Pamela Zúñiga

Diagnosis of mild platelet function disorders. Reliability and usefulness of light transmission platelet aggregation and serotonin secretion assays

Light transmission platelet aggregation (PA), adapted to measure platelet secretion (PS), is the reference test for diagnosing platelet functional disorders (PFD). Problems with these assays include lack of standardisation, unknown reproducibility and lack of universally accepted diagnostic criteria. We addressed these issues in patients with inherited mucocutaneous bleeding (MCB). Normal and abnormal PA tests in 213 patients were reproducible in 93·3% and 90·4% of the cases, respectively. Mean intra‐subject coefficient of variation for PA with strong agonists were <9% and mean intra‐class correlation coefficient for weak agonists were >0·86 (P < 0·0001). Concomitant impaired PA with 10 μmol/l‐adrenaline and 4 μmol/l‐ADP was observed in 13·7% of the controls. This combination was not considered per se a criterion for PFD. PA with adrenaline ≥42% or irreversible aggregation with 4 μmol/l ADP had 93% and 95% Negative Predictive Value for diagnosing PFD, respectively. PA defects were consistently associated with abnormal PS. In contrast, 14·3% of patients with MCB had isolated PS. Thus, standardized PA/PS assays are highly reproducible and concordant in normal and patient populations. Normal PA with adrenaline and low ADP concentration robustly predict a normal PA. Simultaneous PA/PS assays enable the diagnosis of isolated PS defects. This study confirmed that hereditary PA–PS defects are highly prevalent.

Nuevos anticoagulantes orales

Thromboembolic disease (TED) is the leading cause of morbidity and mortality worldwide. The hallmark of oral long-term anticoagulant therapy has been the use of vitamin K antagonists, whose anticoagulant effect is exerted inhibiting vitamin K epoxide reductase. Warfarin and acenocoumarol are the most commonly used. In the last five years several new drugs for long term anticoagulation have been developed, which can inhibit single clotting factors with the purpose of improving drug therapeutic range and, ideally, minimizing bleeding risks. This review addresses the state of the art on the clinical use of inhibitors of activated factor X and thrombin.

Quantitative impact of using different criteria for the laboratory diagnosis of type 1 von Willebrand disease

Only ± 50% of patients with type 1 von Willebrand disease (VWD) have recognized molecular defects and diagnosis still rests on demonstrating low plasma von Willebrand factor (VWF) protein/function. However, no generalized consensus exists regarding the type and number of VWF variables that should be considered for diagnosis.

Factors associated with thrombotic complications in pediatric patients with vascular malformations

BACKGROUND AND OBJECTIVES Thrombosis is an uncommon disorder in children. Patients with slowflow vascular malformations have higher risk of developing localized intravascular coagulation, which is closely related to the presence of thrombotic events. These episodes cause pain, can be recurrent and determine a clear deterioration in the quality of life. Moreover, serious complications such as pulmonary thromboembolism and eventually death have been described. The aim of the present study is to identify clinical and laboratory risk factors associated with thrombotic events in pediatric patients with vascular malformations. METHODS Case-Control study. Clinical records of patients who consulted the vascular anomalies study group (VASG). This group carries out interdisciplinary assessment of patients with vascular malformations. From June 2008 to December 2014, 110 patients were assessed of whom 46 patients met the inclusion criteria, with half of them presenting a thrombotic complication and the others not, these latter serving as controls. Statistical analysis included multivariate logistic regression analysis to determine major risk factors for thrombosis. RESULTS In the bivariate analysis we found a significant association between increased levels of Ddimer and thrombotic complications (OR 17.1 [95% CI 3.95-73.95; p<0.01]). In addition, a surface area≥10cm2 (OR 6.18 [95% CI 1.59-23.99; p<0.01]) and the presence of palpable phleboliths (OR 20.17 [95% CI 2.32-165.77; p<0.01]) were associated with a significant higher risk of thrombosis. Multivariate analysis identified older age (OR 1.33; p=0.013), a surface area≥10cm2 (OR 8.19; p=0.042) and palpable phleboliths (OR 85.29; p<0.01) as significant risk factors. CONCLUSIONS Our study suggests the existence of clinical factors associated with higher risk of thrombotic complications, such as the extent of the malformation, palpable phleboliths and increased age among children with vascular malformations.

Uso de desmopresina en niños con disfunción plaquetaria congénita sometidos a adeno y/o amigdalectomía

INTRODUCTION AND GOALS Adenotonsillar surgery represents a major haemostatic challenge in paediatric patients with mild inherited platelet dysfunction. While there are recommendations for perioperative haemostatic management, there are no reports of the outcomes with the different recommendations in these children when undergoing adenotonsillectomy. Our objective was to evaluate the management of perioperative bleeding with desmopressin in children with mild platelet dysfunctions who underwent adenotonsillar surgery in our hospital. METHODS We performed a retrospective study aimed at determining the perioperative bleeding and complication rate in children with mild inherited platelet dysfunction in whom desmopressin was used while undergoing adenotonsillar procedures. RESULTS Between 2004 and 2010, 27 children with mild inherited platelet dysfunction underwent adenotonsillar procedures in our hospital and were treated with desmopressin. One patient developed perioperative bleeding (3.7%) and there was 1 child (3.7%) who presented transitory hypotension as a side effect of desmopressin. CONCLUSIONS The use of desmopressin allowed adequate perioperative bleeding prophylaxis management in children with mild inherited platelet dysfunction who underwent adenotonsillar procedures without presenting severe complications.

Correlación de valores de TTPa con anti factor Xa para establecer rango terapéutico en tratamiento anticoagulante con heparina sódica

Background: The therapeutic range (TR) of activated partial thromboplastin time (aPTT) for unfractionated heparin (UFH) dosing was established in the 1970 decade. Since then aPTT determination has changed. Current TR may be sub or supra-therapeutic depending on the reagents of the test, and therefore, responsible for complications of therapy. Aim: To establish the TR for UFH dosing in our institution using antifactor Xa analysis as reference standard. Material and methods: After obtaining an informed consent, 43 blood samples were obtained for aPTT determination and antifactor Xa assay in 23 patients treated with intravenous UFH. Samples were processed at Emergency and Hemostasis Labs. We excluded patients receiving other anticoagulants, with thrombophilia, pregnancy or liver disease. Results: Mean aPTT values in the Hemostasis and Emergency labs were 57.1±18.9 and 56.6±18.3 seconds, respectively (p=0.77). The squared correlation coefficients between aPTT and antifactor Xa at hemostasis and emergency labs were R2 0.5 and 0.45 respectively, p<0.001. Using a linear regression analysis, therapeutic aPTT range values in our laboratory were established between 50 and 80 seconds, corresponding to antifactor Xa values of 0.3 to 0.7 IU/mL. Conclusions: According to current recommendations, validation of aPTT determination with reference techniques should be done in every institution.BACKGROUND The therapeutic range (TR) of activated partial thromboplastin time (aPTT) for unfractionated heparin (UFH) dosing was established in the 1970 decade. Since then aPTT determination has changed. Current TR may be sub or supra-therapeutic depending on the reagents of the test, and therefore, responsible for complications of therapy. AIM To establish the TR for UFH dosing in our institution using antifactor Xa analysis as reference standard. MATERIAL AND METHODS After obtaining an informed consent, 43 blood samples were obtained for aPTT determination and antifactor Xa assay in 23 patients treated with intravenous UFH. Samples were processed at Emergency and Hemostasis Labs. We excluded patients receiving other anticoagulants, with thrombophilia, pregnancy or liver disease. RESULTS Mean aPTT values in the Hemostasis and Emergency labs ​​were 57.1 ± 18.9 and 56.6 ± 18.3 seconds, respectively (p = 0.77). The squared correlation coefficients between aPTT and antifactor Xa at hemostasis and emergency labs were R2 0.5 and 0.45 respectively, p < 0.001. Using a linear regression analysis, therapeutic aPTT range values ​​in our laboratory were established between 50 and 80 seconds, corresponding to antifactor Xa values of 0.3 to 0.7 IU/mL. CONCLUSIONS According to current recommendations, validation of aPTT determination with reference techniques should be done in every institution.

Diagnosing type 1 von Willebrand disease: good for patient's health or for doctor's prestige?: comment

We read with interest the commentary by Rodeghiero et al. [1] regarding the diagnosis of type 1 von Willebrand disease (VWD), which affects 80–90% of all patients with VWD. More than 40 years ago, Zimmerman and Edgington [2] reported that the molecules possessing factor VIII coagulant activity (FVIII:C) and von Willebrand factor (VWF) properties could be physically segregated and identified as different molecular entities. This pivotal discovery made it possible to diagnose type 1 VWD and the different VWD subtypes. The advent of mAbs, molecular biology approaches, cell adhesion under flow conditions, and transgenic animal models, among other developments, made it possible to understand the pathophysiology of VWD, the molecular diagnosis of the VWD subtypes, and the treatment alternatives for this disorder. Pathophysiologically, type 1 VWD is defined by partial quantitative deficiency of plasma VWF antigen level with a parallel decrease in VWF activity (usually measured as VWF ristocetin cofactor activity [VWF:RCo] and/or VWF collagen binding). Although this seems simple and straightforward, there is still no global consensus on the plasma VWF cut-off values for the laboratory diagnosis of this frequent subtype. A recent survey [3] showed that only 27% of North American specialized hemostasis laboratories adhered to National Heart, Lung and Blood Institute (NHLBI) expert panel guidelines [4]. The objective of our study [5], referred to in the commentary by Rodeghiero et al., was to compare the various laboratory criteria recommended by authoritative groups for the diagnosis of type 1 VWD, which were based largely on expert opinion and little experimental evidence. We analyzed 4298 laboratory evaluations of patients referred to us during a 5-year period. Succinctly, the same data analyzed according to four proposed criteria led to an almost three-fold difference in the diagnostic rate of type 1 VWD, ranging from between 2.8% (NHLBI recommendation; similar to that recently adopted by the British Committee for Standards in Haematology [6]) to 8.3% (Zimmerman Program for the Molecular and Clinical Biology of VWD criterion) [7]. Therefore, adopting either choice leads to disparate outcomes. We think that these laboratory ranges are unacceptably broad and that their accurate demarcation should be a priority. Rodeghiero correctly criticizes the lack of bleeding score (BS) and family history in our patients; we share this concern, because, without these components, there is no disease to investigate. Diagnosing type 1 VWD on the basis of laboratory values only, without considering bleeding symptoms and inheritance, is as unreasonable as predicting VWD on the basis of the BS only. The authors are also correct in suspecting that an uncertain number of the patients included in our report could have had no abnormal bleeding had they had completed a comprehensive bleeding questionnaire. Nevertheless, this lack of information does not invalidate the central message: the wide variation in diagnotic yield obtained with the isolated analysis of the laboratory results would have not significantly changed had we incorporated the BS and family history in the laboratory diagnosis of VWD in this population. Therefore, we reanalyzed the previously published values [8] of 280 patients with unequivocal pathologic Correspondence: Diego Mezzano, Department of HematologyOncology, School of Medicine, Pontificia Universidad Cat olica de Chile, PO Box 114-D, Santiago, Chile. Tel.: +56 2 2354 3774; fax: +56 2 23543776. E-mail: dmezzano@med.puc.cl

Análisis de las inversiones del intrón 1 y 22 y secuenciación del gen F8 para el diagnóstico genético-molecular de hemofilia A en Chile

Background: Hemophilia A is an inherited disorder caused by alterations in factor VIII gene (F8) located on the X-chromosome, the intron 22 inversion being the most common mutation. The rest are predominantly point mutations distributed along this large gene of 26 exons. Aim: To implement a molecular diagnostic test to detect mutations in the F8 gene in Chilean patients with Hemophilia A. Material and Methods: To validate the testing methods, we analyzed samples with intron 22 and intron 1 inversion, and with point mutations previously studied, as well as one subject without Hemophilia. We also studied unrelated Chilean patients with Hemophilia A and their female relatives for carrier testing. Intron 22 and intron 1 inversions were studied by long distance polymerase chain reaction (PCR) and point mutations by sequencing the coding and promoter regions of the F8 gene. Results: The results obtained in all samples used for validation were concordant with those obtained previously. In the Chilean patients, the intron 22 inversion and point mutations previously described were observed. In 6 out of 9 patients with mild Hemophilia A we found the same mutation (Arg2159Cys) in exon 23, which has been described as prevalent in mild Hemophilia A. Conclusions: The analysis of intron 22 and intron 1 inversions, as well as of point mutations in the F8 gene will help us to confirm the diagnosis in patients with severe, moderate and mild Hemophilia A, and also it will allow us to perform carrier testing and to provide better genetic counseling.