Daniele Prati

Updated Definitions of Healthy Ranges for Serum Alanine Aminotransferase Levels

Context Current upper limits (500 nkat/L [30 U/L] for women, 667 nkat/L [40 U/L] for men) for serum alanine aminotransferase (ALT) level were defined in populations that included persons with nonalcoholic fatty liver disease (NAFLD) and persons with hepatitis C virus (HCV) infection. Contribution This study redefined ALT limits in blood donors at low risk for NAFLD and without hepatitis B or C (317 nkat/L [19 U/L] in women, 500 nkat/L [30 U/L] in men). When applied to 209 anti-HCV-positive donors, the new thresholds had 76.3% sensitivity and 88.5% specificity in identifying patients with hepatitis C viremia compared with 55% and 97.4% for old thresholds. Implications Laboratories should consider revising the upper limits of normal for ALT to improve the sensitivity of this test in identifying subclinical liver disease. The Editors Serum alanine aminotransferase (ALT) concentration is the most commonly used variable for assessment of liver disease (1, 2). However, particularly in the case of chronic hepatitis C virus (HCV) infection, ALT measurement often fails to identify patients with minimal to mild necroinflammatory activity (3-7). Current upper limits of normal for ALT level were set, on average, at 667 nkat/L (40 U/L) (range, 500 to 833 nkat/L [30 to 50 U/L]) in studies conducted over the past 10 years (1, 3-5, 7, 8). Such thresholds, however, were mostly computed in the 1980s, when ALT testing was introduced as a surrogate marker for the screening of non-A, non-B hepatitis among blood donors and before anti-HCV testing and restrictive behavioral criteria for donor selection were implemented. Furthermore, so-called reference populations were likely to include many persons with nonalcoholic fatty liver disease, now recognized as the most prevalent cause of chronic liver disease in developed countries (8-10). Current reference ranges for ALT level probably underestimate the frequency of chronic liver disease. Because dietary and behavioral risks for liver disease are widespread in many countries, a critical revision of ALT limits would require the definition of healthy ranges rather than a generic update of normal ranges. Thus far, several factors have hampered this task. For example, to obtain solid data, many clinical, biochemical, and behavioral variables potentially related to liver disease must be investigated, requiring screening of large numbers of persons. Furthermore, repeated blood donors, who currently represent the vast majority of blood-donation candidates, cannot be included in the sampling frame, because they have been selected on the basis of ALT activity during the past two decades. We report the results of a 4-year study of first-time blood-donation candidates. To update the definitions of healthy ranges for serum ALT level, we identified a population at low risk for subclinical liver disease by exploring factors related to enzyme activity in both healthy persons and those with mild abnormalities on liver tests. Next, we tested the sensitivity and specificity of the ranges obtained from these participants in the clinical evaluation of anti-HCVpositive persons with and without chronic liver damage. Methods Participants Figure 1 summarizes the selection of the study participants. Figure 1. Procedures for selection of the study participants. *Donor candidates were not suitable for the following reasons: previous blood transfusion (2%); use of major illicit drugs (1.5%); at-risk sexual exposures (9%); history of hepatitis or other blood borne infections (4%); recent exposure in a malaria-endemic area (5%); low hemoglobin level (15%); use of medication not compatible with blood donation (4%); seizure or central nervous system disorders (12%); hypertension, arrhythmias, or cardiac disease (8%); hypotension (9%); recent surgery (2%) or other medical or behavioral risks (26%); serologic reactivity on screening assays (antiHIV 1, antiHIV 2, hepatitis B surface antigen, antihepatitis C virus [HCV], or syphilis) on the sample collected at blood donation (2.5%). Standard upper limits were 667 nkat/L (40 U/L) in men, and 500 nkat/L (30 U/L) in women. Two trained hepatologists used a recently proposed algorithm (2) to re-evaluate medical history and physical examination. Participants also underwent additional blood testing, including measurement of the following values: aspartate aminotransferase, alanine aminotransferase (ALT), alkaline phosphatase, -glutamyl transpeptidase, total proteins, bilirubin, iron, total iron-binding capacity, serum ferritin, serum protein electrophoresis, creatine kinase, ceruloplasmin, 1-antitrypsin, antibodies to cytomegalovirus and EpsteinBarr virus, and autoantibodies. Participants also underwent ultrasonography of the liver. Anti-HCVNegative First-Time Blood Donors From 1 September 1995 through 26 October 1999, 9221 blood-donor candidates presenting for first-time donation underwent clinical and laboratory examinations as part of procedures for donor selection at Centro Transfusionale e di Immunologia dei Trapianti in Milan, Italy. A blood-bank physician 1) administered a psychosocial questionnaire [11, 12], which was aimed at identifying and excluding from donation persons at high risk for blood-borne infections; 2) took a medical history; and 3) examined all potential participants and measured body weight and height. Donors candidates had blood drawn for laboratory testing. Clinical data and laboratory-test results were recorded in a relational database management system, as described previously (11). We included in the study donor candidates who had no medical or behavioral contraindication to blood donation (12) and who had negative results on tests for hepatitis B surface antigen (HBsAg), anti-HCV, antiHIV 1, antiHIV 2, and hemagglutination (to assess for presence of syphilis). In addition, we used a recently recommended diagnostic algorithm (2) to conduct a diagnostic work-up in donors who repeatedly had abnormal ALT measurements (that is, they had increased values, according to current ALT ranges, in three subsequent measurements taken at 1-month intervals in the absence of HBsAg and anti-HCV reactivity) (2). Anti-HCVPositive Blood Donors We also studied 209 blood-donor candidates with confirmed anti-HCV reactivity between 1990 and 1999 who presented to our outpatient liver disease clinic for regular follow-up. Of these patients, 78 were HCV RNA negative at initial screening (59 patients) or after antiviral treatment (19 patients), and 131 were HCV RNA positive. Serum ALT activity was determined at presentation and in at least two other serial serum samples collected at 1- to 3-month intervals over at least 6 months. In patients with viremia who underwent treatment, the pattern of serum ALT levels was defined during the period of presumed viremia (that is, not during or after therapy). Liver biopsy was performed in 133 anti-HCVpositive blood donors (103 of whom were HCV RNA positive and 30 of whom were HCV RNA negative) for diagnostic reasons (32 patients) or within clinical trials. These clinical trials were conducted between 1993 and 1998 to define the optimal management of anti-HCVpositive patients with normal or slightly altered ALT levels (101 patients) (3, 4, 11, 13, 14). Laboratory Methods A fasting blood sample was collected in the morning and was centrifuged within 30 minutes of collection. The Laboratory of Biochemistry and the Laboratory of Virology of the Centro Trasfusionale e di Immunologia dei Trapianti at IRCCS Ospedale MaggioreMilan, Italy, performed all analyses by using consistent methods throughout the study period. Complete blood counts were performed by using an NE 8000 automatic cell counter (Sysmex, Kobe, Japan). Analyses of serum biochemistry were performed by using an Olympus AU510 analyzer (EppendorfNetheler, Hamburg, Germany). Upper reference limits for serum biochemistry analyses were computed in 1983 on the basis of findings from 5093 women and 9849 men who were apparently healthy donors with negative results on hepatitis B surface antigen and syphilis tests (4, 15). These limits were as follows: for total cholesterol level, 5.70 mmol/L (220 mg/dL); for triglyceride level, 2.26 mmol/L (200 mg/dL); for blood glucose level, 5.83 mmol/L (105 mg/dL) in men and 5.44 mmol/L (98 mg/dL) in women; for ALT level, 667 mmol/L (40 U/L) in men and 500 nkat/L (30 U/L) in women. Virologic tests included an hepatitis B surface antigen test (Wellcozyme HBsAg, Abbott Laboratories, Chicago, Illinois), an anti-HIV test (Ortho HIV1/HIV2, Ortho Diagnostic Systems, Raritan, New Jersey), and an anti-HCV test (Ortho HCV 3.0, Ortho Diagnostic Systems). Anti-HCV reactivity was confirmed by third-generation recombinant immunoblot assay (RIBA-3, Ortho Diagnostic Systems). Qualitative analysis of serum HCV RNA was performed by using the Amplicor HCV kit (Roche Molecular Systems, Basel, Switzerland). Body mass index (BMI) was calculated by dividing the weight (in kg) by the squared height (in m). On the basis of a recent recommendation (16), we considered a BMI of 24.9 kg/m2 the upper limit for healthy weight. The laboratory and the blood donor center were certified according to International Organization for Standardization 9002 standards. The laboratory intra-assay coefficient of variation (CV) for ALT was 1.1%, and the interassay CV over a 2-week period was 2.4%. Within-individual and between-individual variability were estimated on the basis of 20 donors who were randomly chosen among those with two measurements taken at 3-month intervals. Within-individual variability, expressed as CV, was 21.4% (CI, 16.4% to 31.0%); between-individual variability was 49.8% (CI, 37.9%- to 72.8%). Statistical Analysis Statistical analyses were performed by using the SAS package version 6.12 (SAS Institute, Inc., Cary, North Carolina). The 5th, 25th, 50th (median), 75th, and 95th percentiles for ALT level were calculated on the basis of the empirical distribution of the data. We

STATEMENTS FROM THE TAORMINA EXPERT MEETING ON OCCULT HEPATITIS B VIRUS INFECTION

Giovanni Raimondo*, Jean-Pierre Allain, Maurizia R. Brunetto, Marie-Annick Buendia, Ding-Shinn Chen, Massimo Colombo, Antonio Craxi, Francesco Donato, Carlo Ferrari, Giovanni B. Gaeta, Wolfram H. Gerlich, Massimo Levrero, Stephen Locarnini, Thomas Michalak, Mario U. Mondelli, Jean-Michel Pawlotsky, Teresa Pollicino, Daniele Prati, Massimo Puoti, Didier Samuel, Daniel Shouval, Antonina Smedile, Giovanni Squadrito, Christian Trepo, Erica Villa, Hans Will, Alessandro R. Zanetti, Fabien Zoulim

Practice guidelines for the diagnosis and management of nonalcoholic fatty liver disease. A decalogue from the Italian Association for the Study of the Liver (AISF) Expert Committee.

We report the evidence-based Italian Association for the Study of Liver guidelines for the appropriate diagnosis and management of patients with nonalcoholic fatty liver disease in clinical practice and its related research agenda. The prevalence of nonalcoholic fatty liver disease varies according to age, gender and ethnicity. In the general population, the prevalence of nonalcoholic fatty liver disease is about 25% and the incidence is of two new cases/100 people/year. 2-3% of individuals in the general population will suffer from nonalcoholic steatohepatitis. Uncomplicated steatosis will usually follow a benign course. Individuals with nonalcoholic steatohepatitis, however, have a reduced life expectancy, mainly owing to vascular diseases and liver-related causes. Moreover, steatosis has deleterious effects on the natural history of HCV infection. Nonalcoholic fatty liver disease is usually diagnosed in asymptomatic patients prompted by the occasional discovery of increased liver enzymes and/or of ultrasonographic steatosis. Medical history, complete physical examination, etiologic screening of liver injury, liver biochemistry tests, serum lipids and insulin sensitivity tests should be performed in every patient. Occult alcohol abuse should be ruled out. Ultrasonography is the first-line imaging technique. Liver biopsy, the gold standard in diagnosis and prognosis of nonalcoholic fatty liver disease, is an invasive procedure and its results will not influence treatment in most cases but will provide prognostic information. Assessment of fibrosis by composite scores, specific laboratory parameters and transient elastography might reduce the number of nonalcoholic fatty liver disease patients requiring liver biopsy. Dieting and physical training reinforced by behavioural therapy are associated with improved nonalcoholic fatty liver disease. Diabetes and the metabolic syndrome should be ruled out at timed intervals in nonalcoholic fatty liver disease. Nonalcoholic steatohepatitis patients should undergo periodic evaluation of cardiovascular risk and of advancement of their liver disease; those with nonalcoholic steatohepatitis-cirrhosis should be evaluated for early diagnosis of hepatocellular carcinoma.

Recommendations for the management of mixed cryoglobulinemia syndrome in hepatitis C virus-infected patients.

OBJECTIVE The objective of this review was to define a core set of recommendations for the treatment of HCV-associated mixed cryoglobulinemia syndrome (MCS) by combining current evidence from clinical trials and expert opinion. METHODS Expert physicians involved in studying and treating patients with MCS formulated statements after discussing the published data. Their attitudes to treatment approaches (particularly those insufficiently supported by published data) were collected before the consensus conference by means of a questionnaire, and were considered when formulating the statements. RESULTS An attempt at viral eradication using pegylated interferon plus ribavirin should be considered the first-line therapeutic option in patients with mild-moderate HCV-related MCS. Prolonged treatment (up to 72 weeks) may be considered in the case of virological non-responders showing clinical and laboratory improvements. Rituximab (RTX) should be considered in patients with severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis. High-dose pulsed glucocorticoid (GC) therapy is useful in severe conditions and, when necessary, can be considered in combination with RTX; on the contrary, the majority of conference participants discouraged the chronic use of low-medium GC doses. Apheresis remains the elective treatment for severe, life-threatening hyper-viscosity syndrome; its use should be limited to patients who do not respond to (or who are ineligible for) other treatments, and emergency situations. Cyclophosphamide can be considered in combination with apheresis, but the data supporting its use are scarce. Despite the limited available data, colchicine is used by many of the conference participants, particularly in patients with mild-moderate MCS refractory to other therapies. Careful monitoring of the side effects of each drug, and its effects on HCV replication and liver function tests is essential. A low-antigen-content diet can be considered as supportive treatment in all symptomatic MCS patients. Although there are no data from controlled trials, controlling pain should always be attempted by tailoring the treatment to individual patients on the basis of the guidelines used in other vasculitides. CONCLUSION Although there are few controlled randomised trials of MCS treatment, increasing knowledge of its pathogenesis is opening up new frontiers. The recommendations provided may be useful as provisional guidelines for the management of MCS.

Management of chronic viral hepatitis in patients with thalassemia: recommendations from an international panel.

Chelation therapy with new drugs prevents cardiac damage and improves the survival of thalassemia patients. Liver diseases have emerged as a critical clinical issue. Chronic liver diseases play an important role in the prognosis of thalassemia patients because of the high frequency of viral infections and important role of the liver in regulating iron metabolism. Accurate assessment of liver iron overload is required to tailor iron chelation therapy. The diagnosis of hepatitis B virus- or hepatitis C virus-related chronic hepatitis is required to detect patients who have a high risk of developing liver complications and who may benefit by antiviral therapy. Moreover, clinical management of chronic liver disease in thalassemia patients is a team management issue requiring a multidisciplinary approach. The purposes of this paper are to summarize the knowledge on the epidemiology and the risks of transmission of viral infections, to analyze invasive and noninvasive methods for the diagnosis of chronic liver disease, to report the knowledge on clinical course of chronic viral hepatitis, and to suggest the management of antiviral therapy in thalassemia patients with chronic hepatitis B or C virus or cirrhosis.

Benefits and Complications of Regular Blood Transfusion in Patients with Beta-Thalassaemia major

Early and regular blood transfusion therapy in patients with homozygous β-thalassaemia decreases the complications of severe anaemia and prolongs survival. In the long term, however, the beneficial effects of transfusions are limited by the organ damage resulting from iron overload, a consequence of the body’s limited capacity to excrete iron, and by the complications of infection with blood-borne agents. Transfusion regimens for β-thalassaemia have changed substantially during the past four decades. In current protocols, pre-transfusion haemoglobin concentration should not exceed 95 g/l. This allows adequate control of anaemia, with a relatively low rate of iron accumulation. Although iron chelation therapy has successfully improved survival free from cardiac disease, thalassaemic patients continuously present new clinical challenges. In fact, the vast majority of them suffer from post-transfusion chronic hepatitis C, which is expected to significantly contribute to morbidity in the forthcoming years. Furthermore, recent studies demonstrated that thalassaemics are at high risk of acquiring several blood-borne viruses. The potential role of these multiple infections in inducing clinical disease is still uncertain, and needs to be thoroughly clarified in future surveys.

An estimate of the current risk of transmitting blood‐borne infections through blood transfusion in Italy

Summary. We conducted a retrospective cohort study to estimate the incidence of major blood‐borne agents among Italian blood donors and calculated the risk of infection among blood recipients using the ‘incidence/window period model’. The study was conducted among 46 180 blood donors enrolled in six blood centres between 1994 and 1999. During follow‐up, seven new infections were confirmed: three donors seroconverted for anti‐human immunodeficiency virus (HIV); two for anti‐hepatitis C virus (HCV); and two showed hepatitis B surface antigen (HBsAg) reactivity; no cases of syphilis were observed. The incidence rates per 100 000 person/years were: 4·06 (95% CI: 0·82–11·85) for HIV; 2·41 (95% CI: 0·29–8·70) for HCV; and 2·70 (95% CI: 0·32–9·77) for HBsAg; the incidence for total hepatitis B virus (HBV) infection was 9·77 per 100 000 person/years (95% CI: 1·16–35·36). The estimated risk of an infectious blood unit not being detected was: 2·45 (95% CI: 0·13–12·33) per 1 million units for HIV; 4·35 (95% CI: 0·30–22·39) for HCV; and 15·78 (95% CI: 1·16–84·23) for HBV. Overall, an estimated 22·58 per 1 million units are infected. In Italy, the risk of transfusion‐transmitted infections is low and is similar to that in other western countries. The introduction of new more sensitive screening tests could reduce the residual risk of transfusion‐transmitted infection by 40–80%.

Practice guidelines for the treatment of hepatitis C: Recommendations from an AISF/SIMIT/SIMAST expert opinion meeting

It is increasingly clear that a tailored therapeutic approach to patients with hepatitis C virus infection is needed. Success rates in difficult to treat and low-responsive hepatitis C virus patients are not completely satisfactory, and there is the need to optimise treatment duration and intensity in patients with the highest likelihood of response. In addition, the management of special patient categories originally excluded from phase III registration trials needs to be critically re-evaluated. This article reports the recommendations for the treatment of hepatitis C virus infection on an individual basis, drafted by experts of three scientific societies.

White cell-reduced red cells prepared by filtration : a critical evaluation of current filters and methods for counting residual white cells

White cell (WBC) reduction, red cell (RBC) recovery, and filtration time were determined in 1‐day‐old standard and buffy coat‐depleted RBCs filtered in the laboratory through six commercial filters for WBC reduction. Residual WBCs were counted with a Burker chamber (BC), with a Nageotte chamber (NC), and by flow cytometry (FC). Results show that BC counts were 0 in several cases in which WBCs were detected with NC and FC, which indicated that the traditional BC method is too insensitive in use with currently available filters. Calibration curves performed by FC and with NC with samples containing known concentrations of WBCs from 1000 to 1 per microL showed that both FC and NC detected, on average, 67 percent of WBCs present in the samples (efficiency). However, the efficiency of FC showed small variability (61–70%) at different WBC levels, whereas the variability with NC was large (39–91%). This greater variability prevented the correction of NC counts by using a single factor and indicated difficulty in NC standardization. Therefore, because our main aim was to compare different filters rather than to define absolute levels of WBC contamination, uncorrected FC and NC counts were chosen to be reported. True WBC counts per unit should not exceed values that can be obtained by dividing uncorrected counts by the lowest efficiencies (61% for FC and 39% for NC). Uncorrected NC and FC counts were below 2 × 106 per unit in all units processed through three of the filters and below 5 × 106 per unit in all units processed through the other three. Removal of the buffy coat from RBCs before filtration generally but not invariably caused slightly higher WBC removals. Median RBC recovery in CPD RBCs containing the buffy coat, filtered through four of the filters, was greater than 90 percent, which was significantly higher than that shown by the other two filters. Buffy coat removal prior to filtration involved an additional median loss of 12.4 percent of RBCs. Median filtration time ranged from 5 to 23 minutes per unit. Although NC appears to be less expensive than FC, its performance in experimental and routine conditions requires further evaluation and thorough standardization. Because of the continuous development of more effective filters, more sensitive methods for WBC counting in WBC‐reduced blood components are needed.

Tissue transglutaminase antibodies in patients with end-stage heart failure

OBJECTIVE:For celiac disease (CD), screening a trend has recently emerged to measure tissue transglutaminase antibodies (tTGA) by immunoassays instead of the more laborious endomysial antibodies (EmA), as they recognize the same target, tissue transglutaminase (tTG). However, a high rate of false-positive results has been reported in some patient series with diseases known to be associated with CD. Moreover, tTG is a ubiquitous, multifunctional enzyme, overexpressed in experimental models of heart failure. Therefore, we assessed the specificity of tTGA assays in a large series of EmA-negative patients with end-stage heart failure.METHODS:We studied 288 patients with end-stage heart failure and 60 blood donors. No subject had clinical evidence of CD or IgA deficiency, and all were EmA negative. Serum IgA and IgG tTGA were measured by means of commercial kits using as substrate, either guinea pig or recombinant human tTG. Blocking studies and Western blots were also performed using recombinant human tTG.RESULTS:All blood donor sera were IgA tTGA negative. IgA tTGA positivity was observed in 47.6% and 49.1% of patients with heart failure using, respectively, guinea pig tTG and recombinant human tTG as substrates. Preincubation of positive sera with recombinant human tTG resulted in 81% blocking of IgA tTGA in immunoassay. Western blot analysis confirmed the presence of antibodies against recombinant human tTG. IgA tTGA-positive sera were also IgG tTGA positive.CONCLUSION:IgA and IgG tTGA occur in a large number of EmA-negative patients with end-stage heart failure, and their presence is unlikely to be caused by concomitant CD.