Carola E. Henriksson

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis.

Tissue factor (TF)‐positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP‐associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF‐protein and TF‐activity, which have been explained by antibody binding to “encrypted” or degraded forms of inactive TF‐protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte‐derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP‐associated TF‐protein (flow cytometry) and TF‐activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL−1) had ∼14 times higher TF‐activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti‐TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF‐positive events as MPs secernated from LPS‐stimulated monocytes [∼45,000 events mL−1 (American Diagnostica); ∼15,000 events mL−1 (BD)]. These TF‐positive events did not exert any TF‐activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP‐associated TF‐protein, and interpreted as a discrepancy between TF‐protein and TF‐activity.

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock☆ , ☆☆

INTRODUCTION The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis. MATERIALS AND METHODS MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay. RESULTS MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2-2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r(s)=-0.84), time to peak (rs=-0.83), peak (r(s)=0.85) and ETP (r(s)=0.83). CONCLUSIONS MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.

Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

Tissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.

Is the Blood Basophil Count Sufficiently Precise, Accurate, and Specific? Three Automated Hematology Instruments and Flow Cytometry Compared

We compared the performance of the basophil count of 3 hematology instruments with a flow cytometric method (FCM) in which CD123 and CD193 were used as basophil markers. By analyzing 112 patient samples, we found the ADVIA 120 (Siemens Healthcare Diagnostics, Deerfield, IL) and CELL-DYN Sapphire (Abbott Diagnostics, Santa Clara, CA) to underestimate the number of basophils by approximately 50% and the Sysmex XE-2100 (Sysmex, Kobe, Japan) and ADVIA to overestimate the basophil count in some samples with pathologic leukocytes. All 3 instruments had large (25%-50%) analytic within-run coefficients of variation. Compared with the FCM, we found a relatively good correlation for the CELL-DYN basophil count (r = 0.81), an intermediate correlation for the Sysmex (r = 0.64), and a poor correlation for the ADVIA (r = 0.24). When excluding the 52 samples flagged for the presence of pathologic leukocytes, these correlations were found to be 0.84, 0.90, and 0.57, respectively. The basophil count of the 3 instruments is, at least presently, of unsatisfactory quality.

Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay.

There is increasing clinical interest for measuring microparticle (MP)-associated tissue factor (TF) activity owing to its possible role as a prothrombotic biomarker in a variety of diseases. However, the methods used are to various extents hampered by lack of (pre)analytical standardization as well as limited published documentation. The objective of this study was to evaluate the performance of the Zymuphen MP-TF kit and the calibrated automated thrombogram (CAT) assay in measuring MP-associated TF activity in plasma using a Neisseria meningitidis (Nm)-stimulated whole blood model. In addition, (pre)analytical variables like centrifugation procedures, freezing/thawing and the effect of addition of exogenous phosphatidylserine in plasma were evaluated in the CAT assay. Citrate-anticoagulated blood was stimulated with Nm bacteria for 4 h before platelet-poor plasma (PPP) or platelet-free plasma (PFP) were prepared and assayed with either of the two methods. Nm dose-dependently (104–108 bacteria/ml) induced TF-specific activity, measured as decreased lagtimes, in the CAT assay. The Zymuphen MP-TF kit also detected TF activity, although much higher Nm doses (108 bacteria/ml) were required to achieve measurable levels. Neither freezing/thawing nor the use of PPP vs. PFP influenced the TF activity, measured over a broad range of lagtimes, in the CAT assay. In conclusion, changes in lagtime in the CAT assay reflected levels of MP-associated TF activity in a more sensitive manner than the Zymuphen MP-TF kit did, in our Nm-stimulated whole blood system.

Flow cytometry-sorted non-viable endotoxin-treated human monocytes are strongly procoagulant.

Monocytes/macrophages are important in disease states such as gram-negative sepsis and coronary artery disease. Following exposure to lipopolysaccharide (LPS), monocytes express tissue factor (TF), the main initiator of blood coagulation. We previously demonstrated that human monocytes treated with high concentrations of LPS, or with LPS and calcium ionophore, displayed higher TF activity than monocytes treated with only low concentrations of LPS, even though the monocytes under all conditions expressed similar amounts of cell surface TF antigen. Such restrainedTF activity is often referred to as encryption and its release as de-encryption. We also observed that the increase in TF activity, de-encryption, coincided with an increase in cell surface phosphatidylserine (PS) representing apoptosis and necrosis. In the present work, we separated LPS and LPS and calcium ionophore-treated human monocytes into two populations, one of mainly viable, PS negative cells, and one of mainly non-viable, PS positive cells, by sorting flow-cytometry. We observed that non-viable cells expressed considerably less TF antigen than viable cells. Despite this, non-viable cells were clearly more procoagulant than viable cells in two different coagulation assays. Procoagulant activity was dependent on both TF and PS. We consider the higher content of externalized PS in non-viable monocytes as the major reason for the stronger procoagulant activity of these cells. Thus, TF de-encryption appears largely to occur on PS positive, non-viable cells under these conditions. This supports the important role of PS in coagulation, and it suggests that PS expression signifying cell death, may be clinically relevant.

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression:

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.

The reversal effect of prothrombin complex concentrate (PCC), activated PCC and recombinant activated factor VII against anticoagulation of Xa inhibitor

BackgroundAn increasing number of patients are treated with direct-acting oral anticoagulants (DOACs), but the optimal way to reverse the anticoagulant effect is not known. Specific antidotes are not available and prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) are variously used as reversal agents in case of a major bleeding. We aimed to determine the most effective haemostatic agent and dose to reverse the effect of rivaroxaban in blood samples from patients taking rivaroxaban for therapeutic reasons.MethodsBlood samples from rivaroxaban-treated patients (n = 50) were spiked with PCC, aPCC and rFVIIa at concentrations imitating 80%, 100% and 125% of suggested therapeutic doses. The reversal effect was assessed by thromboelastometry in whole blood and a thrombin generation assay (TGA) in platelet-poor plasma. Samples from healthy subjects (n = 40) were included as controls.ResultsIn thromboelastometry measurements, aPCC and rFVIIa had a superior effect to PCC in reversing the rivaroxaban-induced lenghtening of clotting time (CT). aPCC was the only haemostatic agent that shortened the CT down to below the control level. Compared to healthy controls, patients on rivaroxaban also had a prolonged lag time and decreased peak concentration, velocity index and endogenous thrombin potential (ETP) in platelet-poor plasma. aPCC reversed these parameters more effectively than rFVIIa and PCC. There were no differences in efficacy between 80%, 100% and 125% doses of aPCC.ConclusionsaPCC seems to reverse the anticoagulant effect of rivaroxaban more effectively than rFVIIa and PCC by evaluation with thromboelastometry and TGA in vitro.

Microparticle-associated tissue factor activity is reduced by inhibition of the complement protein 5 in Neisseria meningitidis-exposed whole blood.

Neisseria meningitidis causes fulminant meningococcal sepsis with a massive activation of the coagulation and complement cascades. Bacterial cell envelope molecules from N. meningitidis, particularly lipopolysaccharide (LPS), induce tissue factor (TF) expression. In meningococcal sepsis, TF can be detected on circulating monocytes and microparticles (MPs) within the bloodstream. During infection, Nm activates C5 and C5a, which also is able to induce TF. We evaluated the effect of eculizumab, a C5-blocking monoclonal antibodies (mAb), on cell- and MP-associated TF. Using a lepirudin-anticoagulated whole blood model, we activated the coagulation and complement cascades by N. meningitidis, and investigated the interaction between the cascade systems with special focus on cell-associated TF-expression (mRNA and protein) and MP-associated TF-dependent thrombin and fibrin generation in platelet-free plasma. We also examined the ability of TF-positive MPs to support clot formation in whole blood. In addition, the effect of corn trypsin inhibitor and time-dependent changes on MP-associated functional TF activity was examined. Inhibition of C5 reduced cell-associated TF expression at both gene and protein level, and reduced MP-associated TF-dependent thrombin and fibrin generation in platelet-poor plasma, MP-induced TF-dependent clot formation in whole blood, implying that the complement and coagulation cascades are interplayers in N. meningitidis-mediated activation of these cascades.

Absolute Neutrophil Counts From Automated Hematology Instruments Are Accurate and Precise Even at Very Low Levels

Using 106 samples from patients with an absolute neutrophil count (ANC) less than 2.0 × 10(9)/L, two 5-part differential hematology instruments (Sysmex XE-2100, Sysmex, Kobe, Japan, and Advia 2120i, Siemens Healthcare Diagnostics, Deerfield, IL), two 3-part differential hematology instruments (Sysmex K4500, Sysmex, and Advia 60, Siemens Healthcare Diagnostics), and an automated system for examination of microscopic slides (CellaVision DM96, CellaVision, Lund, Sweden) were compared with a flow cytometric (FCM) neutrophil count using monoclonal antibodies for cell classification. The precision and accuracy of the 5-part differential instrument ANC was very good at more than 0.1 × 10(9)/L, although a small systematic difference (10.3%) was found between the 2 instruments. The ANC of the 3-part differential instruments was less reliable, but the WBC count correlated very well with the WBC count from the 5-part differential instruments. Also, the neutrophil count from the CellaVision DM96 compared very well with FCM. When used in the correct laboratory setting, all of the evaluated instruments provide ANCs and WBCs with adequate accuracy and precision.