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Effective treatment of haemophilic arthropathy requires a detailed evaluation of joint integrity. Methodological assessment of magnetic resonance imaging (MRI) scores are needed to assure reproducibility of measurements when comparing results of clinical trials conducted in different centres. We compared the reliability of two MRI scoring systems for assessment of haemophilic arthropathy: one progressive system that displays the most severe change and one additive system that depicts osteochondral and soft tissue-related changes. A total of 47 1.5 T MRI examinations of knees (n = 21) and ankles (n = 26) of 42 haemophilic boys, age range, 22 months to 18 years, performed at different centres (Toronto, n = 20, Europe, n = 12 and Denver, n = 15) were independently reviewed by four radiologists at two occasions. Twenty-two examinations were from children < 9 years and 25 from children G 9. Sagittal and coronal gradient-echo (MPGR, 3D FLASH with fat saturation, GRASS) images were obtained. The MRI examinations of the ankle and knee studies presented with osteochondral abnormalities in 38.5% and 23.8% of the cases respectively. The two scoring systems demonstrated an excellent inter-reader [progressive, 0.88; additive (A, e, s and h components), 0.86] and intra-reader [progressive, 0.92; additive (A, e, s and h components), 0.93] reliability using intraclass correlation coefficients (ICCs). Although ICCs were slightly higher for knees when compared with ankles, and for older children when compared with younger children, all values fell within excellent inter- and intra-reader reliability categories. The two MRI scoring systems demonstrated a comparable reliability. This result constitutes the basis for further development of a combined MRI scoring system for assessment of haemophilic arthropathy, which incorporates progressive and additive components. (Less)

C1 inhibitor deficiency (hereditary angioedema [HAE]) is a rare disorder for which there is a lack of consensus concerning diagnosis, therapy, and management, particularly in Canada. European initiatives have driven the approach to managing HAE with 3 C1-INH Deficiency Workshops held every 2 years in Hungary starting in 1999, with the third Workshop having recently been held in May 2003. The European Contact Board has established a European HAE Registry that will hopefully advance our knowledge of this disorder. The Canadian Hereditary Angioedema Society/Société d'Angioédème Héréditaire du Canada organized a Canadian International Consensus Conference held in Toronto, Ontario, Canada, on October 24 to 26, 2003, to foster consensus between major European and North American HAE treatment centers. Papers were presented by investigators from Europe and North America, and this consensus algorithm approach was discussed. There is a paucity of double-blind placebo-controlled trials in the treatment of HAE, making levels of evidence to support the algorithm less than optimal. Enclosed is the consensus algorithm approach recommended for the diagnosis, therapy, and management of HAE and agreed to by the authors of this article. This document is only a consensus algorithm approach and requires validation. As such, participants agreed to make this a living 2003 algorithm (ie, a work in progress) and agreed to review its content at future international HAE meetings. The consensus, however, has strength in that it was arrived at by the meeting of patient-care providers along with patient group representatives and individual patients reviewing information available to date and reaching agreement on how to approach the diagnosis, therapy, and management of HAE circa 2003. Hopefully evidence to support approaches to the management of HAE will approach the level of meta-analysis of randomized controlled trials in the near future.

Previous data have demonstrated associations between thrombophilia polymorphisms in pregnant women and an increased risk of intrauterine growth restriction in their offspring, but this finding remains uncertain.We performed a hospital-based case-control study and a family-based study including 493 newborns with intrauterine growth restriction (defined by birth weight below the 10th percentile for gestational age and sex according to Canadian norms) and 472 controls (with birth weight at or above the 10th percentile). We determined the presence or absence in newborns and their parents of the following polymorphisms: methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, factor V Leiden G1691A, and prothrombin G20210A. Mothers were interviewed to obtain information on other risk factors for intrauterine growth restriction.The risk of intrauterine growth restriction was not increased among mothers carrying a polymorphism associated with thrombophilia. In the case-control study, the odds ratios associated with two copies of the variant, after adjustment for newborn genotype and other risk factors, were 1.55 for MTHFR C677T (95 percent confidence interval, 0.83 to 2.90) and 0.49 for MTHFR A1298C (95 percent confidence interval, 0.25 to 0.93); heterozygotes for factor V Leiden had an odds ratio of 1.18 (95 percent confidence interval, 0.54 to 2.55), and heterozygotes for prothrombin G20210A had an odds ratio of 0.92 (95 percent confidence interval, 0.36 to 2.35). These polymorphisms in the newborn were not associated with an increased risk. Newborns who were homozygous for the MTHFR C677T variant had a decreased risk of intrauterine growth restriction (odds ratio after adjustment for mother's genotype and other confounders, 0.52 [95 percent confidence interval, 0.29 to 0.94]). The results of the family-based study supported those of the case-control study.Our findings do not indicate that there are associations between maternal or newborn polymorphisms associated with thrombophilia and an increased risk of intrauterine growth restriction.

Background: Typical assay methods for total homocysteine in human plasma involve EDTA-containing whole blood. Unfortunately, rapid increases of the plasma homocysteine concentrations due to cellular export are observed when the EDTA-containing samples are not stored on ice and processed shortly after collection. This is a cumbersome procedure in perinatal settings, whereby delivery usually takes place at unpredictable times. Methods: The stability of homocysteine was assessed from six placental and neonatal blood samples collected in citrate buffer. Samples were stored at 4 °C and tested at regular intervals for the first 24 h. Results: There was no statistical difference in homocysteine concentrations as observed over the study storage period (mean coefficient of variation [CV] 4.9%). Conclusions: Citrated samples can be left in a refrigerator for at least 24 h with no effect on the plasma homocysteine concentrations.

In the Fall of 1994 the majority of Canadian Hemophilia A (Factor VIII (F.VII) deficiency) patients who were receiving replacement therapy were converted to Recombinant Factor VIII (rF.VIII) from plasma derived products. This decision was taken and funded by the Canadian Blood Agency following the advice of the Association of Hemophilia Centre Directors of Canada (AHCDC) who considered this to be the safest replacement therapy available. Although it was the considered opinion of the AHCDC that there was no evidence available to support the theoretical concern that rF.VIII may prove more immunogenic than plasma derived products, patient follow up included intensive surveillance of all patients converted for this complication. A central reference laboratory was established and plasma specimens obtained before and 6–12 months following conversion to rF.VIII were referred for evaluation for inhibitor development by the classical Bethesda Assay. By concensus of the referring centers a Bethesda Unit (BU)/activity of 0.5 or greater was considered to be clinically significant. No increase in the incidence of inhibitor development has been recorded in 478 patients followed for one year after conversion. This pattern has not changed in 339 of these patients followed for a further year. Of interest was the finding by the reference laboratory that 8.0% of patients had BU activity of 0.5 or greater before conversion to rF.VIII. Many of these individuals lost this activity after conversion to rF.VIII as did others who appeared to have developed inhibitory activity during the first year of follow up but became inhibitor free in the second year of therapy. Overall, the incidence of true inhibitor development, i.e. negative pre-/positive post-conversion to rF.VIII, in this population of Hemophilia A patients was 2–3% over 2 years. This is similar to the incidence in patients treated with plasma derived products. These data emphasize the need for rigorous baseline evaluation in such investigations and the heterogeneity of the inhibitor response as assessed by in vitro assay. It was concluded that, although no attempt was made to correlate these in vitro data with clinical observations including F.VIII recovery and survival, the use of rF.VIII in hemophiliacs previously treated with plasma derived products was not associated with an increase in F.VIII inhibitor development.

von Willebrand disease (VWD) is the most commonly inherited bleeding disorder with a symptomatic prevalence of 1:1000. Type 1 VWD comprises ∼80% of VWD cases and is defined by mild to moderate deficiencies of von Willebrand factor (VWF). Aberrant splicing causes 10-15% of type 1 VWD cases and the mutations are primarily found in consensus splice sites. Investigation of the VWF mRNA is required in order to determine whether the sequence variants outside of consensus splice sites affect splicing. In this study, aberrant splicing of VWF was studied using platelet and/or blood outgrowth endothelial cell (BOEC) mRNA from 3 patients from two type 1 VWD families with putative exonic and intronic splicing mutations. Variant splice forms were identified and their effects were further characterized through expression studies in HEK293(T) cells, as well as further investigation of the patient-derived BOEC. Table 1 displays the patient phenotype, mutation status, and the VWF splice forms identified through reverse transcription of the patient mRNA.Table 1Patient phenotype, mutation, and identified splice variantsPatient IDBleeding ScoreVWF: Ag (IU/mL)VWF:RCo (IU/mL)FVIII:C (IU/mL)Nucleotide ChangeVWF Splice VariantsV70110.270.170.45c.3538G>A heterozygouswildtype (wt) VWF exon 23 skipping exon 26 skipping exons 23&26 skippingV447110.400.380.51c.6599-20A>T heterozygouswt VWF exon 38 skippingV44940.440.440.75c.6599-20A>T heterozygouswt VWF exon 38 skipping The skipping of exon 23, 26, or the combined skipping of these two exons are in-frame changes to the VWF mRNA and therefore truncated VWF would be produced from these transcripts. Skipping exon 38 however, introduces a premature termination codon (PTC) which may induce nonsense mediated decay (NMD) of the VWF mRNA. Site-directed mutagenesis was used to create expression vectors for each of the aberrant VWF splice variants, which were then transfected into HEK293T cells at varying ratios mutant:wt VWF (100:0, 50:50, 25:75, 10:90, 0:100). All 4 of these VWF variants displayed a significant dose dependent impairment of VWF secretion relative to the wt transfections (Table 2). This decrease in VWF secretion was only matched by a corresponding increase in VWF in the cell lysates for the exon 26 skipping transfections (p Pseudo Weibel-Palade body (WPB) localization of the VWF was observed through confocal immunofluorescence of HEK293 cells transfected with wt VWF and VWF skipping exon 26. VWF skipping exon 23 and exons 23&26 show some localization of the VWF in rounded organelles; however this is less organized than was seen with wt VWF. Staining of VWF lacking exon 38 appears to resemble retention in the ER, and co-localization with PDI will be investigated. Overall, we have found that the exonic mutation c.3538G>A causes skipping of exons 23 and/or 26 which cause type 1 VWD in the heterozygous patient V70 through the combined action of intracellular retention of VWF lacking exon 26, and decreased secretion of VWF skipping exon 23 and VWF skipping exons 23&26. Additionally, the skipping of exon 38 from the intronic mutation c.6599-20A>T causes type 1 VWD in patients V447 and V449 likely due to NMD of the PTC containing transcript and potentially ER degradation of the protein product of any residual mutant mRNA. Therefore these mutations lead to pathogenic VWF splicing variants which cause type 1 VWD through multiple mechanisms. Disclosures: James: CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as "global assays" of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during "normal" processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.

Abstract 4353 BACKGROUND Hypersensitivity reactions to factor VIII (FVIII) concentrates are rare but well known complications of hemophilia treatment with FVIII concentrates. Occasional reports have suggested IgE mediation. Many cases have presented circumstantial evidence for the implication of FVIII but have failed to clearly identify FVIII-targeting antibodies (Abs). The absence of evidence for IgE contribution to those reactions, but the presence of FVIII specific IgM and/or IgG1 and/or IgG3, could support the hypothesis of complement mediation. These reactions have been called “complement activation-related pseudoallergy” (CARPA). As we did not study complement we call these reactions CARPA-like. We report 3 cases of CARPA-like reactions to FVIII which we believe could contribute to alert clinicians to this potential serious side effect of FVIII replacement therapy and could shed some light on its pathophysiology. PATIENTS / METHODS Subject A, 5 y. old Caucasian previously untreated patient (PUP) with mild hemophilia A (FVIII 0.12 IU/mL, 6955C>T exon 26 P2300S, H1 haplotype, no inhibitor with Nijmegen < 0.4 unit), developed a CARPA-like reaction with his sixth injection of a von Willebrand factor-containing FVIII concentrate (Wilate®, Octapharma) for traumatic knee hemarthrosis. He never needed further FVIII replacement. Subject B, 5 y. old African PUP with severe hemophilia A (FVIII < 0.01 IU/mL, intron 22 inversion, H1 haplotype, no inhibitor with Nijmegen < 0.4 unit) developed a CARPA-like reaction with his sixth injection of Wilate®. He was switched to a B-domain deleted recombinant FVIII concentrate (Xyntha™, Pfizer) and has yet to relapse with CARPA-like reaction after more than 100 exposure days. Subject C, 15 y. old Caucasian PUP with severe hemophilia A (FVIII < 0.01 IU/mL, intron 22 inversion, H1 haplotype, inhibitor with Nijmegen 10 units) developed a CARPA-like reaction with his second dose of a full length recombinant FVIII concentrate (Kogenate®,Bayer) while on ITI induction program. Blood samples were obtained from each patient shortly before and after their CARPA-like reaction. ELISA assays (AM. Vincent, Haemophila 2009) were used to test the presence of IgG, IgM, IgE Abs reacting with different types of FVIII (Baxter’s Advate, Wilate®, Kogenate®, Xyntha™) in patients’ plasma. The x-MAP technology was used to confirm the presence of IgG and IgM Abs, and to better define their isotypic profile. For that purpose, Abs specific for the heavy chain (HC; MAb 8860) or light chain (LC; MAb ESH8) of the FVIII were first immobilized on magnetic fluorescent beads, and then incubated with EDTA-dissociated FVIII. After incubation with patients’ plasma samples, binding of IgG subclasses (IgG1 to IgG4) and IgM were revealed by specific anti-human phycoerythrin-labeled Abs. RESULTS For patients A and B (with negative Bethesda titre), we identified an immune response characterized by the presence of IgM, as well as IgG (patient A IgG1; patient B IgG1, IgG3), simultaneously directed against the HC and the LC. ELISA assays on these 2 patients showed the presence of IgM and IgG, as well as the absence of IgE compatible with a CARPA-like reaction. Similar results with the 4 different FVIII concentrates are in favour of an immune response specific to FVIII rather than an immune response to anything else than FVIII. To further support this contention, ELISA assays were all negative for patient A’s mother and for his PUP hemophilic brother, as well as for subject B’s mother and for six normal control plasma tested on the same ELISA plate. Patient C (with positive Bethesda titre) presented anti-FVIII IgG (IgG1 to IgG4) and IgM Abs also targeting the HC and the LC simultaneously. These 3 patients developed a CARPA-like reaction associated with injection of FVIII concentrates. Subjects A and C were not rechallenged with FVIII after their reaction and progressively lost their anti-FVIII Abs. Subject B was rechallanged over 100 times with the B-domain deleted Xyntha™ without reaction and with progressive lost of his anti-FVIII Abs, suggesting that his reaction might have been mediated by Abs reacting to the B-domain of FVIII. CONCLUSION CARPA-like reactions to FVIII concentrates can be associated with anti-FVIII Abs. ELISA and x-MAP are useful technologies to investigate this complication of hemophilia treatment and could help select an optimal choice of FVIII concentrate for further replacement therapy. Disclosures: No relevant conflicts of interest to declare.

Shift work has been associated with many health complaints, but its relation with pregnancy loss has rarely been studied. We compared the work schedule during pregnancy of 331 women with a pregnancy loss with that of 993 pregnant women matched on gestational age. The risk of pregnancy loss was four times higher among women with fixed evening work schedules in comparison with women on a fixed day schedule (odds ratio = 4.17; 95% confidence interval = 2.19-7.92), and more than twice as high among those on a fixed night schedule (odds ratio = 2.68; 95% confidence interval = 0.53-13.43).

Lupus anticoagulants and anticardiolipin antibodies are antiphospholipid antibodies that have been associated with fetal loss, but they have not been shown unequivocally to be a risk factor for this event.To estimate the risk of fetal loss in association with these antibodies, we conducted a hospital-based case-control study of 331 women with spontaneous abortion or fetal death (case patients) and 993 controls. The subjects were included in the study only if they reported that they had had no previous spontaneous fetal loss. Each control was a pregnant woman who, in the same period of pregnancy as a case patient, had not had a fetal loss. Lupus anticoagulants were identified in blood samples through a series of coagulation tests, and IgG anticardiolipins by an enzyme-linked immunosorbent assay. Each subject was interviewed in person to obtain information on potential confounding variables, such as sociodemographic characteristics and medical conditions.Lupus anticoagulants were found in blood from 17 case patients (5.1 percent) and 38 controls (3.8 percent). The crude odds ratio for the association between lupus anticoagulants and fetal loss was 1.36 (95 percent confidence interval, 0.75 to 2.43); the odds ratio adjusted for confounders was 1.42 (95 percent confidence interval, 0.72 to 2.80). An IgG anticardiolipin level of 5 units or more was found in 4 case patients (1.2 percent) and 15 controls (1.5 percent). The crude and adjusted odds ratios for fetal loss were 0.80 (95 percent confidence interval, 0.26 to 2.41) and 1.28 (95 percent confidence interval, 0.38 to 4.21), respectively.There is not apparent justification for considering lupus anticoagulants or IgG anticardiolipins to be risk factors for fetal loss among women who present with spontaneous abortion or fetal death and have had no previous spontaneous fetal loss.

The modification of glutamic acid residues to g-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent γ-glutamylcarboxylase enzyme. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-proteins after the liver, we sought to identify Gla proteins secreted by bone-marrow derived mesenchymal stromal cells (MSCs), a precursor to all non-hematopoietic cells in bones. We used a proteomics approach to screen the secretome of MSCs with a combination of 2D gel electrophoresis and tandem mass spectrometry. The most abundant Gla-protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico aminoacid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation using anti-Gla antibodies and could be inhibited by warfarin in MSCs. In conclusion, periostin is a novel vitamin K-dependent γ-carboxylated protein distinguished from other Gla-proteins by the presence of multiple γ-carboxylase recognition sites and MSCs are an abundant source of periostin including its γ-carboxylated variant.

The evaluation of thrombin generation during blood coagulation has become the preferred clinically relevant approach to assess the global integrity of this complex process. In order to give the full picture of the physiology of this multifactorial reaction, all cellular and soluble plasma participants to this reaction have to be present in their natural whole blood environment. Whole blood thrombin generation tests are best suited for this purpose but are somewhat cumbersome for use in real time clinical practice. Thrombelastography has long been recognized as a clinically useful technology for assessment of whole blood coagulation. The purpose of this study was to evaluate the possibility of linking the tracing generated by clotting of whole blood in a Thrombelastograph® (TEG®) Hemostasis System with the generation of thrombin as assessed by thrombin/antithrombin complex (TAT). 320μL of citrated whole blood (3.2% buffered citrate and 1000μg/ml of corn trypsin inhibitor; 1 part to 9 parts of blood) from volunteers was clotted in the presence of 20μL CaCl2 (200mM) and 20μL of Innovin (1/100 diluted in PBS/albumin 4%, pH 7.4). Clotting was monitored with 8 channels of the TEG® system. At different time points (4 to 40 min) the whole blood TEG® reaction cups were put in 2640μL of cold quenching solution (EDTA 50mM, L-benzamidine 10mM, NaCl 150mM, HEPES 2mM, pH 7.4, and 10μL of 10mM D-Phe-Pro-Arg CH2Cl chloromethylketone diluted in 0.01M HCl), vortexed, and centrifuged, and the supernatants were kept at −80°C until assayed for TAT by ELISA. Total Thrombus Generation (TTG) was calculated from the first derivative of the TEG® waveform and was compared to the thrombin generation as measured by TAT as described above. The two vectors of values - the TAT thrombin generation data and the corresponding TEG® TTG - were analyzed using Pearson correlation coefficients and linear, non-linear, and natural log (Ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is the exponential curve (Fig. 1) and its corresponding natural log transformation of TAT value as shown by Fig. 2, with R2 = 0.8787 (P < 0.0001). The prediction equation for TAT thrombin generation is y = 3122.7e0.0005x, where x is the TEG® TTG variable (P < 0.0001). Linearizing using the natural log of the TAT thrombin generation variable (Fig. 2), produces the same R2 as the exponential curve. The prediction equation is y = 8.0465 + 0.0005x, significant at P < 0.0001, where y is the TAT thrombin generation in the Ln transformation variable and x is the TEG® TTG variable. The high magnitude of R2 and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation using the TEG® TTG.

Background. Lupus anticoagulants and anticardiolipin antibodies are antiphospholipid antibodies that have been associated with fetal loss, but they have not been shown unequivocally to be a risk factor for this event. Methods. To estimate the risk of fetal loss in association with these antibodies, we conducted a hospital-based case–control study of 331 women with spontaneous abortion or fetal death (case patients) and 993 controls. The subjects were included in the study only if they reported that they had had no previous spontaneous fetal loss. Each control was a pregnant woman who, in the same period of pregnancy as a case patient, had not had a fetal loss. Lupus anticoagulants were identified in blood samples through a series of coagulation tests, and IgG anticardiolipins by an enzyme-linked immunosorbent assay. Each subject was interviewed in person to obtain information on potential confounding variables, such as sociodemographic characteristics and medical conditions. Results. Lupus antico...

All patients 65 years and older who had been living in an institution for at least six months had a measurement of red blood cell folate (RBCF), serum folate (SF), vitamin B12, hemoglobin, and mean corpuscular volume. Twenty-two percent of the 97 studied subjects were found deficient in RBCF, although only 6% had anemia. Length of stay at the institution was significantly associated with deficiency in RBCF and this association was uncon-founded by age. The proportion of RBCF deficiency among the studied subjects attributable to length of stay is 80%. The prophylactic administration of folate among long-term nursing home type of patients should be considered.