Liver infection and COVID-19: the electron microscopy proof and revision of the literature.

OBJECTIVE: COVID-19, the newly emerging infectious disease, has been associated with acute liver injury, often related to progression to severe pneumonia. The association between moderate-severe liver injury and more severe clinical course of COVID-19 has suggested that liver injury is prevalent in severe than in mild cases of COVID-19, while no difference in liver involvement has been reported between survivors and non-survivors. The spectrum of liver involvement during COVID-19 ranges from an asymptomatic elevation of liver enzymes to severe hepatitis. Only rarely, cases with acute hepatitis have been reported in the absence of respiratory symptoms. Both epithelial and biliary cells possess the angiotensin-converting enzyme-2 receptors that SARS-CoV-2 uses to be internalized. However, to our knowledge, no ultrastructural identification of the virus in liver cells has been reported to date. Here we provide evidence of SARS-CoV-2 in the liver of two patients, a 34-year-old woman and a 60-year-old man with COVID-19. PATIENTS AND METHODS: We investigated two patients with COVID-19 showing several virions within cytoplasmic vacuoles of cholangiocytes and in endothelial cells of hepatic sinusoids. In both patients, we performed histological and ultrastructural examinations by liver biopsy. After two months, both patients were free of symptoms, and the SARS-CoV-2 infection had resolved. RESULTS: Liver biopsy histological and ultrastructural examination showed liver injury and several virions within cytoplasmic vacuoles of cholangiocytes and in endothelial cells of hepatic sinusoids. CONCLUSIONS: Although most studies in COVID-19 have been focused on the lungs, recently, cholestatic liver pathology has been introduced in the spectrum of pathological changes related to COVID-19. To the best of our knowledge, those presented in this paper are the first images of hepatic SARS-CoV-2 infected liver cells. Our findings suggest a role for cholangiocytes and biliary structures in the COVID-19.

Association between red cell distribution width and response to methotrexate in rheumatoid arthritis.

Red cell distribution width (RDW) is an unconventional biomarker of inflammation. We aimed to explore its role as a predictor of treatment response in rheumatoid arthritis (RA). Eighty-two RA patients (55 females), median age [interquartile range] 63 years [52-69], were selected by scanning the medical records of a rheumatology clinic, to analyze the associations between baseline RDW, disease activity scores and inflammatory markers, as well as the relationship between RDW changes following methotrexate (MTX) and treatment response. The lower the median baseline RDW, the greater were the chances of a positive EULAR response at three months, 13.5% [13.0-14.4] being among those with good response, vs 14.0% [13.2-14.7] and 14.2% [13.5- 16.0] (p=0.009) among those with moderate and poor response, respectively. MTX treatment was followed by a significant RDW increase (p<0.0001). The increase of RDW was greater among patients with good EULAR response, becoming progressively smaller in cases with moderate and poor response (1.0% [0.4-1.4] vs. 0.7 [0.1-2.0] vs. 0.3 [-0.1-0.8]; p=0.03). RDW is a strong predictor of early response to MTX in RA. RDW significantly increases after MTX initiation in parallel to treatment response, suggesting a role as a marker of MTX effectiveness.

Daily life changes and adaptations investigated in 154 families with a child suffering from a rare disability at a public centre for rare diseases in Northern Italy.

BACKGROUND: Living with a disabled child has profound effects on the entire family. With a prevalence of developmental disabilities around 2,5 %, there is a considerable need to promote improvements in the health care system. Little is known about changes and adaptations in the lives of affected families and this paucity of information hinders the improvement of services. This study sought to explore the needs and changes in the everyday life of families with children suffering from rare diseases of varying severity, with and without mental disability. The aim was to measure the socio-demographic characteristics, health care problems and living conditions of a large cohort of families with an affected member. METHODS: A sample of 154 families was recruited between September 2011 and April 2013 to respond to a 136 item questionnaire that explored different areas of concern (diagnosis and follow-up of clinical specialists, relationship with pediatrician, rehabilitation, school, work, institutional and/or private support, child care needs and family relationships). RESULTS: All parents answered the questionnaire. They were satisfied with the services provided in particular for diagnosis and follow-up, relationships with the family pediatrician, rehabilitation services and school, regardless of the severity of condition, presence of intellectual disability (ID) or absence of diagnosis. Negative scores were reported for institutional and/or private support and family relationships in severe conditions. CONCLUSIONS: The Health Care System should maintain a family-centered care and a multi-agency working, improving quality of life of families with disabled child to allow adaptation. At present these services are uncoordinated and financial support is poor, resulting in a heavy burden for these families.

Long-term maintenance of sustained virological response in liver transplant recipients treated for recurrent hepatitis C.

BACKGROUND: The recurrence of hepatitis C viral infection is common after liver transplant, and achieving a sustained virological response to antiviral treatment is desirable for reducing the risk of graft loss and improving patients' survival. AIM: To investigate the long-term maintenance of sustained virological response in liver transplant recipients with hepatitis C recurrence. METHODS: 436 Liver transplant recipients (74.1% genotype 1) who underwent combined antiviral therapy for hepatitis C recurrence were retrospectively evaluated. RESULTS: The overall sustained virological response rate was 40% (173/436 patients), and the mean follow-up after liver transplantation was 11±3.5 years (range, 5-24). Patients with a sustained virological response demonstrated a 5-year survival rate of 97% and a 10-year survival rate of 93%; all but 6 (3%) patients remained hepatitis C virus RNA-negative during follow-up. Genotype non-1 (p=0.007), treatment duration >80% of the scheduled period (p=0.027), and early virological response (p=0.002), were associated with the maintenance of sustained virological response as indicated by univariate analysis. Early virological response was the only independent predictor of sustained virological response maintenance (p=0.008). CONCLUSIONS: Sustained virological response achieved after combined antiviral treatment is maintained in liver transplant patients with recurrent hepatitis C and is associated with an excellent 5-year survival.

Feasibility and reproducibility of spleen transient elastography and its role in combination with liver transient elastography for predicting the severity of chronic viral hepatitis.

Liver transient elastography (L-TE) is a reliable, noninvasive predictor of disease severity in chronic liver disease of viral aetiology (CLD). Owing to the relationships among severity of CLD, portal hypertension and spleen involvement, the assessment of splenic stiffness (S-TE) may have an added value in staging CLD. Of 132 CLD patients of viral aetiology, 48 with myeloproliferative disorders (MD) and 64 healthy volunteers (HV), were concurrently investigated by both L-TE and S-TE. Liver disease severity was staged by liver biopsy (LB; Metavir) taken concurrently with TE examination and upper gastrointestinal tract endoscopy for gastro-oesophageal varices. The S-TE inter-observer agreement was analysed by an intra-class correlation coefficient (ICC); L-TE and S-TE accuracy was evaluated by receiver operating characteristic (ROC) curve analysis. Logistic regression analysis assessed the independent effect of L-TE and S-TE as predictors of hepatic fibrosis stage. S-TE failed in 22 CLD (16.6%), 12 (25%) MD and 12 (18%) HV. In the three groups, the ICC was 0.89 (0.84-0.92), 0.90 (0.85-0.94) and 0.86(0.80-0.91), respectively. In the CLD group, L-TE and S-TE independently predicted significant fibrosis (OR 5.2 and 4.6) and cirrhosis (OR 7.8 and 9.1), but at variance from L-TE, S-TE was independent from liver necroinflammation and steatosis. The NPV of S-TE for gastro-oesophageal varices was 100% using a 48 kPa cut-off. In CLD, spleen stiffness alone or in combination with hepatic stiffness can be reliably and reproducibly assessed by TE with the added value of improving the noninvasive diagnosis of severe liver disease and excluding the presence of oesophageal varices.

Biochemical criteria at 1 year are not robust indicators of response to ursodeoxycholic acid in early primary biliary cirrhosis: results from a 29-year cohort study.

BACKGROUND: In primary biliary cirrhosis (PBC), biochemical criteria at 1 year are considered surrogates of response to ursodeoxycholic acid (UDCA). However, due to the slow natural history of PBC, evaluation at 1 year may be suboptimal to assess the therapeutic response, particularly in early disease. AIM: To determine whether evaluation of biochemical criteria at 1 year is a reliable surrogate of UDCA response in early PBC. METHODS: We analysed the prospectively collected data of 215 patients (untreated = 129; UDCA-treated = 86) with early PBC (normal baseline bilirubin/albumin) and a median follow-up of 8 years (range: 1-29.1). The 1-year attainment rates of the Barcelona, Paris-I, Paris-II and Toronto definitions, and their predictive relevance for a poor outcome (death, transplantation, complications of cirrhosis), were assessed either as a result of UDCA or no treatment. Independent associations with attaining each UDCA response definition were identified by multivariate analysis. RESULTS: Untreated patients displayed 1-year biochemical features compatible with 'treatment response' at rates (Barcelona: 36.4%, Paris-I: 66.7%, Toronto: 59.7%, Paris-II: 40.3%) similar to those obtained under UDCA. Depending on the definition, baseline ALP≤3xULN (OR: 4.80-35.90), AST≤2xULN (OR: 5.63-9.34) and early histological stage (OR: 3.67-3.87) were the stronger predictors for attaining the criteria. UDCA treatment was associated with attaining Barcelona (OR = 2.16) and Paris-II (OR = 2.84), but not Paris-I, and not Toronto definition when excluding late histological cases. Paris-I criteria were significantly predictive of long-term outcomes (HR = 2.83) in untreated patients. CONCLUSIONS: In early PBC, biochemical criteria at 1 year reflect severity of the disease rather than the therapeutic response to UDCA.

Dual cut-off transient elastography to assess liver fibrosis in chronic hepatitis B: a cohort study with internal validation.

BACKGROUND: Transient elastography has gained popularity to stage liver fibrosis in chronic viral hepatitis, however, diagnostic cut-offs for severe fibrosis in chronic hepatitis B are poorly defined. AIM: To evaluate an algorithm with two distinct cut-offs for positive and negative prediction of significant fibrosis and cirrhosis in chronic hepatitis B patients. METHODS: Two cohorts of treatment-naïve patients with chronic hepatitis B (125 training and 92 validations) were consecutively and concurrently examined by percutaneous liver biopsy and transient elastography. Fibrosis was staged by Metavir (significant fibrosis = F ≥ 2; cirrhosis = F4) in ≥ 2 cm long liver tissue cores. RESULTS: A >13.1 kPa positive and a ≤ 9.4 kPa negative cut-off for cirrhosis had a >90% sensitivity and specificity, with an accuracy of 94%. The corresponding cut-offs for F ≥ 2 were >9.4 and ≤ 6.2 kPa, thus classifying 56% of patients with an overall accuracy of 90%. In the validation cohort, F4 and F ≥ 2 were predicted by the above transient elastography cut-offs with an overall accuracy >90%. In 165 patients with higher than upper limit of normal transaminase activity the dual cut-off algorithm of transient elastography was as accurate as in the 52 patients with normal alanine aminotransferase values in the prediction and exclusion of cirrhosis, only. CONCLUSIONS: A dual cut-off algorithm allowed for correctly classifying both significant fibrosis and cirrhosis in the majority of the patients with chronic hepatitis B, independent of alanine aminotransferase values, thus reducing the need for liver biopsy investigations.

Predicting the advent of ascites and other complications in primary biliary cirrhosis: a staged model approach.

BACKGROUND: Current survival models for primary biliary cirrhosis have limited precision for medium and long-term survival. Aim To describe a prognostic model for the advent of complications in primary biliary cirrhosis as the first approach to a staged prognostic model. METHODS: From an established database of 289 consecutive primary biliary cirrhosis patients referred to Royal Free Hospital over 12 years (mean follow-up of 4.1 years), baseline characteristics at referral were evaluated by Cox-proportional hazards regression modelling. RESULTS: The following complications occurred de novo: 85 ascites/peripheral oedema, 40 oesophagogastric varices, 63 encephalopathy, 29 spontaneous bacterial peritonitis and/or septicaemia, 59 symptomatic urinary tract infections. Age, albumin, log(10)(bilirubin), presence of ascites at referral, variceal bleeding within 6 weeks before referral, detection of oesophagogastric varices at or before referral were significant at multivariate analysis with different combinations and coefficients for each complication. The model for predicting ascites and/or peripheral oedema best fitted the observed data (ROC = 0.7682, S.E. = 0.0385). CONCLUSIONS: The known prognostic factors in primary biliary cirrhosis also model the advent of complications. In view of the prognostic importance of ascites and its more robust statistical model, ascites and/or peripheral oedema could represent, following validation, the most suitable staged model in primary biliary cirrhosis to improve precision in survival modelling.

Serum autoantibodies against cytochrome P450 2E1 (CYP2E1) predict severity of necroinflammation of recurrent hepatitis C.

We previously reported that autoantibodies against cytochrome P4502E1 (CYP2E1) are frequent in patients with chronic hepatitis C. As autoimmune reactions are increasingly detected after orthotopic liver transplantation (OLT), this study investigates prevalence and significance of anti-CYP2E1 autoantibodies in 46 patients with post-OLT recurrent hepatitis C. IgG against recombinant human CYP2E1 above the control threshold was detected in 19 out 46 (41%) sera collected immediately before OLT and in 15 out 46 (33%) sera collected at the time of the 12 months follow-up liver biopsy. Although anti-CYP2E1 reactivity was not modified by OLT, the patients with persistently elevated anti-CYP2E1 IgG (n = 12; 26%) showed significantly higher prevalence of recurrent hepatitis with severe necroinflammation and fibrosis than those persistently negative or positive only either before or after OLT. Moreover, the probability of developing severe necroinflammation was significantly higher in persistently anti-CYP2E1-positive subjects. Multivariate regression and Cox analysis confirmed that the persistence of anti-CYP2E1 IgG, together with a history of acute cellular rejection and donor age >50 years, was an independent risk factor for developing recurrent hepatitis C with severe necroinflammation. We propose that autoimmune reactions involving CYP2E1 might contribute to hepatic damage in a subgroup of transplanted patients with recurrent hepatitis C.

ERCP and short-term stent-trial in patients with anastomotic biliary stricture following liver transplantation.

BACKGROUND: Anastomotic biliary stricture represents one of the possible factors leading to liver dysfunction after transplantation. PURPOSE: Our aims were to evaluate the role of endoscopic retrograde cholangio-pancreatography and a short-term stenting (stent-trial) in assessment of the clinical relevance of the biliary stricture. MATERIALS AND METHODS: Thirty transplanted patients for HCV (n=17) or non-HCV (n=13)-related cirrhosis (27M, median age 53 yr, range 24-67 yr) who developed persistently abnormal liver function tests and presented with an anastomotic biliary stricture suggested by non-invasive cholangiography, underwent endoscopic retrograde cholangio-pancreatography. If the stricture was confirmed, dilation was performed and a plastic stent was placed. Clinical and biochemical evaluation was done one and two months later. Resolution of symptoms and normalization or > 50% reduction of at least one liver function test were needed to consider the stricture as clinically relevant. Patients were followed up for a median of 19 months. RESULTS: Endoscopic retrograde cholangio-pancreatography was successful in 29 patients and confirmed the anastomotic biliary stricture in 19 (66%); 14 patients underwent endoscopic dilation and stenting and five patients underwent surgery. The stent-trial suggested the stricture to be clinically relevant in 7 of 14 patients, confirmed by prolonged stenting and follow-up. A trend towards a higher likelihood of a clinically relevant stricture was observed in HCV-negative compared to HCV-positive patients (5 of 7, 71% vs 2 of 7, 29% , respectively; p=0.1). CONCLUSIONS: Our data suggest that endoscopic retrograde cholangio-pancreatography is a valuable tool to evaluate the clinical relevance of an anastomotic stricture, when coupled with a short-term stent-trial.

Transient elastography predicts fibrosis progression in patients with recurrent hepatitis C after liver transplantation.

OBJECTIVE: Transient elastography (TE) allows non-invasive evaluation of the severity of liver disease in patients with chronic hepatitis C. This procedure, however, warrants further validation in the setting of liver transplantation (LT), including patients under follow-up for recurrent hepatitis C. SETTING: Tertiary referral hospital. PATIENTS: 95 patients (75 males) transplanted for end-stage liver disease due to hepatitis C virus. INTERVENTIONS: Paired liver biopsy (LB) and TE were carried out 6-156 (median, 35) months after LT. 40 patients with recurrent hepatitis C sequentially evaluated 6-21 months apart. MAIN OUTCOME MEASURES: Clinical, laboratory and graft histological features influencing TE results. RESULTS: Median TE values were 7.6 kPa in the 90 patients with a successful TE examination, being 5.6 kPa in the 30 patients with Ishak fibrosis score (S) of 0-1, 7.6 kPa in the 38 with S2-3; 16.7 kPa in the 22 with S4-6, (p < 0.0001). Areas under the ROC curves were 0.85 (95% CI, 0.76 to 0.92) for S > or = 3, 0.90 (95% CI, 0.82 to 0.95) for S > or = 4 with 7.9 and 11.9 kPa optimal TE cut-off (81% and 82% sensitivity, 88% and 94% negative predictive value, respectively). Fibrosis, necroinflammatory activity and higher than 200 IU/l gamma-glutamyl transpeptidase levels independently influenced TE results. During post-LT follow-up, TE results changed in parallel with grading (r = 0.63) and staging (r = 0.71), showing 86% sensitivity and 92% specificity in predicting staging increases. CONCLUSIONS: TE accurately predicts fibrosis progression in LT patients with recurrent hepatitis C, suggesting that protocol LB might be avoided in patients with improved or stable TE values during follow-up.

Clinical features and prognosis of primary biliary cirrhosis associated with systemic sclerosis.

BACKGROUNDS AND AIMS: To evaluate the prognosis of primary biliary cirrhosis (PBC) together with systemic sclerosis (SSc), as this is unknown. METHODS AND RESULTS: A PBC database of 580 patients identified 43 with PBC and SSc: two patients with PBC alone were matched to each PBC-SSc patient for serum bilirubin concentration at the initial visit. Forty (93%) patients had limited cutaneous SSc. At diagnosis of PBC, median values were: 49.7 years, bilirubin 17 micromol/l, and albumin 40.5 g/l. Liver diagnosis occurred a median 4.9 years after SSc in 24 (56%) patients. In matched patients, median values at diagnosis were: 53.2 years, bilirubin 12 micromol/l, and albumin 41 g/l. Median follow up was similar: 3.16 years (PBC-SSc) and 4.8 years (PBC alone). The risk of transplantation or death from diagnosis, adjusting for sex, age, log bilirubin, and alkaline phosphatase was significantly lower in PBC-SSc (hazard ratio 0.116, p=0.01) due to less transplantation (hazard ratio 0.068, p=0.006). The rate of bilirubin increase was less in PBC-SSc (p=0.04). Overall survival was similar (hazard ratio 1.11, p=0.948); there were nine deaths (21%) in PBC-SSc (six SSc related and two liver related) and nine (11%) in PBC alone (six liver related). CONCLUSIONS: Liver disease has a slower progression in PBC-SSc compared with matched patients with PBC alone.

Gender and liver fibrosis in chronic hepatitis: the role of iron status.

BACKGROUND: The role of gender in the progression of fibrosis in chronic hepatitis C is still under investigation. AIM: To investigate whether gender affects the progression of liver disease and/or hides other risk factors. METHODS: A prospective series of 121 consecutive patients with chronic hepatitis C underwent liver biopsy. Grading and staging for chronic hepatitis were each evaluated according to Ishak's classification. RESULTS: In univariate and multivariate analysis on the whole group of patients, male gender was not associated either with significant liver fibrosis (Ishak's score > 2) or with cirrhosis (Ishak's score > 4). On the contrary, in univariate analysis on patients aged < or = 50 years, male gender was nearly significantly (P = 0.06) predictive of liver fibrosis, whereas it was not in patients > 50 years. Hepatic iron grading, along with age, was an independent factor associated with fibrosis. Moreover, the values of all the variables which describe iron status were significantly higher in males aged < or = 50 years in comparison with females of the same age. CONCLUSIONS: In chronic hepatitis C, male gender may be predictive of liver fibrosis only in patients aged < or = 50 years. Among fibrogenetic factors hidden by gender, iron status could play a major role.

Long-term ursodeoxycholic acid therapy for primary biliary cirrhosis: a follow-up to 12 years.

BACKGROUND: It is uncertain whether ursodeoxycholic acid therapy slows down the progression of primary biliary cirrhosis, according to two meta-analyses. However, the randomized trials evaluated had only a median of 24 months of follow-up. AIM: To evaluate long-term ursodeoxycholic acid therapy in primary biliary cirrhosis. METHODS: We evaluated 209 consecutive primary biliary cirrhosis patients, 69 compliant with ursodeoxycholic acid and 140 untreated [mean follow-up 5.79 (s.d. = 4.73) and 4.87 (s.d. = 5.21) years, respectively] with onset of all complications documented. Comparison was made following adjustment for baseline differences according to Cox modelling, Mayo and Royal Free prognostic models. RESULTS: Bilirubin and alkaline phosphatase concentrations improved with ursodeoxycholic acid (at 36 months, P = 0.007 and 0.018, respectively). Unadjusted Kaplan-Meier analysis showed benefit (P = 0.028), as 44 (31%) untreated and 15 (22%) ursodeoxycholic acid patients died or had liver transplantation. However, there was no difference when adjusted by Cox modelling (P = 0.267), Mayo (P = 0.698) and Royal Free models (P = 0.559). New pruritus or fatigue or other complications were not different, either before or after adjustment for baseline characteristics. CONCLUSIONS: Long-term ursodeoxycholic acid therapy did not alter disease progression in primary biliary cirrhosis patients despite a significant improvement in serum bilirubin and alkaline phosphatase consistent with, and similar to, those seen in ursodeoxycholic acid cohorts in randomized trials.

Antiphospholipid antibodies associated with alcoholic liver disease specifically recognise oxidised phospholipids.

BACKGROUND: Circulating antiphospholipid antibodies (aPL) are often detected in patients with alcoholic liver disease (ALD) but little is known about the causes of their formation. AIMS: We have evaluated whether ethanol mediated oxidative injury might promote the development of aPL in ALD. PATIENTS AND METHODS: IgG against beta(2) glycoprotein 1 (beta(2)-GP1), cardiolipin, and human serum albumin (HSA) complexed with either oxidised arachidonic acid (HSA-APP) or malondialdehyde (HSA-MDA) were assayed by ELISA in heavy drinkers with or without ALD and in healthy subjects. RESULTS: Circulating IgG recognising cardiolipin were significantly higher in ALD patients than in controls. However, anticardiolipin reactivity of ALD sera was only evident using, as the antigen, oxidised cardiolipin but not oxidation protected cardiolipin. In ALD patients, individual values of IgG antioxidised cardiolipin were associated with the titres of antibodies against HSA-MDA and HSA-APP (r=0.68 and 0.72, respectively; p<0.0001) used as markers of oxidative stress. ALD patients also displayed increased levels of antibodies against phospholipid binding protein beta(2)-GP1, and individual reactivity towards oxidised cardiolipin and beta(2)-GP1 were highly correlated (r=0.85; p<0.0001). IgG binding to oxidised cardiolipin, HSA-MDA, and HSA-APP was also significantly higher in beta(2)-GP1 positive than in beta(2)-GP1 negative sera. However, preadsorption of beta(2)-GP1 positive sera on beta(2)-GP1 coated ELISA plates reduced reactivity to oxidised cardiolipin by 80%, without affecting that to HSA-APP or HSA-MDA. CONCLUSIONS: Ethanol induced oxidative injury is associated with the development of antibodies targeting complexes between oxidised cardiolipin and beta(2)-GP1. These antibodies might account for high aPL titres observed in patients with severe ALD.

Chronic hepatitis C treated with phlebotomy alone: biochemical and histological outcome.

BACKGROUND: In patients with chronic hepatitis C, the histological outcome of long term phlebotomy is unknown. AIM: To investigate biochemical and histological findings before and after phlebotomy in chronic hepatitis C. PATIENTS: Twenty-four non-haemochromatotic patients with chronic hepatitis C were treated with long-term phlebotomy alone. RESULTS: Hepatic iron concentration had decreased in all patients who underwent a second liver biopsy, two years after iron depletion was attained and maintained. Histological grading score decreased in four patients, was unchanged in two, and increased in five. Histological staging score decreased in two patients, was unchanged in five, and increased in four. Pretreatment high serum selenium level predicted the reduction of the inflammatory grading score in univariate analysis (p=0.008, while low serum aspartate aminotransferase (p=0.02) and low propeptide of procollagen III (p=0.08) levels predicted the lack of progression of liver fibrosis. Furthermore, when iron depletion was reached, significant reductions of serum levels of aminotransferase, gamma glutamyl transferase (-47%), propeptide of procollagen III, alpha foetoprotein, selenium were observed in 24 patients. No changes in serum hepatitis C virus-RNA levels were found. CONCLUSIONS: Phlebotomy alone seems to be efficacious in suppressing progression of chronic hepatitis C in some patients. Phlebotomy not only induces iron depletion, but it even modifies serum levels of other trace elements involved in the balance between oxidant and antioxidant processes.