WO1995014788A1

WO1995014788A1 - Blood coagulation retardants and devices

Info

Publication number: WO1995014788A1
Application number: PCT/US1994/013537
Authority: WO
Inventors: Martha E. Lyon; Paul Henderson; Sohail Malik; Margaret A. Kenny; Andrew W. Lyon
Original assignee: University Of Washington
Priority date: 1993-11-24
Filing date: 1994-11-23
Publication date: 1995-06-01
Also published as: AU1186295A

Abstract

The invention provides methods of using anticoagulants to retard the coagulation of blood, so that properties and functions of blood, plasma, and blood cells may be determined analytically. The methods do not interfere with electrochemichal techniques use to detect divalent cations and permit accurate analysis of many analytes within a single blood sample, which currently require separately anticoagulated blood samples. The serine protease inhibitors used may be combined with each other or blood cell activation, aggregation, and adhesion inhibitors in mixtures that provide anticoagulant activity. The methods permit, for the first time, the possibility of using a single blood sample to perform a full range of blood, plasma, and blood cell analyses.

Description

BLOOD COAGULATION RETARDANTS AND DEVICES

Field of the Invention

The present invention provides blood coagulation retarding compositions and devices for containing blood that are coated with these compositions that retard the coagulation of blood, without significantly binding calcium or magnesium ions present in the blood, and without interfering with the detection of these ions by commonly used analytical techniques, such as the use of ion selective electrodes. Further, the coagulation retardant properties of the compositions permit the analysis of functions, compositions, and coagulation-independent properties from a single specimen of blood, plasma, and blood cells, whether the analyte be of physiological, pathological, or of external origin.

Background of the Invention

Medical and veterinary diagnoses, management, monitoring, and evaluation frequently rely on chemical, biochemical, and cellular analyses performed on human or animal whole blood and its plasma, cellular, or serum constituents. After blood is drawn from a patient into a tube, the blood is altered by the process of coagulation which transforms fluid blood into a semi-solid heterogeneous clot that is incompatible with most analytical methods. Historically, analyses have often been performed on the serum. However, the composition of the blood sample is frequently altered by cell lysis during the coagulation process leading to inaccurate analytical results. Also, preparation of blood serum requires time for complete clot formation and centrifugation, which extends the turn-around time of analysis and contributes to analyte deterioration. Further, the preparation of a serum matrix is inconsistent with the design and philosophy of near-patient testing technology. In order to overcome the disadvantages produced by the coagulation of blood samples, anticoagulants are added to blood samples. However, anticoagulants currently available in clinical laboratories cause interference with analytical methods used to determine certain analytes. Consequently, it is frequently necessary to obtain more than one blood sample, using different types of anticoagulants in each sample, so that the sample to be analyzed for a particular analyte does not contain an interfering anticoagulant.

The most common currently available anticoagulants fall into two general classes: the thrombin inhibitors and the calcium chelators. Of the thrombin inhibitors, heparin is the most commonly used today. It is a known inhibitor of acid phosphatase, lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, gamma- glutamyl transferase, creatine kinase, and restriction endonucleases. Specific heparin salts bias the determination of sodium, potassium, ionized calcium, ammonia, albumin, lithium, cholesterol, free fatty acids, fructosamine, insulin, phosphate, free thyroxine, amino acids, carcinoembryonic antigen, corticosteroids, amikacin, gentamicin, tobramycin, lysozyme, mucopolysaccharide, and glucose 6-phosphate dehydrogenase.

Heparin is a mucopolysaccharide and is not readily dissolved in blood, depending upon its physical form. For example, dispersion problems occur with heparin introduced into a syringe as a lyophilized pellet whereas heparin sprayed onto the internal surface of syringes seems to partially overcome this problem. Heparinized samples therefore require rolling of blood samples for a significant length of time to obtain mixing and anticoagulation, a requirement that is often limiting in certain medical circumstances.

Of the calcium chelators, ethylenediaminetetraacetic acid (EDTA), sodium citrate, and oxalate salts are the best known.

The sequestration of divalent ions or the presence of the chelating compounds renders both whole blood and plasma unsuitable for the analyses of acid phosphate, albumin, alkaline phosphatase, alpha-tocopherol, amino acids, ammonia, aminolevulinic acid dehydratase, amylase, angiotensin converting enzyme, bicarbonate, calcium, ionized calcium, camitine, chloride, copper, cholesterol, creatine kinase, creatinine, erythrocyte sedimentation rate, free fatty acids, beta- hydroxybutyrate dehydrogenase, glucose, insulin, iron, lactate dehydrogenase, lithium, leucine amino peptidase, magnesium, nonprotein nitrogen, osmolality, pH, phosphate, protein, potassium, pyruvate kinase, retinol binding protein, salicylate, selenium, sodium, sorbitol dehydrogenase, triglycerides, urea nitrogen, vitamin A, and zinc. In an effort to address some of the deficiencies associated with heparin, modified versions of heparin have been produced. These include "calcium-balanced heparin" which is designed to counteract the anticoagulant-induced biases in determination of whole blood ionized calcium. In vitro tests on blood collected in syringes coated with calcium-balanced heparin showed an error as great as 7% in the concentration for detected ionized calcium which could be attributed to "calcium dilution" problems. Further, an increase in the measurement of whole blood potassium concentration has also been observed with the use of this type of syringe. Whether this is an issue of syringe design or a consequence of heparin remains to be determined.

As a further alternative to heparin, U.S. patent 5,187,102 describes the use of hirudin, another thrombin inhibitor. Hirudin, isolated from a leech hirudo medicinalis. is an expensive anticoagulant and is only available in small quantities. For example, following the instructions for preparation of anticoagulated blood in U.S. patent 5,187,102, 3 ml of blood would require 600 antithrombin units of hirudin activity at a cost of approximately $200. An additional problem with the hirudin preparation is standardization, especially when low doses are required.

Low hirudin dosage levels are preferred because of its presently high cost, although there is nonlinearity in the relationship between dosage and clotting time at such low hirudin levels. Each batch of hirudin, like heparin, must be individually tested to determine its potency response curve since anticoagulant capability varies significantly from batch to batch.

Hirudin is a proteinacious composition and there is a risk that it will denature and foul analytical instruments resulting in lost time and increased costs. The utility of hirudin as a broad spectrum anticoagulant for chemistry and biochemical analyses of whole blood or plasma has never been established. The '102 patent significantly only refers to the testing of blood cell properties and functions, not whole blood or plasma.

In a further approach to reduce anticoagulant-induced biases in the measurement of whole blood electrolytes and blood gases, syringes containing lower concentrations of heparin have been marketed. While these syringes do not significantly interfere with the measurement of whole blood ionized calcium, the coagulation retarding property of the syringes is substantially reduced. As a result, there is an increased incidence of blood specimen clotting before they can be analyzed by a clinical laboratory. This translates into requirements for additional blood samples from patients, increased turn-around times for analyzing blood samples, increased analytical instrument downtime as a result of microclots forming the instruments, additional expenses for recalibrating instruments, and increased chemical reagents costs.

Given the high cost, limited availability, and large number of anticoagulant- induced interferences in the analysis of blood components, there exists a need for an anticoagulant which provides minimal interference with analytical techniques, maximizes the number of potential analytes suitable for analysis, is inexpensive, has a long shelf life, can withstand lyophilization, resolubilization, and gas sterilization, and is available in commercial quantities. Desirably, the anticoagulant should be readily standardizable without requiring testing of each batch to establish required dosage rates. The anticoagulant should overcome the limitations of heparin (interference with divalent ion analyses) and hirudin (interference with protein analyses, for example). Such an anticoagulant should desirably be useful for in vitro anticoagulation of clinical samples, surface coating of intravascular or indwelling monitoring devices, micromachining of fluidics for new instrumentation (such as fiber optic and silicon chip devices), and in vivo anticoagulant intervention for coagulopathic and hypercoagulable states as well as anticoagulant therapy for heart and vascular associated surgeries.

Summary of the Invention The invention provides methods of using readily standardizable and sterilizable compositions that are effective in retarding coagulation of blood, while not significantly binding calcium or magnesium ions in the blood or interfering with protein analyses in measuring composition of blood, plasma, and blood cells; functions of blood, plasma, and blood cells; and coagulation-independent properties of blood, plasma, and blood cells; whether of physiological, pathological, or external origin, from a single blood sample.

The compositions include serine protease inhibitors such as irreversible or reversible inhibitors of thrombin and/or irreversible or reversible inhibitors of factor Xa, alone or in combination with blood cell aggregation and adhesion inhibitors such as inhibitors of platelet activation, platelet-platelet interactions, phospholipid binding of proteases, fibrinogen, as well as other cell adhesion molecules, to retard the rate of blood coagulation.

The invention also provides devices coated with a blood coagulation retarding composition, or mixture of such compositions, that are suitable for use in receiving or storing blood for analyses. The coating of the devices of the invention does not interfere with divalent cations, such as calcium and magnesium ions in blood or their analyses, analytes which are dependent on the presence of these ions, analytes which react with heparin, EDTA, or other anticoagulants either chemically or electrochemically, cellular functions dependent upon the presence of calcium and/or magnesium or directly impaired by the presence of chelators and cellular measurements other than function. The coatings of the invention are useful for in vitro anticoagulation of clinical samples, surface coating of intravascular or indwelling monitoring devices, and micromachining of fluidics for instrumentation. In particular, the invention provides blood gas analyzers with coated blood contacting surfaces that retard blood coagulation.

As a result of the lack of interference with alkaline earth ions, such as calcium ions, in the blood, the invention provides the capability to analyze blood more accurately for these ions and blood constituents containing and requiring these ions. This allows accurate determination of acid phosphatase, lactate dehydrogenase, gamma glutamyl transferase, creatine kinase, sodium, potassium, ionized calcium, ionized magnesium, ammonia, albumin, lithium, cholesterol, free fatty acids, fructosamine, insulin, phosphate, free thyroxine, amino acids, carcinoembryonic antigen, corticosteroids, amikacin, gentamicin, tobramycin, lysozyme, mucopolysaccharide, and glucose 6-phosphate dehydrogenase. Heparin and calcium chelator anticoagulants are unsuitable for the analyses of the aforementioned analytes since they either inhibit or bias the analytic determinations. The invention offers, for the first time, the possibility of virtually interference- free analyses of functions of blood, plasma, and blood cells; composition of blood, plasma, and blood cells; coagulation-independent properties of blood, plasma, and blood cells, whether physiological, pathological, or of external origin from a single blood sample doped with the mixtures of the invention compositions, or retained in a device coated with the invention compositions prior to analysis to retard coagulation. The anticoagulant compositions useful in the invention are readily standardizable in that they do not vary in anticoagulant effectiveness from batch to batch, as is the case for hirudin or heparin.

Unlike hirudin, the serine protease inhibiting compositions of the invention have predictable potency and a known mass versus potency relationship.

The anticoagulant compositions useful in the invention are blood soluble and are, in this respect also, superior to heparin that may not be readily dispersed in blood. Thus, the invention permits preservation of blood samples with a reduction in mixing time relative to heparin. Finally, the invention offers the prospect of reducing blood requirements as currently practiced. Not only does this reduce labor for gathering and processing samples from individual patients but also the inherent risks in obtaining and handling multiple blood samples. Consolidation of analyses to a single tube could -minimize the phlebotomy-induced suppression of hematocrit in acute care patients which are often subjected to frequent laboratory testing. When used to preserve blood samples for coagulation, the costs of this invention will be comparable or less than currently available anticoagulants on a per-tube basis. Also, since mixtures of anticoagulant compositions may be used according to the invention, each of which operates by a different mechanism to retard coagulation, interference with analyte determinations may be reduced to insignificance. Detailed Description of the Preferred Embodiments

The invention provides methods for preparing blood to provide measurement of compositions of blood, plasma, and blood cells; functions of blood, plasma, and blood cells; and coagulation-independent properties of blood, plasma, and blood cells, whether physiological, pathological, or of external origin, from a single blood sample. The methods require the contacting of blood with a composition or mixture of compositions effective for retarding the rate of coagulation of a blood sample, without introducing interference with methods for subsequently measuring functions of blood, plasma, and blood cells; compositions of blood, plasma, and blood cells; and coagulation-independent properties of blood, plasma, and blood cells, whether physiological, pathological, or of external origin.

The invention also provides blood coagulation retarding devices for receiving and containing blood samples (for example, syringes (sometimes known as arterial samplers, and the like); devices for insertion into arteries and veins of a patient (such as catheters, sensors, drug delivery devices, and the like); and analytical devices that contact blood, for example, blood gas analyzers, and the like; the blood contacting surfaces of these devices being coated with or containing coagulation-retarding compositions or mixtures of compositions described above. The analytical devices include, for example, the blood gas analyzers sold, for example, by Radiometer, that perform several analyses and that have blood contacting surfaces. The blood coagulation-retarding compositions or mixtures may be doped into the blood sample or may be coated onto blood contacting surfaces of the device or surfaces inserted into the device.

In a typical method for preparing blood to allow measurement of functions of blood, plasma, and blood cells; composition of blood, plasma, and blood cells; and coagulation-independent properties of blood, plasma, and blood cells, according to the invention, a blood sample is first introduced into a means for receiving blood, such -1-

as a syringe. This means for receiving blood contains blood contacting surfaces, such as the internal surfaces of the syringe or a capsule with surfaces contained within the syringe. The coagulation-retarding compositions or mixtures of compositions of the invention are then contacted with the blood sample, through the coating on the blood contacting surfaces. Alternatively, coagulation is retarded by doping the blood sample with the individual compositions or mixtures of compositions.

The suitably contained and preserved blood sample, now admixed with the blood coagulation-retarding composition or mixture of compositions, may then be transported and stored, without coagulating, for analysis. In the specification and claims the term "readily standardizable" in reference to an anticoagulant means that the anticoagulant has a fixed dosage response curve in that once the dosage is determined that produces a certain coagulation time, this dosage will be invariant and not dependent upon the particular production batch that the composition is selected from. The compositions useful in the invention are "readily standardizable." Heparin, calcium balanced heparin (CBH), and hirudin are not, by this definition, readily standardizable since each batch must be tested for dosage response and the dosage adjusted to achieve the desired anticoagulant effect.

In a highly significant aspect of the invention, the compositions and mixtures of compositions useful in the invention do not significantly interfere with divalent ions, such as calcium or magnesium ions present in blood. Therefore, the invention overcomes the disadvantage present with heparin which interferes with electro¬ chemical methods usually used for detecting calcium cations and produces a falsely reduced reading. Even CBH, developed in an effort to overcome this problem, affects calcium determination: when sample calcium level is low, CBH produces a positive bias; when sample calcium level is high, CBH produces a negative bias. This bias may range from 60 to 120% of the ionized calcium reference range. The invention, by not substantially interfering with the charge, and hence electro-chemical property of divalent cations that is used in their detection as analytes, provides methods for obtaining an accurate analysis for calcium or other divalent ion. Indeed, variation from a control standard, using the invention, is within the level of accuracy of most commercially available ion-selective membrane analytical instruments (15-20% of reference range or 0.015 to 0.02 mmol/liter). Thus, the invention provides an accurate determination of, for instance, calcium, magnesium, and the like.

Also, the invention, due to the anticoagulant compositions or mixtures of serine protease inhibitors are amenable to standardization. It may be sterilized by a wide variety of techniques without incurring denaturation. This extends the range of materials useful for blood exposure and allows more precise estimates of efficacy for coated surfaces.

In another important aspect of the invention, the useful blood coagulation- retardant compositions are combined into mixtures containing at least two individual serine protease inhibitors or at least one serine protease inhibitor in combination with blood cell activation, aggregation, and adhesion inhibitors. The serine protease inhibitors may be selected, for example, from the irreversible thrombin inactivators, the reversible thrombin inactivators, and the Xa factor inhibitors. It being understood that other inhibitors that block or retard a reaction in the cascade of reactions that conclude with blood coagulation are also useful. Further, blood cell activation, aggregation, and adhesion inhibitors include monoclonal antibodies to the ARG-GLY-ASP-NAL (SEQ. ID NO. 1), triflavin, α, α'-bis[3-(N,N-diethylcarbomyl)- p-xylene dihydrobromide (GT-12), platelet activation inhibitors, platelet-platelet interaction inhibitors, phospholipid binding inhibitors, fibrinogen inhibitors, and the like.

When mixtures according to the invention are used to retard coagulation, several anticoagulation mechanisms are utilized, each of these acting to a limited extent to produce an overall minimal interference with methods for detecting particular analytes in the blood. As a result, for the first time, the invention provides a method for preparing specimens for accurate analyses of functions of blood, plasma, and blood cells; composition of blood, plasma, and blood cells; and coagulation- independent properties of blood, plasma, and blood cells, whether of physiological, pathological, or external origin, from a single blood specimen.

In general, an anticoagulant should not chemically react with or chelate analytes. It should not inhibit enzymatic or chemical reactions utilized as analytical methods. An anticoagulant should not bias analyte quantisations by electrochemical, spectrophotometric, electrophoretic, immunological, or other analytical methodologies. It should also be stable for long periods of time and be stable to sterilization processes. Finally, an anticoagulant should be present in excess, to prevent clot formation and analytical or instrumentation bias in hypercoagulable samples.

In order to select suitable anticoagulants for use in the invention's methods and devices, certain criteria must be met. To avoid interference with positively charged analytes, such as divalent cations, to be detected by ion-selective electrodes, the anticoagulant composition should be non-polyanionic so that it will not neutralize the charge of the analyte. Also, the anticoagulant should be of reasonably low molecular weight, preferably less than about 2500 daltons, more preferably less than about 1000 daltons, to provide reduced molecular surface and thereby minimize potential interaction with analytes that interfere with their detection.

The preferred compositions useful for retarding blood coagulation according to the invention include: D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK); 1,5-dansyl-glu-gly-arg chloromethyl ketone (GGACK); D-methyl- phenylalanyl-prolyl-arginal (GYKI- 14766) (Eli Lilly); D-phenylalanyl-L-prolyl-L- arginyl chloromethyl ketone (FPRCH2C1); (2R,4R)-4-methyl-l-[N2](3-methyl- l,2,3,4-tretrahydro-8-quinolinesulfonyl-L-arginyl)-2-piperidine carboxylic acid monohydrate (MD805) (Central Laboratory, Hyogo Prefecture Awaji Hospital Sumoto, Japan); 4-methyl-l-(N2-[(3-methyl-l,2,3,4-tetrahydro-8-quinolinyl (MCI- 9038); (Ac-(D) phe-pro-boroArg) (DuP 714); alpha-[(acetylthio)acetyl]-D-phe-pro- arg-CH2Cl and N-alpha-[(acetylthio)acetyl]-D-phe-phe-arg-Ch2Cl; fumaryl-L-arginyl leucyl arginal (Ro 09-1679) (Nippon Roche Research Center, Kanagawa, Japan); α, α'-bis[3-(N,N-diethylcarbomyl)-p-xylene dihydrobromide (GT-12); and a synthetic arg-gly-asp-containing peptide known as G4120 (University of Leuven, Belgium).

Of these, the most preferred are PPACK and GGACK. PPACK is an irreversible thrombin inactivator, but can also be synthesized in its aldehyde form, which is a reversible inactivator of thrombin. GGACK, on the other hand, is an irreversible inhibitor of factor Xa.

PPACK is useful as a coagulation retardant when commingled with blood at a concentration that will allow preservation (i.e., non-coagulation) of the blood for the desired length of time. Usually, in modern clinical testing procedures, it is desired that blood should be preserved for from about 5 minutes to about 3 days. The invention readily achieves preservation from coagulation for up to about 3 days, or more, if desired. In order to achieve preservation for this period of time, a blood sample should be commingled with PPACK to provide from about 10 to about 1000 μmoles of PPACK per liter of blood. Similarly, GGACK is preferably commingled with blood in the range from about 10 to about 1000 μmoles of GGACK per liter of blood. It is understood that the commingling of the compositions with blood may take place through dosing the compositions directly into the blood or through coated blood contacted surfaces in receptacles for the blood.

Dosage levels for other serine protease inhibitors according to the invention may be readily ascertained by testing and developing a relationship between dosage level and time for blood coagulation. Once established, this relationship is invariant, unlike heparin or hirudin which require testing of each batch to establish the relationship between dosage level and coagulation time.

In a further embodiment of the invention, mixtures of the preferred compositions may be prepared. Desirably, these mixtures have component compositions selected to further minimize interference with analyte detection, retard different steps in the serine protease cascade of reactions leading to coagulation, and produce the desired level of coagulation inhibition. The most preferred compositions, PPACK and GGACK, may be combined to produce a mixture. This mixture may be more effective than either PPACK or GGACK individually. Consequently, to achieve preservation of a blood sample for from about 5 minutes to about 3 days or more, a mixture of these compositions is preferably commingled with blood in concentrations ranging from about 10 to about 500 μmoles of PPACK per liter of blood, and from about 10 to about 500 μmoles of GGACK per liter of blood.

Further enhancement of the coagulation-retardant activity of these compositions or mixtures of compositions are achieved by adding blood cell activation, aggregation, and adhesion inhibitors to PPACK or GGACK either singularly or a PPACK-GGACK mixture. Such blood cell aggregation and adhesion inhibitors may include: annexins, monoclonal antibodies to the ARG-GLY-ASP and the ARG-GLY-ASP-NAL (SEQ. ID NO. 1) sequences, triflavin, and GT-12. An advantage of devices according to the invention, coated with the preferred coagulation-retarding compositions or mixtures of compositions, is that they may be gas-sterilized without loss of coagulation-retarding activity. This provides patient safety, especially in drawing arterial blood samples, while also providing cost-effective device preparation. The following examples are intended to illustrate the invention and do not in any way restrict the scope of the invention as described above and as claimed below.

EXAMPLE 1

Determination of Anticoagulation Effect of PPACK

Plastic tubes with the capacity to collect 1.0 ml of whole blood were used to examine which concentration(s) of PPACK would provide effective anticoagulation.

To each tube, 0.1 ml of an aqueous solution of PPACK (concentrations between 100 μM and 1 mM) was added. Then, 0.9 ml of whole blood was collected directly into each tube. The tubes were gently swirled by hand. Clot formation was monitored by dipping a wooden stick into the sample. The samples were examined at room temperature and the results are tabulated below: Clotting Time. Mins.

0.9 ml Blood Sample with: no additives 17 0.1 ml of0.9% NaCl 17 0. l ml of I mM PPACK > 4 days 0.1 ml of 100 μM PPACK 133 0. l ml of 10 μM PPACK 38 0.1 ml of l μM PPACK 28

0. l ml of 100 μM PPACK 22

1. 1.0 ml blood sample.

EXAMPLE 2 Gas Sterilization of Lyophilized PPACK Syringes

Plastic syringes with the capacity to collect 3.0 ml of whole blood were used to determine whether gas sterilization of lyophilized PPACK syringes affected the measurement of hematocrit, pH, pCO2, pθ2, sodium, potassium, ionized calcium, and glucose.

Two sets of syringes were selected. To each of the syringes, an aqueous solution of PPACK (75 μM) was added. Both sets of syringes were frozen at -70°C and then lyophilized. One set of syringes was then gas sterilized, the other was not. Three ml of whole blood was collected directly into each of the non-gas sterilized syringes containing lyophilized PPACK and each of the gas sterilized syringes containing PPACK. The syringes were mixed between the palms of the hands for 20 seconds and then placed on wet ice for 20 minutes. As can be seen from the table below, gas sterilization of the syringes did not affect the measurement of hematocrit, pH, CO2, pθ2, sodium, potassium, ionized calcium, and glucose.

pC0₂ Pθ2 Na K Ca¹

Syringe pH (mmHg) (mmHg) HCT (μM) (μM) (μM) Glucose

0.9% NaCl 7.339 48.6 22.2 40 156 4.3 1.18 64

75 μM PPACK 7.317 49.1 22.3 40 158 4.4 1.17 72 (not gas sterilized)

75 μM PPACK 7.327 49.1 21.3 40 157 4.3 1.17 67 (gas sterilized)

1. Calcium concentrations are pH corrected to pH 7.4.

EXAMPLE 3 Demonstration of Lack of Time Dependent Bias in the Measurement of Hematocrit and Whole Blood pH. pCO?. and pO? Plastic syringes with the capacity to collect 3.0 ml of whole blood were used to determine whether a time dependent bias in the measurement of hematocrit and whole blood pH, pCO2, and pθ2 would exist using PPACK (75 μM) as an anticoagulant.

Whole blood (2.0 ml) was collected into PPACK-containing syringes and then either analyzed immediately or following 5, 15, 30, 45, 60, and 90 minutes incubation on wet ice. The results are tabulated below:

TIME pCO₂ pθ2 (min) HCT (%) PH (mmHg) (mmHg)

5 40 ± 0 7.34 ± 0 48.6 ± 0 22.2 ± 0

15 40 ± 0 7.31 ± .01 49.1 ± 1.2 22.3 ± 2.2

30 39 ± 1 7.32 ± .01 48.2 ± 2.9 22.4 ± 1.6

45 39.7 ± 1.5 7.31 ± .01 49.0 ± 2.3 22.5 ± 2.3

60 39.7 ± 1.5 7.31 ± .01 48.5 ± 2.3 22.8 ± 1.7

90 37.7 ± .6 7.31 ± .01 48.6 ± 1.8 22.9 ± 1.6

No time dependent bias in the measurement of hematocrit and whole blood pH, pCO2, and pθ2 were observed with PPACK-containing syringes.

EXAMPLE 4 Demonstration of Lack of Time Dependent Bias in the Measurement of Serum Sodium, Potassium, Ionized Calcium, and Ionized Magnesium Plastic syringes with the capacity to collect 3.0 ml of serum were used to determine whether a time dependent bias in the measurement of serum sodium, potassium, ionized calcium, and ionized magnesium would exist using PPACK (50 μ M) as an anticoagulant.

Serum (2.0 ml) was collected into PPACK-containing syringes and then either analyzed immediately or following 15, 30, 45, 60, and 90 minutes incubation in wet ice. The sodium, potassium, ionized calcium, and ionized magnesium were compared with serum collected into syringes containing lithium heparin (100 units); syringes containing zinc heparin (100 units); syringes containing calcium balanced lithium/sodium heparin (120 units); and syringes containing no heparin. The results were as follows:

TIME Na⁺ K⁺ Ca²⁺ Mg²⁺

SYRINGE (min) mmol/l mmol/l mmol/l mmol/l

No Heparin 0 141 4.6 .59 .41

II 15 142 4.6 .59 .41

II 30 141 4.6 .60 .41

II 45 141 4.6 .60 .41

II 60 141 4.6 .60 .41

II 90 139 4.5 .61 .42

PPACK 0 142 4.6 .60 .41

II 15 142 4.6 .60 .41

II 30 142 4.6 .60 .41

II 45 142 4.6 .61 .41 " 60 141 4.6 .61 .42 " 90 142 4.6 .62 .42

Calcium Heparin 0 142 4.6 .61 .41

II 15 142 4.6 .61 .41

II 30 143 4.6 .62 .41

I. 45 142 4.6 .62 .40

II 60 143 4.6 .62 .41

II 90 143 4.6 .63 .41

Lithium Heparin 0 142 4.6 .57 .41

II 15 142 4.6 .58 .41

II 30 141 4.6 .58 .41

II 45 141 4.6 .58 .41

II 60 141 4.6 .59 .41

II 90 141 4.6 .59 .42

Zinc Heparin 0 142 4.6 .65 .54

II 15 142 4.6 .67 .48

II 30 141 4.6 .68 .46

II 45 141 4.6 .69 .45

II 60 141 4.6 .69 .45

II 90 141 4.6 .70 .45 (Magnesium & Calcium concentrations are pH corrected to pH 7.4.) No time dependent bias in the measurement of serum sodium, potassium, ionized calcium, and ionized magnesium was observed with PPACK-containing syringes. EXAMPLE 5

Demonstration of Lack of Concentration Dependent Bias in the Measurement of Serum Sodium, Potassium. Ionized Calcium, and Ionized Magnesium Plastic syringes with the capacity to collect 3.0 ml of serum were used to determine whether a concentration dependent bias in the measurement of serum sodium, potassium, ionized calcium, and ionized magnesium would exist using PPACK as an anticoagulant. The final concentrations of PPACK examined were 1 μ M, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM.

Serum (2.0 ml) was collected into PPACK-containing syringes. The syringes were mixed between the palms of the hands for 20 seconds and then placed on wet ice for 20 minutes. The sodium, potassium, ionized calcium, and ionized magnesium were compared with serum collected into syringes containing either lithium heparin with potencies of 7 units, 50 units, 100 units, 150 units, and 200 units or zinc heparin with potencies of 50 units, 75 units, 100 units, and 300 units. The results were as follows:

Concentration Νa⁺ K⁺ Ca²⁺ Mg²⁺

Syringe or Potency (mmol/l) (mmol/l) (mmol/l) (mmol/l)

No Heparin — 147 4.8 .58 .39

PPACK l μM 147 4.8 .59 .40

PPACK 10 μM 147 4.8 .59 .40

PPACK 25 μM 146 4.8 .58 .40

PPACK 50 μM 147 4.9 .59 .40

PPACK 75 μM 147 4.9 .60 .40

PPACK 100 μM 146 4.9 .58 .40

Lithium Heparin 7 units 147 4.8 .57 .38

Lithium Heparin 50 units 146 4.8 .55 .38

Lithium Heparin 100 units 147 4.8 .53 .38

Lithium Heparin 150 units 146 4.7 .53 .41

Lithium Heparin 200 units 146 4.7 .51 .41 Zinc Heparin 50 units 147 4.8 .71 .52

Zinc Heparin 75 units 148 4.8 .85 .67

Zinc Heparin 100 units 146 4.7 1.10 1.83

Zinc Heparin 300 units 146 4.8 .57 .39

(Calcium and magnesium concentrations are pH corrected to pH 7.4.)

No concentration dependent bias in the measurement of serum sodium, potassium, ionized calcium, and ionized magnesium was observed with PPACK- containing syringes.

EXAMPLE 6

Demonstration of Lack of a PPACK Effect on

Assays Performed Using a Discrete Random Access

Multi-Channel Clinical Chemistry Analyzer

Glass collection tubes with the capacity to collect 7.0 ml of whole blood were used to determine whether gas sterilized and lyophilized PPACK (50 μM) in the tubes affected the measurement of sodium, potassium, chloride, carbon dioxide, glucose, blood urea nitrogen, creatinine, total bilirubin, direct bilirubin magnesium, phosphate, albumin, total protein, total and pancreatic amylase, and ethanol.

Whole blood (7.0 ml) was collected into PPACK-containing glass collection tube. The specimens were mixed by gentle inversion of the tubes and then were centrifuged to obtain plasma. The PPACK treated samples were compared with serum and lithium heparin (100 units) treated plasma obtained from the same individual. The samples were analyzed using a Beckman CX-7 analyzer and a Dupont Dimension AR analyzer.

The results, tabulated below, indicated that PPACK did not affect the measurement of sodium, potassium, chloride, carbon dioxide, glucose, blood urea nitrogen, creatinine, total bilirubin, direct bilirubin magnesium, phosphate, albumin, total protein, total and pancreatic amylase, and ethanol by these analyzers.

Test Analyzer Serum Lithium Heparin PPACK

Sodium CX-7 141 (mmol/l) 142 (mmol/l) 141 (mmol/l)

Potassium 4.5 (mmol/l) 4.4 (mmol/l) 4.3 (mmol/l)

Chloride 105 (mmol/l) 105 (mmol/l) 105 (mmol/l)

CO, 31 (mmol/l) 31 (mmol/l) 30 (mmol/l) Glucose 87 (mg/dl) 85 (mg/dl) 86 (mg/dl)

Blood Urea Nitrogen 12 (mg dl) 12 (mg/dl) 12 (mg/dl)

Creatinine 0.8 (mg/dl) 0.8 (mg/dl) 0.8 (mg/dl)

T. Bilirubin 1.3 (mg/dl) 1.3 (mg/dl) 1.5 (mg dl)

Direct Bilirubin 0 (mg dl) 0 (mg/dl) 0.1 (mg/dl)

Magnesium 1.5 (mEg 1) 1.5 (mEg 1) 1.5 (mEg/1)

Phosphate 3.1 (mg/dl) 3.0 (mg/dl) 2.9 (mg/dl)

Albumin 4.1 (g dl) 4.1 (g/dl) 4.1 (g dl)

Total Protein 7.5 (g/dl) 7.8 (g/dl) 7.9 (g/dl)

Total Amylase 46 (IU/1) 47 (IUΛ) 47 (IU/1)

Pancreatic Amylase 35 (KM) 35 (IU/1) 35 (IU/1)

Ethanol 0.3 (mg dl) 0.4 (mg/dl) 0.4 (mg/dl)

EXAMPLE 7 Demonstration of Lack of a PPACK Effect on Enzyme Assays Glass collection tubes with the capacity to collect 7.0 ml of whole blood were used to determine whether gas sterilized and lyophilized PPACK (50 μM) in the tubes affected the measurement of gamma-glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, and creatine kinase MB (mass). Whole blood (7.0 ml) was collected into PPACK-containing glass collection tube. The specimens were mixed by gentle inversion of the tubes and then were centrifuged to obtain plasma. The PPACK treated samples were compared with serum and lithium heparin (100 units) treated plasma obtained from the same individual. The samples were analyzed using the Technicon Axon Enzyme Analyzer as well as the Ciba-Coming Chemiluminescent Magic Lite Analyzer and Roche Fara Analyzer.

The results, tabulated below, indicated that PPACK did not affect the measurement of gamma-glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, and creatine kinase MB (mass). Lithium

Serum Heparin

Test (units/1) (units/1) PPACK

Gamma Glutamyl 12 14 13 Transferase

Aspartate Amino 15 15 14 Transferase

Alanine Amino 10 11 12 Transferase

Alkaline Phosphatase 38 38 39

Lactate Dehydrogenase 140 144 146

Creatine Kinase 48 41 49

Creatine Kinase <1 <1 <1 MB Mass

EXAMPLE 8 Demonstration of Lack of a PPACK Effect in the Measurement of Non-Esterified Fatty Acids, Cholesterol. Triglycerides. and Transthyretin Glass collection tubes with the capacity to collect 7.0 ml of whole blood were used to determine whether gas sterilized and lyophilized PPACK (50 μM) in the tubes affected the measurement of nonesterified fatty acids, cholesterol, triglycerides, and transthyretin.

Whole blood (7.0 ml) was collected into PPACK-containing glass collection tube. The specimens were mixed by gentle inversion of the tubes and then were centrifuged to obtain plasma. The PPACK treated samples were compared with serum and lithium heparin (100 units) treated plasma obtained from the same individual.

The results, tabulated below, indicated that PPACK did not affect the measurement of nonesterified fatty acids, cholesterol, triglycerides, and transthyretin.

Lithium

Test Serum Heparin PPACK

Non-Esterified Fatty Acids (mEq/1) .09 .10 .06

Cholesterol (mg/dl) 153 152 156

Triglycerides (mg/dl) 71 68 72

Transthyretin (mg dl) 35.2 36.2 39 Although the invention has been described with reference to its preferred embodiments, those of ordinary skill in the art may, upon reading this disclosure, appreciate changes and modifications which may be made and which do not depart from the scope and spirit of the invention as described above and claimed below.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lyon, Martha E. Henderson, Paul Malik, Sohail Kenny, Margaret A. Lyon, Andrew W.

(ii) TITLE OF INVENTION: BLOOD COAGULATION RETARDANTS AND DEVICES

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Christensen, O'Connor, Johnson & Kindness

(B) STREET: 1420 Fifth Avenue, Suite 2800

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: USA

(F) ZIP: 99201-2347

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Word for Windows, Version 2.0c

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Karjeker, Shaukat A.

(B) REGISTRATION NUMBER: Reg. No. 34,049

(C) REFERENCE/DOCKET NUMBER: WRFO18026

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 206-224-0728

(B) TELEFAX: 206-224-0779

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Arg Gly Asp Val 1

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of measuring analytes selected from compositions, functions, and properties of whole blood, plasma, and blood cells, the method comprising: selecting a blood sample; retarding coagulation of the sample with at least one serine protease inhibitor effective for retarding blood coagulation rate, said inhibitor substantially free of interfering effects with detecting of divalent cations by ion selective electrodes and said inhibitor readily standardizable for anti-coagulant dosage response; and measuring multiple analytes in a single coagulation retarded sample using analytical methods with accuracy dependent upon divalent cation concentration.

2. The method of Claim 1, wherein said inhibitor comprises at least one anticoagulant selected from the group consisting of:

1,5-dansyl-glu-gly-arg chloromethyl ketone; D-methyl-phenylalanyl-prolyl- arginal; D-phenylalanyl-L-prolyl-L-arginal chloromethyl ketone; (2R, 4R)-4-methyl- l-[N2](3-methyl-l,2,3,4-tetrahydro-8-quinolinesulfonyl-L-arginyl)-2-piperdine carboxylic acid monohydrate; (Ac-(D) phe-pro-boroArg); alpha- [(acetylthio) acetyl]- D-phe-pro-arg-CH2Cl; and fumaryl-L-arginyl leucyl arginal.

3. The method of Claim 1, wherein said retarding comprises retarding with a mixture comprising a serine protease inhibitor; and a blood-cell activation, aggregation, and adhesion inhibitor.

4. The method of Claim 3, wherein said mixture comprises a serine protease inhibitor selected from the group consisting of:

1,5-dansyl-glu-gly-arg chloromethyl ketone; D-methyl-phenylalanyl-prolyl- arginal; D-phenylalanyl-L-prolyl-L-arginal chloromethyl ketone; (2R, 4R)-4-methyl-l- [N2](3-methyl-l,2,3,4-tetrahydro-8-quinolinesulfonyl-L-arginyl)-2-piperdine carboxylic acid monohydrate; (Ac-(D) phe-pro-boroArg); alpha- [(acetylthio) acetyl]- D-phe-pro-arg-CH2Cl; and fumaryl-L-arginyl leucyl arginal; and a blood cell activation, aggregation, and adhesion inhibitor selected from the group consisting of: annexins; monoclonal antibodies to the ARG-GLY-ASP and the ARG-GLY-ASP-VAL (SEQ. ID NO. 1) sequences; triflavin; and α, α-bis{3-(N, N- diethylcarbomyl) } -p-xylene dihydrobromide .

5. A method of preparing blood for measurement of compositions, functions, and properties of whole blood, plasma, and blood cells, the method comprising: introducing a blood sample into a means for receiving a blood sample; and retarding coagulation of the blood sample with at least one blood cell activation, aggregation, and adhesion inhibitor effective for retarding the blood coagulation rate, said blood cell activation, aggregation, and adhesion inhibitor substantially free of interfering effects with electro-chemical methods for detecting divalent cations.

6. The method of Claim 5, wherein said inhibitor is selected from the group consisting of: annexins; monoclonal antibodies to the ARG-GLY-ASP and the ARG-GLY- ASP-NAL (SEQ. ID NO. 1) sequences; triflavin; and α, α-bis{3-(N, N- diethylcarbomyl) }-p-xylene dihydrobromide.

7. A method of preserving blood, plasma or blood cells without coagulation for subsequent analysis of analytes selected from the group consisting of functions, properties and compositions of whole blood, plasma and blood cells, the method comprising: collecting a sample of blood in a device comprising a blood coagulation retarding surface for contacting blood, the surface comprising: a coating on the surface of said blood contacting device effective for retarding the coagulation of blood, said coating comprising a serine protease inhibitor substantially free of interfering effects with detection of divalent cations with ion selective electrodes and said inhibitor being readily standardizeable for anticoagulant dosage response; and retaining the collected sample, free of coagulation, and free of interfering effects with detection of divalent cations by ion selective electrodes, for a period of time ranging from about 5 minutes to about 3 days before measuring analytes.

8. The method of Claim 7, wherein said inhibitor is selected from the group consisting of:

1,5-dansyl-glu-gly-arg chloromethyl ketone; D-methyl-phenylalanyl-prolyl- arginal; D-phenylalanyl-L-prolyl-L-arginal chloromethyl ketone; (2R, 4R)-4-methyl-l- [N2](3-methyl-l,2,3,4-tetrahydro-8-quinolinesulfonyl-L-arginyl]-2-piperdine carboxylic acid monohydrate; (Ac-(D) phe-pro-boroArg); alpha-[(acetylthio) acetyl]- D-phe-pro-arg-CH2Cl; and fumaryl-L-arginyl leucyl arginal.

9. The method of Claim 7, wherein the coating of the surface further comprises a blood cell activation, aggregation and adhesion inhibitor.

10. The method of Claim 9, wherein the serine protease inhibitor is selected from the group consisting of:

1,5-dansyl-glu-gly-arg chloromethyl ketone; D-methyl-phenylalanyl-prolyl- arginal; D-phenylalanyl-L-prolyl-L-arginal chloromethyl ketone; (2R, 4R)-4-methyl-l- [N2] (3 -methyl- 1 ,2,3 ,4-tetrahydro-8-quinolinesulfonyl-L-arginyl)-2-piperdine carboxylic acid monohydrate; (Ac-(D) phe-pro-boroArg); alpha-[(acetylthio) acetyl]- D-phe-pro-arg-CH2Cl; and fumaryl-L-arginyl leucyl arginal.

11. A method of preserving whole blood, plasma, or blood cells without coagulation for subsequent analysis of analytes selected from compositions, functions and properties of the whole blood, plasma or blood cells, the method comprising: collecting a sample of blood in a blood coagulation retarding surface of a device for contacting blood, the surface comprising: a coating on the surface of said blood contacting device, the coating effective for retarding the coagulation of blood, the coating comprising a blood cell activation, aggregation, and adhesion inhibitor composition free of interfering effects with detection of divalent cations with ion selective electrodes and protein analytes; and retaining the collected sample free of coagulation for a period of time ranging from about 5 minutes to about 3 days.

12. The method of claim 11, wherein said inhibitor is selected from the group consisting of: annexins; monoclonal antibodies to the ARG-GLY-ASP and the ARG-GLY-ASP-VAL (SEQ. ID NO. 1) sequences; triflavin and α,α,-bis{3-(N, N- diethylcarbomyl) } -p-xylene dihydrobromide.

13. The method of claim 1, wherein the serine protease inhibitor is selected from the group consisting of D-phenylalanyl-prolyl-arginyl chloromethyl ketone and 1,5-dansyl-glu-gly-arg chloromethyl ketone.

14. The method of Claim 13 further comprising addiing a cell activation, aggregation, and adhesion inhibitor to the blood sample.

15. The method of claim 7, wherein the serine protease inhibitor is selected from the group consisting of D-phenylalanyl-prolyl-arginyl chloromethyl ketone and 1,5-dansyl-glu-gly-arg chloromethyl ketone.

16. The method of Claim 15 further comprising adding a cell activation, aggregation, and adhesion inhibitor to the blood sample.

17. The method of Claim 9, wherein at least one said inhibitor is selected from the group consisting of: annexins; monoclonal antibodies to the ARG-GLY-ASP or the ARG-GLY- ASP-NAL (SEQ. ID NO. 1) sequences; triflavin; and α, α-bis{3-(N, N- diethylcarbomyl)}-p-xylene dihydrobromide.

18. The method of Claim 7, wherein the surface for contacting blood is a blood contacting surface of a blood gas analyzer.

19. (Amended) The method of Claim 11, wherein the coagulation retarding surface is a blood contacting surface of a blood gas analyzer.