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Xylum CSA[R]: automated system for assessing hemostasis in simulated vascular flow.

Nearly 20 years ago it became clear that analysis of hemostasis function in blood was affected by sample shear rate and anticoagulation [1]. Yet, present technologies still perform hemostasis analysis on anticoagulated samples under static conditions. In 1984, Gorog and Ahmed developed a test system that analyzed hemostasis function of untreated whole blood under physiological flow [2], a technology potentially useful for assessing patients with bleeding disorders [2, 3]. This approach has been automated in a user-friendly benchtop instrument, the Clot Signature Analyzer (CSA[R]; Xylum Corp.). From a single venipuncture, the CSA provides information on platelet adhesion induced by high shear, platelet aggregation, and coagulation attributable to Immoral factors [4]. Measurements are performed on untreated nonanticoagulated whole blood under the physiological conditions of nonrecirculating flow at 37 [degrees]C.

[FIGURE 1 OMITTED]

The single-use, disposable CSA cassette consists of two perfusion channels, the "punch" and "collagen" channels. In each channel, blood is perfused in tubing under conditions simulating vascular flow. Blood is drawn into two 3-mL syringes, which are attached to luer fittings on the cassette. When the cassette is loaded onto the instrument, low-density oil is delivered into the syringes from a reservoir on the instrument. The imiscible oil rises in the syringes and displaces the blood, which then flows into perfusion lines maintained at 37 [degrees]C. The luminal pressure exerted by the flowing blood is measured continuously in both channels by sensors on the instrument. In the punch channel, vascular injury is simulated by piercing the blood perfusion line with a 0.15-mm-diameter needle to form two small holes. Blood flow is largely diverted through these "punch" holes, causing a sudden decrease in luminal pressure (Fig. 1A). The punch holes close as a result of hemostasis, thereby restoring luminal pressure to its original value (punch recovery in Fig. 1A). The time from punch to recovery is reported as platelet hemostasis time (PHT). The blood flowing in the lumen eventually coagulates, and luminal flow ceases. This causes pressure in the pressure chamber to drop to 0 [clotting time (CT) in Fig. 1A].

The collagen channel is a separate flow path, similar to but distinct from the punch channel. In the collagen channel a 1.9-cm-long collagen fiber (type I bovine collagen; Ethicon, Sommerville, NJ) is immobilized in the lumen and aligned with the direction of flow. As blood flows, platelets adhere to the collagen, initiating thrombus formation. Eventually the thrombus occludes the lumen and causes luminal pressure to drop to 0 (Fig. 1B). The time from start of blood flow to 50% drop in luminal pressure is reported as collagen-induced thrombus formation (CITF).

To determine how hemostasis deficiencies affect the CSA parameters, we mixed blood samples with hemostasis inhibitors before assaying, using the following inhibitors of primary hemostasis: (a) antibody to the platelet receptor GPIIbIIIa, which is involved in platelet aggregation [5, 6] [the anti-GPIIbIIIa, ReoPro[TM] (Centocor)], used at a final concentration of 5 mg/L; (b) antibody to the platelet receptor involved in shear adhesion, GPIb [7, 8] [anti-GPIb (monoclonal AP1; GTI Corp., Brookfield, WI)], used at a final concentration of 5 mg/L; (c) antibody to von Willebrand factor (vWf), which blocks the binding of vWf to the GPIb receptor [9] [monoclonal AVW-3 (GTI Corp.)], used at a final concentration of 2.5 mg/L; and (d) aurintricarboxylic acid (ATA; Aldrich Chemical Co.), a polyanion that inhibits vWf interaction with platelet receptor GPIb [10], used at a final concentration of 400 [micro]mol/L.

For all studies blood was obtained from apparently healthy adult donors of both sexes who had given informed written consent and had refrained from taking any medication influencing platelet function or plasmatic coagulation for at least 1 week. All studies were performed in accordance with the Institutional Review Board for the facility.

With use of a 21-gauge winged blood collection set (Becton Dickinson), blood was drawn from the antecubital vein by a two-syringe technique into 3-mL syringes containing 50 [micro]L of either phosphate-buffered saline, pH 7.4 (PBS; Sigma Chemical Co.), or inhibitor diluted in PBS. Immediately before each assay, the inhibitor was mixed with the blood by inversion of the syringes. Samples from 5 to 14 donors were run for each inhibitor and the paired t-test was used to compare each inhibitor vs control to determine the significance of effect on PHT or CITF. Figs. 1C and 1D show the effect of inhibitors on PHT and CITF, respectively. Both were significantly increased by each of the inhibitors anti-vWf, anti-GPIb, anti-GPIIbIIIa, and ATA (P <0.05, two-tailed).

Treatment of blood with anti-vWf or ATA causes a decrease in vWf function, simulating von Willebrand's disease (vWd). Addition of anti-GPIb to blood causes a decrease in availability of platelet receptor GPIb, mimicking Bernard-Soulier syndrome (BSS), whereas anti-GPIIbIIIa treatment causes a decrease in the availability of platelet receptor GPIIbIIIa, mimicking Glanzmann thrombasthenia (GT). When blood samples from patients with vWd or BSS were perfused at shear rates of 1300 [s.sup.-1] or more, platelet adhesion to subendothelial tissue was much less than in normal blood [1]. When blood from a patient with GT was perfused over a subendothelial substrate, platelet aggregation was markedly subnormal [1]. The significant prolongation of PHT and CITF with anti-GPIb, anti-vWf, or ATA indicates that shear-induced platelet activation, involving interaction between bound vWf and platelet receptor GPIb, is involved in punch hole closure and collagen channel occlusion. The results with anti-GPIIbIIIa further indicate that platelet aggregation is involved in punch hole closure and collagen channel occlusion.

The CSA CT test results were obtained from runs made with and without added unfractionated heparin and demonstrate the ability of the CSA to detect abnormal blood conditions. The 3-mL plastic syringes (Becton Dickinson) were preloaded with 10 mL/L of either isotonic saline or porcine intestine-derived sodium heparin (Elkins-Sinn, Cherry Hill, NJ) diluted in saline and tested at 300 IU/L final concentration. From 11 donors, nonanticoagulated whole blood was drawn into prewarmed syringes, mixed by inversion, and loaded onto the CSA cassette. The CSA has a maximum test time of 1800 s. If the blood failed to coagulate by this time, CT was assigned the value of 1800 s. The average CT with saline was 1217 s, while all samples with added heparin failed to coagulate, i.e., CT = 1800 s (significantly longer by paired t-test: P <0.001, two-tailed, n = 11). This indicates that lumen occlusion is caused by coagulation factors sensitive to heparin inhibition.

In conclusion, the Xylum CSA analyzes hemostasis function in untreated, nonanticoagulated whole blood perfused under nearly physiological conditions, providing information on shear-induced platelet adhesion, platelet aggregation, and activated CT.

We thank Gregory Dehmer and Harvey Weiss for their generous gifts of ReoPro and heparin, respectively.

References

[1.] Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. I. Shear rate-dependent decrease of adhesion in von Willebrand's disease and the Bernard-Soulier syndrome. J Lab Clin Med 1978;92:750-64.

[2.] Gorog P, Ahmed A. Haemostatometer. A new in vitro technique for assessing haemostatic activity of blood. Thromb Res 1984;34:341-57.

[3.] Kovacs IB, Hutton RA, Kernoff PB. Hemostatic evaluation in bleeding disorders from native blood. Am J Clin Pathol 1989;91:271-9.

[4.] Muga KM, Melton LG, Gabriel DA. A flow dynamic technique used to assess global haemostasis. Blood Coagulat Fibrinol 1995;6:73-8.

[5.] Coller BS. Blockade of platelet GPIIb/IIIa receptors as an antithrombotic strategy. Circulation 1995;92:2373-80.

[6.] Fauls D, Sorkin EM. Abciximab (c7E3 Fab). a review of its pharmacology and therapeutic potential in ischaemic heart disease. Drugs 1994;48:583-98.

[7.] George JN, Nurden AT, Phillips DR. Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 1984;311:1084-95.

[8.] Montgomery RR, Kunicki TJ, Pideard D, Corcoran M. Diagnosis of Bernard- Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood. J Clin Invest 1983;71:385-9.

[9.] Alevriadou BR, Moake JL, Turner NA, Ruggeri ZM, Folie BJ, Phillips MD, et al. Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets. Blood 1993;81: 1263-75.

[10.] Strony J, Phillips M, Brands D, Moake J, Adelman B. Aurintricarboxylic acid in a canine model of coronary artery thrombosis. Circulation 1990;81:1106-14.

Conan K.N. Li,* Thomas J. Hoffmann, Pei-Ying Hsieh, Suneil Malik, and William Watson (Xylum Corp., 670 White Plains Rd., Scarsdale, NY 10583; * corresponding author: fax 914-7251158, e-mail Conan Li@Xylum.ccmail.compuserve.com)
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Title Annotation:Poster Session
Author:Li, Conan K.N.; Hoffmann, Thomas J.; Hsieh, Pei-Ying; Malik, Suneil; Watson, William
Publication:Clinical Chemistry
Date:Sep 1, 1997
Words:1422
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