Silvano Milani

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

Italian cross-sectional growth charts for height, weight and BMI (2 to 20 yr)

The aim of this study is to extend to pre-school ages the Italian Society for Pediatric Endocrinology and Diabetes (SIEDP)-2002 growth charts for height, weight and body mass index (BMI), to obtain charts (SIEDP-2006) that apply to the Italian population from 2 to 20 yr of age, taken as a whole, or separately in two geographical areas (Central-North Italy and South Italy). The charts are based on a sample of about 70,000 subjects attending infant, primary and secondary schools, between 1994 and 2004. The distribution of the sample by gender, age and geographic area was roughly similar to that of Italian school population in the last decade of the 20th century. Height and weight were measured using portable Harpenden stadiometers and properly calibrated scales, respectively. SIEDP-2006 references are presented both as centiles and as LMS curves for the calculation of SD scores, and include the extra-centiles for overweight and obesity. Large differences in BMI growth pattern emerged between the SIEDP-2006, 2000 CDC and UK90 references: in Italy, BMI is higher and its distribution is more skewed during childhood and adolescence. At the end of growth, median values of the three references are similar, but the 97th centile of 2000 CDC charts is much higher and increases more steeply than that of SIEDP-2006 charts, which on the contrary reach a plateau. SIEDP-2006 references intend to supply pediatricians with a tool that avoids the use of charts that are outdated or that refer to other populations, and thus should be suitable for adequately monitoring the growth of their patients.

Bone loss in response to long-term glucocorticoid therapy

A number of studies have shown that an excess of glucocorticoids induces osteoporosis, but the mechanism(s) and the time course of the reduction of bone mass remain uncertain. In order to clarify this issue we carried out a longitudinal clinical and histomorphometric study of patients requiring long-term glucocorticoid treatment. In 23 patients (9 men, 10 post- and 4 premenopausal women) biochemical and bone histomorphometric investigations were carried out before and during treatment with 10-25 mg/day of prednisone. Histomorphometric analysis of bone biopsies of the iliac crest showed that the decrease of TBV (up to -27%, P less than 0.001) occurs predominantly within the first 5-7 months of treatment; during the subsequent stages, which include observations after 12 months of treatment, only minor changes were observed. Therefore trabecular bone loss can be satisfactorily described by a negative exponential function. None of the other histomorphometric parameters (osteoid surfaces, resorption surfaces, etc.) showed significant changes. However, the histological features of the bone biopsies during steroid therapy, showing a virtual lack of osteoblastic activity, ruled out an increase of bone resorption. Moreover, the dynamic study of the bone formation by double tetracycline labelling showed, in a small subgroup of patients, a decrease of the apposition rates (from 0.763 +/- 0.053 to 0.305 +/- 0.074 microns/day (mean +/- SE) after treatment). No significant changes, at any time during steroid treatment, were observed in serum alkaline phosphatase, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone or urinary calcium excretion. Serum calcium increased significantly within the first 1-2 months of therapy and then it returned to baseline. Urinary hydroxyproline excretion decreased significantly within the first 1-2 months and continued to fall throughout the treatment. Thus, both biochemical and histological findings suggest that long-term glucocorticoid therapy causes a reduction of bone turnover, that the bone loss occurs predominantly within the first 6 months of treatment and that patients with lower bone mass have a lower rate of bone loss.

Dioxin exposure, from infancy through puberty, produces endocrine disruption and affects human semen quality.

Background Environmental toxicants are allegedly involved in decreasing semen quality in recent decades; however, definitive proof is not yet available. In 1976 an accident exposed residents in Seveso, Italy, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Objective The purpose of this study was to investigate reproductive hormones and sperm quality in exposed males. Methods We studied 135 males exposed to TCDD at three age groups, infancy/prepuberty (1–9 years), puberty (10–17 years), and adulthood (18–26 years), and 184 healthy male comparisons using 1976 serum TCDD levels and semen quality and reproductive hormones from samples collected 22 years later. Results Relative to comparisons, 71 men (mean age at exposure, 6.2 years; median serum TCDD, 210 ppt) at 22–31 years of age showed reductions in sperm concentration (53.6 vs. 72.5 million/mL; p = 0.025); percent progressive motility (33.2% vs. 40.8%; p < 0.001); total motile sperm count (44.2 vs. 77.5 × 106; p = 0.018); estradiol (76.2 vs. 95.9 pmol/L; p = 0.001); and an increase in follicle-stimulating hormone (FSH; 3.58 vs. 2.98 IU/L; p = 0.055). Forty-four men (mean age at exposure, 13.2 years; median serum TCDD, 164 ppt) at 32–39 years of age showed increased total sperm count (272 vs. 191.9 × 106; p = 0.042), total motile sperm count (105 vs. 64.9 ×106; p = 0.036), FSH (4.1 vs. 3.2 UI/L; p = 0.038), and reduced estradiol (74.4 vs. 92.9 pmol/L; p < 0.001). No effects were observed in 20 men, 40–47 years of age, who were exposed to TCDD (median, 123 ppt) as adults (mean age at exposure, 21.5 years). Conclusions Exposure to TCDD in infancy reduces sperm concentration and motility, and an opposite effect is seen with exposure during puberty. Exposure in either period leads to permanent reduction of estradiol and increased FSH. These effects are permanent and occur at TCDD concentrations < 68 ppt, which is within one order of magnitude of those in the industrialized world in the 1970s and 1980s and may be responsible at least in part for the reported decrease in sperm quality, especially in younger men.

Neonatal Anthropometric Charts: The Italian Neonatal Study Compared With Other European Studies

Background and Objective: This was a nationwide prospective study carried out in Italy between 2005 and 2007, involving 34 centers with a neonatal intensive care unit. The study reports the Italian Neonatal Study charts for weight, length, and head circumference of singletons born between 23 and 42 gestational weeks, comparing them with previous Italian data and with the most recent data from European countries. Patients and Methods: Single live born babies with ultrasound assessment of gestational age within the first trimester, and with both parents of Italian origin. Only fetal hydrops and major congenital anomalies diagnosed at birth were excluded. The reference set consists of 22,087 girls and 23,375 boys. Results: At each gestational age, boys are heavier than girls by about 4%. Later-born neonates are heavier than firstborn neonates by about 3%. The effects of sex and birth order on length and head circumference are milder. No differences were observed between babies born in central-north Italy and southern Italy. A large variability emerged among European neonatal charts, resulting in huge differences in the percentage of Italian Neonatal Study neonates below the 10th centile, which is traditionally used to define small-for-gestational-age babies. In the last 2 decades prominent changes in the distribution of birth weight emerged in Italy and in the rest of Europe, in both term and preterm neonates. Conclusions: The existing European neonatal charts, based on more or less recent data, were found to be inappropriate for Italy. Until an international standard is developed, the use of national updated reference charts is recommended.

Success and failure in periradicular surgeryA longitudinal retrospective analysis

The objective of the present study was to compare the success rates of 2 different periapical surgical techniques, the traditional technique with rotary instruments and the ultrasonic technique, which uses ultrasonic retrotips. A longitudinal retrospective study was carried out on 302 apices (181 teeth) that had undergone periapical surgery. Surgical outcome was evaluated by 2 independent operators using standardized periapical radiographs. Each radiographic finding was classified into 1 of 4 groups: complete healing, incomplete healing, uncertain healing, and unsatisfactory outcome (failure). An SAS statistical analysis system was used for data management and analysis. Prognostic factors were determined by means of the Fisher exact test. Complete healing after 4.6 years (the average follow-up period) was observed in 68% of the teeth treated through the use of the standard technique and 85% of those treated through the use of the ultrasonic technique. The success rate increased as the follow-up period lengthened (68.4% at 2 years vs 80% at 6 years). The success rate was higher in maxillary (77.9%) than in mandibular (66.1%) teeth. A comparison between the retrofilling materials was not feasible because all teeth in the standard technique group were retrofilled with amalgam and all teeth in the ultrasonic group were retrofilled with Super-EBA.

Postnatal weight increase and growth velocity of very low birthweight infants

Background: Only a few studies have dealt with postnatal growth velocity of very low birthweight (VLBW) infants. Objective: To analyse weight growth kinetics of VLBW infants from birth to over 2 years of age. Patients: A total of 262 VLBW infants were selected; inaccurate estimate of gestational age, major congenital anomalies, necrotising enterocolitis, death, and loss to follow up within the first year were the exclusion criteria. Methods: Body weight was recorded daily up to 28 days or up to discontinuation of parenteral nutrition, weekly up to discharge, then at 1, 3, 6, 9, 12, 18, and 24 months of corrected age. Individual growth profiles were fitted with a seven constant, exponential-logistic function suitable for modelling weight loss and weight recovery, two peaks, and the subsequent slow decrease in growth velocity. Results: After a postnatal weight loss, all infants showed a late neonatal peak of growth velocity between the 7th and 21st weeks; most also experienced an early neonatal peak between the 2nd and 6th week. VLBW infants who were small for gestational age and those with major morbidities grew less than reference VLBW infants who were the appropriate size for gestational age without major morbidities: at 2 years of age, the difference in weight was about 860 g. The more severe growth impairment seen in VLBW infants with major morbidities is almost entirely due to the reduced height of the late neonatal peak of velocity. Conclusions: The growth model presented here should be a useful tool for evaluating to what extent different pathological conditions or nutritional and medical care protocols affect growth kinetics.

Mouse genome-wide association mapping needs linkage analysis to avoid false-positive loci

We carried out genome-wide association (GWA) studies in inbred mouse strains characterized for their lung tumor susceptibility phenotypes (spontaneous or urethane-induced) with panels of 12,959 (13K) or 138,793 (140K) single-nucleotide polymorphisms (SNPs). Above the statistical thresholds, we detected only SNP rs3681853 on Chromosome 5, two SNPs in the pulmonary adenoma susceptibility 1 (Pas1) locus, and SNP rs4174648 on Chromosome 16 for spontaneous tumor incidence, urethane-induced tumor incidence, and urethane-induced tumor multiplicity, respectively, with the 13K SNP panel, but only the Pas1 locus with the 140K SNP panel. Haplotype analysis carried out in the latter panel detected four additional loci. Loci reported in previous GWA studies failed to replicate. Genome-wide genetic linkage analysis in urethane-treated (BALB/c×C3H/He)F2, (BALB/c×SWR/J)F2, and (A/J×C3H/He)F2 mice showed that Pas1, but none of the other loci detected previously or herein by GWA, had a significant effect. The Lasc1 gene, identified by GWA as a functional element (Nat. Genet., 38:888–95, 2006), showed no genetic effects in the two independent intercross mouse populations containing both alleles, nor was it expressed in mouse normal lung or lung tumors. Our results indicate that GWA studies in mouse inbred strains can suffer a high rate of false-positive results and that such an approach should be used in conjunction with classical linkage mapping in genetic crosses.

Age-specific nomogram for the decline in antral follicle count throughout the reproductive period

OBJECTIVE To investigate the relationship between antral follicle count (AFC) and chronological age and to establish normal values for AFC in women with regular menstrual cycles. DESIGN Cross-sectional study. SETTING University hospital. PATIENT(S) Four hundred fifteen premenopausal women were recruited for the study. Data from 362 patients were available for the statistical analysis. INTERVENTION(S) AFC was measured by transvaginal ultrasound examination. MAIN OUTCOME MEASURE(S) Estimating the relationship between AFC and age and developing the AFC nomogram. RESULT(S) The analysis showed a linear decline in AFC with age; for every year increase in age, the median AFC decreases by 0.4. The AFC corresponding to the 5th, 25th, 50th, 75th, and 95th centiles for each age have been calculated. CONCLUSION(S) A linear relationship of AFC to age was found. For the first time, a nomogram reporting normal and interquartile values for AFC, age by age, throughout the reproductive period has been provided. Until now, the interpretation of the measurement was mainly based on the individual experience of the operator, because no normative data were present. Therefore, the establishment of a nomogram of AFC values is the first step to counsel patients on a scientific basis.

Evaluation of acute ischemic stroke using quantitative EEG: A comparison with conventional EEG and CT scan

The sensitivity of quantitative electroencephalogram (EEG) was compared with that of conventional EEG in patients with acute ischaemic stroke. In addition, a correlation between quantitative EEG data and computerized tomography (CT) scan findings was carried out for all the areas of lesion in order to reassess the actual role of EEG in the evaluation of stroke. Sixty-five patients were tested with conventional and quantitative EEG within 24 h from the onset of neurological symptoms, whereas CT scan was performed within 4 days from the onset of stroke. EEG was recorded from 19 electrodes placed upon the scalp according to the International 10-20 System. Spectral analysis was carried out on 30 artefact-free 4-sec epochs. For each channel absolute and relative power were calculated for the delta, theta, alpha and beta frequency bands and such data were successively represented in colour-coded maps. Ten patients with extensive lesions documented by CT scan were excluded. The results indicated that conventional EEG revealed abnormalities in 40 of 55 cases, while EEG mapping showed abnormalities in 46 of 55 cases: it showed focal abnormalities in five cases and nonfocal abnormalities in one of six cases which had appeared to be normal according to visual inspection of EEG. In a further 11 cases, where the conventional EEG revealed abnormalities in one hemisphere, the quantitative EEG and maps allowed to further localize abnormal activity in a more localized way. The sensitivity of both methods was higher for frontocentral, temporal and parieto-occipital cortical-subcortical infarctions than for basal ganglia and internal capsule lesions; however, quantitative EEG was more efficient for all areas of lesion in detecting cases that had appeared normal by visual inspection and was clearly superior in revealing focal abnormalities. When we considered the electrode related to which the maximum power of the delta frequency band is recorded, a fairly close correlation was found between the localization of the maximum delta power and the position of lesions documented by CT scan for all areas of lesion excepting those located in the striatocapsular area.