Ri Liesner

Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura

The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

Can early subclinical gait changes in children with haemophilia be identified using the GAITRite walkway.

Summary.u2002 Development of haemophilic arthropathy has long‐term implications for functional mobility in people with haemophilia, but early manifestations are often asymptomatic and difficult to identify. Earlier identification of joint damage may improve outcomes. The aim of this case note review was to determine whether the GAITRite® system (electronic pressure sensitive walkway) could identify early changes in gait patterns in boys with haemophilia compared with their peers.

Discrepancies between ADAMTS13 activity assays in patients with thrombotic microangiopathies

ADAMTS13 activity assays are sometimes useful in confirming the clinical diagnosis or to distinguish different thrombotic microangiopathies (TMA). We investigated the commonly used clinical assays for ADAMTS13 activity. 159 samples from normal subjects or acquired TMA patients were studied in collagen binding (CBA), Fret and chromogenic peptide substrate assays. Frozen aliquots of pooled normal plasma gave similar values by CBA, Fret-VWF73 peptide, Fret-VWF86 and chromogenic VWF73 ELISA (chr-VWF73). Two lyophilised commercial calibrants gave lower ADAMTS13 activity by CBA than peptide substrate assays. The addition of solid HEPES to normal plasma caused a significant fall in CBA, but not Fret-VWF73 activity and might partly explain the differences, since lyophilised plasmas are often HEPES buffered. Normal plasmas showed good agreement between CBA and Fret assays, although chr-VWF73 gave slightly higher values. In acquired TMA, there was reasonable agreement between assays for samples with <11% ADAMTS13 activity (83% of samples showed agreement between CBA, Fret-VWF73 and chr-VWF73), but samples with moderate deficiency frequently showed lower CBA levels (only 41-52% agreement). However, there were also some discrepancies among the peptide substrate assays, with Fret-VWF86 sometimes giving slightly higher values than the VWF73 substrate assays. An International reference plasma might improve standardisation, but is not the only problem. It is unclear which assay has greatest clinical utility, this may depend on the nature of the sample. If the activity does not match the clinical picture, an alternative method should be performed. Where therapeutic monitoring is required, the same activity assay should be used throughout.

Stem cell transplantation for children with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is a rare autosomal recessive platelet function disorder. Stem cell transplantation (SCT) is curative, but it is only indicated in selected patients with a severe clinical phenotype or who develop anti‐platelet antibodies. SCT have previously been limited to full intensity myeloablative conditioning regimens. This study details the successful outcome of SCT in five consecutive patients with GT, three of whom received reduced intensity conditioning (RIC) with stem cells from non‐sibling donors. This is the first time RIC SCT has been reported in GT, and offers the possibility of curative therapy with reduced late effects.

Similar bleeding phenotype in young children with haemophilia A or B: a cohort study

The bleeding phenotype has been suggested to differ between haemophilia A and B. More knowledge on the bleeding phenotype at initiation of treatment is important to optimize patient care. The aim of this study was to investigate the severity of the bleeding phenotype and the variation in bleeding in children with severe or moderate haemophilia A and B. Consecutive, previously untreated patients with severe or moderate haemophilia A and B (factor VIII or IX activity <0.01 or 0.01–0.05 IU mL−1 respectively) born between January 1st 2000 and January 1st 2010 were included. Primary outcome was severity of bleeding tendency. Secondary outcome was variation in bleeding pattern. A total of 582 patients with severe haemophilia A and 76 with severe haemophilia B did not differ in age at first exposure to clotting factor (0.81 vs. 0.88 years, P = 0.20), age at first bleed (0.82 vs. 0.88 years, P = 0.36), and age at first joint bleed (1.18 vs. 1.20 years, P = 0.59). Patients with moderate haemophilia were older compared to patients with severe haemophilia. In patients with moderate haemophilia there were no clear differences between haemophilia A and B. Severity and variation in bleeding phenotype are similar during the early stage of treatment in patients with severe and moderate haemophilia A and B respectively. The findings imply that children with haemophilia B should be observed and treated as vigilantly as those with haemophilia A.

The value of the white precursor cell channel (WPC) on the Sysmex XN-1000 analyser in a specialist paediatric hospital

Background Historically, haematology analyser flags for abnormal white blood cells (WBCs) show good sensitivity but lower specificity, causing unnecessary blood film reviews. While the WBC differential channel on Sysmex XE and XN instruments reports a combined flag for blasts/abnormal lymphocytes, the new white precursor cell channel (WPC) on the XN series has been introduced to separate this into a specific flag for either cell type or, if no abnormality, remove the flag entirely. Aims To compare the efficiency of abnormal WBC flags from the XN WPC to our existing analyser and determine whether WPC can reduce false positive flags and blood films required. Methods Abnormal WBC flags from the Sysmex XE-5000 and XN-1000 were compared to manual differential and blood film morphology on 300 K2EDTA samples from infants and children. Results The XN WPC flag for blasts was more sensitive and specific than flags indicating blasts on the XE-5000, with a reduction in false positives from 64% (XE) to 36% (XN). Overall efficiency of the WPC flag for abnormal lymphocytes was 94% vs 79% on the XE. WPC reduced false positive flags for blasts and abnormal lymphocytes on neonatal samples by 50%. Automatic reflex analysis by WPC correctly removed a false positive flag from the white cell differential channel on 46% of samples. Total abnormal WBC flags from XN WPC were less (73) than the XE-5000 (92). Conclusions XN WPC demonstrated superior efficiency of abnormal WBC flags on paediatric samples, compared to the XE-5000, with greater sensitivity and specificity of flagging, reducing blood films for review.

Intracranial haemorrhage in children and adolescents with severe haemophilia A or B - the impact of prophylactic treatment

The discussion of prophylactic therapy in haemophilia is largely focused on joint outcomes. The impact of prophylactic therapy on intracranial haemorrhage (ICH) is less known. This study aimed to analyse ICH in children with haemophilia, with a focus on different prophylaxis regimens and sequelae of ICH. We conducted a multicentre retrospective and prospective study that included 33 haemophilia centres from 20 countries. Inclusion criteria were children and adolescents born between 1993 and 2014, with severe haemophilia A or B without inhibitors. Participants were categorized by prophylaxis regimen: full, partial or none, based on dose and dose frequency of regular infusions. The cohort study included 1515 children: 29 cases of ICH over 8038 patient years were reported. The incidence of ICH in the prophylaxis group, 0·00033 cases of ICH/patient year, was significantly lower compared to the no prophylaxis group, 0·017 cases of ICH/patient year (RR 50·06; P < 0·001) and the partial prophylaxis group, 0·0050 cases of ICH/patient year (RR 14·92; P = 0·007). In the on‐demand‐group, 8% (2/24) children with ICH died and 33% had long‐term sequelae, including intellectual and behavioural problems, paresis and epilepsy. Children on regular, frequent prophylaxis have a low risk of ICH compared to those using non‐frequent or no prophylaxis.

Wilate use in 47 children with von Willebrand disease: the North London paediatric haemophilia network experience.

Children with von Willebrand disease (VWD) in whom DDAVP is ineffective or contraindicated require treatment with a coagulation factor concentrate containing von Willebrand factor (VWF) and factor VIII (FVIII). The aim of this study was to monitor the safety, efficacy and tolerability of Wilate® (a VWF:FVIII concentrate with a 1:1 ratio) used across the North London Paediatric Haemophilia Network since May 2010. In total, 47 children (aged 0.0–17.0 years) with type 1 (n = 28), type 2 (n = 7), type 3 (n = 10) and acquired VWS (n = 2) have been treated for bleeds, surgery and/or prophylaxis using 260 000 IU Wilate®. Analysis of dose and frequency of treatment show expected responses to treatment with mean doses of 55, 50 and 50 IU kg−1 for bleeds, surgery and prophylaxis respectively. Most bleeds responded to a single treatment. Surgical procedures were covered with clinician approved dosing schedules with 95% (39/41) reported as having excellent or good efficacy. There was no accumulation of FVIII or VWF and no thromboembolic events. This case series confirms the efficacy, safety and tolerability of Wilate® in neonates, children and adolescents when used on‐demand, prophylactically and in the surgical setting.

First-line immune tolerance induction for children with severe haemophilia A: A protocol from the UK Haemophilia Centre Doctors' Organisation Inhibitor and Paediatric Working Parties

1Arthur Bloom Haemophilia Centre, University Hospital of Wales, Cardiff, UK 2Haemophilia Centre, Royal Hospital for Children, Glasgow, UK 3Haemophilia Centre, Evelina London Children’s Hospital, London, UK 4Haemophilia Centre, University Departmentt of Haematology, Manchester, UK 5Haemophilia Centre, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK 6Sheffield Haemophilia and Thrombosis Centre, Royal Hallamshire Hospital, Sheffield, UK 7Departement of Haematology, Sheffield Children’s Hospital, Sheffield, UK 8Haemophilia Centre, Hampshire Hospitals NHS Foundation Trust, Basingstoke, UK 9Haemophilia Centre, Department of Paediatric Haematology, Leeds Children’s Hospital, Leeds, UK 10Haemophilia Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust (NUTH), Newcastle, UK 11Department of Paediatric Haematology, Bristol Royal Hospital for Children, Bristol, UK 12Department of Haematology, Birmingham Children’s Hospital, Birmingham, UK 13Barts and The London School of Medicine & Dentistry, QMUL, London, UK

Paradoxical association between the 316 Trp to Ser beta 2-glycoprotein I (Beta2GPI) polymorphism and anti-Beta2GPI antibodies.

Summary. We report a woman with an obstetric history ofa stillbirth at 28u2003weeks, associated with hypertension and severe intrauterine growth restriction and a miscarriage at 9u2003weeks. She was persistently positive for immunolgobulin G (IgG) anticardiolipin antibodies and IgG anti‐Beta‐2‐glycoprotein I (anti‐Beta2GPI) antibodies. She has delivered three healthy babies when managed antenatally with aspirin and low‐molecular‐weight heparin prophylaxis. Genotyping revealed that she was homozygous for the 316 Trp to Ser Beta2GPI polymorphism. Studies examining the binding of her plasma Beta2GPI to purified cardiolipin showed markedly reduced binding in comparison with Beta2GPI in pooled normal plasma.