Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

An automated chemiluminescence immunoassay may detect mostly relevant IgG anticardiolipin antibodies according to revised Sydney criteria.

BACKGROUND: Detection of anticardiolipin antibodies (ACA) is an independent laboratory criterion for diagnosis of antiphospholipid syndrome (APS). Alternative methods to ELISA were recently developed such as automated chemiluminescence immunoassay (CLIA). PATIENTS AND METHODS: We compared a CLIA to an ELISA kit for the detection of IgG isotype of ACA. 87 routine samples from 75 patients suspected of having APS were tested using each method. Cut-off values were calculated in our laboratory for each test using 99th percentile of 50 normal controls. RESULTS: Cut-off values were >20 GPL for ELISA and > 2 GPL for CLIA. Overall agreement (OA), agreement for positive (AP) and agreement for negative (AN) cases were 56.3%, 49.2% and 77.2% respectively. Most discrepant results were positive with ELISA and negative with CLIA. However, OA, AP and AN increased to 82.1%, 84.6% and 80% respectively when CLIA was compared to the repeated ELISA performed at least 12 weeks later. When correlated with APS-related clinical background, CLIA showed lower sensitivity, higher specificity and higher likelihood ratio (LR) as compared to first ELISA whereas these parameters were similar to those of the repeated ELISA. No association was found between any test results and APS-related clinical background of the patients. Using our own cut-off value (> 2GPL), sensitivity, specificity and LR of CLIA to identify patients with APS were respectively 100%, 72.3% and 3.6. A ROC curve showed that at 7.5 GPL cut-off value, specificity and LR improved to 91.1% and 11.25 respectively, without affecting sensitivity. A strong correlation was observed between CLIA results and APS (Chi2 = 12.25; p < 0.001). CONCLUSION: The performance of CLIA is as good as a repeated ELISA test to detect IgG ACA in suspected APS patients. It is fully automated, which represents several advantages over semi-manual ELISA techniques for its implementation in a routine laboratory.

[Thrombin generation test: establishment of reference values according to age and tissue factor concentration is essential before implementation into the laboratory]. / Test de génération de thrombine: importance d'établir les valeurs de référence en fonction de l'âge et des concentrations en facteur tissulaire avant l'implémentation au laboratoire.

The calibrated and automated thrombinography (CAT) developed by H.C. Hemker is a simple and reproducible technique that can be potentially used in coagulation laboratories. This test is able to record the complete thrombin generation in vitro, giving an interesting approach in the evaluation of the haemostatic potential at the individual level. We aimed to implement this test in our laboratory to follow patients with haemorrhagic or thrombotic pathologies. Haemorrhagic and thrombotic disorders are incompletely explored by the coagulation tests used presently in routine labs. These tests don't indeed reflect the real haemostatic phenotype of the patient neither the individual response to haemostatic treatments. Furthermore, they don't have any predictive value for the occurrence of haemorrhage and/or thrombosis. We report here reference values we established in a population of children and adults in pre-analytical conditions easily applicable in coagulation labs. Platelet poor plasma is prepared by a double centrifugation and analyzed immediately or frozen at -80 degrees C for delayed analysis.

Osteoprotegerin and progression of coronary and aortic calcifications in chronic kidney disease.

Vascular calcifications (VCs) are important predictors of cardiovascular mortality in patients with chronic kidney disease (CKD). We have shown previously that osteoprotegerin (OPG), a potential early biomarker for VC, was an independent predictor of mortality in CKD patients. The aim of our study was to follow longitudinally coronary and aortic VCs. VCs were measured using Siemens 16 detector CT in a group of predialysis and hemodialyzed patients before and after a follow-up of 4 years. Some of these patients were transplanted in the meantime. Renal function, calcium, phosphate, iPTH, hs-CRP (high sensitive protein C reactive), and OPG serum levels were also compared. VCs progressed in predialysis, hemodialyzed, and transplanted patients but the progression was not the same in all arterial beds. A progression of coronary calcifications was observed in predialysis and transplanted patients, while aortic calcifications worsened significantly only in hemodialyzed patients. OPG serum levels and hs-CRP were significantly lower among transplanted patients. We concluded that VC depends on the severity of the kidney disease. Transplanted patients are not protected from VC, yet their OPG serum levels were significantly lower, suggesting that there is no link between between OPG levels and severity of VC. Longer follow-up of these patients would be necessary to assess whether a decline in OPG correlates with better survival.

[Pediatric venous thromboembolism: review of the literature and news]. / La thrombose veineuse de l'enfant: revue de la littérature et actualités.

Pediatric venous thromboembolism is a multifactorial disorder with an annual incidence of 1/100.000 patients (0-18 years). The presence of central venous catheters is a major trigger for development of venous thrombosis, especially in neonates. As a consequence, thrombosis in the upper extremities are much more frequent in children than in adults. Congenital prothrombotic disorders may play a role in children thrombosis, often in combination with several acquired risk factors like immobilization, serious illness and/or hospitalization. Diagnosis is most frequently made by noninvasive radiologic techniques, like ultrasonography. Prophylaxis and treatment are extrapolated from adult patient protocols or small observational studies performed in small groups of patients.

Hydroxyurea treatment for sickle cell disease: impact on haematopoietic stem cell transplantation's outcome.

Since 1988, 24 children have undergone haematopoietic stem cell transplantation (HSCT) for severe sickle cell disease (SCD) in our unit, 13 being grafted after having been exposed to hydroxyurea (HU) to control SCD-related complications. Different pre-transplant conditioning regimens were given over time: Bu14/Cy200 in six patients (group 1), Bu16/Cy200/antithymocyte globulin (ATG) in five (group 2) and Bu16/Cy200/ATG with HU prior to HSCT in 13 (group 3). The aim of this study is to compare the outcome after HSCT of these groups of patients, which differ according to pre-transplant drug exposure. Overall, 20 of the 24 transplanted children had stable engraftment and have remained free of SCD-related symptoms after HSCT; 19 of them are currently alive and cured of SCD. In group 1 (HU-, ATG-), we observed one unexplainable late death, one absent engraftment, one late rejection and one mixed stable chimerism. In group 2 (HU-, ATG+), we observed the absence of engraftment in two patients and one early rejection. In group 3 (HU+, ATG+), we observed no cases of either absent engraftment, mixed stable chimerism or late rejection. In our experience, pre-transplant treatment with HU seems to be associated with a lower incidence of rejection/absent engraftment in severe SCD patients. These results need to be confirmed with a larger number of patients.

[Chronic subdural hematoma and extramedullary erythropoiesis in an infant]. / Cellules érythropoïétiques et hématome sous-dural chronique chez un nourrisson.

We report the case of a six-month old child with bilateral chronic subdural hematoma of unknown origin containing erythroblasts, metamyelocytes and blast-like cells. No such cells were found in venous blood. No primary neoplastic disorder was found. Throughout a 19-month follow-up period, general and neurodevelopmental examination remained normal with complete resolution of the subdural haematoma in the presence of macrocephaly. We discuss the origin and role of these cells.

Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

Effects of CGRP on human osteoclast-like cell formation: a possible connection with the bone loss in neurological disorders?

Osteoclast-like cell (OCL-like) differentiation is increased in long term cultures of bone marrow taken from paralyzed areas of paraplegic patients. Among the neuropeptides recently described in bone, calcitonin gene-related peptide (CGRP) has been shown in animal studies to inhibit bone resorption in vivo and OCL-like differentiation in vitro: its deficiency could thus be a link between the neural lesion and increased OCL-like production in paraplegia and some other neurologic disorders. We therefore investigated in this study the effects of CGRP on human OCL-like formation and found that it indeed has an inhibitory effect mediated at least in part via cAMP.

Recurrent viral associated hemophagocytic syndrome in a child with Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) with subsequent viral-associated hemophagocytic syndrome (VAHS) or secondary hemophagocytic lymphohistiocytosis (HLH) is extremely rare. A 15-month-old girl with disseminated LCH experienced three episodes of VAHS during maintenance therapy. Viral infection, with influenza A, herpes simplex, and adenovirus, respectively, was documented at each episode. She recovered each time after interruption of maintenance therapy. The occurrence of fever and pancytopenia in patients with chemotherapy-treated LCH can be associated with VAHS and not with relapsing LCH.

Cell biology of Paget's disease.

Paget's disease is characterized by markedly increased osteoclast formation and bone resorption followed by excessive new bone formation. Osteoclasts in Paget's disease are increased both in number and size, contain paramyxoviral-like nuclear inclusions, and can have up to 100 nuclei per cell. Marrow culture studies have identified several abnormalities in osteoclast formation in Paget's disease. Osteoclast-like multinucleated cells formed more rapidly in marrow cultures from patients with Paget's disease, produced increased levels of interleukin-6 (IL-6), and expressed high levels of IL-6 receptors compared to normals. IL-6 levels were also increased in bone marrow and peripheral blood of patients with Paget's disease. In addition, osteoclast precursors from patients with Paget's disease are hyperresponsive to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and calcitonin. The increased sensitivity of osteoclast precursors to 1,25(OH)2D3 is mediated through the vitamin D receptor (VDR), since 24-hydroxylase activity is also up-regulated at concentrations of 1,25(OH)2D3 that are one log less than that needed to induce 24-hydroxylase activity in osteoclast precursors from normals. However, VDR numbers and affinity for 1,25(OH)2D3 do not differ in osteoclast precursors from Paget's patients compared to those from normals. Synergistic interactions between cytokines such as IL-6 and 1,25(OH)2D3 also cannot explain the enhanced sensitivity of osteoclast precursors from patients with Paget's disease to 1,25(OH)2D3. Interestingly, coculture studies of osteoclast precursors and cells from the marrow microenvironment of patients with Paget's disease and normals have demonstrated that the marrow microenvironment is more osteoclastogenic than normal. Thus, studies of the cell biology of osteoclasts in Paget's disease have demonstrated an increased rate of osteoclast formation and abnormalities in both osteoclast precursors and the marrow microenvironment. Enhanced IL-6 production by osteoclasts in Paget's disease may further amplify the increased osteoclast formation already ongoing in the pagetic lesion, and may explain the increased bone turnover at uninvolved sites distant from the pagetic lesion.

Increased osteoclast-like cells formation in long-term bone marrow cultures from patients with a spinal cord injury.

Patients with a spinal cord section loose a significant amount of bone. After paraplegia, bone loss occurs below the lesional level and is the more dramatic in iliac bones and in the metaphyseal area of long bones. A peak of urinary calcium and hydroxyprolinuria is observed approximately 6 weeks after their lesion. To further understand the mechanisms underlying the bone damage, we used long-term bone marrow cultures to compare osteoclast-like (OCL-like) cell formation above and below the lesional level. Seven paraplegic, one quadriparetic, one quadriplegic patients and five normal subjects were investigated. Six weeks after their spinal cord section, the number of OCL-like cells formed in iliac bone marrow cultures was significantly greater than those formed in sternal bone marrow cultures for all paraplegic patients tested. No significant differences were seen between iliac and sternal bone marrow cultures for the quadriparetic, the quadriplegic patient, or for the five normal subjects. Conditioned media (CM) from iliac marrow of paraplegic patients increased OCL-like cell formation in normal bone marrow cultures. IL-1, TNF-alpha, IL-6, and PGE2 were measured in the CM after 3 weeks of culture. IL-6 was found to be significantly higher in iliac CM compared with sternal CM in six out of seven paraplegic patients. In two patients, addition of an anti-IL-6 monoclonal antibody to the marrow cultures significantly decreased the number of OCL-like cells formed at 3 weeks. We conclude that paraplegia caused by a cord section locally induces an increase in the capacity of progenitors to form OCL-like cells in long-term bone marrow cultures. A locally increased IL-6 production in the marrow below the lesional level could be partly responsible for this observation.

Pagetic osteoclasts formed in vitro: absence of paracrystalline inclusions.

In Paget's disease of bone, osteoclasts are increased in number and size and contain intracellular paramyxoviral-like inclusions which cross-react with antibody against measles, respiratory syncytial, and canine distemper viral nucleocapsid antigens. Moreover, measles virus nucleocapsid transcripts are present in pagetic osteoclasts and their mononuclear precursors formed in vitro. The present study was undertaken to morphologically assess pagetic osteoclasts formed in culture; special attention has been directed towards the ultrastructural identification of nuclear and cytoplasmic inclusions. Pagetic osteoclasts were produced in long-term cultures of non-adherent bone marrow mononuclear cells derived from involved bone of patients with Paget's disease. These cultured osteoclasts had many of the ultrastructural features of pagetic osteoclasts in vivo. Of interest, no viral-like inclusions were observed in either the multinucleated osteoclasts or mononuclear cell precursors in these cultures. These data suggest that other factors in the bone microenvironment are required for viral nucleocapsid formation in pagetic osteoclasts.

[Preventive management of patients presenting with an increased tendency to venous thrombosis]. / Attitudes préventives chez les patients présentant une tendance accrue aux thromboses veineuses.

The thrombophilias are conditions characterized by an increased tendency to thrombosis. This paper aims at presenting the actual guidelines concerning the preventive attitudes in the thrombophilias that mainly expose to venous thromboembolism. The identification of these thrombophilias resides on one hand on the patients' and their family's history of venous thrombosis, and on the other hand on the diagnosis of disorders known to be associated with an increased risk of venous thrombosis. Heparin-associated thrombocytopenia of type II is a still too often underdiagnosed syndrome in which the prevention of thrombosis requires a specific approach. The authors discuss thoroughly the preventive attitudes for patients that do not require a long-term anticoagulation, for patients in whom a long-term anticoagulation is generally recommended, and for patients in whom it is sometimes recommended. The practical use of anticoagulation is described. Lastly, a special attention is paid to situations in which the thrombotic risk is increased, such as prolonged immobilization, surgery, traumas, pregnancy and postpartum, contraception and oestrogen therapy.

[Value of in vitro study of osteoclast precursors in the case of severe infantile osteopetrosis treated by bone marrow graft]. / Intérêt de l'étude in vitro des précurseurs ostéoclastiques dans un cas d'ostéopétrose infantile sévère traité par greffe médullaire.

BACKGROUND: Pathogenesis of osteopetrosis is still debated. Testing the ability of osteoclastic progenitors to support the proliferation of functional cells may be useful in understanding pathogenesis. CASE REPORT AND METHODS: A diagnosis of osteopetrosis was made in a girl 1 month-old, born to consanguuineous parents. Bone marrow transplantation was uneffective at the age of 3 months but a second engraftment was successful at 5 months. Unfortunately, the patient died from severe thrombocytopenia at the age of 8 months. Long-term cultures of mononucleated cells from the patient's blood were performed before and after the bone marrow transplantation, with or without growth factors such as vitamin D3, IL-6 and IL-1. Similar studies were made from the patient's marrow obtained after transplantation; all results were compared with those obtained after culturing control cells from cord blood umbilical. RESULTS: Production of osteoclastic cells was mild in peripheral blood cultures; it was important in bone marrow cultures in presence of growth factors. CONCLUSION: These results suggest that osteopetrosis in our patient resulted from an intrinsic defect in progenitors of osteoclasts.

Multinucleated cells formed in vitro from Paget's bone marrow express viral antigens.

Paget's disease of bone is characterized by large numbers of osteoclasts that have viral-like nuclear and/or cytoplasmic inclusions. Pagetic osteoclasts express respiratory syncytial viral (RSV) and measles viral (MV) nucleocapsid antigens. The data suggest a possible viral etiology for Paget's disease. However, studies to characterize further the putative viral inclusions in Paget's osteoclasts have been severely hampered by the extreme difficulty in isolating large numbers of osteoclasts from pagetic bone. The recent demonstration that osteoclast-like multinucleated cells (MNC), that had certain characteristics of pagetic osteoclasts formed in marrow cultures from Paget's patients, may permit studies to describe this virus further. Therefore, we have cultured marrow samples from involved and uninvolved bones from Paget's patients and from normal subjects to determine if the MNC formed in these cultures express viral antigens. RSV and/or MV antigens were expressed in the mononuclear cells and/or the MNC formed in 12 of 12 marrow cultures from active lesions of patients with Paget's disease, with 40-50% of the cells expressing viral antigens. In contrast, less than 5% of cells isolated from cultures from normal subjects expressed RSV and/or MV. These results suggest that MNC formed in long-term marrow cultures from patients with Paget's disease frequently express paramyxoviral antigens and are very similar to pagetic osteoclasts. Thus, these marrow cultures may be useful for further characterizing the virus in Paget's disease.

Lithium inhibits calcitriol-stimulated formation of multinucleated cells in human long-term marrow cultures.

We observed that lithium (3 mM) blocked the 1,25-dihydroxyvitamin D [1,25-(OH)2D3]-stimulated bone resorption in fetal rat long bones in culture. Because this inhibitory effect was not seen when bone resorption was stimulated by parathyroid hormone or interleukin-1, we reasoned that Li specifically inhibited events involved in the 1,25-(OH)D3-stimulated bone resorption. The increased bone resorption induced by vitamin D in culture is associated with differentiation and/or fusion of osteoclast progenitors. In the present work, we studied the effect of Li on the basal and 1,25-(OH)2D3-stimulated generation of multinucleated osteoclast-like cells (MNC) and MNC containing tartrate-resistant acid phosphatase (TRAP+) in long-term human bone marrow cultures. Total MNC and TRAP+ cells were counted after 3 weeks of culture. In the absence of both lithium and 1,25-(OH)2D3, total MNC and TRAP+ cell numbers were 146 +/- 22 and 110 +/- 18 per well, respectively (mean +/- SEM); in the presence of Li, corresponding figures were 79 +/- 17 and 59 +/- 14. When the generation of MNC and TRAP+ cells was stimulated with 1,25-(OH)2D3, (10(-8) M), total MNC and TRAP+ cells were 521 +/- 66 and 473 +/- 63, respectively, in the absence of Li and 251 +/- 44 and 155 +/- 27 in the presence of Li (p < 0.05). The inhibitory effect of Li was dose dependent and was not observed when the cultures were exposed to parathyroid hormone instead of 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities in osteoclast precursors and marrow accessory cells in Paget's disease.

Paget's disease of bone is characterized by increased numbers of abnormal osteoclasts. To determine if osteoclast precursors were increased or abnormal in this disease, we examined CFU-GM, the committed granulocyte-macrophage progenitor and the most likely precursor for osteoclasts. In cultures of unfractionated marrow mononuclear cells, CFU-GM colony formation was significantly increased in Paget's marrow cultures compared to that in normal cells (356 +/- 44 vs. 271 +/- 15/10(5) cells; P < 0.05). However, when we enriched hematopoietic precursors from Paget's and normal marrow samples using an antibody that recognizes the CD34 antigen present on most hematopoietic precursors, we found that similar numbers of CFU-GM colonies were formed (87 +/- 13/10(4) cells plated vs. 83 +/- 13). Coculture experiments with highly purified hematopoietic precursors (CD34+ cells) and nonhematopoietic marrow accessory cells (CD34- cells) revealed that the growth of Paget's precursors was significantly enhanced above expected levels by normal or Pagetic CD34- cells (P < 0.05). CFU-GM colony formation was also significantly enhanced when normal CD34+ cells were cocultured with Pagetic, but not with normal, CD34- cells. In addition, CFU-GM colony-derived cells from Paget's patients were hyperresponsive to 1,25-dihydroxyvitamin D3 and could form osteoclast-like multinucleated cells with 1,25-dihydroxyvitamin D3 concentrations one tenth of that required for normal multinucleated formation (10(-11) vs. 10(-10) M). These data suggest that osteoclast precursors may be abnormal in Paget's disease, and other cells in the Pagetic marrow microenvironment may further enhance the growth and differentiation of these abnormal precursors.