Tiziana Tison

High incidence of anti‐FVIII antibodies against non‐coagulant epitopes in haemophilia A patients: a possible role for the half‐life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post‐transfusion antibodies may also arise against non‐coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII‐induced antibodies by use of an enzyme‐linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non‐inhibitory antibodies was evaluated in three patients; although half‐life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti‐FVIII antibodies by ELISA when routinely assessing haemophilia A patients.

Plasma exchange for the management of the catastrophic antiphospholipid syndrome : importance of the type of fluid replacement

Dear Sir, Therapeutic plasma exchange (TPE) is actually a reliable treatment option for catastrophic antiphospholipid syndrome (CAPS), as beneficial effects have been clearly demonstrated, with improved patient survival. In fact, TPE can remove pathological antiphospholipid antibodies and cytokines, TNF-alpha and complement activation products. Adverse effects, mostly reversible, have been shown in about 5% of patients [1]. In 2003, a treatment algorithm for CAPS was proposed, where TPE (and ⁄or IVIG) was indicated in the presence of a life-threatening condition and as an adjunctive therapy after effective anticoagulation with intravenous heparin and high-dose steroids [2]. Application of this protocol could be the main reason to account for the significant reduction in mortality in CAPS from 2001 [3]. The note, which had been added to the treatment algorithm [2], points out that TPE should be performed with fresh frozen plasma (FFP) and it is specially indicated if schistocytes are present, so very likely emphasizing the possibility of an overlapping syndrome with thrombotic microangiopathy. This new subset, recently named ‘microangiopathic antiphospholipid syndrome’ [4], consists of CAPS plus some clinical features of thrombotic thrombocytopenic purpura (TTP), where TPE using FFP or cryo-depleted plasma is actually the gold standard treatment [5]. FFP contains natural anticoagulants, such as AT-III, but also a quantity of clotting factors which could worsen the thrombotic storm of CAPS. In a recent review, Erkan [6] highlighted a series of therapeutic controversies in the management of CAPS without evidence-based answers, in particular on the exact role of FFP during TPE. Thus, it has been concluded that ‘it is unknown if TPE with FFP or with a different replacement fluid such as human albumin solution would result in different outcomes in CAPS’. Indeed, a literature review on the replacement fluid of TPE in CAPS shows very discordant options, including FFP [7, 8], solvent ⁄detergent plasma [9] and albumin solution [10, 11]. In this regard, Koenig et al. [12] described a 19-year old female affected with primary antiphospholipid syndrome, who developed HELLP (haemolytic anaemia, elevated liver enzymes and low platelets) syndrome during pregnancy, complicated by CAPS with hepatic and intestinal infarctions, bone marrow necrosis and severe thrombocytopenia in the puerperium. In this patient no response to TPE with FFP was recorded and she recovered by means of IVIG together with anticoagulant therapy. Moreover, Espinosa et al. [13] described a 32-year-old man showing a well-defined microangiopathic disease with cerebral, renal and cardiac involvement and markedly raised antiphospholipid antibody levels, who fully recovered after TPE using 5% albumin as a replacement fluid. The FFP use in TPE for CAPS has been suggested also in the clinical applications of therapeutic aphaeresis proposed by the American Society for Apheresis in 2007 [14], where CAPS was first included in category III indication, along with diseases with insufficient evidence, conflicting results or unclear risk-to-benefit of aphaeresis treatment. Subsequently, CAPS has been considered as a paradigmatic example of such a category indication, besides discussing whether ‘we do more harm than good by exposing the patient to blood donors through the administration of plasma products’ [15].

Detection of B-Cell Monoclonality in Fine Needle Aspiration by PCR Analysis

Cytologic examination of fine-needle aspiration (FNA) sometimes fails to diagnose the malignant nature of B-cell proliferations. In this study we analyzed the Ig gene rearrangement of 49 FNA samples by polymerase chain reaction (PCR) in order to evaluate whether molecular analyses can improve the accuracy of FNA. Twenty-six patients had non-Hodgkins lymphoma, 11 had reactive lymphoid diseases, 5 had chronic inflammation and 7 had carcinoma. A semi-nested PCR was performed using an oligoprimer specific for consensus sequences of the V regions (FR3A) and two oligoprimers derived from conserved sequences of the J regions (LJH and VLJH). Histologic examination always followed the molecular and cytologic analysis. The sensitivity of PCR and FNA morphological examination in detecting a neoplastic pattern was 92% and 78%, respectively. When samples were considered inadequate for cytologic examination, PCR always reached a diagnosis consistent with the histologic features. Our results demonstrate that PCR analysis of FNA specimens is a reliable and sensitive method capable of enhancing the diagnostic accuracy of cytologic examination.

Plasma exchange and immunoadsorption effectively remove antiphospholipid antibodies in pregnant patients with antiphospholipid syndrome.

Conventional therapy with aspirin and/or heparin is at times incapable of preventing complications in high risk pregnancies of patients with antiphospholipid syndrome (APS). In those cases, a so‐called second‐line treatment protocol is used in addition to conventional therapy strategies. This manuscript is a report on three APS pregnant patients who were successfully treated with plasma exchange (PE) (two cases) or with immunoadsorption (IA) (one case) as a second‐line treatment strategy. The efficacy of these procedures in removing anticardiolipin (aCL) and anti‐β2glycoprotein I (aβ2GPI) antibodies from blood was evaluated. Serum samples were collected before and after 87 apheretic treatment sessions. Serum IgG/M aCL and IgG/M aβ2GPI antibodies were determined using an “in‐house” enzyme‐linked immunosorbent assay and showed that all three patients had medium/high IgG aCL and aβ2GPI titers. All three women had a successful pregnancy. A significant decrease in IgG aCL (P = 0.0001) and aβ2GPI (P = 0.0001) antibody titers was observed after PE and IA sessions. There was moreover a significant, steady fall in serum IgG aCL pretreatment levels during the course of all three pregnancies (P = 0.0001, P = 0.0001, P = 0.001). The fall in IgG aβ2GPI was significant in two of the patients (P = 0.0001, P = 0.0001) both with high antibody titers, but not in one with medium antibody titers, who was treated with PE (P = 0.17). J. Clin. Apheresis 2012.

The Italian Registry of Pediatric Therapeutic Apheresis: A report on activity during 2005

The results of the 2005 Survey of the Italian Society for Apheresis and Cell Manipulation (SIdEM) reporting on the pediatric procedures carried out in 18 Italian Apheresis Units are presented here. Utilizing a standardized questionnaire, the survey collected data on techniques, types of blood separators, clinical indications, and adverse events. A total of 1,693 apheresis procedures were carried out in 355 pediatric patients: 219 plasma‐exchange, 291 peripheral blood stem cell collections, 791 extracorporeal photochemotherapy (ECP), 265 LDL‐apheresis, 71 erythro‐exchange, 9 cytoreductive apheresis, 47 immunoadsorption sessions. Adverse events were registered in 94 procedures (5.6%), most of which of mild entity, e.g., insufficient flow rate (50.0%) and symptomatic hypocalcemia (24.4%). Our data indicate that all types of apheresis procedures can be safely carried out in children. ECP, utilized primarily for the treatment of graft versus host disease (GvHD) and rejection of solid organ transplantation, are burgeoning procedures in pediatric patients, whereas plasma exchange, which is a common treatment in adults, is infrequently utilized in pediatric medicine. J. Clin. Apheresis, 2009.

Case reports of the use of immunoadsorption or plasma exchange in high-risk pregnancies of women with antiphospholipid syndrome.

Conventional treatment of antiphospholipid syndrome (APS) pregnancies with aspirin and/or heparin is sometimes unable to counteract maternal and/or fetal complications. In this article we report the cases of two patients who were unresponsive to conventional treatment for APS during their first pregnancy, and who were treated in the following pregnancy with plasma exchange and immunoadsorption respectively, in addition to conventional therapy. Both patients had a history of thrombotic events, a previous pregnancy loss at the 11th week of gestation and the same antiphospholipid antibody profile (lupus anticoagulant activity and high titers of immunoglobulin G (IgG) anti‐β2 glycoprotein I and IgG anticardiolipin antibodies). Patient 1 was treated from the fourth week of her second pregnancy with weekly plasma exchange. Due to fetal growth restriction and oligohydramnios in the 26th week she delivered, by cesarean section, a healthy female infant weighing 730 g who survived. Patient 2 was treated from the seventh week of her second pregnancy with twice a week protein A immunoadsorption. The pregnancy proceeded normally until the 36th week, when, due to slight intrauterine growth restriction, she delivered a healthy baby girl weighing 2375 g by cesarean section. Anti‐β2 glycoprotein I antibody trends were similar during both types of treatment. On the basis of our findings obtained from only two cases it is impossible to define the best aphaeretic treatment of APS high risk pregnancies. Nevertheless, as a whole these data suggest better disease control using the immunoadsorption technique as compared to plasma exchange, despite their apparently similar anti‐β2 glycoprotein I antibody removal capabilities.

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.

Plasma exchange in pediatric anti-NMDAR encephalitis: A systematic review

OBJECTIVE To clarify the most frequent modalities of use of plasma exchange (PE) in pediatric anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis and to establish the most effective association with other immunotherapies. METHODS Systematic literature review on PE in pediatric anti-NMDAR encephalitis (2007-2015). RESULTS Seventy-one articles were included (mostly retrospective), reporting a total of 242 subjects (73.2%, 93/127 females; median age at onset 12years, range 1-18). Median time to immunotherapy was 21days (range 0-190). In most cases, PE was given with steroids and IVIG (69.5%, 89/128), or steroids only (18%, 23/128); in a minority, it was associated with IVIG only (7%, 9/128), or was the only first-line treatment (5.5%, 7/128). In 54.5% (65/119), PE was the third treatment after steroids and IVIG, in 31.1% (37/119) the second after steroids or IVIG; only in 14.3% (17/119) was it the first treatment. Second-line immunotherapies were administered in 71.9% (100/139). Higher rates of full/substantial recovery at follow-up were observed with immunotherapy given ⩽30days from onset (69.4%, 25/36) compared to later (59.2%, 16/27), and when PE was associated with steroids (66.7%, 70/105) rather than not (46.7%, 7/15). Significant adverse reactions to PE were reported in 6 patients. CONCLUSION Our review disclosed a paucity of quality data on PE in pediatric anti-NMDAR encephalitis. PE use in this condition has been increasingly reported, most often with steroids and IVIG. Despite the limited number of patients, our data seem to confirm the trend towards a better outcome when PE was administered early, and when given with steroids.

Plasma exchange effectively removes 52- and 60-kDa anti-Ro/SSA and anti-La/SSB antibodies in pregnant women with congenital heart block.

Pregnant women positive for 52‐ and 60‐kDa anti‐Ro/SSA and anti‐La/SSB antibodies can suffer from congenital heart block (CHB), a passively acquired autoimmune disease.

Maternal autoantibody profiles at risk for autoimmune congenital heart block: a prospective study in high-risk patients

Objective This prospective study aimed to identify antibody profiles characterising mothers with fetuses developing congenital heart block (CHB) by comparing their antibody frequencies and levels with those in unaffected mothers. Methods Eighty-one consecutive pregnant patients positive to anti-Ro±anti-La antibodies, at high risk of developing fetal CHB were prospectively studied. The 16 patients with fetal CHB outcome were considered the study population and the 65 patients with normal pregnancy outcomes were considered the control cohort. Anti-Ro52, anti-Ro60, anti-p200 and anti-La antibodies were assayed using home-made ELISA assays. Results The prevalence of anti-p200 antibodies was significantly higher in the fetal CHB affected patients than in the controls (p=0.03). Combinations of anti-p200 with anti-Ro52 and anti-Ro60 antibodies were significantly more frequent in the women with fetuses developing CHB than in the controls (p=0.03 for all combinations). The women with fetal CHB had significantly higher mean anti-Ro52, anti-Ro60 and anti-p200 levels than the controls (p=0.003, p=0.0001 and p=0.04, respectively); mean anti-La/SSB level was not significantly different in the two cohorts (p=0.25). Conclusions Since anti-p200, anti-Ro52 and anti-Ro60 antibodies, especially at high level, seem to identify patients at increased risk of developing fetal CHB, their detection could recognise anti-Ro/La positive women at risk for having an infant with this rare, potentially dangerous disorder.