Giuseppe Tagariello

Clinical heterogeneity and predictors of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308 patients

The clinical outcome, response to treatment, and occurrence of acute complications were retrospectively investigated in 308 primary autoimmune hemolytic anemia (AIHA) cases and correlated with serological characteristics and severity of anemia at onset. Patients had been followed up for a median of 33 months (range 12-372); 60% were warm AIHA, 27% cold hemagglutinin disease, 8% mixed, and 5% atypical (mostly direct antiglobulin test negative). The latter 2 categories more frequently showed a severe onset (hemoglobin [Hb] levels ≤6 g/dL) along with reticulocytopenia. The majority of warm AIHA patients received first-line steroid therapy only, whereas patients with mixed and atypical forms were more frequently treated with 2 or more therapy lines, including splenectomy, immunosuppressants, and rituximab. The cumulative incidence of relapse was increased in more severe cases (hazard ratio 3.08; 95% confidence interval, 1.44-6.57 for Hb ≤6 g/dL; P < .001). Thrombotic events were associated with Hb levels ≤6 g/dL at onset, intravascular hemolysis, and previous splenectomy. Predictors of a fatal outcome were severe infections, particularly in splenectomized cases, acute renal failure, Evans syndrome, and multitreatment (4 or more lines). The identification of severe and potentially fatal AIHA in a largely heterogeneous disease requires particular experienced attention by clinicians.

Antibody to Hepatitis C Virus and Liver Disease in Volunteer Blood Donors

OBJECTIVE To evaluate the specificity of antibodies to hepatitis C virus (anti-HCV) and their relation to liver disease in blood donors. DESIGN Case series of consecutive blood donors found positive for anti-HCV by enzyme-linked immunosorbent assay (ELISA). Patients were evaluated for antibody specificity using a recombinant immunoblotting assay (RIBA) and were evaluated for biochemical evidence of liver disease. Patients showing increased alanine aminotransferase (ALT) levels had a liver biopsy. SETTING University hospital. PARTICIPANTS Fifty consecutive blood donors found to be anti-HCV positive on both an initial and repeat ELISA. Inclusion criteria were as follows: an absence of hepatitis B surface antigens and non-organ-specific autoantibodies; a daily alcohol intake of less than 50 g; no history of recent hepatotoxic drug use; and normal serum levels of alpha 1 antitrypsin, ceruloplasmin, and copper. MAIN RESULTS Anti-HCV positivity was confirmed by RIBA in only 13 of 50 donors (26%) who had positive ELISA results. These 13 donors had an elevated ALT level and histologic evidence of chronic hepatitis, which was active in 8 patients (62%) and had already produced cirrhosis in 2 patients (15%). In contrast, the 17 donors with an intermediate RIBA pattern had only mild and often nonspecific histologic liver abnormalities. The 20 patients with a negative RIBA result had normal ALT levels. CONCLUSION In blood donors, the anti-HCV RIBA is not only more specific than the anti-HCV ELISA, but is also useful in identifying patients who have an underlying chronic liver disease.

ANTIBODY RESPONSE TO PRE-S2 AND HEPATITIS B VIRUS INDUCED LIVER DAMAGE

Antibodies to the pre-S2 encoded sequence of the hepatitis B virus (HBV) envelope were detected in 83% of patients recovering from acute hepatitis B. Such antibodies were absent in cases showing chronic evolution and were found in less than 10% of chronic hepatitis B cases, with no relation to liver disease activity. In acute infection anti-pre-S2 became detectable when maximum liver damage had already occurred and was still detectable in 30% of the cases tested 5-7 years after recovery, as well as in 40% of healthy individuals with naturally acquired immunity to HBV. 10 out of 20 recipients of a plasma-derived, pre-S2-containing, HB vaccine acquired anti-pre-S2 and had no evidence of concurrent liver damage or of autoimmune reactions to human albumin or of suppression of the anti-HBs response. These findings indicate that the antibody response to pre-S2 is a marker of HBV clearance and has no role in the pathogenesis of HBV-related liver damage.

Efficacy and inhibitor development in previously treated patients with haemophilia A switched to a B domain-deleted recombinant factor VIII

There have been recent reports of unexpected poor efficacy of a B‐domain‐deleted recombinant factor VIII (BDD‐rFVIII) in haemophiliacs, and inhibitor development in previously treated patients (PTPs) switched to BDD‐rFVIII. The results of a 6‐month prospective study of 25 PTPs and of a retrospective survey of 94 PTPs, all switched to BDD‐rFVIII, were used to evaluate efficacy and inhibitor development. The prospective study showed that 89% of 362 bleeds were controlled by one to two infusions, reproducing the efficacy profiles of other recombinant products (rFVIIIs). One patient, previously treated with plasma‐derived FVIII only, developed a high titre inhibitor (30 BU) after 5 days of exposure. The retrospective survey, carried out in the total Italian PTP population switched to BDD‐rFVIII, involved 19 PTPs at higher inhibitor risk due to previous exposure of ≤ 50 days and 75 PTPs at lower inhibitor risk due to previous exposure of >50 days. One patient developed an inhibitor: he was a high‐risk, severe PTP previously exposed to another rFVIII for 3 days only. Among the entire low‐risk population of severe Italian PTPs switched to BDD‐rFVIII (25 in the prospective study, 49 in the retrospective cohort) only one developed an inhibitor (1·3%). These data indirectly support the views that BDD‐rFVIII is equivalent to other rFVIIIs in term of efficacy and inhibitor development.

Hepatitis C virus genotypes and severity of chronic liver disease in haemophiliacs

Summary. We studied the activity and stage of chronic liver disease in 45 HCV‐seropositive/HIV‐seronegative patients with severe haemophilia followed for at least 10 years. HCV‐RNA was detected in serum in 36 patients (80%) Viraemic cases were further analysed for HCV genotypes: 10 (28%) were infected by type la, 10 (28%) by type lb, seven (19%) by type 2, four (11%) by type 3, four (11%) had mixed infections (one by la + lb, one by la 4‐ 2, one by type 2+3, and one by la + 2 + 3). ALT levels were within the normal range in 55% of the HCV‐RNA negative patients but in only 11% of the viraemic cases. Results show a trend for higher levels of ALT in HCV‐RNA‐positive patients compared with those without viraemia (98 ± 56 v 60 ± 61), and particularly with patients with type 3 HCV infection (148 ± 44). We suggest that a slow progression of chronic liver disease occurs in haemophilic HCV‐positive/HiV‐negative patients and conclude that presence of HCV‐RNA in serum correlates well with cytolitic damage but, in the time‐scale of our follow‐up period, commonly used clinical‐laboratory parameters cannot predict the chronic evolution of liver infection or identify differences in disease progression in relation to specific HCV subtypes.

Concurrent total hip and knee replacements in a patient with haemophilia with inhibitors using recombinant factor VIIa by continuous infusion

Summary.  The introduction of activated recombinant factor VIIa (rFVIIa) has allowed elective surgery to be safely performed in haemophiliacs with inhibitors. The main problems associated with its use are the short half‐life, necessitating frequent intravenous injections, and its very high cost. Here we describe, for the first time, the performance of total hip and knee replacements in a haemophiliac with inhibitors at the same operation. The amount of rFVIIa concentrate used (8.57 mg) was similar to that normally used for a single joint replacement. The use of continuous infusion allowed for easier administration and further contributed to the reduction in cost as it avoids the peak levels associated with bolus injections.

Intra-articular rifamycin in haemophilic arthropathy.

Synoviorthesis is the intra‐articular injection of chemical or radioactive substances able to produce fibrosis of hypertrophied synovium, which has proved effective in the treatment of chronic haemophilic synovitis. Between September 1999 and October 2001 we treated 28 outpatients (25 with haemophilia A, three with haemophilia B). Our treatment was focused on pain and functional limitation of joints. A schedule was adopted to treat each joint using intra‐articular rifamycin once a week, repeated five times. Patients were covered with factor replacement on demand. Oral analgesia was offered as required because of acute but transient painful inflammatory reaction. Their median age was 34 years (range 15–60 years). The indication for synoviorthesis was chronic synovitis characterized by recurrent haemarthroses, persistent pain and limited range of motion (ROM). Thirty‐five joints were treated with a total of 169 injections, including six joints (20%) in patients with inhibitors. In five patients two joints were treated in the same session. Thirty procedures were completed: 24 (80%) were considered effective (as excellent or good), while six were considered insufficient (20%). Pain was reduced in 96% of cases and in 70% the ROM was improved. In our experience intra‐articular infiltration with rifamycin appears to be effective in reducing joint pain and in improving the ROM. The procedure presents a low risk of bleeding, can be used for patients with inhibitors and multiple joints can be treated without any additional cost.

Prevalence of anti-FVIII antibodies in severe haemophilia A patients with inversion of intron 22

The aim of our study was to investigate whether haemophilia A patients with inversion of intron 22 are at high risk for non‐inhibitory anti‐FVIII antibodies development detected by ELISA. It is known that patients with severe forms of haemophilia A are more likely to develop anti‐FVIII antibodies. The incidence of inhibitory anti‐FVIII antibodies in patients with factor VIII gene inversion has been extensively evaluated, but if this defect has to be considered a predisposing factor is still debatable. Non‐inhibitory anti‐FVIII antibodies are attracting interest, due to the potential influence on FVIII half‐life. Our data show that FVIII gene inversion was a major predisposing factor for anti‐FVIII antibodies development.

Factor VIII gene intron 1 inversion: lower than expected prevalence in Italian haemophiliac severe patients.

Haemophilia A is characterized by a wide heterogeneity as many different mutations have been detected in the coding, intronic and regulatory regions of the Factor VIII (F8) gene. So far, the most recurrent molecular defect is the inversion involving the intron 22 (invint22), accounting for almost half of the patients with severe disease [1,2]. Recently, a new inversion mutation has been described, which affects intron 1 [3], resulting from homologous recombination between two 1-kb repeats shown to be opposite in orientation, Int1h-1, which is in F8 intron 1 and the nearly identical to Int1h-2, a more telomeric extragenic region. Homologous pairing followed by recombination results in disruption of the F8 gene with separation and inversion of the 5 end including exon 1 from the rest of the gene, in a very similar way to the invint22 [2]. The recombined F8 gene is presumably unable to produce any F8 protein. The prevalence described by different investigating groups for the intron 1 inversion (invint1) is between 3 and 5% in severe patients [3,4,5,6,7,12] being the most frequent mutation in those patients, after the invint22. We have analysed 327 severe patients referred to our haemophilia centre for invint22 by Long PCR [8] and we have found 137 patients with this mutation. Among the investigated patients, 293 were unrelated and the invint22 accounts for 121 haemophiliacs, with a prevalence of 41%, according to previously published studies [9]. Among the remaining severe unrelated haemophiliacs, we have analysed 71 patients for the causative mutation by using conformation sensitive gel electrophoresis (CSGE), an heteroduplex based method for screening the regulatory and coding gene regions of the F8 gene, followed by sequencing of any DNA fragments suspected to contain a mutation [10]. In five cases (7%) it was not possible to identify the causative mutation. The invint1 has been investigated in 100 unrelated patients negative for invint22 and negative or as yet uninvestigated for other mutations in the F8 gene. The analysis consists of two multiplex PCRs: one reaction uses the primers specific for intronic Int1h-1 (9F and 9cR) and the primers specific for the sequence flanking extragenic Int1h-2 on its centromeric side (int1h-2F); the other reaction contains the

The JAK2(V617F) tyrosine kinase mutation in blood donors with upper-limit haematocrit levels.

BACKGROUND It is not rare to observe in blood donors a level of haematocrit (Hct) above or close to the highest normal limit. In the case of blood donors the diagnosis and clinical evaluation of this alteration may be complicated by regular blood donations that can mask an underlying disease such as polycythaemia vera. Recently a single acquired mutation in the Janus kinase 2 gene (JAK2) on chromosome 9 was identified and it was found that the incidence of this mutation was high in patients with polycythaemia vera. MATERIAL AND METHODS From the January 1, 2006 to December 31, 2006 all consecutive donors with a Hct above 50% if males (n=84) and 46% if females (n=19) underwent JAK2 mutation analysis. Seventy-nine donors (59 males and 20 females) whose Hct was normal at their last blood donation were randomly selected and used as controls. RESULTS Among the group of blood donors with a high Hct, we identified one donor who was positive for the JAK2 mutation. This man had a Hct of 50.6% at his last donation, while his average Hct in the preceding year was 51.7%. The prevalence of the JAK2 mutation could be estimated to be 1%, 0.6% or 0.02% in the three different populations considered: donors with a Hct level above the upper limit of normal, all tested donors or the entire donor cohort attending our transfusion service, respectively. CONCLUSIONS The present study suggests that apparently healthy subjects with repeatedly high levels of Hct may have the acquired mutation in JAK2. Laboratory screening tests for JAK2 may be offered to blood donors at transfusion services with expertise in molecular genetics.