Paolo Carraro

Isolated Hepatocyte Transplantation for Crigler-Najjar Syndrome Type 1:

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 × 109 hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean ± SD bilirubin level was 530 ± 38 μmol/L before and 359 ± 46 μmol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.

The role of cysteine and serine proteases in colorectal carcinoma

Cathepsin B (CATB) and cathepsin L (CATL), which are cysteine proteases, urokinase‐(UPA) and tissue‐type plasminogen activator (TPA), both serine proteases, and their inhibitor type‐1 (PAI‐1) are believed to play an important role in colorectal carcinoma (CRC) invasion and metastasis. The objective of this study was to measure CATB, CATL, UPA, TPA, and PAI‐1 in the same cancerous tissue (CANCER) and in tissues obtained from a tumor free area (NORMAL) to compare their respective prognostic roles in patients with CRC.

Exploring the Initial Steps of the Testing Process: Frequency and Nature of Pre-Preanalytic Errors

BACKGROUND Few data are available on the nature of errors in the so-called pre-preanalytic phase, the initial steps of the testing process. We therefore sought to evaluate pre-preanalytic errors using a study design that enabled us to observe the initial procedures performed in the ward, from the physicians test request to the delivery of specimens in the clinical laboratory. METHODS After a 1-week direct observational phase designed to identify the operating procedures followed in 3 clinical wards, we recorded all nonconformities and errors occurring over a 6-month period. Overall, the study considered 8547 test requests, for which 15 917 blood sample tubes were collected and 52 982 tests undertaken. RESULTS No significant differences in error rates were found between the observational phase and the overall study period, but underfilling of coagulation tubes was found to occur more frequently in the direct observational phase (P = 0.043). In the overall study period, the frequency of errors was found to be particularly high regarding order transmission [29 916 parts per million (ppm)] and hemolysed samples (2537 ppm). The frequency of patient misidentification was 352 ppm, and the most frequent nonconformities were test requests recorded in the diary without the patients name and failure to check the patients identity at the time of blood draw. CONCLUSION The data collected in our study confirm the relative frequency of pre-preanalytic errors and underline the need to consensually prepare and adopt effective standard operating procedures in the initial steps of laboratory testing and to monitor compliance with these procedures over time.

Proteases in gastrointestinal neoplastic diseases

Cysteine and serine proteases are involved in cancer invasion and metastasis. In the past few years we investigated the tissue levels of these proteases in gastric cancer (GC), gastric precancerous changes (CAG), colorectal cancer (CRC) and the plasma and serum levels of proteases in several gastrointestinal tumours, using ELISA methods. Significantly higher antigen levels were found not only in GC tissue but also in CAG with respect to the levels found normal tissue; with respect to CAG, patients with dysplasia had higher levels than patients without dysplasia. The same findings were obtained in CRC. In general protease levels correlated with the major histomorphological parameters, such as grading and histotype in GC as well as in CRC. Tissue protease levels had a strong prognostic impact in GC, in which UPA was singled out by multivariate analysis as the major prognostic factor, and CRC. The plasma levels of urokinase-type plasminogen activator (UPA) and the serum levels of cathepsin B were significantly increased in patients with gastrointestinal tumours. In conclusions, cysteine and serine proteases may have a part not only in GC and CRC invasion and metastasis, but also in the progression of gastric precancerous changes into cancer. They are strong prognostic factors in GC and CRC. These proteases may also have a role as tumour markers in the early diagnosis of gastrointestinal tract tumours.

Different biochemical correlates for different neuropsychiatric abnormalities in patients with cirrhosis.

The diagnosis of hepatic encephalopathy (HE) relies on clinical, neurophysiological, psychometric and laboratory variables. The relationships between such tests remain debated. The aim of this study was to determine the laboratory correlates/prognostic value of neurophysiological/psychometric abnormalities in patients with cirrhosis. Seventy‐two patients and 14 healthy volunteers underwent EEG and paper‐and‐pencil psychometry (PHES). Blood was obtained for C reactive protein (CRP), interleukin 6 (IL6), tumor necrosis factor (TNF)α, ammonia and indole/oxindole. Patients were followed prospectively for a median of 22 months in relation to the occurrence of death, transplantation and HE‐related hospitalizations. Thirty‐three patients had normal PHES and EEG, 6 had abnormal PHES, 18 abnormal EEG and 13 abnormal PHES and EEG. Patients with abnormal PHES had higher CRP (17 ± 22 vs 7 ± 6, P < 0.01), IL6 (32 ± 54 vs 12 ± 13, P < 0.05) and TNFα (17 ± 8 vs 11 ± 7, P < 0.001) levels than those with normal PHES. Patients with abnormal EEG had higher indole (430 ± 270 vs 258 ± 255, P < 0.01) and ammonia (66 ± 35 vs 45 ± 27, P < 0.05) levels than those with normal EEG. Psychometric test scores showed significant correlations with CRP, TNFα and IL6; EEG indices with ammonia and IL6. CRP and TNFα concentrations were independent predictors of abnormal PHES, ammonia and indole of abnormal EEG on multivariate analysis. Seven patients were lost to follow‐up; of the remaining 65, 20 died and 14 underwent transplantation; 15 developed HE requiring hospitalization. PHES and EEG performance were independent predictors of HE and death (P < 0.05). Conclusion: PHES and EEG abnormalities in patients with cirrhosis have partially different biochemical correlates and independently predict outcome. (HEPATOLOGY 2011;53:558‐566)

Hepatocyte transplantation in the treatment of acute liver failure: microencapsulated hepatocytes versus hepatocytes attached to an autologous biomatrix.

A liver transplant is considered today to be the only effective therapeutic solution for many otherwise intractable hepatic disorders. However, liver transplantation is beset by shortage of donors. Over the years, many liver support systems have been developed to supply the liver functions, mostly as a bridge to transplantation. Transplantation of isolated hepatocytes (HcTx) instead of whole liver has constituted one of the most appealing possibilities to treat several diseases. We compared two different models of HcTx in a surgical model of acute liver failure in pigs, using microencapsulated hepatocytes (MHcTx) and hepatocytes attached to a porcine biomatrix (PBMHcTx), both transplanted into peritoneum. The collected data were survival, laboratory findings, hemodynamic parameters, light microscopy, histology, MTT, and glycogen content. The group with PBMHcTx has a better outcome than the group with MHcTx (p < 0.05). Histology showed normal morphology of the hepatocytes, high glycogen content, 75% viability, positive MTT, and 95% adhesion of the hepatocytes to the biomatrix. Our biomatrix (PBM) provides cell-to-cell contact and interaction with extracellular matrix, which have been shown to play major roles in hepatocyte survival and physiologic regulation of gene expression, and guarantee a prompt engraftment and an adequate neovascularization. PBMHcTx is a useful method to treat acute liver failure and it indicates a possible liver-direct gene therapy in the treatment of inherited and acquired disorders.

Urokinase-type plasminogen activator receptor in gastric cancer: tissue expression and prognostic role.

The urokinase-type plasminogen activator (UPA) and its inhibitor PAI-1 are thought to play an important part in gastric cancer (GC) invasion and metastasis. Little is known about the behavior and prognostic impact of the receptor for UPA (UPAR). The aims of the present study were: (1) to measure UPAR, UPA and PAI-1 levels in GC and in non-malignant tissue distant from the tumor (NORM); (2) to evaluate their relationship with histomorphological parameters; and (3) to determine their prognostic value. UPAR, UPA and PAI-1 levels were determined by ELISA in GC and NORM samples from 20 patients with GC undergoing surgery. The GC was also examined in terms of the presence (n=10) or absence (n=10) of metastasis, differentiation (five differentiated, 15 undifferentiated) and histotype. Survival was analysed using life table analysis. UPAR, UPA and PAI-1 were significantly higher in GC vs NORM, in the presence of metastasis (UPAR, UPA) and in undifferentiated GC (UPAR, PAI-1). UPAR significantly correlated with UPA and PAI-1. Low levels of UPAR (P=0.04), UPA (P=0.007) and PAI-1 (P=0.02) were associated with a better survival. Our results demonstrate a sharp increase in UPAR in GC and suggest a prognostic role for it. The concomitant activation of UPAR, UPA and PAI-1 in GC confirm the important role of the plasminogen activator system in the process of invasion and metastasis.

Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts.

Background. Fibrin deposition is central to the acute humoral rejection process occurring in the presence of consumptive coagulopathy when pig organs are transplanted into primates. Methods. To assess whether strategies aimed at preventing fibrin formation may extend xenograft survival, we administered high daily doses of recombinant human antithrombin (rhAT) (500 U/kg twice daily) to obtain both anticoagulant and anti-inflammatory effects in immunosuppressed primate recipients of porcine kidneys. Results. Some degree of consumptive coagulopathy developed in both rhAT-treated (n=3) and untreated (n=3) primates. No major differences in the coagulation parameters analyzed were observed between the 2 groups. Similarly, no difference in survival was seen between rhAT-treated (20.6±4 days; range: 15–23 days) and untreated animals (17.3±11.6 days; range: 7–30 days), although the rhAT-treated primates had a higher bleeding tendency. Despite the high daily dose of rhAT, considerable fibrin deposition was observed in the graft as early as 2 weeks after transplantation. Conclusions. These results suggest that a high daily dose of rhAT fails to influence survival or prevent fibrin formation and deposition in the graft in our pig-to-primate model. However, the potential role of rhAT administered in combination with heparins or other clotting inhibitor concentrates in this model remains to be determined.

Point-of-care testing of cardiac markers: results from an experience in an Emergency Department.

AIM An experimental approach to the use of point-of-care testing for cardiac markers in the Emergency Department (ED) of our Institution has been carried out using two devices (SCS, Dade Behring and Triage Cardiac Panel, Biosite Diagnostics) for the measurement of cardiac markers. RESULTS (1) From the analytical point of view, a fundamental tool for an efficient management of patients was the agreement between results from point-of-care testing and from the instruments located in STAT lab and/or central laboratory: in about 5% of patients, a lack of comparability of data, resulted in an inappropriate admission of patients (medical vs. intensive care unit). (2) The actual total turnaround time (TAT) in the management of samples sent to STAT lab was estimated to be equal to 82.5 min (50th percentile). (3) In the same organizational setting, the use of a point-of-care device produced a turnaround time equal to 17 min (50th percentile). (4) The reduction in turnaround time resulted in a faster discharge for five patients who had normal ECG findings and cardiac marker values, the Delta time (POCT-STAT lab) ranging from -10 to -70 min. CONCLUSIONS The point-of-care option evaluated also in relation to personnel issues for staff working in the ED, brought some interesting questions about the characteristics of POCT devices (easy to use 100%, safety for operator 91%) and the obtained results (quantitative and correlated to STAT lab, 91%), as well as the need of other options such as the implementation of rapid tube sample delivery.

Tissue factor pathway inhibitor release induced by defibrotide and heparins.

We evaluated the release of tissue factor pathway inhibitor (TFPI) induced by defibrotide (DF), a single-stranded, negatively charged polydeoxyribonucleotide extracted from mammalian organ. Ten normal volunteers were injected with an intravenous bolus of 400 mg DF and 2,000 IU unfractionated heparin (UFH). In addition, three volunteers were also injected with an intravenous bolus of 2,000 anti-Xa U of two low-molecular-weight heparins (LMWHs), enoxaparin and nadroparin. UFH caused a 4-fold increase in plasma TFPI at 5 minutes, with a decrease that was parallel to the heparin level measured by the anti-Xa assay. However, at 80 minutes, although the plasma anti-Xa activity of UFH was almost undetectable, the level of TFPI remained 2-fold baseline. DF induced an increase of TFPI that was 2-fold higher than the baseline level, with a steady state achieved between 5 and 20 minutes. At 40 minutes, the TFPI levels returned to basal level. This pattern was not coincident with the clearance of DF and at 40 minutes, the concentration of DF was still one third of the levels at 5 minutes (25.4 ± 4.04 μg/mL). Both of the LMWHs induced a similar TFPI peak level at 5 minutes (1.5-fold increase) and at 40 minutes the TFPI levels returned to the initial levels, At 5 minutes, both LMWHs showed a higher plasma anti-Xa activity than UFH, which was detectable even at 80 minutes. The current study demonstrated that one of the mechanisms of the antithrombotic activity of DF is mediated via TFPI. Furthermore, the release of TFPI by heparin is mediated by non-antithrombin III binding fragments. Thus, polyanionic electrolytes are capable of releasing TFPI irrespective of their antithrombin III effect.