Point-of-care fibrinolytic tests: the other side of blood coagulation.

Point-of-care (POC) coagulation tests with paramagnetic iron oxide particles have provided alternatives to testing previously done only in the laboratory. With this technology, POC fibrinolytic tests have followed quietly the trail blazed by POC clotting tests and have found specific applications. These include rapid verification of in vivo thrombolytic drug action by in vitro testing with subsequent quantitative drug monitoring of the systemic lytic state, and also the determination of in vitro thrombolytic drug response before the drug is actually administered, to individualize therapy by selection of the most appropriate drug. Other applications include POC coagulation factor assays associated with fibrinolysis, and most recently the POC screening of patients with fibrinolytic defects. In this latter application, plasma from cardiac catheterization (n = 19) and venous thrombosis (n = 47) patient groups were tested. Controls consisted of two independent donor pools (n = 10, n = 21) as negatives and two plasma samples with known genetic defects in the fibrinogen molecule (A alpha 554 Arg-->Cys) as positives.

Thrombosis and hemostasis testing at the point of care.

Point-of-care testing provides current, accurate information relating to thrombosis and hemostasis in patients. Several forces are driving point-of-care testing, particularly economic factors and improved technology. Point-of-care testing has the potential to improve patient management and to decrease integrated costs, although this remains to be shown. For such testing to be successful, the technology must be complemented by hospital-wide point-of-care testing programs. The role of the laboratorian and pathologist will become important for coordinating programs, maintaining quality assurance, and promoting quality improvement.

Homogeneous immunoassay of whole-blood samples.

Proof of principle has been shown for a rapid, quantitative, homogeneous immunoassay capable of analyzing whole-blood samples. The assay was performed with test cards and a small instrument designed for use at the point of care. The immunoassay has an immunological "front end" combined with a coagulation cascade chemistry "back end" and is made possible by combining two patented technologies: (a) a serine protease inhibitor [Porter and Bruhnke, Photochem and Photobiol 1990; 51(1):37] and (b) paramagnetic iron oxide particles (PIOP) in a mixture of buffers and coagulation assay components supplied as a dry film in a test-card reaction chamber [Oberhardt et al., Clin Chem 1991;37:520]. A model steric-hindrance immunoassay based on these technologies was established for the measurement of biotin. The calibration curve was developed by measuring plasma samples supplemented with biotin. The reagents were inhibited biotinylated thrombin, anti-biotin monoclonal antibody, and PIOP.

Differences in clot lysis among patients demonstrated in vitro with three thrombolytic agents (tissue-type plasminogen activator, streptokinase and urokinase).

This study compares the ability of 3 thrombolytic drugs to promote clot lysis using a new in vitro testing procedure. Whole blood samples from 132 patients were tested using 5 different concentrations of tissue-type plasminogen activator (t-PA), streptokinase (SK) and urokinase. A mixture of blood and thrombolytic drug was placed on a dry-reagent test card containing reptilase, buffers and paramagnetic particles where clot formation occurred. Analysis of the motion of the clot-embedded paramagnetic particles caused by an oscillating magnetic field was used to define the lysis onset time. The slope of the linear regression plot of lysis onset time versus 1/[drug concentration] defined the kinetic rate constant (k) for each drug in each patient. Higher values of k indicated greater resistance to in vitro clot lysis. In the patients studied, there was a large range of k values for t-PA and SK (coefficient of variation 143 and 137%, respectively) but a smaller range of k for urokinase (coefficient of variation 32%). The coefficients of variation for t-PA and SK observed in the study group were five- to 10-fold greater than the coefficients of variation determined for replicate test measurements. Resistance to all SK concentrations tested was found in 9% of the patients. In vitro sensitivity to thrombolysis was compared among the drugs by correlating the derived k values. These comparisons indicated no relation for any of the drugs; many patients had a relatively low k value for 1 drug, while having a relatively high k value for a different drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Decentralized testing for prothrombin time and activated partial thromboplastin time using a dry chemistry portable analyzer.

Previous work has established the precision and accuracy of a portable blood coagulation analysis system using paramagnetic particles contained in a dry reagent on a disposable test card. We examined the deployment of this technology in decentralized hospital settings and compared test results obtained in the surgical intensive care unit, coronary care unit, and outpatient cardiology clinic with those obtained in the central laboratory. Nursing personnel were instructed in the use of the system, and quality control testing was performed daily by the laboratory staff. In the intensive care units, patient subjects included those on whom tests of prothrombin time and activated partial thromboplastin time had been ordered. Immediate determinations were performed by the intensive care unit nursing staff on the same citrated, whole-blood samples that were subsequently sent to the central laboratory. In the outpatient cardiology clinic, fingerstick blood samples were obtained for prothrombin time determinations with the dry chemistry system. Paired prothrombin time samples obtained by venipuncture were run in the hospital laboratory. The study involved multiple users, multiple locations, two lots of activated partial thromboplastin time cards, and several different instruments, over an extended period. Correlation coefficients between the dry chemistry system and the hospital laboratory under these conditions were in an acceptable range in all sites studied. We concluded that, with appropriate training and quality assurance, the dry chemistry system provides an acceptable alternative to the hospital laboratory for prothrombin time and activated partial thromboplastin time determinations.

Exploration of rapid bedside monitoring of coagulation and fibrinolysis parameters during thrombolytic therapy.

Monitoring coagulation parameters during thrombolytic therapy could be useful for prediction and treatment of haemorrhagic episodes. Technology based on dry reagent chemistry has been developed that allows rapid (less than 10 min) assays on small samples of whole blood. The assay principle is based on the restriction of motion of paramagnetic particles during fibrin polymerization, and subsequent liberation of particle motion during fibrinolysis. This technology was used to monitor prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen levels and fibrinolysis profiles during thrombolytic therapy with tissue plasminogen activator for acute myocardial infarction. The PT and aPTT obtained with the COAG-1 correlated well with conventional assays (r = 0.93 and 0.92 for PT and aPTT, respectively; p = 0.0001). Fibrinogen estimates, obtained by COAG-2 also correlated well with modified Clauss assays (r = 0.86, p = 0.0001). The rapid determination of the aPTT may improve management of adjunctive anticoagulant therapy following thrombolysis. The fibrinolysis profile may be useful during thrombolytic therapy to verify that a lytic state has been achieved, to monitor the lytic state throughout therapy, and to verify that the lytic state normalizes once therapy has been completed.

Dry reagent technology for rapid, convenient measurements of blood coagulation and fibrinolysis.

Rapid coagulation and fibrinolysis assays suitable for use with an imprecisely measured sample volume (either whole blood or plasma) have been developed, utilizing a technology based on paramagnetic iron oxide particles (PIOP) that move in response to an oscillating magnetic field. PIOP are combined with appropriate test reagents for clotting and thrombolysis assays and formulated as dry reagents within a capillary test chamber. The minima and maxima of the PIOP oscillations define a two-sided waveform that provides kinetic information on fibrin polymerization and lysis. Subject to the chemistry of the dry reagent formulation, the resulting waveform can be used to define clotting time, lysis onset time, or fibrinogen variables. Applications to one-stage prothrombin time and one-stage activated partial thromboplastin time tests have yielded assays with consistently good correlations with other test methods. Applications to fibrinolysis studies have yielded global assays of thrombolytic activity, in that the assay results reflect the interactions of multiple factors associated with the effectiveness of thrombolytic therapy. Depending on the components utilized in a particular reagent formulation, one can derive information about the activity of such factors as fibrinogen, plasminogen, and related inhibitors, as well as the lytic agent being administered. Use of these assays in a clinical setting should provide a rapid, convenient alternative to conventional testing of coagulation variables and a reliable method for monitoring thrombolytic therapy.

Glucose sensor characteristics for miniaturized portable closed-loop insulin delivery: a step toward implantation.

An implantable glucose sensor must be free of baseline and sensitivity drift. To our knowledge, a drift-free glucose sensor with fast response time is not currently available. We are therefore proposing a miniature extracorporeal glucose sensor with access to the vascular system as a practical step toward implantation. This sensor could use existing technology to form the basis of a patient-wearable closed-loop system.

Kinetic studies of a doubly bound red cell antigen-antibody system.

The Polybrene method for detection of red cell antibodies which utilizes continuous flow equipment was modified so that kinetic studies could be performed on red cell antibodies doubly bound between adjacent red cells. In the anti-Rh(o)-Rh(o) erythrocyte system, deaggregation by temperature was studied over an antibody concentration range of from approximately 1 to 500 antibody molecules per erythrocyte, a residence time range of approximately eightfold, and a temperature range of from 10 to 55 degrees C. The rate of dissociation of antigen-antibody complex, as determined from deaggregation of antibody-dependent red cell aggregates, was found to be of apparent zero order. The apparent activation energy for the antigen-antibody reaction under the experimental conditions was determined and found to be higher than would be expected for singly bound antigen-antibody systems. Possible explanations are considered for these findings in terms of an antigen-antibody bond-breaking model.