ALBERT

Description: Epstein Barr Virus (EBV) associated Lymphoproliferative Disease (LPD) is a complication of allogeneic haemopoietic stem cell tranplantation (HSCT). In certain groups (congenital immunodeficiency, unrelated and mismatched donor transplants, T cell depletion) the risk may be as high as 25% with significant morbidity and mortality. Strategies to predict such illlness and allow early intervention have therefore assumed importance. We have routinely screened peripheral blood of paediatric, transplanted patients by quantitiative PCR. We report the results of such analysis of 28 successive patients and the EBV serial quantitation in 4 patients with EBV LPD. The median age at time of transplant was 6.5 years. 17 patients received an unrelated donor transplant and one patient received a haplo-identical transplant. The remainder (n=9) received a matched family donor transplant. 23 patients received either Alemtuzumab (n=19) or ATG (n=4). 13 patients had leukaemia, 5 had mucopolysaccharide syndrome, 4 for congential immune deficiency and 6 for non malignant haeamtological conditions. 9 (32%) patients showed no evidence of EBV reactivation using serial PCR monitoring. 10 patients had low level EBV reactivation as defined with a PCR log[copy number] 6, whilst the highest level without disease was 5.2). All 4 patients responded to therapy for EBV LPD, with a combination of rituximab, withdrawal of immune suppression or administration of donor lymphocytes - DLI). At higher EBV levels the quantitative PCR had increasing positive predictive value for clinical LPD. We therefore conclude that EBV serial quantitative PCR is useful in discriminating those who will develop LPD from those that will not. Our data suggest that it is possible to use EBV PCR quantitation further to discriminate asymptomatic EBV reactivation that will resolve without therapy from EBV reactivation that will require intervention. This prevents over exposure of patients to treatments (rituximab, DLI, immune suppression withdrawal) with significant associated toxicity (prolonged B cell aplasia, graft versus host disease).

Description: This retrospective study looked at all patients with Haemophilia A who attended Manchester Pendlebury Childrens Hospital for their first and subsequent Factor replacement therapy between January 1980 and May 2005. The aim was to determine :- Does an increased number of different products used influence inhibitor formation. Does using a B domain deleted factor (Refacto) concentrate influence inhibitor formation. Does changing from plasma derived to recombinant factor influence inhibitor formation. A total of 107 patients who received a cumulative total of 65,102 trearment days were identified and the following information was recorded for each patient :-severity of haemophilia (mild, moderate or severe) and baseline factor VIII level, date and age of first product, all types of products used, number of treatment days used of each product, detection of inhibitor and if present date of ocurrence and age of patient. The policy of the unit was to test for inhibitors every six months, pre operatively or when clinically indicated. Thirteen different products were in use during the period of the study :- AHFC(anti haemophilic factor concentrate), Cryoprecipitate, 8Y, 8SM, Alphanate, Replenate, Monoclate P, Helixate, Refacto, Recombinate, Hemophil M, Advate and Haemate P. Only 4 patients out of 107 developed inhibitors and all were exposed to less than 4 products. Inhibitor patient demographics Haemophilia type and baseline factor VIII Number of treatment days before inhibitor Products used with respective treatment days Severe (

Description: β thalassemia carriers are usually symptom free with microcytic hypochromic red cells and a raised HbA2 level. However, an increased output of α globin through co-inheritance of extra α globin genes, converts a typically asymptomatic β thalassemia carrier state to that of thalassemia intermedia. We describe 3 families with 3 unique head-to-tail duplications of the a-globin cluster in which all the probands have thalassemia intermedia ranging from moderately severe anemia with splenomegaly, to transfusion-dependence. In Family 1, both father (Chinese) and son (Chinese and Anglo-Saxon English) were heterozygous for the HBB:c.316-197C〉T β thalassemia variant but had moderately severe anemia (Hb 67 to 91 g/L) with splenomegaly; they were both transfusion-independent. In Family 2, the father (Syrian) had normal hematology, while mother (Iraqi) had a hematologic profile (Hb 110 g/L, RBC 5.68x1012/L, MCH 19.4 pg, MCV 58.9 fL and HbA2 4.8%) typical of heterozygous β0 thalassemia. The proband presented at age 5 years with severe anemia precipitated by an infection, that needed a blood transfusion. She continued to need intermittent blood transfusion while an older sister with a hematologic profile of Hb 75 g/L, RBC 3.04x1012/L, MCH 19.6 pg, MCV 65.6 fL, and mild spenomegaly, remained transfusion-independent. In Family 3 (Greek Cypriot), both parents were asymptomatic; the father was heterozygous for the HBB:c.93-21G〉A β thalassemia variant with a normal a globin genotype (aa/aa), and the mother had normal HbA2 levels. In contrast, both their daughter and son who had inherited father's β thalassemia, had moderately severe anemia and needed intermittent blood transfusion. In all 3 families, MLPA suggested duplication of the whole alpha globin cluster but could not differentiate the different duplications. Next generation sequencing using Agilent SureSelect bait capture, targeted sequence analysis to the two globin loci. Sequence alignment to the reference sequence was performed using NextGene (SoftGenetits, USA). Analysis of the β globin gene sequence identified the β thalassemia-causing variant in each family. Comparison of the sequence coverage across the a loci on chromosome 16 between each case and normal controls, showed that where duplications had occurred, there was proportionally more sequence captured, similar to SNP or CGH array analysis. At the point where the sequence coverage increased, a duplication breakpoint was suspected, and the aligned sequence reads were examined in detail. In Family 1, individual sequence reads matched part of the reference sequence, BLAT query in UCSC showed that the two halves of the read aligned at either end of the duplication, indicating they were sequences that spanned the head-to-tail breakpoint. This was confirmed by gap-PCR and Sanger sequence analysis. In the other two families, breakpoints were identified within repetitive regions which could not be captured by the baits and were therefore not covered by the captured sequence. The high resolution of the coverage map allowed precise characterisation of the duplications by gap-PCR and Sanger sequencing analysis of the breakpoint amplicons. Duplication of the a globin cluster was encompassed in 188.7 kb in family 1, 120.5 kb in family 2, and 274 kb in family 3 (Figure 1). Both father and son in Family 1 were heterozygous for the duplicated a globin cluster (aa/aa, aa) and HBB:c.316-197C〉T mutation. Both siblings in Family 2 were heterozygous for mother's β thalassemia c.135delC variant and father's duplicated a globin cluster. In family 3, the mother had a 3.7 kb a deletion variant and a duplicated a globin cluster (-a3.7/aa, aa), a total of 5 a globin genes. Both the daughter and son had inherited mother's duplicated a globin cluster with father's β thalassemia variant. These families clearly show that a duplicated a globin cluster does not have a discernible phenotype on its own but is readily detectable when co-inherited with a β thalassemia variant. In all 3 cases, the a globin cluster duplications are in a head-to-tail orientation and occurred in repeats. These have most likely formed by non-homologous recombination events involving repetitive Alu sequences interspersed throughout the region. Whether these events occur more commonly in this region or if the region tolerates these changes better, allowing them to accumulate, remains to be resolved. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Direct sequencing of VWF genomic DNA in 21 patients with type 3 von Willebrand disease (VWD) failed to reveal a causative homozygous or compound heterozygous VWF genotype in 5 cases. Subsequent analysis of VWF mRNA led to the discovery of a deletion (c.221-977_532 + 7059del [p.Asp75_Gly178del]) of VWF in 7 of 12 white type 3 VWD patients from 6 unrelated families. This deletion of VWF exons 4 and 5 was absent in 9 patients of Asian origin. We developed a genomic DNA-based assay for the deletion, which also revealed its presence in 2 of 34 type 1 VWD families, segregating with VWD in an autosomal dominant fashion. The deletion was associated with a specific VWF haplotype, indicating a possible founder origin. Expression studies indicated markedly decreased secretion and defective multimerization of the mutant VWF protein. Further studies have found the mutation in additional type 1 VWD patients and in a family expressing both type 3 and type 1 VWD. The c.221-977_532 + 7059del mutation represents a previously unreported cause of both types 1 and 3 VWD. Screening for this mutation in other type 1 and type 3 VWD patient populations is required to elucidate further its overall contribution to VWD arising from quantitative deficiencies of VWF.

Description: Bristol has previously published extensively on the use of Campath antibodies during the unrelated donor cell transplantation of children with relapsed or otherwise high risk Acute Lymphoblastic Leukaemia (A.L.L.). These former reports (Br J Haematol1996;94(3):574–8; Blood1999; 94(7) 2236–46) concerned the use of Campath IG (a rat monoclonal antibody directed at CD52) given pre-transplant to the patients by intravenous infusion during conditioning therapy that also included cyclophosphamide (120 mgs/kg) and fractionated total body irradiation (1440 Gy). During this treatment the unrelated donor marrow was T cell depleted in vitro using Campath IM (also monoclonal and of rat origin). The results of such treatment demonstrated results comparable with HLA-matched sibling BMT, with low levels of acute graft versus host disease. In this communication transplant centres in 3 UK hospitals (Bristol, Great Ormond Street, London and Manchester) report their combined experience of using Campath IH (Alemtuzumab, a humanised monoclonal antibody - also directed at CD52) in the conditioning therapy of children with relapsed or otherwise high risk A.L.L.. The conditioning was otherwise again with Cyclophosphamide and fractionated total body irradiation. In this protocol a T cell replete graft was given and there was no in vitro T cell depletion of the unrelated donor marrow. We report 34 successive patients where follow up for at least 12 months was available. The median age was 6.5 years and 28 patients received fully matched grafts (matched at HLA-A, -B, -C, -DRB1 and DQB1). The remaining patients received a graft that was mismatched at one of these class I loci. All patients engrafted and the median time to neutrophil count sustained above 0.5x10e9 / l was 20 days (range 12–58 days). Only tow patients experienced grade III acute GvHD and no patient experienced grade IV acute GvHD. 22 patients survive at a median follow up of 23.8 months including 4 of the patients who received a mismatched graft. There were only 3 deaths in the first 100 days following transplant (2 due to disseminated, invasive adenovirus and one to aspergillus). Beyond 100 days 3 further patients have died of adenovirus, 3 have relapsed, 2 have died of chronic GvHD and one died of a pulmonary haemorrhage of uncertain aetiology. We therefore conclude the combination of Aletuzumab with cyclophosphamide and TBI is an effective and safe conditioning therapy for children with relapsed A.L.L. All patients engraft and there are low rates of acute and chronic GvHD.

Description: Blood loss during corrective surgery for scoliosis and the requirement for homologous blood is an important issue in paediatric spinal surgery. This is particularly pertinent in cases of scoliosis secondary to underlying neuromuscular disease where anaemia may be less well tolerated, both intra-operatively and post-operatively, due to multiple organ dysfunction. Despite the introduction of techniques to minimise the necessity to transfuse homologous blood (pre-deposit autologous donation, cell salvage and isovolaemic haemodilution), it is often required. This brings with it the potential risks of allo-immunization and infective complications. In the UK homologous blood is an increasingly precious resource following the recent exclusion of donors who have themselves received blood products in the last ten years in view of the potential risk of new variant CJD. Aprotinin, a serine proteinase inhibitor (now produced by recombinant technology) with antifibrinolytic properties and the ability to preserve platelet function has been shown to reduce blood loss in major cardiac surgery and liver transplantation. Its effectiveness in the orthopaedic setting has generated more varied reports, however in the setting of spinal surgery for idiopathic scoliosis it has recently been shown to reduce intra-operative blood loss and the requirement for homologous transfusion. We report here our experience with the use of aprotinin in a group of paediatric patients undergoing surgery for scoliosis secondary to an underlying neuromuscular disorder and its effect on intra-operative blood loss. 33 successive patients referred to our institution were allocated to receive aprotinin (n=18) or not (n=15). For those who received the drug a test dose of 50,000KIU was followed by a loading dose of 10,000KIU/kg over 30 minutes. A maintenance infusion was set up at 5,000KIU/hour for the duration of the procedure. The demographics of the two groups were not significantly different. The median age of those children receiving aprotinin was 12 years (7–15) and those who did not 13 years (7–17), [p=0.09]. The median weight of the children receiving aprotinin was 40kg (25–55) and those who did not 38.5kg (21–55), [p=0.74]. The procedures undertaken were posterior spinal fusion (n=26), anterior and posterior fusion (n=3), insertion of growing rods (n=2) and correction of kyphoscoliosis (n=2). All procedures were performed by one of three consultants. The operative time in the two groups was not significantly different, 6.5 hours (4.1–8.9) in those who received aprotinin and 5.7 (4.4–7) in those who did not [p=0.54]. There was however a significant reduction in blood loss in the group who received aprotinin, with an average reduction of 51.6%. Median losses for those who received aprotinin were 1380ml (380ml–2380ml) and 2849ml (1047ml–4651ml) for those who did not [p=0.01]. No adverse events in the form of allergic reactions were observed in the children who received the drug. We conclude that aptotinin is a safe and effective pharmacological intervention which can be used in spinal surgery to reduce operative blood loss. This should translate into a reduced requirement for homologous blood although this was not determined in this study. Evaluation of this secondary endpoint is ongoing.