Diagnostic utility of the lymphoid screening tube supplemented with TRBC1 for the assessment of T-cell clonality.

INTRODUCTION: Flow cytometric panels for the investigation of lymphoproliferative disorders, such as the EuroFlow Lymphoid Screening Tube (LST), often fail to demonstrate T-cell clonality, as a suitable clonality marker was unavailable until recently. Aim of this study was to evaluate the added value of supplementing TRBC1, a flow cytometric T-cell clonality marker, to the LST. METHODS: Flow cytometric analysis was performed on 830 routine samples referred to our lab for suspicion of hematological malignancy. T-cells with monotypic TRBC1-expression were additionally characterized with a 12-color T-cell tube and molecular T-cell receptor gamma gene rearrangement (TRG). RESULTS: LST analysis revealed 97 (11.7%) samples with the presence of a monotypic T-cell population according to TRBC1, including 21 (2.5%) "high-count" (≥500 cells/µL blood or ≥15% of lymphocytes) and 76 (9.2%) "low-count" (<500 cells/µL blood or <15% of lymphocytes) populations. Clinical symptoms indicative for T-CLPD could be correlated to 11/21 "high-count" and 17/76 "low-count" monotypic T-cell populations. Molecular TRG analysis demonstrated a monoclonal result in 76% (16/21) of "high-count" samples and in 64% (42/66; 10 samples not tested) of "low-count" samples, but also in 9/20 samples with polytypic TRBC1 results. CONCLUSION: Analysis of an LST tube supplemented with TRBC1 led to the detection of a high number of monotypic T-cell populations. The detection of numerous small monotypic T-cell populations raises the question of their clinical significance. A possible flowchart for assessment of these populations, based on the available literature, is proposed. Molecular TRG analysis is complementary and cannot be omitted from T-cell clonality assessment.

Evaluation of two automated cell counters for the analysis of hematopoietic progenitor cell apheresis products.

INTRODUCTION: Routine hematology parameters in hematopoietic progenitor cell apheresis products (HPC-A) are usually determined using automated cell counters. These instruments, however, are designed to analyze whole blood samples, that differ considerably from HPC-A in blood cell composition. This study evaluates the performance of two automated cell counters for the analysis of HPC-A. METHODS: Routine hematology parameters [red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (MCV), white blood cells (WBC), WBC differentiation, and platelets (PLT)] were determined on the Unicel DxH 800 instrument (Beckman Coulter) and the XN-350 instrument (Sysmex). Correlations with the reference methods, intrarun precision, and linearity of the analyses were studied. RESULTS: Good correlations were found for almost all parameters. However, RBC count was overestimated by XN-350, using the impedance technique, as was neutrophil percentage using DxH 800. Coefficients of variation for intrarun precision were below 10% on both analyzers for all parameters, except for neutrophil percentage (14.7%) and PLT (10%) on DxH 800. Both instruments showed good linearity for all parameters, except for RBC and HCT on DxH 800. CONCLUSION: With the exception of the measurement of neutrophils on DxH 800 and RBC by the impedance technique on the XN-350, routine hematology parameters in HPC-A can safely be determined using automated cell counters.

Evaluation of the Red Blood Cell Advanced Software Application on the CellaVision DM96.

INTRODUCTION: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBCs on a digital microscopy system. Upon automated precharacterization into 21 categories, the software offers the possibility of reclassification of RBCs by the operator. We aimed to define the optimal cut-off to detect morphological RBC abnormalities and to evaluate the precharacterization performance of this software. METHODS: Thirty-eight blood samples of healthy donors and sixty-eight samples of hospitalized patients were analyzed. Different methodologies to define a cut-off between negativity and positivity were used. Sensitivity and specificity were calculated according to these different cut-offs using the manual microscopic method as the gold standard. Imprecision was assessed by measuring analytical within-run and between-run variability and by measuring between-observer variability. RESULTS: By optimizing the cut-off between negativity and positivity, sensitivities exceeded 80% for 'critical' RBC categories (target cells, tear drop cells, spherocytes, sickle cells, and parasites), while specificities exceeded 80% for the other RBC morphological categories. Results of within-run, between-run, and between-observer variabilities were all clinically acceptable. CONCLUSION: The CellaVision Advanced RBC Software Application is an easy-to-use software that helps to detect most RBC morphological abnormalities in a sensitive and specific way without increasing work load, provided the proper cut-offs are chosen. However, evaluation of the images by an experienced observer remains necessary.

Bing-Neel syndrome: Two unexpected cases and a review of the literature.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by the proliferation of small B-lymphocytes in the bone marrow that produce monoclonal immunoglobulin M (IgM). We describe two patients with WM who presented with neurological symptoms due to infiltration of lymphoplasmacytoid tumor cells in the central nervous system, a condition known as Bing-Neel syndrome. A literature review revealed that this syndrome is rare and commonly missed in clinical practice due to its variable presentation and a lack of awareness or knowledge. Brain and spinal magnetic resonance imaging may show a focal mass or diffuse infiltration. The diagnosis of Bing-Neel syndrome requires proof of IgM or lymphoplasmacytoid cells in cerebrospinal fluid or in a brain biopsy. Treatment with intravenous and/or intrathecal chemotherapy and cranial radiotherapy is described in literature with generally poor outcome, although a combination of these therapies seems to improve outcome. Nevertheless, insufficient data are currently available to make general treatment recommendations.

Evaluation of schistocyte analysis by a novel automated digital cell morphology application.

INTRODUCTION: The CellaVision Advanced Red Blood Cell (RBC) Software Application is a new software for advanced morphological analysis of RBC, which automatically performs a preliminary characterization and grouping of RBC into 21 morphological categories, including schistocytes. Upon automated classification, the software offers the possibility of reclassification of RBC by the operator. The aim of this study was to evaluate the schistocyte analysis by the CellaVision Advanced RBC Application. METHODS: Schistocyte counts were evaluated comparing the automated count on a CellaVision DM96, both before and after reclassification, with the reference manual microscopic method according to the ICSH criteria. Thirty-six samples of hospitalized patients and 40 samples of controls were analyzed. RESULTS: Within-run, between-run and between-observer coefficients of variation were lower when counted with the CellaVision compared to the manual microscopic count. The very high sensitivity but rather poor specificity implicates the need for reclassification by the operator, following automated analysis. After reclassification, method comparison studies revealed good agreement with the manual microscopic method, with however slightly higher values of schistocytes for the automated analysis. CONCLUSION: The CellaVision Advanced RBC Software Application provides a sensitive and reproducible measurement of schistocytes in peripheral blood, but still requires manual revision. Furthermore, it is an easy-to-use software and an excellent teaching tool that might contribute to standardization in the investigation of schistocyte-related conditions.

Antidote strategies to reverse anticoagulation with TB-402, a long-acting partial inhibitor of factor VIII.

BACKGROUND: TB-402 is a partially inhibiting antibody of factor VIII that is under development as a long-acting anticoagulant. PATIENTS AND METHODS: The reversibility of FVIII inhibition by TB-402 was evaluated in vitro after spiking with recombinant human FVIII (rhFVIII), human plasma-derived FVIII (hpdFVIII), recombinant activated human FVII (rhFVIIa), FVIII inhibitor bypassing activity (FEIBA), and prothrombin complex concentrate (PCC). Twelve subjects were randomized to placebo or 35 or 70 IU kg(-1) rhFVIII 48 h after a single dose of 620 µg kg(-1) TB-402. TB-402 concentrations, FVIII activity (FVIII:C), activated partial thromboplastin time (APTT) and thrombin generation were measured over a period of 8 weeks. RESULTS: In spiked samples, TB-402 inhibited FVIII:C by 30%, prolonged APTT by 4.5 s, and reduced the peak height in the thrombin generation assay to 56% ± 13% of the control value. In the presence of 10 µg mL(-1) TB-402, rhFVIII restored FVIII:C and APTT to the values obtained in the absence of TB-402. The inhibitory effect of TB-402 on thrombin generation was entirely reversed by rhFVIII, hpdFVIII, rhFVIIa, FEIBA, and PCC. In men, the mean half-life (t(1/2) ) of TB-402 was 14.2 days. TB-402 lowered the endogenous thrombin potential by 23% for ~ 35 days. Infusion of 35 IU kg(-1) rhFVIII had a marginal effect, whereas 70 IU kg(-1) rhFVIII restored FVIII:C, reduced APTT back to baseline for 9 h, and restored thrombin generation for ~ 3 h. CONCLUSIONS: TB-402 resulted in a stable long-term anticoagulant effect. rhFVIII and other procoagulants counteracted the effect of TB-402 temporarily, and may be effective antidotes for future clinical practice.

The effect of air pollution on haemostasis.

Ambient environmental air pollutants include gaseous and particulate components. In polluted air, especially particulate matter seems responsible for cardiovascular complications: It consists of a heterogeneous mixture of solid and liquid particles with different diameters ranging from large thoracic to ultrafine particles, with a diameter <100 nm. Ultrafines can penetrate deeply into the lung to deposit in the alveoli. Cardiovascular manifestations result both from short-term and long-term exposure and have been linked to interference with the autonomic nervous system, direct translocation into the systemic circulation, pulmonary inflammation and oxidative stress. Thrombotic complications associated with air pollution comprise arterial and probably venous thrombogenicity. This review describes the existing epidemiological and experimental evidence to explain the rapid induction of myocardial infarction within 1-2 hours after exposure to polluted air and advances several explanations as to why more chronic exposure will lead to enhanced venous thrombogenicity. Mechanisms such as platelet activation, endothelial dysfunction, coagulation factor changes and microvesicle production are discussed.

Partial versus complete factor VIII inhibition in a mouse model of venous thrombosis.

INTRODUCTION: Partial inhibition of Factor VIII (FVIII) may provide antithrombotic efficacy whilst avoiding excessive anticoagulation. MATERIALS AND METHODS: We studied the anticoagulant effects of a partial (TB-402) and a complete (BO2C11) FVIII-inhibiting monoclonal antibody (MAb) on FVIII, aPTT, thrombin generation and fibrin deposition in a flow chamber model. The antithrombotic efficacy of TB-402 and BO2C11 was compared in a mouse model of venous thrombosis. RESULTS: Both in vitro and ex vivo, the maximally achievable FVIII inhibition by TB-402 was about 25 to 30%. The degree of inhibition reached a plateau in vitro at 0.316 µg/mL and ex vivo after administering 0.1mg/kg and higher doses. BO2C11 strongly inhibited FVIII:C, up to 91% at 100 µg/mL in vitro, and by 88% ex vivo 1 hour after administering 1mg/kg to the mice. Whereas BO2C11 also markedly prolonged the aPTT and completely inhibited thrombin generation in vitro and ex vivo, the effect of TB-402 on the aPTT and on thrombin generation was limited. Similarly, in a dynamic flow chamber model, TB-402 and BO2C11 inhibited tissue factor-induced human fibrin deposition by 40% and 76%, respectively. In a mouse model of FeCl(3)-induced venous thrombosis, TB-402 (1mg/kg) inhibited thrombus formation to the same extent as BO2C11 (2mg/kg) and enoxaparin (5mg/kg), with a mean (±SD) occlusion time of 51 ± 13 minutes for TB-402, compared to 28 ± 6 minutes for the controls, 51 ± 13 minutes for BO2C11 and 55 ± 11 minutes for enoxaparin. CONCLUSIONS: In this mouse model of venular thrombosis, partial FVIII inhibition yielded similar antithrombotic effects as nearly complete FVIII inhibition. These preclinical data are indicative of a therapeutic potential of partial FVIII inhibition in the management of venous thromboembolism.

Air pollution-associated procoagulant changes: the role of circulating microvesicles.

BACKGROUND: Epidemiological studies suggest an association between exposure to particulate matter (PM) in air pollution and the risk of venous thromboembolism (VTE). OBJECTIVES: To investigate the underlying pathophysiological pathways linking PM exposure and VTE. PATIENTS AND METHODS: We assessed potential associations between PM exposure and coagulation and inflammation parameters, including circulating microvesicles, in a group of 233 patients with diabetes. RESULTS: The numbers of circulating blood platelet-derived and annexin V-binding microvesicles were inversely associated with the current levels of PM(2.5) or PM(10), measured on the day of sampling. Recent past exposure to PM(10), up to 1 week prior to blood sampling, estimated at the patients' residential addresses, was associated with elevated high-sensitivity C-reactive protein (CRP), leukocytes and fibrinogen, as well as with tissue factor (TF)-dependent procoagulant changes in thrombin generation assays. When longer windows of past exposure were considered, up to 1 year preceding blood sampling, procoagulant changes were evident from the strongly increased numbers of red blood cell-derived circulating microvesicles and annexin V-binding microvesicles, but they no longer associated with TF. Past PM exposure was never associated with activated partial thromboplastin time (aPTT), prothrombin time (PT), or factor (F) VII, FVIII, FXII or D-dimers. Residential distance to a major road was only marginally correlated with procoagulant changes in FVIII and thrombin generation. CONCLUSIONS: Increases in the number of microvesicles and in their procoagulant properties, rather than increases in coagulation factors per se, seem to contribute to the risk of VTE, developing during prolonged exposure to air pollutants.

Short-term exposure to particulate matter induces arterial but not venous thrombosis in healthy mice.

BACKGROUND: Epidemiological findings suggest an association between exposure to particulate matter (PM) and venous thrombo-embolism. OBJECTIVES: To investigate arterial vs. venous thrombosis, inflammation and coagulation in mice, (sub)acutely exposed to two types of PM. METHODS: Various doses (25, 100 and 200 µg per animal) of urban particulate matter (UPM) or diesel exhaust particles (DEP) were intratracheally (i.t.) instilled in C57Bl6/n mice and several endpoints measured at 4, 10 and 24 h. Mice were also repeatedly exposed to 100 µg per animal on three consecutive days with endpoints measured 24 h after the last instillation. RESULTS: Exposure to 200 µg per mouse UPM enhanced arterial thrombosis, but neither UPM nor DEP significantly enhanced venous thrombosis. Both types of PM induced dose-dependent increases in broncho-alveolar lavage fluid (BALF) total cell numbers (mainly neutrophils) and cytokines (IL-6, KC, MCP-1, RANTES, MIP-1α), with peaks at 4 h and overall higher values for UPM than for DEP. Systemic inflammation was limited to increased serum IL-6 levels, 4 h after UPM. Both types of PM induced similar and dose-dependent but modest increases in factor (F)VII, FVIII and fibrinogen. Three repeated instillations did not or only modestly enhance the proinflammatory and procoagulant status. CONCLUSIONS: Compared with DEP, UPM induced more pronounced pulmonary inflammation, but both particle types triggered similar and mild short-term systemic effects. Hence, acute exposure to PM triggers activation of primary hemostasis in the mouse, but no substantial secondary hemostasis activation, resulting in arterial but not venous thrombogenicity.