An Unusual Compound Heterozygosity for Hb O-Arab (HBB: c.364G>A) and Hb D-Los Angeles (HBB: c.364G>C).

We report a newborn with a compound heterozygosity for Hb O-Arab (HBB: 364G>A) and Hb D-Los Angeles (HBB: 364G>C). To the best of our knowledge, the combination of these two hemoglobin (Hb) variants has not been identified and reported before. The variants of the proband and parents were identified by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). DNA analysis was performed to confirm the variants. The levels of Hb variants of the proband were determined post-partum, at 3 months and 1 year after birth. Blood count analysis after 1 year revealed that the proband had a mild microcytic anemia. Furthermore, HPLC and CE analysis revealed an equal distribution of Hb D-Los Angeles compared to Hb O-Arab at the age of 1 year. The follow-up of the patient, suggested that the Hb combination is clinically silent or mild.

Analytical Performance and Clinicopathologic Correlation of Four Fecal Calprotectin Methods.

OBJECTIVES: Calprotectin is a noninvasive biomarker that can distinguish inflammatory bowel disease from irritable bowel syndrome. We investigated four automated fecal calprotectin methods on five different platforms for their preanalytical process, analytical performance, and clinicopathologic correlation. METHODS: Four calprotectin methods (Bühlmann, EliA CN, EliA CN2, and DiaSorin) were performed on five platforms (Cobas 8000 E502, Phadia Immunocap 100 and 250, and Liaison and Liaison XL) in two hospital laboratories. RESULTS: Overall variation for the different extraction devices was less than 19% when feces were of normal consistency. Freeze-thawing of samples resulted in comparable results compared with fresh samples. The different methods had a good analytic correlation (R = 0.83-0.95). Their clinicopathologic correlation was comparable, but the Bühlmann method showed significantly higher calprotectin values in every patient category. CONCLUSIONS: The automated calprotectin methods showed a good performance and comparable clinicopathologic correlation. Due to lack of standardization, the numerical values differ for the various methods.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Differences in the mechanism of blood clot formation and nanostructure in infants and children compared with adults.

INTRODUCTION: Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. MATERIALS AND METHODS: We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. RESULTS: Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. CONCLUSIONS: This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults.

Hypoxia Induces a Prothrombotic State Independently of the Physical Activity.

Hypoxia (oxygen deprivation) is known to be associated with deep vein thrombosis and venous thromboembolism. We attempted to get a better comprehension of its mechanism by going to high altitude, thereby including the potential contributing role of physical activity. Two groups of 15 healthy individuals were exposed to hypoxia by going to an altitude of 3900 meters, either by climbing actively (active group) or transported passively by cable car (passive group). Both groups were tested for plasma fibrinogen, von Willebrand factor and factor VIII levels, fibrinolysis, thrombin generating capacity, heart rate, oxygen saturation levels and blood pressure. As a control for the passive group, 7 healthy volunteers stayed immobile in bed for 7 days at normoxic conditions. The heart rate increased and oxygen saturation levels decreased with increasing altitude. Fibrinolysis and fibrinogen levels were not affected. Factor VIII and von Willebrand factor levels levels increased significantly in the active group, but not in the passive group. Plasma thrombin generation remained unchanged in both the active and passive group with increasing altitude and during 7 days of immobility in healthy subjects. However, by applying whole blood thrombin generation, we found an increased peak height and endogenous thrombin potential, and a decreased lagtime and time-to-peak with increasing levels of hypoxia in both groups. In conclusion, by applying whole blood thrombin generation we demonstrated that hypoxia causes a prothrombotic state. As thrombin generation in plasma did not increase, our results suggest that the cellular part of the blood is involved in the prothrombotic phenotype induced by hypoxia.

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups.

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and α2Macroglobulin (α2M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and α2M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, α2M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of α2M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network.

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

Cardiac remodeling during and after renin-angiotensin system stimulation in Cyp1a1-Ren2 transgenic rats.

This study investigated renin-angiotensin system (RAS)-induced cardiac remodeling and its reversibility in the presence and absence of high blood pressure (BP) in Cyp1a1-Ren2 transgenic inducible hypertensive rats (IHR). In IHR (pro)renin levels and BP can be dose-dependently titrated by oral administration of indole-3-carbinol (I3C). Young (four-weeks old) and adult (30-weeks old) IHR were fed I3C for four weeks (leading to systolic BP >200 mmHg). RAS-stimulation was stopped and animals were followed-up for a consecutive period. Cardiac function and geometry was determined echocardiographically and the hearts were excised for molecular and immunohistochemical analyses. Echocardiographic studies revealed that four weeks of RAS-stimulation incited a cardiac remodeling process characterized by increased left ventricular (LV) wall thickness, decreased LV volumes, and shortening of the left ventricle. Hypertrophic genes were highly upregulated, whereas in substantial activation a fibrotic response was absent. Four weeks after withdrawal of I3C, (pro)renin levels were normalized in all IHR. While in adult IHR BP returned to normal, hypertension was sustained in young IHR. Despite the latter, myocardial hypertrophy was fully regressed in both young and adult IHR. We conclude that (pro)renin-induced severe hypertension in IHR causes an age-independent fully reversible myocardial concentric hypertrophic remodeling, despite a continued elevated BP in young IHR.

Variability in exposure of epitope G40-R43 of domain i in commercial anti-beta2-glycoprotein I IgG ELISAs.

BACKGROUND: A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-ß2glycoprotein I (ß2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces ß2GPI opens up, thereby exposing G40-R43. OBJECTIVES: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. METHODS: Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes ß2GPI irrespective of its conformation. These antibodies were tested in commercial anti-ß2GPI assays (A-E). RESULTS: In assay A, both antibodies showed equal reactivity towards ß2GPI, indicating that all the ß2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. CONCLUSIONS: Exposure of epitope G40-R43 on ß2GPI is highly variable between commercial anti-ß2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.