Cristina Tassinari

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.

Impaired cytokine production by neutrophils isolated from patients with AIDS

Objectives:To determine the ability of neutrophils isolated from HIV-seropositive patients to produce proinflammatory cytokines. Design:The in vitro responsiveness of polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) to lipopolysaccharide (LPS), used in the presence or absence of interferon (IFN)-γ, was determined in 47 HIV-positive patients with advanced stages of virus infection. Methods:Cytokines in cell-free supernatants were measured by enzyme-linked immunosorbent assay or radioimmunoassay. Results:Cell-free supernatants from PMN isolated from the peripheral blood of HIV-positive patients and stimulated with LPS contained increased amounts of tumour necrosis factor (TNF)-α and interleukin (IL)-8 with respect to supernatants obtained from PMN of normal donors. In contrast, release of IL-1β and IL-1ra (IL-1 receptor antagonist) in response to LPS, or LPS plus IFN-γ, was found to be lower in PMN from HIV-positive patients than in PMN from controls, but was significant only in the case of IL-1ra. Furthermore, the release of IL-12 induced by LPS or LPS plus IFN-γ did not significantly differ between PMN from HIV-positive patients and healthy donors. Concerning PBMC, the production of TNF-α and IL-12 in response to LPS, or LPS plus IFN-γ, was found to be significantly higher in cells isolated from HIV-positive patients, whereas the release of IL-1β was significantly lower. In the case of IL-8, no statistically significant difference was found between PBMC isolated from HIV-positive patients and healthy donors. Conclusions:Collectively, the data suggest that in HIV-positive patients with advanced stages of disease, the ability of PMN to produce specific cytokines in response to LPS is significantly altered. Alterations in this ability might contribute to the evolution of HIV disease.

Skewing of the T-cell receptor repertoire in the lung of patients with HIV-1 infection

ObjectivesA bias of the use of T-cell receptor (TCR) Vβ regions has been reported both in peripheral blood and in several tissues in patients with AIDS, including lymph nodes, spleen and salivary glands. Although the disease is frequently characterized by an infiltration of T cells in the lung interstitium, no information is presently available on the configuration of the TCR repertoire in this microenvironment. This study was performed to address the question of whether a bias in T-cell selection occurs in the lung of patients with AIDS. MethodsTCR β-chain variable region (TCR-Vβ) repertoire was analysed by flow cytometry and polymerase chain reaction (PCR) analyses in blood and lung lymphocytes of 13 patients with HIV-1 infection at different stages of the disease. Blood and lung lymphocytes were also assessed for their responsiveness to different superantigenic stimuli represented by staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED and SEE). ResultsFlow cytometry analysis in AIDS patients demonstrated an overexpression of cells bearing Vβ2 and Vβ3 gene segments in the lung compared with peripheral blood of the same subjects, as well as to lung and blood lymphocytes of normal controls. PCR analysis performed in AIDS patients extended these observations and demonstrated a significant bias also in the use of T cells bearing Vβ7 and Vβ9 gene regions in the lung compartment with respect to the blood. Virtually all T cells bearing the over-represented Vβ segment belong to the CD8 subset. Interestingly, T-lymphocyte response to different superantigens demonstrated a low proliferative rate in the lung with respect to the blood in HIV-1-infected patients. ConclusionsThese findings indicate a compartmentalization of cells bearing discrete Vβ gene products in the pulmonary microenvironment of patients with AIDS and suggest that the expansion of specific-Vβ region subsets occurring in the lung might result from triggering by a superantigen.

Lung Lymphocytes: Origin, Biological Functions, and Laboratory Techniques for Their Study in immune-Mediated Pulmonary Disorders

Different types of immunocompetent cells, including T lymphocytes and alveolar macrophages, account for pulmonary host defense. Taking advantage of the availability of the monoclonal antibody technique, cell culture facilities, pure recombinant cytokines, and molecular probes for their genes, in the last few years it has been possible to keenly study the different steps that lead to the compartmentalization of immune response in human lung. Furthermore, the immunological analysis of cells retrieved from bronchoalveolar lavage (BAL) allowed recognition of the importance of immune mechanisms in the evolution of immune-mediated pulmonary disorders. The purpose of this review is to summarize recent advances on the immunologic characterization of lung lymphocytes in health and disease. Following a brief description of the pathways through which the pulmonary lymphoid system contributes to removing potentially harmful inhaled antigenic materials, available laboratory techniques to evaluate the lymphoid component of the pulmonary immune system and their byproducts are discussed. These techniques cover methods for preparing lymphocytes from the BAL fluid and for characterizing lung lymphocytes both in cell suspensions and pulmonary tissue biopsies. Other sections of this review describe the techniques for measuring the immunologic effector functions of lung lymphocytes. We also provide the reader with a flavor of the molecular biology methods used to characterize lymphocytes in the pulmonary microenvironment. The final sections of the review article highlight the pathogenetic role envisaged for lymphoid cells in pulmonary disease states and emphasize the importance of the BAL analysis in the clinical management of the most relevant immune-mediated lung disease.

Total ankle replacement for end-stage arthropathy in patients with haemophilia

Dear Editor, In Haemophilia November 2006 van der Heide [1] and colleagues reported on the feasibility of a total ankle prosthesis (TAP) in two patients with coagulation disorders. Even though it is a recognized fact that the ankle is one of the most affected joints, in haemophiliacs prostheses are uncommon, at least in this group of patients, being considered to be an innovative surgical approach. It is well known that from an early age the ankle joint is frequently affected by bleeding in haemophiliacs and standard haemarthrosis treatment, including replacement therapy administered as primary prophylaxis or on-demand, is not always able to prevent joint damage. Physical therapy, footwear modification or orthoses may be useful but when chronic haemophilic synovitis occurs and recurrent haemarthroses become unresponsive to substitution therapy, synoviorthesis is to be considered using intra-articular injections of radioactive material or chemical substances [2]. These procedures, although effective in reducing the frequency of bleeding, are not able to alter the natural history of a degenerative arthropathy. When advanced ankle arthropathy develops arthrodesis is considered the standard treatment for pain relief [3] but total ankle replacement may also be considered for persistent pain and/or motion improvement [4]. As only limited reports have been published on this subject to date it is hoped that the following description of surgical intervention performed on two of our patients affected by severe haemophilia A (HA) will be helpful. Patient 1 was a 30-year-old male with severe HA and chronic HCV infection. The World Federation of Haemophilia joint score developed by Pettersson and Gilbert was used as the assessment instrument, according to this scale the total ankle score was 8 at the time of orthopaedic evaluation. Surgery was indicated because of limited motion and severe pain but in the year 2000 the patient, being worried about the possibility of further movement loss, refused ankle fusion. In the past he had been identified as being low responder anti-factor VIII (FVIII) inhibitor (max. titre 2 BU), before the surgery in question, however, the inhibitor was not detected. At the beginning of the operation, performed under general anaesthesia, replacement therapy was started with a bolus infusion of 100 IU kg of high-purity plasma-derived FVIII concentrates (Emoclot D.I., Kedrion, Italy) to obtain 100% plasma level. The prosthesis was implanted according to the instructions given by manufacturer, all the components were inserted without cement. Substitution treatment was continued by bolus injections every 12 h and the total amount of FVIII concentrates was 50 000 IU (684 IU Kg). PTT (partial thromboplastin time) and plasma FVIII levels were measured daily and concentrate dosage was adjusted in order to obtain >70% plasma activity. After the surgery the ankle was placed in a splint for 30 days. The postoperative period was uneventful, morphine was given to control the pain, no drainage was applied, there were no haemorrhages and the substitution treatment lasted a total of 10 days. This patient has had a 7-year followup, during which time he has had painless ankle movement and improved walking ability. There have not been any radiographic signs of prosthetic loosening or infection. A STAR prothesis cementless was used; Figs 1 and 2 show postoperative radiographs of this patient. Patient 2 was a 43-year-old male with severe HA, co-infected with HCV and HIV. HIV infection was kept under control by using highly Correspondence: Paolo Radossi, Transfusion Service, Haemophilia and Regional Blood Diseases Centre, Italy. Tel.: +39 0423 732336; fax: +39 0423 732337; e-mail: radossi.p@alice.it

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.

Dramatic post-splenectomy onset of malaria caused by latent Plasmodium vivax in a female immigrant with severe immunological anaemia

Immune-mediated anaemia is characterized by the production of autoantibodies against different erythrocyte antigens resulting in red blood cells being destroyed, mainly in the spleen. First-line therapy consists of immune suppression by steroids, while the second choice of treatment, in resistant or relapsed patients, is splenectomy. The World Health Organisation (WHO) World Malaria Report 2012 summarises data received from 104 malaria endemic countries and territories and updates the 2011 report. According to these latest estimates, there were about 219 million cases of malaria in 2010 and an estimated 660,000 deaths. The disease is still highly endemic in the tropics but has been completely eradicated in some temperate areas, including southern Europe, and partially eradicated in northern Africa1–3. In some malaria cases a concomitant, positive direct antiglobulin test (DAT) can increase disease severity and interfere with a correct diagnosis4. Plasmodium vivax is unique among human malarias in that erythrocyte invasion is almost entirely dependent on the red cell surface receptor, known as the Duffy blood group antigen (Fy)5. When steroids fail, the treatment of choice for immune-mediated chronic anaemia is splenectomy, although this operation can carry the risk of reactivating the infection in the case of underlying silent forms of malaria6,7.

One‐stage replacement of infected knee prosthesis in a patient with haemophilia A and high titre of inhibitors

Dear Editor, Total joint replacement is indicated for end-stage haemophilic arthropathy when pain becomes not responding to medical treatment and impaired function limits normal life activities. In the general population, the risk of postsurgical infections is around 1–2% after knee or hip replacement but this percentage is much higher in haemophiliacs, despite the normal haemostasis obtained during and after surgery [1]. When conservative therapies (antibiotics) fail to control infections, different surgical approaches can be utilized, including debridement with retention of the prosthesis, removing of the prosthesis with arthrodesis, prosthesis exchange by using one or two-stage replacement or amputation [2]. A well-balanced haemostasis is the basic requirement for successful operative interventions and recombinant factor VIIa (rFVIIa; NovoSeven , NovoNordisk, Denmark) has been shown to be effective for haemostasis in haemophiliacs with hightitre factor VIII (FVIII) or factor IX (FIX) inhibitors. A major problem of this therapy is the high cost, which makes this treatment extremely expensive. For this reason and because of the potential advantages of the one-stage replacement of prosthesis, including a single rFVIIa administration, a single anaesthetic session, a shorter hospital stay and finally correspond to patient s desire, we have chosen this approach on this case. Continuous infusion (CI) with coagulation factors has been demonstrated to provide some advantages avoiding peaks and troughs, cheaper due to lower usage of clotting factors and, in the case of rFVIIa, avoids the problem of the very frequent drug administrations due to its short halflife. Other advantages are the possibility to use tranexamic acid and to avoid the anamnestic response to FVIII or FIX which occurs with other alternative treatments such as activated prothrombin complex concentrates or porcine FVIII. In our case, a 47-year-old patient with severe sporadic haemophilia A (FVIII:C < 1%, FVIII intron 22 gene inversion), mild chronic hepatitis C and HIV negative developed severe haemophilic arthropathy and, in 2003, he underwent total right knee replacement. The postoperative course was regular from the haemostatic point of view, but a skin ulcerative lesion persisted on the site of the intervention since 1 month after the surgery. Sixteen months later, after a haemorrhagic event, he had progressive swelling of the knee with deterioration of the function and constant pain. Subsequently, purulent secretion was obtained by arthrocentesis and microbiological tests were positive for Enterococcus faecalis. Therefore, antibiotic therapy was started, driven by antibiogram, with ampicillin and gentamicin, but repeatedly microbiological tests demonstrated E. faecalis persistence. Because of the failure of the treatment with antibiotics, the persistence of swelling and pain, and the progressive loss of function, 21 months later, we decided to remove and replace the prosthesis during the same operative session. One week before the intervention, we treated the patient with vancomycin 500 mg four times daily, rifampicin 500 mg once daily and ciprofloxacin 500 mg twice daily and these antibiotics were maintained for 3 weeks after the surgery. This one-stage replacement was planned by using rFVIIa in CI as major haemostatic agent since in our previous experiences including a concurrent double-joint replacement [3]. At the time of operation, the Oxford knee score (OKS) was 48 and the titre of the FVIII inhibitor was 3 BU with a peak level in the past of 66 BU. The implant was a Correspondence: Giuseppe Tagariello, Transfusion Service, Haemophilia and Regional Blood Diseases Centre, Castelfranco Veneto Hospital, 31033 Castelfranco Veneto (TV), Veneto, Italy. Tel.: +39 0423 732336; fax: +39 0423 732337; e-mail: giuseppe.tagariello@ulssasolo.ven.it

Prenatal diagnosis of haemophilia A by using intron 1 inversion detection

Haemophilia A (HA) is characterized by a wide allelic heterogeneity as mutations are spread out into the exonic, intronic and regulatory regions of the factor VIII (F8) gene. 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], followed by the more recently reported inversion affecting intron 1 (invint1) [3]. Both types of inversions result from homologous recombination between intron 22 and 1 repeats, Int22 h-1 and Int1 h-1, respectively, shown to be opposite in orientation when compared with the identical more telomeric extragenic regions. Homologous pairing followed by recombination results in the disruption of the gene [2], therefore the recombined F8 genes are presumably unable to produce any FVIII protein. After the identification of invint1 in 2002, several papers appeared reporting in severe patients different prevalence between 0% and 5%, as recently summarized by Schroder et al. [4], including 5 out of 356 positive patients (1.4%) found at our institution [5]. The prevalence for inhibitor development in invint1-positive patients ranges from 0% to 26% in different cohorts [4]. Our data [5] report one inhibitor out of five invint1-positive patients (20%) and this prevalence seems in keeping with that found for inhibitor in invint22 [6,7]. Because of the risk of this severe side effect which turns haemophilia into a more severe disease, genetic counselling requires special attention and accuracy. Starting from 1997 in our centre we have performed 49 prenatal diagnoses (PND) on 45 carrier women related to HA (26) or haemophilia B (HB) (19). Recently, a pregnant niece of a haemophilic patient, whose invint1 mutation was identified in our laboratory, came to our attention for a PND. She was obliged carrier as she already had a haemophilic son and another case was present in her family. She was not investigated earlier and the presence of the invint1 was confirmed before chorion villus sampling (CVS). During her twelfth week of gestation, CVS was performed and foetal DNA was analysed for gender determination using polymerase chain reaction (PCR) for amelogenin gene, followed by direct mutation identification, as the foetus was a male. In a previous report [8] we have outlined that some PCR conditions for invint22, such as the amount of DNA and MgCl2 concentration, should be modified when DNA from CVS is used, possibly in relation to the need for high quality and weight DNA in longrange PCR such as that used for invint22 detection. As results can vary remarkably with foetal DNA samples, we optimized a panel with variable conditions in order to increase the rate of success during invint22 analysis from CVS. As this was our first experience of PND by using the invint1 mutation, we decided to arrange the PCR in a similar way as the invint22 detection. DNA analysis for the invint1 mutation consists of two multiplex PCR: reaction 1 uses the primers for intronic Int1 h-1 (9 F and 9cR) and the primers for the sequence flanking extragenic Int1 h-2 on its centromeric side (int1 h-2 F); reaction 2 contains the primers for extragenic Int1 h-2 (int1 h-2 F and int1 h-2R) and the primer for the sequence flanking Int1 h-1 at the telomeric side (9 F). F8 gene in wild-type and mutated X chromosome are shown in Fig. 1, where details of primers sequences are given as well. Some changes have been introduced compared with what referred in the original paper by Bagnall et al. [3], using AmpliTaq Gold Correspondence: Giuseppe Tagariello, Transfusion Service, Haemophilia and Regional Blood Diseases Centre, Castelfranco Veneto Hospital, 31033 Castelfranco Veneto (TV), Italy. Tel.: ++39 0423 732336; fax: ++39 0423 732337; e-mail: giuseppe.tagariello@ulssasolo.ven.it

Acquired haemophilia A as a blood transfusion emergency.

INTRODUCTION Acquired haemophilia is a rare autoimmune disorder caused by autoantibodies directed in the majority of the cases against clotting factor VIII. This disorder is characterised by the sudden onset of bleeding that not rarely may be life-threatening and need transfusion support. Most reports on this condition describe the need for blood transfusions during the acute, haemorrhagic phase, but the number of transfused red cell units is often unknown. PATIENTS AND METHODS In the last 5 years, 14 patients with acquired haemophilia A were identified in the transfusion and haemophilia centres of Verona and Castelfranco Veneto. The transfusion support for these 14 patients was analyzed in this retrospective survey. RESULTS The 14 patients required a total of 183 red cell units. The average transfusion requirement was 13 red cells units/patient, with a range from 0 to 38 units. CONCLUSIONS Eleven of the 14 patients studied needed strong transfusion support to enable any further management of the haemorrhages, as well as for eradication treatment of the autoantibodies to factor VIII. A relevant part of the management of haemorrhagic symptoms as well as the first choice for any further treatment (bleeding or the cure of the underlying disease) is transfusion of red blood cells.