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Increasing evidence supports the link between ABO(H) blood group determinants and hemostasis. In particular, the ABO-related different glycosylation patterns of von Willebrand factor strongly influence its clearance and functional levels, and this may contribute to the inter-individual variations in the half-life of infused Factor VIII (FVIII) in hemophilia A (HA) patients. We investigated the role of ABO blood groups in regulating FVIII immunogenicity by evaluating their distribution in patients with severe (FVIII

Background Center-specific TTR (c-TTR) is a measure reporting the mean patient TTR within an anticoagulation clinic describing the quality of anticoagulant monitoring offered by that clinic. c-TTR has a considerable between-center variation, but its determinants are poorly understood. Objectives We aimed at evaluating which clinical, procedural or laboratory factors could be associated with c-TTR variability in a multicenter, observational cross-sectional study over a five-year period. Patients/Methods Data from 832,204 individual patients followed for VKA therapy in 292 Centers affiliated with the Italian Federation of Anticoagulation Clinics (FCSA) were analyzed. c-TTR was computed based on the TTR of patients followed at each Center, and a mixed linear regression model was used for a predefined set of explanatory variables. Results The Center next-visit interval ratio (the mean number of days after a visit with an INR outside the therapeutic range, divided by the days after a visit with an INR within the therapeutic range), the Center mean patient INR and the Center laboratory performance at EQA proficiency testing were the only variables that were independently associated with c-TTR (β-coefficients -17.32, 9.67, and -0.11, respectively; r2 = 0.635). Conclusions These findings suggest that c-TTR associates with proactive strategies aimed at keeping patients very close to their target INR with a prompt re-evaluation of those patients with under- or over-therapeutic INR.

Summary. The treatment of haemophilia in developed countries is based on home self-infusion of concentrates. Improving communication between haemophilia centres (HC) and patients is very important. The Hub Centre (Parma) designed a new outpatient clinical record, ‘xl’Emofilia®’, as part of a project ‘Web Connections of the Region’s HC’ funded by Emilia-Romagna Health Authority. It is a web-based application suited to the needs of HC, which shares the databases of the region’s HC, integrated with regional and national registries that can be accessed from anywhere. Data are managed with the ‘https’ protocol. Significant innovations are ‘pathways’ that help with the entry of data and ‘problem list’, which is a summary (updated automatically) of the patient’s clinically significant data that can be consulted at a glance. With a ‘web identity’ (a personal USB key for secure web access), patients can record bleeds and home infusions, consult their own data and allow access to their general practitioners or in emergency departments anywhere in the world (also in English language). In December 2006, the HC started to use ‘xl’Emofilia®’ and 673 clinical records are now active. Since April 2007, 50 pilot patients have been trained and are successfully using the system. A questionnaire administered to these patients on their level of satisfaction with the system and its ease of use gave excellent results. Our web-based system facilitates communication between patients and HC, improves the quality of care and enables patients to use these information at any time and from anywhere in the world.

Background Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.

Emicizumab has been approved in several countries for regular prophylaxis in patients with congenital haemophilia A and FVIII inhibitors because it substantially reduces their bleeding risk and improves quality of life. However, although significantly less frequent, some breakthrough bleeds may still occur while on emicizumab, requiring treatment with bypassing or other haemostatic agents. Thrombotic complications have been reported with the associated use of activated prothrombin complex concentrates. In addition, when surgery/invasive procedures are needed while on emicizumab, their management requires multidisciplinary competences and direct supervision by experts in the use of this agent. Given this, and in order to expand the current knowledge on the use of emicizumab and concomitant haemostatic agents, and reduce the risk of complications in this setting, the Italian Association of Haemophilia Centres (AICE) here provides guidance on the management of breakthrough bleeds and surgery in emergency situations in patients with haemophilia A and inhibitors on emicizumab prophylaxis. This paper has been shared with other National Scientific Societies involved in the field.

// Margherita Perricone 1 , Francesca Palandri 1 , Emanuela Ottaviani 1 , Mario Angelini 2 , Laura Bagli 3 , Enrica Bellesia 4 , Meris Donati 5 , Donato Gemmati 6 , Patrizia Zucchini 7 , Stefania Mancini 8 , Valentina Marchica 9 , Serena Trubini 10 , Giovanna De Matteis 11 , Silvia Di Zacomo 12 , Mose Favarato 13 , Annamaria Fioroni 14 , Caterina Bolzonella 15 , Giorgia Maccari 16 , Filippo Navaglia 17 , Daniela Gatti 18 , Luisa Toffolatti 19 , Linda Orlandi 20 , Veronique Laloux 21 , Marco Manfrini 1 , Piero Galieni 2 , Barbara Giannini 3 , Alessia Tieghi 4 , Sara Barulli 5 , Maria Luisa Serino 6 , Monica Maccaferri 7 , Anna Rita Scortechini 8 , Nicola Giuliani 9 , Daniele Vallisa 10 , Massimiliano Bonifacio 11 , Patrizia Accorsi 12 , Cristina Salbe 13 , Vinicio Fazio 14 , Milena Gusella 15 , Eleonora Toffoletti 16 , Marzia Salvucci 3 , Mirija Svaldi 18 , Filippo Gherlinzoni 19 , Francesca Cassavia 20 , Francesco Orsini 20 , Giovanni Martinelli 1 1 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology ‘L. and A. Seragnoli’, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy 2 Molecular Hematology Laboratory U.O.C of Hematology Hospital Mazzoni, Ascoli Piceno, Italy 3 Medical Genetics Unit- Hub Laboratory AUSL Romagna, Pievesestina di Cesena, Italy 4 Imaging and Laboratory Diagnostic Department, Clinical Chemistry and Endocrinology Laboratory, Hematology Unit, Oncology and Technology Department, Hospital S. Maria Nuova, IRCCS, Reggio Emilia, Italy 5 Clinical Pathology Laboratory, A.O. Ospedali Riuniti Marche Nord, Pesaro, Italy 6 Center Hemostasis and Thrombosis, Section of Medical Biochemistry, Molecular Biology and Genetics, Department of Biomedical and Specialty Surgical Sciences, University of Ferrara, Ferrara, Italy 7 Department of Medical and Surgical Sciences, Division of Hematology, University of Modena and Reggio Emilia, Modena, Italy 8 Clinical Hematology Laboratory, Department of Molecular and Clinical Sciences, Polytechnic University of Marche, Ancona, Italy 9 Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy 10 Clinical Pathology, Molecular Biology Laboratory, and Hematology/Bone Marrow Transplantation Unit, AUSL Piacenza, Piacenza, Italy 11 Section of Clinical Biochemistry and Section of Hematology, Azienda Ospedaliera Universitaria Integrata di Verona, Verona, Italy 12 Department of Hematology, Blood Bank and Biotechnology, Ospedale Civile Pescara, Pescara, Italy 13 UOS Molecular Diagnostics, Department of Clinical Pathology, ULSS12 Venetian, Venice, Italy 14 UOC laboratory medicine, P.O. San Salvatore, Sulmona, L’Aquila, Italy 15 Department of Oncology, Laboratory of Pharmacology and Molecular Biology, ULSS 18, Rovigo, Italy 16 Clinical Hematology, Department of Experimental and Clinical Medical Sciences, University of Udine, Udine, Italy 17 Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy 18 Department of Haematology and BMT, Healthcare Company of South Tyrol, District of Bolzano, Bolzano, Italy 19 Department of Pathology and Haematology, Treviso General Hospital, Treviso, Italy 20 Werfen, Milano, Italy 21 QIAGEN GmbH, Hilden, Germany, member of the European LeukemiaNet (ELN) Foundation Circle Correspondence to: Margherita Perricone, email: margherita.perricon2@unibo.it Keywords: JAK2 V617F mutation, myeloproliferative neoplasms, qPCR standardization, molecular diagnosis Received: November 01, 2016 Accepted: February 22, 2017 Published: March 06, 2017 ABSTRACT To date, a plenty of techniques for the detection of JAK2 V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory. A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2 V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2 V617F allele burden (AB) using both quantitative and qualitative assays. The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2 V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples. In conclusion, a qualitative approach is not sensitive enough to detect the JAK2 V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2 V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2 V617F .

Macular degenerations represent leading causes of central blindness or low vision in developed countries. Most of these severe visual disabilities are due to age-related macular degeneration (AMD) and pathologic myopia (PM), both of which are frequently complicated by subfoveal choroidal neovascularization (CNV). Photodynamic therapy with verteporfin (PDT-V) is still employed for CNV treatment in selected cases or in combined regimen. In Caucasian patients, the common polymorphism G185T of factor XIII-A gene (FXIII-A-G185T, rs5985) has been described as predictor of poor angiographic CNV responsiveness to PDT-V. Nevertheless, the prognostic implications of this pharmacogenetic determinant on long-term visual outcome after a PDT-V regimen have not been evaluated. We retrospectively selected Caucasian patients presenting with treatment-naive CNV and receiving standardized PDT-V protocol for two years. The study population included patients affected by subfoveal CNV secondary to AMD or PM. We assessed the correlations between the polymorphic allele T of FXIII-A-G185T and: (1) total number of photodynamic treatments, and (2) change in visual acuity from baseline to the end of the follow-up period. Considering a total study population of 412 patients with neovascular AMD or PM, the carriers of 185 T-allele of FXIII-A (GT or TT genotype) received a higher number of photodynamic treatments than patients without it (GG wild-type genotype) (p &lt, 0.01, mean number of PDT-V: 5.51 vs. 3.76, respectively). Moreover, patients with 185 T-allele of FXIII-A had a more marked worsening of visual acuity at 24 months than those with the GG-185 wild genotype (p &lt, mean difference in logMAR visual acuity: 0.22 vs. 0.08, respectively). The present findings show that the G185T polymorphism of the FXIII-A gene is associated with significant differences in the long-term therapeutic outcomes of patients treated with standardized PDT-V protocol. The comprehensive appraisal of both anti-thrombophilic effects due to FXIII-A G185T variant and photo-thrombotic action of PDT-V toward CNV provides several clues about the rationale of this intriguing pharmacogenetic correlation. Further investigations are warranted to outline the appropriate paradigm for guiding PDT-V utilization in the course of the combined therapeutic protocol for neovascular macular degeneration.