International Society on Thrombosis and Haemostasis core curriculum project: Core competencies in laboratory thrombosis and hemostasis.

BACKGROUND: Laboratory analyses of blood samples are essential for diagnostics and therapy monitoring of patients with bleeding and thromboembolic diseases. Following publication of the core curriculum for clinical thrombosis and hemostasis, the International Society on Thrombosis and Haemostasis (ISTH) recognized that thrombosis and hemostasis laboratory specialists require distinct competencies that differ from medical doctors working clinically with patients. To address this gap the ISTH formed a working group of international hemostasis and thrombosis laboratory specialists to develop an evidence-based core curriculum for laboratory specialists. OBJECTIVE: This research sought consensus from the international community on core competencies required for laboratory specialists in thrombosis and hemostasis. METHODS: A draft list of 64 competencies was developed and an online stakeholder survey was circulated electronically to 15 302 ISTH members and contacts in the wider international community. The results were analyzed and used to develop the final approved core curriculum. RESULTS: Three hundred and thirty responses contained meaningful data, with broad international representation of specialists. No draft competencies were excluded, and 58 were rated as "does" or "shows how." The Leik measure of consensus for most competences was "moderate" (n = 30) or "fair" (n = 32). CONCLUSIONS: The development of an international core curriculum for laboratory specialists provides a foundation for the development and enhancement of education and quality management of the laboratory. Although there is no formal designation for laboratory specialists, international governing bodies and regulatory organizations are encouraged to consider the diagnostic core curriculum for development and accreditation of more standardized educational programs and formal assessment across jurisdictions.

Global public awareness about atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is an important cause of ischemic stroke that often remains undetected until stroke occurs. Awareness of the risk factors and symptoms is important so that AF can be diagnosed and thromboprophylaxis given. However, the extent of public awareness of AF is uncertain. We assessed public awareness of AF across six continents and compared it with that of other thrombotic and non-thrombotic disorders. METHODS: In collaboration with Ipsos-Reid, we conducted an internet-based, cross-sectional survey between September and October of 2016 in 10 countries: Argentina, Australia, Canada, Germany, Japan, Thailand, the Netherlands, Uganda, United Kingdom, and United States. Participants were selected from survey panels in weighted, age-stratified categories (40-60, 61-74, and ≥75 years). The survey included 11 questions about demographics and assessed awareness about AF, as well as that of other thrombotic and non-thrombotic disorders. Proportions and 95% confidence intervals (CI) were calculated. RESULTS: Of a total of 6312 respondents, overall awareness of AF was 48% (95% CI, 46-50%), which was lower than awareness about other thrombotic and non-thrombotic disorders except for deep vein thrombosis (awareness 43%, 95% CI, 41-45%). Awareness about AF ranged from 25% to 69% across countries, while awareness of the risk factors for AF ranged from 8% to 52%, and awareness that AF leads to stroke ranged from 36% to 46%. Among those reporting awareness of AF, 82% correctly identified palpitations as an AF symptom. CONCLUSIONS: Global public awareness of AF is low. Improving awareness may empower patients to seek timelier stroke preventive care.

Combined effects of two mutations in von Willebrand disease 2M phenotype.

BACKGROUND: Type 2M von Willebrand disease (VWD2M) is usually characterized by VWF:RCo/VWF:Ag<0.6 and normal multimeric profile; desmopressin (DDAVP) challenge test commonly shows poor response of VWF:RCo. OBJECTIVE: We describe the bleeding tendency and the laboratory phenotype in a patient carrying two heterozygous mutations affecting VWF-A1 domain and VWF-A2 domain. SUBJECTS/METHODS: A 12-year-old patient (O blood group) with severe hemorrhagic tendency was phenotypically and genotypically analyzed; his parents were also studied. RESULTS: The proband showed decrease FVIII:C, VWF:RCo/VWF:Ag, and VWF:CB6/VWF:Ag ratios, but normal platelet count, VWF:CB1/VWF:Ag ratio, VWFpp and multimeric pattern, suggesting a VWD2M phenotype. The DDAVP challenge test, compared to controls (VWD2M patients with mutations in VWF-A1 domain), showed lower increase of FVIII:C and VWF:Ag than in heterozygous, but very similar to homozygous control. Two mutations were found in heterozygous and trans presentation: p.Pro1648fs*45 and a novel missense mutation, p.Arg1426Cys. The mother was p.Arg1426Cys heterozygous carrier, with few clinical symptoms. The father was asymptomatic, with no mutations. The p.Pro1648fs*45 was considered an apparent de novo mutation; proband's AS-PCR revealed mosaicism in the paternal allele. According to the predicted models, p.Arg1426Cys would not be affecting the binding of GPIbα to A1 domain, whereas p.Pro1648fs*45 seems to modify the folding of A2 domain, and in this way, it would affect the binding to GPIbα and type VI collagen. We believe that the combination of these two heterozygous mutations, in a child with O blood group, could result in a defective phenotype enhancer.

Thrombosis: a major contributor to global disease burden.

Thrombosis is a common pathology underlying ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE). The Global Burden of Disease Study 2010 (GBD 2010) documented that ischemic heart disease and stroke collectively caused one in four deaths worldwide. GBD 2010 did not report data for VTE as a cause of death and disability. We performed a systematic review of the literature on the global disease burden due to VTE in low-, middle-, and high-income countries. Studies from Western Europe, North America, Australia, and Southern Latin America (Argentina) yielded consistent results with annual incidences ranging from 0.75 to 2.69 per 1,000 individuals in the population. The incidence increased to between 2 and 7 per 1,000 among those 70 years of age or more. Although the incidence is lower in individuals of Chinese and Korean ethnicity, their disease burden is not low because of population aging. VTE associated with hospitalization was the leading cause of disability-adjusted-life-years (DALYs) lost in low- and middle-income countries, and second in high-income countries, responsible for more DALYs lost than nosocomial pneumonia, catheter-related blood stream infections, and adverse drug events. VTE causes a major burden of disease across low-, middle-, and high-income countries. More detailed data on the global burden of VTE should be obtained to inform policy and resource allocation in health systems, and to evaluate if improved utilization of preventive measures will reduce the burden.

Diagnosis and management of von Willebrand disease in a single institution of Argentina.

Von Willebrand disease (VWD) is a bleeding disorder with variable clinical expression. In this article we describe types, clinical features, genetic testing when needed, genotype/phenotype relationships, and the response to desmopressin (DDAVP) testing, according to our experience. Our findings are possible type 1, 69.6%; type 1, 13.5%; severe type 1, 0 .35%; type 3, 0.55%; type 2A, 9.5%; probable 2B, 0.6%; type 2M, 2.5%; and probable type 2N, 3.4%. The most frequent symptoms are ecchymoses-hematomas and epistaxis, and, in females >over 13 years also menorrhagia. In pregnant patients, assessment of laboratory parameters in months 7 and 8 is recommended to plan the need for prophylaxis at term. DDAVP merits to be considered as the first-choice therapy, including pregnant women and children, and no patient showed significant unwanted effects. Because this is a safe, effective, and affordable therapy, we hope to encourage clinicians, mainly pediatricians and obstetricians, to a wider use of DDAVP, especially in developing countries. We also report two patients with prophylactic treatment.

Antiphospholipid antibodies and hyperhomocysteinaemia in patients with vascular occlusive disease.

Hyperhomocysteinemia (HHcy), lupus anticoagulant (LA) and anticardiolipin antibodies (ACA) are independent risk factors for thrombosis. Even though risks are cumulative, the clinical impact of the association is unknown. Preliminary data suggested that HHcy might be associated with transient LA and ACA, disappearing after lowering HHcy. We prospectively evaluated the association of HHcy and LA/ACA, the effect of lowering HHcy with folic acid in LA behavior, and the correlation of the initial dRVVT with LA behavior after folic acid in 210 patients with thrombosis and adverse pregnancy outcomes. Prevalence of HHcy among patients with LA/ACA was 40%. Thirty-one patients exhibited only HHcy (15%; Group 1), 106 (50%; Group 2) had only LA/ACA, while 73 (35%; Group 3) had both. After therapy, 63% and 64% of LA/ACA remained positive in Group 3 and 2, respectively. We observed a trend towards a more positive dRVVT in persistent LA after lowering HHcy. No differences in clinical presentation or in outcomes after two years of followup were observed among the groups. Even though the association of HHcy and LA/ACA is common in patients with thrombosis, it might have no prognostic implications if Hcy levels are lowered. Currently, no laboratory findings correlate with LA behavior, which is independent of homocysteine levels and vitamin treatment.

Endogenous or exogenous coagulation factor level and the response to activated protein C.

BACKGROUND: The abnormal response to activated protein C could be the mechanism to explain the prothrombotic role of elevated coagulation factor levels. OBJECTIVE: We evaluated the effect of factor VIII, II, or X (FVIII, FII, or FX) levels on activated protein C resistance technique and its association with the resistant phenotype. MATERIALS AND METHODS: The correlation between APCR and FVIII was assessed in 36 samples, after Desmopressin infusion and the correlation between FII or FX and APCR in 15 patients with plasma levels between 100-125 U/dl. Also, the effect of the addition of purified human factors (FII, FX) to a normal plasma pool (final concentration: 100, 120, 140, 180, 220 U/dl) was estimated on the APCR technique. RESULTS: APCR values correlated with FVIII increase (r(Spearman) = 0.839; p < 0.001); APCR was abnormal (<2.4) in 9/36 samples, showing higher FVIII values in the abnormal group (VIII(abnormalAPCR) = 176.7 +/- 14.2; VIII(normalAPCR) = 103.5 +/- 8.0). APCR did not correlate with endogenous FII (r(Spearman) = 0.423) or FX (r(Spearman) = -0.169). However, the addition of human FII or FX to the normal plasma pool caused a decrease in APCR (r(SpearmanFII) = -0.843; r(SpearmanFX) = -0.958) without reaching abnormal (<2.4) results. FVIII levels may be associated with a resistant phenotype at values >153.0 U/dl, according to the linear regression analysis. Exogenous FII or FX levels greater than 120 U/dl would affect the APCR, without obtaining abnormal results. CONCLUSIONS: The data do not allow the direct association of the FII or FX increase with a defect in the protein C system in the current conditions.

Morbidity of lupus anticoagulants in children: a single institution experience.

INTRODUCTION: The lupus anticoagulant (LA) and the anticardiolipin antibodies (ACA) are the antiphospholipid antibodies more relevant clinically. Their clinical manifestations are diverse with most patients being asymptomatic while others present venous or arterial thrombosis, and more rarely, bleeding. Our objectives were to evaluate clinical presentation of LA in children and to correlate it to LA behavior. PATIENTS AND METHODS: A retrospective cohort of patients (under 18 years old) who had a positive determination of LA followed by at least another determination of LA at a variable period was evaluated. Personal and family history, including infectious diseases temporally related to the event, were recorded. The screening of other coagulation disorders was performed according to symptoms, family history or laboratory results. RESULTS: Thirty-six patients were evaluated, median age was 10.8 years old, and 52.8% were female. Asymptomatic patients were 19.4% (7/36) of study population. Bleeding and thrombosis were found in 52.8% and 27.8%, respectively. Median LA determinations per patient were 3. von Willebrand disease was diagnosed in 66.7% of patients consulting for bleeding. A concomitant hemostatic defect was found in 8/10 patients with thrombosis (p = 0.003). LA behavior was not uniform and not correlated to symptoms. CONCLUSIONS: Most LA found in children is incidental and asymptomatic. In children with bleeding, LA might be a fortuitous finding associated with VWD. The persistence of LA does not imply a higher risk of thrombosis.

Primary upper-extremity deep vein thrombosis: high prevalence of thrombophilic defects.

Primary deep venous thrombosis of the upper extremity (UEDVT) is an unusual disorder. Limited data are available on the contribution of hypercoagulable status in the pathogenesis of this disease. This study aims to report the prevalence of inherited and acquired thrombophilic risk factors (TF) in patients with primary (effort-related and spontaneous) UEDVT. From 1993 to 2002, 31 patients (17 females, median age 38.8 years, range 16-60 years; and 14 males, median age 31.4 years, range 20-56 years) with primary UEDVT (n = 15 effort-related and n = 16 spontaneous) were referred for screening of hypercoagulable status. Nineteen (61.3%) patients had at least one coagulation abnormality. The most common acquired TF were antiphospholipid antibodies (31% lupus anticoagulant and 12.9% anticardiolipin antibodies). Factor V Leiden (12.9%) and prothrombin G20210A mutation (20%) were the most prevalent genetic risk factors. Five patients (16.1%) had high plasma homocysteine levels, and one patient (4.7%) had protein S deficiency. Effort-related UEDVT was associated with male gender (P = 0.04) and younger age (P = 0.02). There was no significant difference in the prevalence of acquired or inherited TF between patients with effort-related or spontaneous UEDVT. A local anatomic abnormality was detected in seven patients (22.5%), and the prevalence of TF was significantly lower within this group (P = 0.006). The incidence of TF in patients without an anatomic abnormality was 75% (RR 5.25). This study found a high prevalence of an underlying thrombophilic status in spontaneous and effort-related UEDVT. Hypercoagulable status may play a significant role in both groups. Screening for local anatomical abnormalities and thrombophilia should be included in the evaluation of primary UEDVT.

Antiphospholipid antibodies impact the protein C (PC) pathway behavior.

Antiphospholipid antibodies may interfere with the PC pathway, displaying a resistance to the activated PC (resistant phenotype). This effect was evaluated by the APCR and the ProCG systems in 36 lupus anticoagulant samples, yielding abnormal results in 47% of APCR(original), 17% of APCR(modified), and 22% of ProCG test. ProCG values correlated with APCR(original) but not with APCR(modified). Most of lupus anticoagulants affecting the PC pathway showed abnormal APCR(original) results but not abnormal ProCG values. The different behavior between both systems may be due to the heterogeneity of the antibodies or could be attributed to the fact that, in the ProCG, a PC activator is added, while the APCR employs already activated exogenous PC.

Estudio interlaboratorio de los ensayos para anticuerpos antifosfolípidos / Cooperative study for Interlaboratory antiphospholipid antibodies detection

La presencia de anticuerpos antifosfolípidso (aFL) en pacientes con trombosis venosa y/o arterial, pérdidas fetales o abortos recurrentes caracteriza al síndrome antifosfolípido (SAF). Los aFL son evaluados por métodos convencionales como ensayos de coagulación para la actividad de anticoagulante lípico (AL) y ensayos inmunológicos para los anticuerpos anticardiolipina (aCL). Aún existen muchos inconvenientes cuando se comparan los resultados informados por distintos laboratorios a pesar de los avances logrados en la estandarización de estos ensayos. El comité de Síndrome Antifosfolípido del grupo CLART realizó un estudio interlaboratorio que consistió en dos etapas: 1) distribución de un cuestionario para conocer la metodología utilizada por los centros participantes en la evaluación de los aFL, 2) distribución y análisis de 12 muestras de sueros para determinar la concordancia de los resultados de aCL dados por los participantes con aquellos considerados de referencia determinados por el centro coordinador. Se recibieron en la primera etapa 27 respuestas y en la segunda, los resultados de 20 de los 24 centros que acordaron participar. Se observó una gran dispersión de los resultados para a CL, IgG e IgM (principalmente usando equipos comerciales) en comparación con los de referencia (ELISA desarrollado), considerando tanto los valores en unidades como en títulos. La concordancia entre los resultados mejoró significativamente cuando los datos de aCL de los participantes eran agrupados como resultado negativo (título negativo o positivo débil) o positivo (título positivo moderado o alto) para el SAF. Esto remarca la necesidad de una mejor estandarización y de urgentes recomendaciones internacionales para la realización del ensayo de aCL, parte fundamental de los criterios de laboratorio para el SAF.

Estudio interlaboratorio de los ensayos para anticuerpos antifosfolípidos / Cooperative study for Interlaboratory antiphospholipid antibodies detection

La presencia de anticuerpos antifosfolípidso (aFL) en pacientes con trombosis venosa y/o arterial, pérdidas fetales o abortos recurrentes caracteriza al síndrome antifosfolípido (SAF). Los aFL son evaluados por métodos convencionales como ensayos de coagulación para la actividad de anticoagulante lípico (AL) y ensayos inmunológicos para los anticuerpos anticardiolipina (aCL). Aún existen muchos inconvenientes cuando se comparan los resultados informados por distintos laboratorios a pesar de los avances logrados en la estandarización de estos ensayos. El comité de Síndrome Antifosfolípido del grupo CLART realizó un estudio interlaboratorio que consistió en dos etapas: 1) distribución de un cuestionario para conocer la metodología utilizada por los centros participantes en la evaluación de los aFL, 2) distribución y análisis de 12 muestras de sueros para determinar la concordancia de los resultados de aCL dados por los participantes con aquellos considerados de referencia determinados por el centro coordinador. Se recibieron en la primera etapa 27 respuestas y en la segunda, los resultados de 20 de los 24 centros que acordaron participar. Se observó una gran dispersión de los resultados para a CL, IgG e IgM (principalmente usando equipos comerciales) en comparación con los de referencia (ELISA desarrollado), considerando tanto los valores en unidades como en títulos. La concordancia entre los resultados mejoró significativamente cuando los datos de aCL de los participantes eran agrupados como resultado negativo (título negativo o positivo débil) o positivo (título positivo moderado o alto) para el SAF. Esto remarca la necesidad de una mejor estandarización y de urgentes recomendaciones internacionales para la realización del ensayo de aCL, parte fundamental de los criterios de laboratorio para el SAF. (AU)

Acquired von Willebrand factor abnormalities in myeloproliferative disorders and other hematologic diseases: a retrospective analysis by a single institution.

BACKGROUND AND OBJECTIVES: Acquired von Willebrand syndrome (AVWS) is a rare acquired disorder. In most cases it is associated with lymphoproliferative disorders and monoclonal gammopathies, while less frequently myeloproliferative disorders (MPD) are involved. Although bleeding is the most important symptom, thrombotic complications have also been observed in cases associated with MPD. Our aim was to review the clinical and laboratory findings in AVWS patients from a single institution. DESIGN AND METHODS: The records of 99 patients with AVWS were reviewed to identify the underlying diseases, the symptoms and the laboratory parameters. RESULTS: In 75% of cases the AVWS was associated with MPD. The most frequent pattern was type 2 (67.7%). Abnormalities of bleeding time, factor VIII levels or platelet retention to glass beads were observed in 83.8% of cases. Bleeding was present in 38.4% of patients, more frequently in the not-MPD-associated (58.3%) vs. MPD-associated cases (32%) (p=0.022), with a significant predominance in females, irrespective of the underlying disease (p=0.0007). In 32% of patients with MPD, thrombotic manifestations, mostly microvascular and arterial episodes, were observed. INTERPRETATION AND CONCLUSIONS: AVWS in MPD seems to be mainly a laboratory diagnosis, without clinical symptoms in most cases, although bleeding as well as ischemic events can be present. In contrast, AVWS in not-MPD-associated cases is most frequently associated with severe bleeding symptoms. Performing appropriate laboratory tests may be useful for screening for AVWS.

A chromogenic substrate method for detecting and titrating anti-factor VIII antibodies in the presence of lupus anticoagulant.

BACKGROUND AND OBJECTIVES: The development of neutralizing anti-factor VIII antibodies (a-fVIII) is a major clinical complication. Lupus anticoagulant (LA) might affect detection of a-fVIII, since both inhibitors may act on the same coagulation pathway. Our aim was to accomplish unequivocal detection and titration of a-fVIII even in the presence of LA. DESIGN AND METHODS: We evaluated a-fVIII activity by a chromogenic substrate (CS) method in samples with a-fVIII (n=6), LA (n=12) and presumably both LA+a-fVIII (n=5). The inhibition index before (Ii) and after incubation at 37 C (Ii(37)) was estimated. We also performed factor VIII assays (one-stage and CS) and titration methods (Bethesda and CS) in parallel. RESULTS: Inhibition in the a-fVIII group (Ii=5-3200) was potentiated by incubation (Ii(37)=27-5200) as it was in LA+a-fVIII (Ii=9-21; Ii(37)=50-903). LA samples showed no or meaningless inhibitory effect (Ii=0-7; Ii(37)=0-4) or a-fVIII activity (0.00-0.06 CSU/ml) by the CS method; on the contrary, very low to moderate (0.52-7.00 BU/ml) a-fVIII activity was recorded by the Bethesda method. The two titration methods did not correlate (p>0.100) in the presence of LA, or LA+a-fVIII. Differences between factor VIII:C and factor VIIIcs were significant only in LA samples (p=0.005); however, patients with residual factor VIII activity from the LA+a-fVIII group also showed higher factor VIIIcs values than factor VIII:C ones. INTERPRETATION AND CONCLUSIONS: Results indicate the possibility of detecting and titrating a-fVIII without interference of LA by the CS method. This marks a difference with respect to the Bethesda method, in which a measurable effect can be expected in the presence of a strong LA.

Utilidad de la detección de anticuerpos anti-factor VIII por Elisa en hemofílicos / Usefulness of detection of afVIII antibodies by Elisa in haemophiliacs

Los hemofílicos pueden desarrollar en forma concomitante inhibidor lúpico (LA) e inhibidor neutralizante anti-factor VIII (a-fVIII). El diagnóstico diferencial es complejo ya que el LA interfiere en la identificación de los a-fVIII. A fin de detectar a-fVIII sin interferencia de LA, desarrollamos un ELISA utilizando fVIII recombinante libre de fosfolípidos como antígeno con la muestra y revelamos la IgG unidad mediante un anticuerpo anti-IgG humana-fosfatasa. El sistema permitió detectar un anticuerpo monoclonal a-fVIII al ser revelado con el antisuero anti-IgG ratón. Como control inespecífico se utlizaron anticuerpos monoclonales anti-GPIIIa y anti-factor von Willebrand. Para evaluar la utilidad del ELISA, se analizaron plasmas de pacientes hemofílicos con a-fVIII, con o sin LA y hemofílicos sin inhibidor. Como controles negativos se incluyeron plasmas con LA y normales. Los plasmas de hemofílicos con a-fVIII dieron valores de absorbancia >X Normalñ3DE; algunos plasmas hemofílicos sin inhibidor también mostraron absorbancias superiores al normal, aunque inferiores a las observadas para a-fVIII. Las lecturas de los controles no fueron significativas. No observamos interferencias por la presencia de LA. El ELISA a-fVIII sería un elemento diagnóstico complementario, al permitir evidenciar anticuerpos capaces de unirse al fVIII, aún en plasmas LA. (AU)

Utilidad de la detección de anticuerpos anti-factor VIII por Elisa en hemofílicos / Usefulness of detection of afVIII antibodies by Elisa in haemophiliacs

Los hemofílicos pueden desarrollar en forma concomitante inhibidor lúpico (LA) e inhibidor neutralizante anti-factor VIII (a-fVIII). El diagnóstico diferencial es complejo ya que el LA interfiere en la identificación de los a-fVIII. A fin de detectar a-fVIII sin interferencia de LA, desarrollamos un ELISA utilizando fVIII recombinante libre de fosfolípidos como antígeno con la muestra y revelamos la IgG unidad mediante un anticuerpo anti-IgG humana-fosfatasa. El sistema permitió detectar un anticuerpo monoclonal a-fVIII al ser revelado con el antisuero anti-IgG ratón. Como control inespecífico se utlizaron anticuerpos monoclonales anti-GPIIIa y anti-factor von Willebrand. Para evaluar la utilidad del ELISA, se analizaron plasmas de pacientes hemofílicos con a-fVIII, con o sin LA y hemofílicos sin inhibidor. Como controles negativos se incluyeron plasmas con LA y normales. Los plasmas de hemofílicos con a-fVIII dieron valores de absorbancia >X Normalñ3DE; algunos plasmas hemofílicos sin inhibidor también mostraron absorbancias superiores al normal, aunque inferiores a las observadas para a-fVIII. Las lecturas de los controles no fueron significativas. No observamos interferencias por la presencia de LA. El ELISA a-fVIII sería un elemento diagnóstico complementario, al permitir evidenciar anticuerpos capaces de unirse al fVIII, aún en plasmas LA.