Immunological screening for tumor cells in serous body fluids has added value with the CELL-DYN Sapphire.

BACKGROUND: Conventional cytological examination has limited sensitivity for detecting tumor cells in serous body cavity effusions and therefore, adjuvant techniques are necessary for a reliable diagnosis. Flow cytometry has proven benefit in these circumstances. The aim of our study was to explore the feasibility of CELL-DYN Sapphire, an advanced hematology analyzer with flow cytometric capabilities, for detecting tumor cells in serous body fluids, using CD326 monoclonal antibodies, which are directed against the epithelial marker EpCAM. METHODS: One hundred and five serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using monoclonal antibody combinations CD3/CD19 and CD45/CD326. Of all samples a cytospin preparation was made and microscopically examined; the pathology findings served as a reference. RESULTS: Using a threshold of 1% CD326+ cells, CELL-DYN Sapphire identified nine out of 12 cases with tumor cells in the serous effusions (sensitivity 75%), whereas routine cytology found eight cases (sensitivity 67%). The combination of immunophenotyping and cytology identified all 12 cases with tumor cells in the effusion fluid (sensitivity 100%). The specificities were 92% and 100%, respectively. CONCLUSIONS: We demonstrated that it is feasible to run an immunophenotypic assay on CELL-DYN Sapphire for detecting tumor cells in serous body fluids. In addition, this study confirmed that a combination of conventional cytology and flow cytometry had a very high diagnostic yield in cases of carcinomatous effusions.

Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer.

BACKGROUND: Correct cell enumeration and differential analysis of body fluids are important in the diagnosis and management of several diseases. Currently, microscopic analysis is still considered the "gold standard". The aim of the present study was to evaluate the analytical performance of the CELL-DYN Sapphire hematology analyzer for automated differentiation of cells in serous fluids and to explore whether manual analysis of the raw data files could improve the differential count compared with reference microscopy. METHODS: A total of 105 serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using standard whole-blood algorithm. Additionally, we performed optimized manual gating of the Sapphire raw data file using standard flow cytometry software. RESULTS: The standard Sapphire algorithm showed substantial deviations from the reference microscopic differentiation: polymorphonuclear cell counts were too high because they contained some monocytic cells. However, when optimized manual gating strategy is used, a good correlation and negligible bias were found. CONCLUSIONS: We have demonstrated that with a modified algorithm, CELL-DYN Sapphire will provide reliable identification and enumeration of blood cells in peritoneal and pleural fluids.

Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1.

OBJECTIVE: In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin. METHODS AND RESULTS: We demonstrate that procoagulant activity is related to a sustained increased [Ca2+]i, which in turn depends on extracellular Ca2+ influx. Increased PtdSer exposure coincides with increased [Ca2+]i and was observed in a subpopulation (approximately 14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbalpha made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca2+]i and procoagulant activity induced by collagen plus thrombin. CONCLUSIONS: The interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced procoagulant activity by inducing a sustained elevation in [Ca2+]i in a subpopulation of platelets.

Covalently-bound heparin makes collagen thromboresistant.

OBJECTIVE: Blood compatibility of artificial surfaces depends on their immunogenic and thrombogenic properties. Collagen's weak antigenicity makes it an attractive candidate for stent coatings or fabrication of vascular grafts. However, the thrombogenic nature of collagen limits its application. We examined whether heparinization can make collagen more thromboresistant. METHODS AND RESULTS: Collagen was heparinized by crosslinking collagen with extensively periodate oxidized heparin and/or by covalently bonding of mildly periodate oxidized heparin. Both ways of heparinization have no effect on platelet adhesion and could not abolish induction of platelet procoagulant activity. However, thrombin generation was completely prevented under static and flow conditions. The functionality of immobilized heparin was confirmed by specific uptake of antithrombin, 13.5+/-4.7 pmol/cm2 and 1.95+/-0.21 pmol/cm2 for mildly and heavily periodated heparin, respectively. CONCLUSIONS: These results indicate that immobilization of heparin on collagen, even as a crosslinker, is a very effective way to prevent surface thrombus formation. These data encourage the application of heparinized collagen as stent-graft material in animal and eventually human studies.

Fibrinogen adsorption, platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood.

Thrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anti-coagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.

Thrombin-induced hyperactivity of platelets of young stroke patients: involvement of thrombin receptors in the subject-dependent variability in Ca2+ signal generation.

Activated platelets are implicated in the development of premature arterial vascular diseases, in particular ischemic stroke. Since elevated cytosolic [Ca(2+)](i) is an integrative marker of platelet activation, we determined the generation of Ca(2+) signal in stimulated platelets from 26 young patients recuperating from stroke, 20 patients with symptomatic peripheral arterial disease, and 56 healthy volunteers. Even in the presence of aspirin, the platelets from various individuals showed highly different thrombin-induced Ca(2+) responses. On average, the thrombin-induced Ca(2+) response was increased for platelets from either patient group in comparison to the controls (P <0.04). Relatively more stroke patients had high-responsive platelets (27%, 7/26) than patients with peripheral arterial disease (10%, 2/20) or healthy subjects (4%, 2/56). The average prothrombinase activities of platelets from patients and controls were similar, but 3 out of 6 patients with increased thrombin-induced Ca(2+) responses also exhibited high prothrombinase activity. In a follow-up study, the subject-dependent thrombin-induced Ca(2+) response was found to correlate strongly with the platelet response to protease-activated receptor 1 (PAR1) agonist (r = 0.91), but was not linked to the Pl(A1/2) polymorphism. It is concluded that a significant part of young patients with stroke have platelets with hyperactivity toward thrombin, which is not normalised by aspirin treatment. Furthermore, the subject-dependent variation in thrombin-induced signalling is likely to involve PAR1-mediated platelet activation.

Thrombogenicity of polysaccharide-coated surfaces.

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.

The pro- and anticoagulant role of blood-borne phagocytes in patients with acute coronary syndrome.

This study was performed to gain further insight in pro- and anticoagulant characteristics of leukocytes in acute coronary syndrome (ACS). For this purpose, patients presenting on the emergency department (ED) with anginal chest pain were included in this study. In peripheral blood, procoagulant tissue factor (TF) expression was measured in the different blood-borne phagocytes, i.e. neutrophilic granulocytes and the three different monocyte subsets based on expression of CD14 and CD16. Simultaneously, intracellular presence of platelet-(CD41) and/or endothelial cell-remnants (CD62e) was analysed in these different leukocyte subsets. Neutrophils showed a weak intracellular staining of CD62e and CD41 that increased with severity of ACS. Monocytes, and especially the classical (CD14++CD16-) and intermediate monocytes (CD14++CD16+) showed a clear and significant increase in intracellular CD41-staining after coronary damage. The different monocyte subsets showed an increase in expression of TF in severe ACS. Finally, it appeared that also neutrophils showed a significant increase in expression of TF on their membrane. In conclusion, this study showed an increased intracellular staining in blood-borne phagocytes for CD62e and CD41 in patients with ACS compared to non-cardiac related control patients. This indicates that at least in the acute phase of ACS phagocytosis of platelet and endothelial cell-remnants is increased. These data support the recent hypothesis that neutrophils protect against further thrombotic processes by clearing platelet and endothelial cell-remnants. In addition, this study shows that the different monocyte subsets are also involved in this process. Furthermore, both monocytes and neutrophils show increased TF expression in ACS.

[Application of tumour markers in clinical practice]. / Toepassing van tumormarkers in de klinische praktijk.

Usefully requesting and applying serum tumour markers in diagnosis and treatment can be difficult. It should be noted that tumour markers are used for varying purposes: screening, diagnosis, staging and prognostic evaluation, detection of recurrence and treatment monitoring. Due to the poor sensitivity and specificity of current tumour markers, most are not suitable for screening an asymptomatic population. Further, the benefits of an improved prognosis by early detection should be weighed against a poorer quality of life and the cost of substantial over-diagnosis and over-treatment. Serum tumour markers are particularly applicable in treatment monitoring and detection of recurrence. Sometimes they can be used to support the diagnostic process and give useful prognostic information.

[Laboratory input. General practitioners positive about reflective testing]. / Eigen inbreng van het laboratorium. Huisartsen positief over 'reflecterend testen'.

OBJECTIVE: To investigate the opinion of general practitioners on reflective testing, i.e. the practice of additional tests being performed and comments added to the results by laboratory staff when appropriate. DESIGN: Descriptive. METHOD: In the eastern South Limburg region 155 general practitioners received a list of 10 fictitious patient cases, each involving the possible addition of a specific test. The general practitioners could choose what they preferred the laboratory to do: add tests, phone the general practitioner, add a written comment or do nothing. In addition the general practitioners were asked to judge the effect of additional tests and comments on patient management with respect to diagnosis, treatment and referral, using 200 laboratory reports from their own patients. RESULTS: The response to the fictitious cases was 45%. Most general practitioners favoured the laboratory taking the initiative by adding on tests and commenting on the results in the given clinical scenarios. 78% of the questionnaires accompanying the lab reports were returned by 87% of the general practitioners. In nearly all cases (99%) the service was marked as useful. In more than half of the cases (53%) reflective testing affected the measures taken by the general practitioners. CONCLUSION: Reflective testing was in general welcomed by the general practitioners. In the majority of cases this led to an improvement in the diagnosis or adjustment of treatment.

Factor Va, bound to microparticles released during platelet storage, is resistant to inactivation by activated protein C.

BACKGROUND: Microparticles (MPs) support coagulation and can be helpful in restoring the hemostatic system in thrombocytopenic patients. The anticoagulant properties of MPs shed during storage of platelets (PLTs) have not been studied yet. STUDY DESIGN AND METHODS: Storage-induced MPs were harvested from outdated PLT concentrates. Whether factor (F)Va was present on the surface of these MPs was investigated. The activated protein C (APC)-catalyzed inactivation of MP-bound FVa was further determined. Also, inactivation of FVa at the surface of thrombin-activated PLTs and synthetic vesicles was determined. RESULTS: MPs in stored PLT products carry FVa at their surface. APC-catalyzed inactivation of MP-bound FVa resulted in 42 +/- 2 percent residual FVa activity after 20 minutes. The residual activity of FVa on thrombin-activated PLTs was 25 +/- 3 percent. Plasma-derived FVa was rapidly inactivated in the presence of synthetic vesicles, with 5 +/- 4 percent residual FVa activity. When synthetic vesicles were added to the inactivation mixture of MP- or thrombin-activated PLTs, a residual activity of 5 to 10 percent was found. Furthermore, addition of excess plasma-FVa to storage-induced MPs resulted in a residual activity of 26 +/- 2 percent. Moreover, the APC-resistant phenotype of MPs was confirmed in plasma in which thrombin generation was measured in the absence and presence of APC. Residual FVa activity in the presence of MPs, PLTs, or synthetic vesicles was 87 +/- 6, 65 +/- 3, and 8 +/- 19 percent, respectively. CONCLUSION: Together, these results suggest that the MP surface environment renders FVa resistant to APC. It is further concluded that the APC resistance of FVa at the surface of storage-induced MPs enhances their procoagulant nature.

Platelet ADP response deteriorates in synthetic storage media.

BACKGROUND: During storage under blood bank conditions, platelets (PLTs) are known to secrete ADP. PLT stimulation by ADP results in refractoriness to restimulation, making this response one of the most unstable PLT reactions. The goal of this study was to evaluate the ADP-induced responses of PLTs stored in full plasma or in plasma and additive solution (AS). STUDY DESIGN AND METHODS: Surface expression of P-selectin, ADP-induced aggregation, and reconstituted whole-blood thrombus formation were determined on collagen surfaces in a perfusion model with PLTs that were stored for 4 days either in plasma or in the presence of plasma with PAS-II or Composol. RESULTS: After 4 days of storage in PAS-II but not in Composol, the percentage of PLTs that had secreted granule contents (P-selectin) was increased, when compared to PLTs stored in full plasma. Maximal aggregation in response to ADP was reduced for PLTs stored in PAS-II or Composol. Resuspension of these PLTs in plasma at 37 degrees C for 1 hour caused partial recovery of the aggregation response. Addition of apyrase to PLTs in AS preserved the responsiveness toward ADP. Titration experiments indicated that this response gradually decreased with decreasing plasma concentration. The functional significance of these findings was demonstrated by perfusion experiments. Thrombus formation on collagen was significantly higher for PLTs stored in full plasma than for PLTs stored in PAS-II or Composol. CONCLUSIONS: Storage of PLTs in the presence of AS under blood bank conditions induces deterioration of the PLT responsiveness to ADP compared to PLT concentrates in 100 percent plasma. Higher plasma-to-AS ratios result in better preserved responses.

Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation.

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage-induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib-alpha. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF-driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle-associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF-activated plasma was 15-fold higher when platelet-free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin-activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.

Integrin alphaIIbbeta3 and shear-dependent action of glycoprotein Ibalpha stimulate platelet-dependent thrombin formation in stirred plasma.

Under conditions of arterial-wall shear rates, platelets bind to von Willebrand factor (vWf) by way of the glycoprotein Ib (GP Ib) complex and integrin alpha(IIb)beta(3). Both adhesive receptors may also play roles in the development of procoagulant activity of platelets. Here, we investigated the effect of shear stress, as provided by a rotating cylinder, on GP Ib- and integrin alpha(IIb)beta(3)-dependent thrombin generation in coagulating platelet-rich plasma (PRP). We measured thrombin continuously with the use of fluorometry from the cleavage rate of a fluorescent low-affinity substrate. The integrin alpha(IIb)beta(3) antagonist abciximab progressively reduced the peak of thrombin formation up to 43% when rate of stirring and shear stress were increased (estimated shear rates of 105-420 s(-1)). Abciximab did not lower the peak of thrombin formation in stirred PRP from patients with Glanzmann's thrombasthenia lacking alpha(IIb)beta(3) but, surprisingly, shortened the time until onset. In PRP from control subjects, antibodies specifically directed against vWf-binding epitopes on GP Ibalpha reduced thrombin formation, with 25% to 30% at the high but not at the low stirring rate. In combination with the anti-GP Ib antibody, abciximab retained its strong inhibitory effect only at the high stirring rate. We conclude that thrombin formation and coagulation in stirred PRP depend, to a large extent, on platelet adhesion to integrin alpha(IIb)beta(3) and, in a shear-dependent way, on GP Ib.

von Willebrand factor C1C2 domain is involved in platelet adhesion to polymerized fibrin at high shear rate.

Fibrin is actively involved in platelet reactions essential for thrombus growth, in which von Willebrand factor (VWF) might be an important mediator. The aim of this study was to localize VWF domains that bind to fibrin and to determine their relevance in platelet adhesion. VWF binds specifically to fibrin with an apparent Kd of 2.2 microg/mL. Competition in the presence of 2 complementary fragments, SpIII (residues 1-1365) and SpII (residues 1366-2050), indicated that the high affinity binding site for fibrin is located in the C-terminal part, thus distinct from the A domains. Comparison of 2 deleted rVWF (DeltaD4B-rVWF, DeltaC1C2-rVWF) suggested that the C1C2 domains contained a fibrin binding site. This site is distinct from RGD, as shown by binding of D1746G-rVWF to fibrin. Perfusion studies at high shear rate demonstrated that C1C2 domains were required for optimal platelet adhesion to fibrin. With the use of a VWF-deficient mouse model, it was found that plasma VWF is critical for platelet tethering and adhesion to fibrin. These results suggest a dual role of fibrin-bound VWF in thrombus formation: first, fibrin-bound VWF is critical in the recruitment of platelets by way of glycoprotein (GP) Ib, and, second, it contributes to stationary platelet adhesion by way of binding to activated alphaIIbbeta3.