BRCA carriers after risk-reducing bilateral salpingo-oophorectomy: menopausal hormone therapy knowledge gaps, and the impact of physicians' recommendations.

OBJECTIVE: Female carriers of BRCA1/2 gene mutations are at an increased lifetime risk for breast and ovarian cancers. They are recommended to undergo risk-reducing surgery, including bilateral salpingo-oophorectomy (RR-BSO), upon completion of childbearing. RR-BSO surgery decreases morbidity and mortality but results in early menopause. Menopausal hormone therapy (MHT) is under-utilized despite being shown as safe for carriers. We aim to evaluate the factors associated with decision-making regarding MHT use following RR-BSO in healthy BRCA mutation carriers. METHODS: Female carriers aged <50 years who underwent RR-BSO and were followed in a multidisciplinary clinic completed online multiple-choice and free-text questionnaires. RESULTS: A total of 142 women met the inclusion criteria and filled the questionnaire: 83 were MHT users and 59 were non-users. MHT users underwent RR-BSO earlier than non-users (40.82 ± 3.91 vs. 42.88 ± 4.34; p < 0.0001). MHT usage was positively associated with MHT explanation (odds ratio 4.318, 95% confidence interval [CI] [1.341-13.902], p = 0.014), and knowledge regarding the safety of MHT and its effects on general health (odds ratio 2.001, 95% CI [1.443-2.774], p < 0.0001). MHT users and non-users retrospectively evaluated their comprehension of RR-BSO consequences as significantly lower than before surgery (p < 0.001). CONCLUSION: Post-RR-BSO outcomes, including the effects on women's quality of life and its possible mitigation through MHT use, need to be addressed pre surgery by healthcare providers.

Chitosan as capping agent in a robust one-pot procedure for a magnetic catalyst synthesis.

Here, we report a one-pot solvothermal method for the development of magnetically recoverable catalysts with Ru or Ag nanoparticles (NPs) capped by chitosan (CS), a derivative of natural chitin. The formation of iron oxide NPs was carried out in situ in the presence of CS and iron acetylacetonate in boiling triethyleneglycol (TEG) due to CS solubilization in warm TEG. Coordination with Ru or Ag species and the NP formation take place in the same reaction solution, eliminating intermediate steps. In optimal conditions the method developed allows stabilization of 2.2 nm monodisperse Ru NPs (containing both Ru0 and Ru4+ species) that are evenly distributed through the catalyst, while for Ag NPs, this stabilizing medium is inferior, leading to exceptionally large Ag nanocrystals. Catalytic testing of CS-Ru magnetically recoverable catalysts in the reduction of 4-nitrophenol to 4-aminophenol with excess NaBH4 revealed that the catalyst with 2.2 nm Ru NPs exhibits the highest catalytic activity compared to samples with larger Ru NPs (2.9-3.2 nm). Moreover, this catalyst displayed extraordinary shelf-life in the aqueous solution (up to ten months) and excellent reusability in ten consecutive reactions with easy magnetic separation at each step which were assigned to its conformational rigidity at a constant pH. These characteristics as well as favorable environmental factors of the catalyst fabrication, make it promising for nitroarene reduction.

Facile Synthesis of Magnetically Recoverable Pd and Ru Catalysts for 4-Nitrophenol Reduction: Identifying Key Factors.

This paper reports the development of robust Pd- and Ru-containing magnetically recoverable catalysts in a one-pot procedure using commercially available, branched polyethyleneimine (PEI) as capping and reducing agent. For both catalytic metals, ∼3 nm nanoparticles (NPs) are stabilized in the PEI shell of magnetite NPs, whose aggregation allows for prompt magnetic separation. The catalyst properties were studied in a model reaction of 4-nitrophenol hydrogenation to 4-aminophenol with NaBH4. A similar catalytic NP size allowed us to decouple the NP size impact on the catalytic performance from other parameters and to follow the influence of the catalytic metal type and amount as well as the PEI amount on the catalytic activity. The best catalytic performances, the 1.2 min-1 rate constant and the 433.2 min-1 turnover frequency, are obtained for the Ru-containing catalyst. This is discussed in terms of stability of Ru hydride facilitating the surface-hydrogen transfer and the presence of Ru4+ species on the Ru NP surface facilitating the nitro group adsorption, both leading to an increased catalyst efficiency. High catalytic activity as well as the high stability of the catalyst performance in five consecutive catalytic cycles after magnetic separation makes this catalyst promising for nitroarene hydrogenation reactions.

Clinico-demographical profile of keratomycosis in Delhi, North India.

This study was undertaken to evaluate the clinico-demographical profile of keratomycosis. (January 2004 to January 2012). The corneal scrapings were processed by direct microscopic methods and standard culture techniques. Of 209 cases of keratitis studied, culture yielded growth in 80 cases (38.3%). Out of these 80 cases of growth, fungi were isolated in 77.5% and bacteria in 22.5%. The spectrum of keratomycosis was Aspergillus flavus (22.5%), Fusarium solani (16.1%), A. fumigatus (11.3%), Candida albicans (6.4%), etc., Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent use of un-warranted anti-microbial therapy.

Antiphospholipid antibody syndrome: the flow cytometric annexin A5 competition assay as a diagnostic tool.

The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient's aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73.3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72.2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97.0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

The deacylation mechanism of AmpC beta-lactamase at ultrahigh resolution.

Beta-lactamases confer bacterial resistance to beta-lactam antibiotics, such as penicillins. The characteristic class C beta-lactamase AmpC catalyzes the reaction with several key residues including Ser64, Tyr150, and Lys67. Here, we describe a 1.07 A X-ray crystallographic structure of AmpC beta-lactamase in complex with a boronic acid deacylation transition-state analogue. The high quality of the electron density map allows the determination of many proton positions. The proton on the Tyr150 hydroxyl group is clearly visible and is donated to the boronic oxygen mimicking the deacylation water. Meanwhile, Lys67 hydrogen bonds with Ser64Ogamma, Asn152Odelta1, and the backbone oxygen of Ala220. This suggests that this residue is positively charged and has relinquished the hydrogen bond with Tyr150 observed in acyl-enzyme complex structures. Together with previous biochemical and NMR studies, these observations indicate that Tyr150 is protonated throughout the reaction coordinate, disfavoring mechanisms that involve a stable tyrosinate as the general base for deacylation. Rather, the hydroxyl of Tyr150 appears to be well positioned to electrostatically stabilize the negative charge buildup in the tetrahedral high-energy intermediate. This structure, in itself, appears consistent with a mechanism involving either Tyr150 acting as a transient catalytic base in conjunction with a neutral Lys67 or the lactam nitrogen as the general base. Whereas mutagenesis studies suggest that Lys67 may be replaced by an arginine, disfavoring the conjugate base mechanism, distinguishing between these two hypotheses may ultimately depend on direct determination of the pK(a) of Lys67 along the reaction coordinate.

Autoimmune thrombocytopenia: flow cytometric determination of platelet-associated autoantibodies against platelet-specific receptors.

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. We here describe a feasible diagnostic method using the commonly available technique of flow cytometry. An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls. The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads. The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8-31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7-1.2), and in the healthy controls 1.0 (range 0.7-1.3). The average assay coefficient of variation was 0.218 [95% confidence interval (CI) 0.213, 0.221]. The difference between the ITP patients and both groups was highly significant (P < 0.001), using a stringent non-parametric analysis. A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R2 = 0.71, 95% CI 0.39, 0.53). The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033. We conclude that the present FCM assay is clinically useful for routine diagnosis and follow-up of ITP.

Platelet activation as a marker for in vivo prothrombotic activity: detection by flow cytometry.

Circulating platelets play a pivotal role in hemostasis. The platelet hemostatic function involves the direct interaction with damaged vessel walls, and circulating coagulation factors, primarily thrombin resulting in platelet activation, aggregation and formation of hemostatic plug. Flow cytometry is a useful technique for the study of platelet activation in circulating blood. Platelet activation markers for ex vivo analysis may include a) activation-dependent epitopes of the membrane glycoprotein (GP) IIb/IIIa (CD41a) receptor, as demonstrated by the binding of activation-specific monoclonal antibodies (MoAbs) PAC1, anti-LIBS1 and anti-RIBS); b) the expression of P-selectin (CD62p), the alpha-granule GP translocated to the platelet surface following release reaction; and c) platelet procoagulant activity, as demonstrated by the binding of i) annexin V protein to the prothrombinase-complex (prothrombin, activated factor X (Xa) and V (Va)) binding sites on the surface of activated platelets, and of ii) MoAbs against activated coagulation factors V and X bound to the surface of activated platelets. Using this method, platelet activation as a marker for in vivo prothrombotic activity can be demonstrated in various clinical conditions including coronary angioplasty, orthostatic challenge in primary depression, sickle cell disease in clinical remission and during pain episode, and in pregnancy-related hypertension with marked increase during preeclampsia. The finding of platelet procoagulant activity is corroborated by increased levels of plasma markers for thrombin generation and fibrinolytic activity.

Postmenopausal HRT is not independent risk factor for dural sinus thrombosis.

While a dural sinus thrombosis (DST), is a well-known consequence of the use of oral contraceptives, the role of hormone replacement therapy (HRT) in DST was not previously evaluated. We report two postmenopausal women, presenting with DST under HRT. Antiphospholipid antibodies in one case and borderline protein S deficiency in another were diagnosed. Only five cases of DST under HRT were previously reported and in two of them additional prothrombotic risk factors were found. According to these and previous cases, HRT is not an independent risk factor for DST.

Increased coagulation factor levels leading to allograft renal vein thrombosis.

A patient with end-stage kidney disease is described, who lost his renal allograft in the early post-transplant period due to allograft renal vein thrombosis. Prior to transplantation, he had been treated by hemodialysis and lost several vascular accesses because of thrombosis. A search for potential thrombophilic factors disclosed a unique combination of increased clotting factor levels, i.e. FVIII, FIX, FXI and homocysteine. More common hereditary and acquired hypercoagulability factors have been excluded in this patient. While clotting factor deficiencies are well known causes of hemophilia, their levels should also be measured in the workup of transplant candidates with a history of multiple vascular access thrombosis.

Thermodynamic cycle analysis and inhibitor design against beta-lactamase.

Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase. To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure. The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases. The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes.

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle.

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.

A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle.

The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.

Reduction of pain episodes and prothrombotic activity in sickle cell disease by dietary n-3 fatty acids.

The effects of dietary n-3 fatty acids (n-3FAs) on the frequency of pain episodes and ex vivo blood tests of thrombosis have been evaluated in patients with sickle cell disease (SCD) utilizing a double-blind, olive oil-controlled clinical trial. Dietary n-3FA therapy (0.1 g/kg/d) was provided as menhaden fish oil (0.25 g/kg/d) containing 12% eicosapentaenoic acid (EPA), and 18% docosahexaenoic acid (DHA). Within 1 month dietary n-3FAs exchanged with n-6FAs in plasma and erythrocyte membrane phospholipids (p <0.01 in all cases). Treatment with dietary n-3FAs for 1 year reduced the frequency of pain episodes requiring presentation to the hospital from 7.8 events during the preceding year to 3.8 events/year (p <0.01; n = 5). By contrast, subjects receiving control dietary olive oil (n = 5) experienced 7.1 pain events/year, compared to 7.6 during the previous year (p >0.4). The reduction in episodes in n-3FA-treated subjects was also significant when compared to control subjects (p <0.01). Dietary n-3FA therapy was not associated with hemorrhagic, gastrointestinal or other adverse effects. Compared to 10 asymptomatic African-American controls, sickle cell subjects demonstrated significantly increased pretreatment: 1) flow cytometric expression of platelet membrane P-selectin (CD62p; p <0.01) and annexin V binding sites (p = 0.02); 2) plasma levels of platelet-specific secretory proteins platelet factor 4 (PF4) and beta-thromboglobulin (betaTG) (p <0.01 in both cases); 3) plasma products of thrombin generation, prothrombin fragment 1.2 (F1.2) and thrombin:antithrombin (TAT) complex (p <0.01 in both cases); and 4) plasma levels of thrombolytic products, D-dimer and plasmin:antiplasmin (PAP) complex (p <0.01 in both cases). Treatment with dietary n-3FAs concurrently decreased plasma levels of F1.2, D-dimer, and PAP (p <0.05, compared to olive oil controls), implying that the reduction in pain events was related to n-3FA-dependent inhibition of thrombosis. We conclude that dietary n-3FAs reduce the frequency of pain episodes perhaps by reducing prothrombotic activity in sickle cell disease.

Thrombogenesis in sickle cell disease.

Thirty-three subjects with sickle cell disease (SCD), 11 during episodes of pain and 22 during periods without pain, were evaluated for in vivo thrombogenic activities as compared with 10 normal black control subjects. Measurements were performed for (1) platelet surface activation, assessing flow cytometric expression of activated integrin alpha(IIb)beta(3) receptor (GPIIb/IIIa, CD41a) and P-selectin (CD62p); (2) platelet and erythrocyte surface procoagulant activities, measuring flow cytometric binding of activated factor (FVa) and annexin V; (3) plasma levels of platelet-specific secreted proteins platelet factor 4 (PF4) and beta-thromboglobulin (betaTG); (4) plasma markers of thrombin generation, prothrombin activation fragment (F(1.2)), and thrombin: antithrombin complex (TAT); and (5) plasma markers of fibrinolysis, D -dimer, and plasmin:antiplasmin complex (PAP). As compared with control subjects, asymptomatic subjects with SCD demonstrated significantly increased platelet activation (P <.01 for P-selectin and annexin V binding), elevated plasma levels of PF4 and betaTG (P <.01 and P <.03, respectively), and increased plasma concentrations of F(1.2), TAT, PAP, and D -dimer (P <.05 in all cases). During episodes of SCD pain, platelet activation was increased as compared with periods without pain (P <.01 for expression of activated integrin alpha(IIb)beta(3) receptor and P-selectin and binding of FVa and annexin V), erythrocytes expressed procoagulant activities (P <.01 for FVa and annexin V binding), and platelet microparticles appeared in the circulation (3% to 30%; P <.001). SCD pain episodes were associated with elevated plasma levels of F(1.2), TAT, PAP, and D -dimer (P <.05 as compared with asymptomatic intervals). The frequency of pain episodes correlated with enhanced platelet procoagulant activity (r = 0.61, P <.05) and elevated plasma fibrinolytic activity (r = 0.74, P <.01) measured during periods without pain. Plasma fibrinolytic activity was inversely correlated with time to the next pain episode (r = -0.50, P <.05). Thus, asymptomatic subjects with SCD exhibit ongoing platelet activation, thrombin generation, and fibrinolysis that increases during episodes of pain. These changes are predictive of frequency of pain and interval to next pain episode, thereby implicating thrombogenic activity in the development of SCD pain episodes.

Severe Coombs'-negative autoimmune hemolytic anemia in a kidney transplant patient.

BACKGROUND/AIM: Few cases are found in the literature regarding autoimmune hemolytic anemia which is Coombs' test positive in kidney transplant patients, although hemolytic uremic syndrome due to cyclosporin and FK506 has been well described. In the following, we describe a case of severe life-threatening Coombs' test negative autoimmune hemolytic anemia after kidney transplantation. METHODS: Soon after undergoing renal transplantation, the patient presented with hemolytic anemia. Kidney biopsy, routine Coombs' test, gel filtration and flow-cytometric assay were undertaken. RESULTS: Kidney biopsy ruled out hemolytic uremic syndrome; although Coombs' test and gel filtration assay were negative, flow cytometry revealed circulating antierythrocytic autoantibodies. CONCLUSIONS: Our findings indicate that flow cytometry may be an efficient method in the diagnosis of hemolysis of unknown origin in transplant patients. We further hypothesize that the underlying mechanism of autoimmune hemolytic anemia is related to the passenger B lymphocytes in the graft.

Neurodevelopmental outcome in children with intrauterine growth retardation: a 3-year follow-up.

The study was designed to detect early clinical predictors of developmental outcome in children with intrauterine growth retardation. Eighty-five children with intrauterine growth retardation were followed up prospectively to 3 years of age, using biometric parameters, perinatal risk questionnaires, and neurodevelopmental evaluations. Forty-two children served as controls. A significant difference in neurodevelopmental score at 3 years of age was noted between the intrauterine growth retardation and control groups (P < .001). In the intrauterine growth retardation group, the clinical parameters that most significantly correlated with outcome were cephalization index (head circumference:birthweight ratio), neonatal risk score, and birthweight. The best predictor of 3-year outcome was the cephalization index (P < .01). The children with intrauterine growth retardation with neonatal complications had significantly lower IQ scores (P < .05) and a poorer neurodevelopmental outcome (P < .01) than those without complications. Children with intrauterine growth retardation are at higher risk for developmental disabilities than are controls, especially in the presence of neonatal complications and a high cephalization index.

Determination of heparin-induced thrombocytopenia: a rapid flow cytometric assay for direct demonstration of antibody-mediated platelet activation.

Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the alpha-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment.

High-dose intravenous immunoglobulin in transient neonatal myasthenia gravis.

A preterm newborn had transient neonatal myasthenia gravis and was mechanically ventilated for 9 days. In addition to the usual supportive care, she was treated with neostigmine and underwent two exchange transfusions. High-dose intravenous immunoglobulin therapy (2 gm/kg) was used for the first time in transient neonatal myasthenia gravis to the best of our knowledge. The clinical and laboratory responses are presented.

A sensitive and specific functional flow cytometric assay for the diagnosis of heparin-induced thrombocytopenia.

A functional flow cytometric assay (FCA) for the immediate diagnosis of heparin-induced thrombocytopenia (HIT), with simultaneous compatibility testing for alternative anticoagulant therapies, has been developed to provide rapid and reliable results which effectively support patient management. The assay provides results within 1-2 h, uses readily available non-radioactive reagents, and employs standard equipment. Using the highly sensitive annexin V protein probe, the method detects activated platelets induced by heparin immune-complexes, with 300-fold increased binding to activated platelets. Twenty-five samples from patients clinically-suspected of having HIT (131 tests) and 10 normal control (NG) samples (36 tests) were simultaneously tested with unfractionated heparin (UH) and low-molecular-weight heparin (LMWH), and by the radioactive serotonin-release assay (SRA) (62 and 16 tests respectively). The FCA highly correlated with the SRA, showing 100% specificity and 95% sensitivity. Moreover, the FCA exhibited higher resolution between positive and negative samples (an average value of 8.6-fold the NC versus 4.0-fold the NC by SRA). The LMWH showed concordant results with UH (r = 0.95). We conclude that the functional FCA for HIT is practical, specific and sensitive, thereby permitting the rapid diagnosis of HIT and the suitability of alternative therapies.