Anti-protein C antibodies are associated with resistance to endogenous protein C activation and a severe thrombotic phenotype in antiphospholipid syndrome.

BACKGROUND: Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). AIMS: To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). METHODS: APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. RESULTS: APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. CONCLUSION: Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-ß2 -glycoprotein I antibodies are responsible for APCr.

The automation of routine light transmission platelet aggregation.

INTRODUCTION: The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser. METHODS: We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5-10 µm), epinephrine (0.5-10 µm), collagen (0.5-10 mg/µL), ristocetin (0.75-1.25 mg/mL) and arachidonic acid (0.12-1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument. RESULTS: CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150-480 x 10(9) /L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2-5 µm ADP MA and slope varying between 3-12%. CONCLUSION: Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200-300 x 10(9) /L; aggregation with a PRP count <100 x 10(9) /L showed poor sensitivity. Imprecision and detection of antiplatelet drug effects was similar between the CS-2000i and AggRAM. These data demonstrate that CS-2000i is comparable to a stand-alone aggregometer, although CS-2000i has the advantages of walk-away technology and also required a smaller sample volume than the AggRAM (44% less).

A phenotype-genotype correlation of ADAMTS13 mutations in congenital thrombotic thrombocytopenic purpura patients treated in the United Kingdom.

BACKGROUND: ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis. OBJECTIVES: To establish a phenotype-genotype correlation in a cohort of congenital TTP patients. PATIENTS/METHODS: Clinical history and ADAMTS13 activity, antigen and anti-ADAMTS13 antibody assays were used to diagnose congenital TTP, and DNA sequencing and in vitro expression were performed to identify the functional effects of the ADAMTS13 mutations responsible. RESULTS: Seventeen (11 novel) ADAMTS13 mutations were identified in 17 congenital TTP patients. All had severely reduced ADAMTS13 activity and antigen levels at presentation. Six patients with pregnancy-associated TTP and six patients with childhood TTP were homozygous or compound heterozygous for ADAMTS13 mutations located in the metalloprotease (MP), cysteine-rich, spacer and/or distal thrombospondin type 1 domains. The adults had TTP precipitated by pregnancy, and had overall higher antigen levels (median, 30 ng mL(-1) ; range, < 10-57 ng mL(-1) ) than the children (median, 14 ng mL(-1) ; range, < 10-40 ng mL(-1)). Presentation in the neonatal period was associated with more intensive treatment requirements. The two neonates with the most severe phenotype had mutations in the first thrombospondin type 1 motif of ADAMTS13 (p.R398C, p.R409W, and p.Q436H). Using transfected HEK293T cells, we have shown that p.R398C and p.R409W block ADAMTS13 secretion, whereas p.Q436H allows secretion at reduced levels. CONCLUSIONS: This study confirms the heterogeneity of ADAMTS13 defects and an association between ADAMTS13 genotypes and TTP phenotype.

Evaluation of an automated platelet-based assay of ristocetin cofactor activity.

von Willebrand's disease (VWD) is regarded as the most common congenital bleeding disorder, and although not available in all laboratories von Willebrand factor (VWF) activity is most frequently assessed as ristocetin cofactor (VWF:RCo). This test can be technically challenging, is subject to poor sensitivity (∼20 IU dL(-1) VWF:RCo) and has a high degree of inter- and intra-assay imprecision [coefficient of variation (cv) > 25%]. We studied an automated assay using a combined fixed platelet/ristocetin reagent (BC von Willebrand reagent, Siemens Healthcare Diagnostics) on the CS-2000i analyser (Sysmex UK Ltd). Initially inter- and intra-assay imprecision was assessed. The automated method showed good day-to-day reproducibility and linearity of standard curves. This technique, also gave low intra- and inter-assay imprecision using commercial normal (cv < 4.5%) and pathological (cv < 8.1%) control plasmas. We then compared automated technique results from 30 healthy normal subjects and 39 VWD patients to those obtained using standard aggregometry (Bio/Data, PAP4) with lyophilised fixed platelets (Helena BioSciences) and ristocetin (American Biochemical and Pharmaceutical Ltd). The automated method had a sensitivity limit of approximately 10 IU dL(-1) vs. 20 IU dL(-1) for aggregometry. Samples giving results within the aggregometry measurable range (n = 50) exhibited good correlation with the automated technique (median 70 IU dL(-1), range 7-184 IU dL(-1); and 64 IU dL(-1), 6-138 IU dL(-1) respectively; R(2) = 0.85). We subsequently compared 3 different batches of BC von Willebrand reagent, using a second group of normal subjects and VWD patients (n = 35, 55-139 IU dL(-1) and n = 30, <10-50 IU dL(-1)). The CS-2000i results exhibited no clinically significant variation between batches (mean cv = 7%). The automated VWF:RCo assay offers a more sensitive, reproducible, robust and less laborious alternative to standard aggregometry.

Factor XIII--an under diagnosed deficiency--are we using the right assays?

BACKGROUND: The clot solubility test is the most widely used method for detection of factor (F)XIII deficiency. However, it will only detect severe deficiencies; consequently mild deficiencies and heterozygous states are probably under diagnosed. OBJECTIVE: As an alternative first-line screening test, we assessed an automated quantitative ammonia release assay (QARA). PATIENTS/METHODS: Inter-assay imprecision was evaluated with commercial normal and pathological control plasmas (10 replicates on each of 5 days). Using the QARA and other commercial assays a comparative assessment of congenital (FXIII range < 1-70 u dL(-1), n = 9) and acquired (n = 43) deficiencies was made. We also investigated the prevalence of acquired deficiencies in hospitalized patients using residual samples from adult patients (n = 1004) and from a paediatric intensive care unit (ICU, n = 56). RESULTS: Assay imprecision was acceptably low (normal control: mean 86.6 u dL(-1); cv = 2.0%; pathological control: mean 27.5 u dL(-1); cv = 3.8%). Using an iodoacetamide blanking procedure, the QARA results (FXIII range < 1-70 u dL(-1)) exhibited close agreement with those from an immuno-turbidometric FXIII A-subunit (FXIII-A) method. There was also good correlation (R(2) ≥ 0.89) between the QARA (range 20-180 u dL(-1)), a second chromogenic assay, the FXIII-A and FXIII A+B-subunit ELISA. We found that 21% of samples from adult patients had FXIII levels < 70 u dL(-1) (mean normal ± 2 SD 73-161 u dL(-1)) with 6% < 50 u dL(-1). Within the paediatric ICU samples, 52% were < 70 u dL(-1), with 21% < 50 u dL(-1). CONCLUSIONS: Our data demonstrates that the automated assay is sensitive, highly reproducible and the results from clinical samples suggest that acquired FXIII deficiency is a relatively common phenomenon in hospital patients after surgery and in ICU.

Berend Houwen Memorial Lecture: ISLH Las Vegas May 2009: the pathogenesis and management of thrombotic microangiopathies.

Thrombotic microangiopathies are a relatively rare group of congenital and inherited disorders caused by defects in processing the ultra large forms of von Willibrand factor which pathologically give rise to platelet rich microthrombi in the micro arterial circulation leading to end organ damage particularly in the brain, heart and kidneys. Identification of the ADAMTS 13 gene has led to the definition of congenital deficiency of its activity or failure of activity due to the development of an inhibitory IgG antibody. The idiopathic autoimmune form of the disease is the most common. There are various subgroups of acquired TTP associated with HIV infection, pregnancy, pancreatitis, associated with bone marrow transplantation, various disseminated malignancies and certain drugs, particularly Clopidogrel. Diagnostic assays are now becoming widely available to identify ADAMTS 13 activity and also acquired antibodies to the enzyme. Mainline treatment is associated with daily plasma exchange with associated other immunosuppressant treatments particularly steroids and recently the use of Rituximab, a monoclonal anti- CD20 antibody. Despite improvement in treatment modalities there is still significant mortality of 10-20%, particularly if there is a delay in initiating plasma exchange. Relapse also occurs in 20-50% of patients although this may be improved by Rituximab therapy.

Immature platelet fraction measurement: a future guide to platelet transfusion requirement after haematopoietic stem cell transplantation.

summary. The decision to prophylactically transfuse platelets is dependent on the platelet count, careful regular clinical assessment and agreed local protocol. The ability to predict when platelet recovery will occur should allow a more reasoned approach to platelet transfusion. An increase in reticulated platelets demonstrates impending platelet recovery. A new rapid automated method to assess reticulated platelets, the immature platelet fraction (IPF), is described, and its clinical utility assessed. The IPF is identified by flow cytometry with the use of a nucleic acid specific dye in the reticulocyte channel on the Sysmex XE-2100. Fifty healthy adult volunteers were used to establish the normal range. Patients where platelet marrow production or destruction might be abnormal were studied, and some patients followed serially during treatment. Thirty patients receiving cytotoxic chemotherapy were tested, and 13 of these patients followed serially. Fifteen patients post-autologous or allogeneic transplant were followed daily for platelet count and IPF percentage to monitor platelet recovery. The method demonstrates good reproducibility and stability. The recovery phase of thrombocytopenia in most chemotherapy and transplant patients was preceded by a rise in IPF percentage several days prior to platelet recovery. In particular, patients undergoing autologous transplantation (n = 8) using peripherally collected stem cells have a characteristic IPF percentage motif, with a rise one or two days prior to engraftment. The automated IPF is a useful parameter in the clinical evaluation of the thrombocytopenic patient and has the potential to allow optimal transfusion of platelet concentrates.

Thrombotic thrombocytopaenic purpura in HIV-infected patients.

Thrombotic thrombocytopaenic purpura (TTP) results from deficiency of von Willebrand factor-cleaving protease (vWF-cp) activity. Eight HIV-infected patients presented with TTP, representing 12.5% of all TTP treated at this centre. In four patients presentation with TTP revealed underlying HIV infection, the other four patients were previously known to be HIV infected, with plasma exchange and highly active antiretroviral therapy (HAART) all recovered. Normalization of vWF-cp activity was associated with recovery. Relapse occurred in two patients who discontinued HAART against medical advice, suggesting that HIV has a causal role in this condition. Given the clear benefit from HAART in addition to plasma exchange, these data suggest that all patients presenting with TTP should undergo HIV testing.

The anticardiolipin assay is required for sensitive screening for antiphospholipid antibodies.

The importance of testing for anticardiolipin antibodies (aCL) in the diagnosis of antiphospholipid syndrome (APS) in patients with thrombosis has recently been challenged (ISTH SSC meeting, Boston 2002). We have analyzed the antiphospholipid serology of 123 patients with persistent antiphospholipid antibodies (aPL) attending our hematology department. The cohort was tested for anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies and aCL of IgG and IgM class and for lupus anticoagulant (LA). Ninety-six of these patients fulfilled Sapporo clinical criteria for APS and 70 of these patients had venous and/or arterial thrombosis. Patients with LA plus anti-beta(2)-GPI antibodies had significantly higher levels of IgG aCL and anti-beta(2)-GPI antibodies than those exhibiting positivity for only LA or anti-beta(2)-GPI antibodies (P < 0.05). Patients with aCL IgG levels over 60 GPLU were found in all cases to be positive for LA and anti-beta(2)-GPI antibodies; 25.2% (31/123) of all patients and 26.04% (25/96) of patients fulfilling Sapporo clinical criteria for APS were positive for aCL only. The mean IgG aCL level in the Sapporo clinical criteria positive patients who had aCL only was 11.5 GPLU (normal < 5 GPLU). These data indicate that omission of aCL testing from the clinical investigation of APS could lead to a failure to diagnose the syndrome in a proportion of patients.

Performance evaluation of a new compact hematology analyzer, the Sysmex pocH-100i.

Medical practitioners are searching for ways to improve the quality and outcome of care while decreasing cost. One technological advance that may facilitate this is point-of-care testing. The pocH-100i hematology analyzer (Sysmex Corporation, Kobe, Japan) is a compact device designed specifically for point-of-care testing environments. The analyzer provides a full blood count and a 3-part differential leukocyte count. A distinct advantage is that neutrophils are reported separately, which is useful for monitoring oncology patients. The device was evaluated in accordance with International Council for Standardization in Haematology (ICSH) guidelines for precision, linearity, carryover, and effects of sample aging. The pocH-100i analyzer was compared with the Sysmex KX-21N device. The results for all parameters tested were almost identical. When samples including blast cells, immature granulocytes, and nucleated red blood cells were excluded, the pocH-100i automated differential compared well with a 400-cell manual differential. Results for neutrophils (r2 = 0.996), lymphocytes (r2 = 0.999), and the "mixed" population of cells (r2 = 0.611) indicated the pocH-100i analyzer would be highly suitable for low-volume laboratories and near-patient services.

Patients with essential thrombocythaemia have an increased prevalence of antiphospholipid antibodies which may be associated with thrombosis.

A significant proportion of patients with Essential Thrombocythaemia (ET) have thrombotic complications which have an important impact upon the quality, and duration of their life. We performed a retrospective cross sectional study of the prevalence of antiphospholipid antibodies (APA) in 68 ET patients. Compared to 200 "elderly" controls (>50 years) there was a significant increase in anticardiolipin IgM (p < 0.0001) and anti beta2 glycoprotein I (anti-beta2GPI) IgM (p < 0.0001) antibodies in ET. Thrombosis occurred in 10/20 with APA and 12/48 without, p = 0.04, relative risk 2.0 (95% confidence intervals 1.03-3.86): these patients did not differ in terms of other clinical features. The prevalence of thrombosis in patients with dual APA (6/7) was significant when compared to those with single APA (p = 0.02) and the remaining patients (p < 0.0002). Also anti-beta2GP1 IgM antibodies either alone, or in combination with another APA, were associated with thrombosis (p = 0.02). These results suggest that the prevalence of APA in ET and their influence upon thrombotic risk merit investigation in a larger study.

An evaluation of screening tests for defects in the protein C pathway: commercial kits lack sensitivity and specificity.

A comparative evaluation of four commercial coagulation test kits for screening the protein C pathway kits was performed at two centres. The tests were Acticlot V-OUT (V-OUT), the PCA test (PCA), the GradiThrom PCP test (PCP) and ProC Global (ProC). Reference ranges were established in 40 normal subjects and, with the exception of V-OUT and ProC, significant differences were observed between males and females. Consequently, sex-specific normal cut-off values (fifth percentile) were used that led to greatly improved sensitivity when compared with the manufacturers' recommended cut-off values. Plasma from patients with factor V Leiden (n = 23), congenital protein S deficiency (n = 19), congenital protein C deficiency (n = 11), lupus anticoagulant (n = 17) and thrombophilia with no demonstrable protein C pathway defect (n = 20) were tested. All kits showed 100% sensitivity to factor V Leiden, but sensitivity was variable for protein C deficiency (27-73%), and poor for protein S deficiency (29-35%). The lupus anticoagulant affected all kits to some degree, with 29-35% giving values below the fifth percentile of normal, whereas all kits gave 1/20 unexpected abnormal results in the thrombophilia group, with the same sample accounting for the abnormal results in three of the four kits. Overall sensitivity and specificity, respectively, for defects in the protein C pathway were: V-OUT, 60 and 91%; PCA, 70 and 86%; PCP 75 and 94%; and ProC, 66 and 88%. We conclude that all four kits lack the sensitivity and specificity required for routine laboratory screening for defects in the protein C pathway and cannot replace assays for the individual proteins of this system.

Lack of pathogenic mutations in the 5'-untranslated region of the thrombopoietin gene in patients with non-familial essential thrombocythaemia.

Thrombopoietin (TPO) is thought to be the major physiological regulator of thrombopoiesis, and, in general, circulating levels are inversely proportional to megakaryocyte and platelet mass. However, normal or elevated TPO levels are found in patients with essential thrombocythaemia (ET) and the reason for this is not fully understood. Recent studies have shown that four kindreds with hereditary thrombocythaemia (HT) have point mutations in the 5'-untranslated region (UTR) of the TPO gene which lead to increased TPO translation. In order to determine whether similar mutations are present in apparently acquired ET, in particular in those patients with polyclonal myelopoiesis, we have studied this region in 50 ET patients using neutrophil DNA. The known HT mutations were investigated using polymerase chain reaction with mismatch primers and restriction enzyme digestion; only wild-type alleles were detected. Single-stranded conformation polymorphism (SSCP) analysis of exons 1-4 identified a C-->T substitution at nucleotide 3767. However, this appears to be a common polymorphism, as it was present at the same frequency in haematologically normal controls and is unlikely to be of pathological significance. These results demonstrate that mutations in the 5' UTR of the TPO gene are not the cause of the normal or elevated TPO levels in acquired ET.

Platelet c-mpl expression is dysregulated in patients with essential thrombocythaemia but this is not of diagnostic value.

Essential thrombocythaemia (ET) can be difficult to discriminate from an occult case of reactive thrombocytosis (RT). Since thrombopoietin (TPO) is the primary regulator of thrombopoiesis, we have investigated whether levels of TPO and/or its receptor, c-mpl, are of value in the differential diagnosis of ET. Plasma TPO levels in patients with ET, RT and other myeloproliferative disorders (MPDs) did not differ significantly from normal controls. However, surface c-mpl expression was significantly reduced in platelets from 18 ET patients, 0-65.5% of controls (P < 0.001). Immunoblots on five of these and five additional patients were consistent with absent or reduced c-mpl protein levels. The surface c-mpl expression results were significantly different from those in eight RT patients (21. 3-95.5%, P = 0.0015), but there was considerable overlap between the two groups and a reduced level was not restricted to ET. Furthermore, c-mpl expression in ET patients was not different from eight patients with other MPDs (0-87.6%, P = 0.06), nor could it differentiate between ET patients with monoclonal and polyclonal haemopoiesis. Although a low or absent c-mpl level is suggestive of a primary rather than a secondary thrombocytosis, it is insufficiently discriminatory to be used as a diagnostic marker for ET.

Use of the haemopoietic progenitor cell count of the Sysmex SE-9500 to refine apheresis timing of peripheral blood stem cells.

The Sysmex SE-9500 automated cell counter provides an estimate of immature cells referred to as 'haemopoietic progenitor cells' (HPC). The aim of this study was to relate the HPC count to CD34+ cell levels in mobilized peripheral blood and to determine whether the HPC count was valuable in predicting apheresis yields of CD34+ cells. Studies were performed on 114 samples from 67 patients undergoing progenitor cell mobilization. HPC cells were undetectable in the steady state. On the day of apheresis the HPC and CD34 counts were weakly correlated, with the median HPC count being 2.3-fold greater than the CD34+ cell count. The HPC count did not include the CD34+ cells as immunomagnetic depletion of CD34+ cells did not significantly reduce the HPC count. CD34+ cell counts predicted for apheresis yield (r = 0.773) on that day as did the HPC count (r = 0. 623). The optimal strategy to prevent unnecessary harvesting while minimizing the risk of missing an adequate harvest, and minimizing laboratory investigations, was to screen all samples for HPC and limit CD34+ cell measurements to those with an HPC count <10 x 106/l (19/114 samples). If the CD34+ cell count was also <10 x 106/l then harvesting should not be carried out.

Clopidogrel: a novel antiplatelet agent.

Clopidogrel is a novel antiplatelet agent that has a different mechanism of action to aspirin. Clopidogrel is chemically related to ticlopidine, both of which are more effective than aspirin in preventing some thrombotic events, but it has a safer side-effect profile than ticlopidine. It is likely to add to the antithrombotic armoury and reduce vascular mortality and morbidity more than current therapies.

Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single ascending oral doses of sibrafiban, an oral platelet aggregation inhibitor, in healthy male volunteers.

AIMS: This study constituted the first administration of the oral platelet inhibitor, sibrafiban, to humans. The aim was to investigate the pharmacokinetics and pharmacodynamics of Ro 44-3888, the active principle of sibrafiban, after single ascending oral doses of sibrafiban. Particular emphasis was placed on intersubject variability of the pharmacokinetic and pharmacodynamic parameters of Ro 44-3888. METHODS: The study consisted of three parts. Part I was an open ascending-dose study to determine target effect ranges of sibrafiban. Part II, a double-blind, placebo-controlled, parallel-group study, addressed the intersubject variability of pharmacokinetic and pharmacodynamic parameters of the active principle at a sibrafiban dose achieving an intermediate effect. Part III was a double-blind, placebo-controlled, ascending-dose design covering the complete plasma concentration vs pharmacodynamic response curve of sibrafiban. RESULTS: At sibrafiban doses between 5 mg and 12 mg, the pharmacokinetics of free Ro 44-3888 in plasma were linear whereas those of total Ro 44-3888 were non-linear because of the saturable binding to the glycoprotein IIb-IIIa receptor. Saturation of the GP IIb-IIIa receptor was reached at plasma concentrations of 15.9 ng ml-1. At sibrafiban doses up to 2 mg, ADP-induced platelet aggregation was inhibited by 50%, whereas the inhibition of TRAP-induced platelet aggregation was about 20-30%. At the higher doses, ADP-induced platelet aggregation was almost completely inhibited while a clear dose-response could be observed with TRAP-induced inhibition of platelet aggregation at sibrafiban doses of 5 to 12 mg. Ivy bleeding time increased very steeply with dose with a significant prolongation observed at doses of 5 to 7 mg of sibrafiban (5-7 min, >30 min in one case). At a sibrafiban dose of 12 mg, the stopping criterion for dose escalation (prolongation of the Ivy bleeding time >30 min in three out of four subjects per dose group) was reached. The interindividual coefficients of variation of the integrated pharmacokinetic and pharmacodynamic parameters (AUC and AUE) were below 20%, thus lying well within the pre-set level of acceptance. CONCLUSIONS: With a low intersubject variability of its pharmacokinetic and pharmacodynamic parameters, linear pharmacokinetics and pharmacodynamic effects closely related to its plasma concentrations, Ro 44-3888 has good pharmacological prerequisites for a well controllable therapy of secondary prevention of arterial thrombosis in patients with acute coronary syndrome.

Hextend, a physiologically balanced plasma expander for large volume use in major surgery: a randomized phase III clinical trial. Hextend Study Group.

UNLABELLED: Hextend (BioTime, Inc., Berkeley, CA) is a new plasma volume expander containing 6% hetastarch, balanced electrolytes, a lactate buffer, and physiological levels of glucose. In preclinical studies, its use in shock models was associated with an improvement in outcome compared with alternatives, such as albumin or 6% hetastarch in saline. In a prospective, randomized, two-center study (n = 120), we compared the efficacy and safety of Hextend versus 6% hetastarch in saline (HES) for the treatment of hypovolemia during major surgery. Patients at one center had a blood sample drawn at the beginning and the end of surgery for thromboelastographic (TEG) analysis. Hextend was as effective as HES for the treatment of hypovolemia. Patients received an average of 1596 mL of Hextend: 42% received >20 mL/kg up to a total of 5000 mL. No patient received albumin. Hextend-treated patients required less intraoperative calcium (4 vs 220 mg; P < 0.05). In a subset analysis of patients receiving red blood cell transfusions (n = 56; 47%), Hextend-treated patients had a lower mean estimated blood loss (956 mL less; P = 0.02) and were less likely to receive calcium supplementation (P = 0.04). Patients receiving HES demonstrated significant prolongation of time to onset of clot formation (based on TEG) not seen in the Hextend patients (P < 0.05). No Hextend patient experienced a related serious adverse event, and there was no difference in the total number of adverse events between the two groups. The results of this study demonstrate that Hextend, with its novel buffered, balanced electrolyte formulation, is as effective as 6% hetastarch in saline for the treatment of hypovolemia and may be a safe alternative even when used in volumes up to 5 L. IMPLICATIONS: Hextend (BioTime, Inc., Berkeley, CA) is a new plasma volume expander containing 6% hetastarch, balanced electrolytes, a lactate buffer, and a physiological level of glucose. It is as effective as 6% hetastarch in saline for the treatment of hypovolemia but has a more favorable side effects profile in volumes of up to 5 L compared with 6% hetastarch in saline.

A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications.

Essential thrombocythemia (ET) is traditionally considered to be a clonal disorder. No specific karyotypic abnormalities have been described, but the demonstration of clonality using X-chromosome inactivation patterns (XCIPs) has been used to differentiate ET from a non-clonal reactive thrombocytosis. However, these assays may be difficult to interpret, and contradictory results have been reported. We have studied 46 females with a diagnosis of ET according to the Polycythemia Vera Study Group (PVSG) criteria. XCIP results in 23 patients (50%) were uninterpretable due to either constitutive or possible acquired age-related skewing. Monoclonal myelopoiesis could be definitively shown in only 10 patients. Thirteen patients had polyclonal myelopoiesis, and in 8, it was possible to exclude clonal restriction to the megakaryocytic lineage. Furthermore, there was no evidence of clonal progenitors in purified CD34(+)CD33(-) and CD34(+)CD33(+) subpopulations from bone marrow of 2 of these 13 patients. There was no difference between patients with monoclonal and polyclonal myelopoiesis with respect to age or platelet count at diagnosis, duration of follow-up, incidence of hepatosplenomegaly, or hemorrhagic complications. However, polyclonal patients were less likely to have experienced thrombotic events (P =.039). These results suggest that ET is a heterogeneous disorder, and the clinical significance of clonality status warrants investigation in a larger study.