo HBV infection after living donor liver transplantation (LDLT) using grafts from anti-HBc(+) donors remains to be elucidated. From June 1994 to August 2000, LDLT was performed in 42 recipients. Twenty-four of the 42 donors were anti-HBc(+) (57%). Pre-transplant HBV vaccination was given to all recipients irrespective of anti-HBc status at monthly intervals for 3 months. Until December 1997, eight recipients received liver grafts from anti-HBc(+) donors without prophylaxis. Since January 1998, prophylaxis with lamivudine monotherapy was given to 16 recipients receiving liver grafts from anti-HBc(+) donors. De novo HBV infection occurred in three of the eight recipients (37.5%) who did not receive prophylaxis, while none of the 16 recipients given lamivudine developed de novo HBV infection after a mean follow-up of 25 months. Two of the three recipients with de novo HBV infection were anti-HBs(-) and one recipient was anti-HBs(+). Lamivudine was well tolerated, and no side effects were noted. These results suggest that lamivudine monotherapy for recipients receiving anti-HBc(+) liver grafts is a simple, relatively inexpensive and effective prophylactic regimen for prevention of de novo HBV infection. The additive protection provided by vaccine-induced or natural immunity is uncertain.