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    22 ABSTRACT An assay to determine anticoagulants in a blood or blood plasmasample, wherein the assay comprises analyses with at least two wetchemistry prothrombine time (PT) methods. The assay comprises measuringPT in a first reaction mixture with a first PT method and measuring PT in asecond reaction mixture with a second PT method, wherein the concentrationof blood or blood plasma in the second reaction mixture differs from theconcentration of blood or blood plasma in the first reaction mixture. The PTmethods are calibrated to give the same or approximately the same PTresults for reference samples which lack anticoagulants of interest for theassay. Further, calculating a difference in PT from the measurements,wherein if the difference in PT is 1) significant, this is indicative of a presenceof anticoagulants in the sample, or 2) non-significant, this is indicative of anabsence of anti-coagulants above detectable level in the sample. Elected figure for publication: Fig. 1
    公开号:SE1450612A1
    申请号:SE1450612
    申请日:2014-05-22
    公开日:2015-11-23
    发明作者:Mats Rånby
    申请人:Zafena Ab；
    IPC主号:
    专利说明:

    AWAPATENTABKontor/Handläggare Linköping/ J cnny C arls son/ SZJ Zafcna AB A nsökningsnr Vår referens SE-210700441 ASSAY TO DETERMINE ANTICOAGULANTS IN BLOOD OR BLOODPLASMA Technical fieldThe present disclosure relates to an assay to determine anticoagulantsin a blood or blood plasma sample.
    BackgroundAnticoagulants are substances that dampen, reduce or abolish the ability of blood, or blood plasma, to coagulate - anticoagulants reduce therate by which the fibrinogen is converted into fibrin.
    Some anticoagulants are naturally occurring, endogenous, and playimportant physiological roles in limiting the onset, the speed of propagationand the spatial extension of blood coagulation in vivo. Other anticoagulantsappear under pathological conditions. There are anticoagulants found insnake-toxins and in excretions of mosquitoes, leaches, bats and ticks. Agrowing group of anticoagulants are man-made substances created for thepurposes of treating and preventing thromboembolic disorders.
    Blood coagulation is a complex phenomenon involving a multitude ofinteracting molecules found in blood plasma or on the surface of engagedcells. There are numerous cellular and molecular interactions, including manyenzymatically catalyzed reactions, which may be down-regulated ordisrupted, by the action of anticoagulants.
    The direct acting anticoagulants inhibit, hamper or abolish theenzymatic activity of one or both of the critical enzymes in the coagulationprocess, thrombin (Flla) or activated coagulation factor X (FXa). lndirectacting anticoagulants work in some other way, one of which is to enhance thepotency of direct acting inhibitors. Anticoagulants of therapeutic use, heparinand the vitamin-K antagonists are viewed as indirect acting anticoagulants.Direct acting anticoagulants are hirudin (a protein of the leach) and therecently created inhibitors of Flla or FXa. lt is these new direct acting anticoagulants, particularly those that can be administered orally, DOACs,that are causing a profound change within clinical, and laboratory medicine.
    The fact that anticoagulants have different modes of action poses achallenge to laboratory medicine in devising methods by which theanticoagulants can be determined. To a degree, this challenge is exasperatedby recent pharmacological successes in creating new substances to treatthromboembolic disorders, see e.g. J. Harenburg, S. Marx and R. Krämer,Determination of the anticoagulation effects of the new oral anticoagulants: anunmet need, Expert Review of Haematology, February 2012, Vol. 5, No. 1,Pages 107-113.
    At central hospital laboratories, where there is a variety of testsavailable and qualified personnel to interpret the results, detection anddetermination of the DOAC appears relatively straightforNard -the Fllainhibitors and FXa inhibitors may be assayed by thrombin time (TT) tests orthe FXa tests, respectively. lf standard variants of these tests do not fulfill theneeds, some modification or “dilution” of the standard variants will. At primarycare centers, and at places, sites, within hospitals but remote from the centrallaboratory, “off-site”, the situation is different. Characteristic for laboratorymedicine at POC-sites (Point of Care) is that the number of differentlaboratory tests is limited because the physical space of such laboratory sitesis limited and because the availability of qualified laboratory personnel islimited. Within coagulation the most available POC test is the prothrombin test(PT-test) with the results expressed in INR (international normalized ratio, aratio between the PT of the sample and a normal PT normalized by beingpowered to normalizing exponent, a sensitivity index). Other “regular” or”common” coagulation test are activated partial thromboplastin time (APTT),activated coagulation time (ACT) and the mentioned TT. lt is the hope ofvisionary experts in the coagulation field that these “regular” or “common”coagulation tests can be adapted, modified, “diluted”, to fulfill the POC-needin assaying the “new” anticoagulants, particularly the DOAC. Suchthoughts/hopes are for example expressed by E.J. Favaloro and G. Lippi, Thenew oral anticoagulants and the future of haemostasis laboratory testing,Biochemia Medica 2012; 22(3):329-41.
    Because PT-INR is the most commonly available POC-test forcoagulation measurements, a modified PT-test or “diluted PT” by which theDOAC can be determined is highly desirable. There is reported work in suchdirection. The low sensitivity of most PT-tests for direct FXa inhibitors, suchas rivaroxaban, is known. C. Kluft discloses in EP 2 405 274 A1 that PT-teststhat are affected by a certain snake venom, RVV-V, also show sensitivitytoward rivaroxaban, and discloses the use of a PT-test that employ suchthromboplastins.
    The hopes of finding a way to determine DOAC by some PT-test,modified or not, has during the recent years declined. ln January of 2014, TLindahl, a member of expert group in coagulation of the external qualityorganization of Sweden, EQUALIS, reported on studies performed by theexpert group on one FXa-inhibiting substance aprixaban. The conclusion wasthat none of the many commercially available PT or APTT tests was of use indetermining aprixaban at clinically relevant concentrations in blood plasma.FXa-tests, on the other hand worked well for this purpose.
    Efforts to correct PT-results for the variable anticoagulant effect of non-functioning coagulation factors, pivka, found in the blood of patients ontreatments with vitamin-K antagonists, warrant to be mentioned. US7,767,459 B2 (J. Horsti) provides for this by measuring PT of blood plasma bythe standard protocol of any given PT method, and also measuring the sameafter a pre-dilution of the plasma with a physiological buffer such as 9 g/lNaCl. The PT results, expressed either in seconds or in INR, are then plottedagainst the degree of final plasma dilution and extrapolated to zero. The PT ofthe plasma at final dilution of zero, reduced by the same for a normal plasma,is taken as a measure of the pivka-effect and is used to correct the originalPT-result.
    New coagulation tests have been devised with the aim to determineseveral different kinds of anticoagulants, particularly heparins and DOACs.
    These efforts demonstrate the clinical importance of determining theseanticoagulants.
    See Calatzis A, Peletz D, Haas S, Spannagl M, Rudin K, Wilmer M,Prothromibnase-induced Clotting Time Assay for Determination of the Anticoagulant Effects of UFH and LMWH, Fondaparinux, and Thrombinlnhibitors, Am J Clin Pathol 2008; 130: 446-454, and Samama MM, MartinoliJL, LeFlem L, Guinet C, Plu-Bureau G, Depasse F, Assessment of laboratoryassays to measure rivaroxaban - an oral, direct factor Xa inhibitor, ThrombHaemost 2010; 103/4: 815-825.
    Relevant background to the present invention is the distinctionbetween wet-chemistry methods and dry chemistry methods. Wet-chemistryis defined by the mixing of a volume of the sample, in a coagulation assay ofblood or blood plasma, and a volume of reagent. The sample is thus diluted toa certain degree in the reaction mixture. ln dry-chemistry this dilution does notoccur. The sample is mixed, or contacted, with reagent substance in a dryform and there is no dilution of the sample. Most POC-tests are dry-chemistrytests because they can often be presented in an easy to use format, e.g. astrip or a chip. The operator needs typically only to add a small volume of thesample. A disadvantage of dry-chemistry methods is that the tests are moredifficult to modify. One dimension of the freedom granted by the wet-chemistry procedures, variation of the degree of sample dilution, is notavailable with dry-chemistry. Every mention of a “diluted” modification of a“regular” test has wet-chemistry as a prerequisite.
    Summarylt is an object of the present disclosure to provide an assay to determine anticoagulants in a blood or blood plasma sample, the assayinvolving analyses with at least two wet chemistry prothrombine time (PT)methods, which assay is designed to be applicable for determination of bothdirect and indirect anticoagulants in blood or blood plasma samples.
    The invention is defined by the appended independent claim.Embodiments are set forth in the dependent claims, in the attached drawingsand in the following description.
    According to a first aspect there is provided an assay to determineanticoagulants in a blood or blood plasma sample, wherein the assaycomprises analyses with at least two wet chemistry prothrombine time (PT)methods. The assay comprises the steps of: a) in a first PT analysis with a first PT method measure PT in a first reaction mixture comprising a firstvolume of blood or blood plasma diluted in a first volume of a liquid reagent,b) in a second PT analysis with a second PT method measure PT in a secondreaction mixture comprising a second volume of the blood or blood plasmadiluted in a second volume of the liquid reagent, wherein the concentration ofblood or blood plasma in the second reaction mixture differs from theconcentration of blood or blood plasma in the first reaction mixture. The atleast two PT methods are calibrated to give the same or approximately thesame PT results when used to analyze reference blood or blood plasmawhich lack anticoagulants of interest for the assay. The assay furthercomprises the step of c) calculating a difference in PT from themeasurements in step a) and b), wherein if the difference in PT is 1) significant, this is indicative of a presence of anticoagulants in the blood orblood plasma sample, or 2) non-significant, this is indicative of an absence ofanti-coagulants above detectable level in the blood or blood plasma sample.
    With analyses with at least two PT methods is here meant that theassay may involve analyses with 2, 3, 4, 5 or up to 6 PT methods.
    The at least two PT methods used in the assay may be fundamentallydifferent PT methods, e.g. one PT method could be a PT method of the Quicktype and the other PT method or alternatively one or more of the other PTmethods could be of Owren type.
    Although it does not appear practical at present one of the PT methodscould even by a dry chemistry method, but this presses the limits of thepresent invention.
    The at least two PT methods used in the assay may be fundamentallythe same PT method, but with some difference that may appear insignificant.Such differences, apart from the difference provided by the invention, i.e.difference in the relationship between sample volume and reagent volume,could be differences in the temperature at which the measurements areperformed and differences in ionic strength or pH of the reaction mixture.
    With wet chemistry PT method is here meant Quick type PT or Owrentype PT methods.
    The ratio between the volume of blood or blood plasma and the volumeof liquid reagent in the reaction mixture may vary depending on the kind of PTmethod used in the assay, i.e. Quick type PT method or Owren type PTmethod, and the concentration in the sample of the anticoagulant to bedetected. A liquid reagent of the Quick type PT method contain no fibrinogenand therefore has an inherent limitation in how much the sample may bediluted. This is because the coagulation to be registered is the conversion ofsample fibrinogen, dispersed in the reaction mixture, to fibrin. ln order for thisconversion to be noticeable, the fibrinogen in the reaction mixture needs to beabove some practical limit, say above about 0.1 g/L. Hence, the blood orblood plasma sample cannot be diluted further than about tenfold in liquidreagent, as blood or blood plasma often contains fibrinogen concentrations of1 g/L or less. ln practicing the invention with Quick type PT methods, the allowableratio between the volume of blood or blood plasma and the volume of liquidreagent in the reaction mixture may be varied between 1:2 and 1:10. ln addition, similar limitations as are set by the presence of fibrinogenare also set by FV (Factor V). FV must be present in the reaction mixture ateffective levels. Since FV is not included in the PT liquid reagent of a QuickPT type method, FV must come from the blood or blood plasma sample itself.
    When an Owren type PT method is used in practicing the assay nolimits are set by fibrinogen or FV since the liquid reagent, apart fromthromboplastin, also contains effective levels of these. However, with theliquid reagent used in the Owren type PT method there are practicallimitations to the final sample dilution due to reaction speeds. At very lowvolumes of the blood or blood plasma sample in the reaction mixture, thelevels of FVll, FX and Fll will be so low that the coagulation time (the PT)becomes so long that detection becomes difficult or impossible. With theOwren type PT method the lower level of blood or blood plasma sampledilution in a liquid reagent is about 1:2, which is the same as with the Quicktype PT method, but the highest possible dilution will be considerably higher,about 1:200. Hence, in practicing the invention with the Owren type PTmethod, the ratio between the volume of blood or blood plasma and the volume of liquid reagent in the reaction mixture may be varied between 1:2and 1:200. A preferred ratio range may be between 1:5 and 1:100 or between1:10 and 1:50. ln the present assay, PT is analyzed in at least two reaction mixtureshaving different concentrations of blood or blood plasma in the reactionmixtures. The difference in concentration of blood or blood plasma in thereaction mixtures when using the Owren type PT method in the assay may beabout 1.5 to 100 times, 1.5 to 50 times, 1.5 to 25 times, 1.5 to 10 times or 1.5to 5 times. A difference in concentration of 1 .5 to 5 times is practical toachieve and is with the present assay shown to allow determination ofanticoagulants in clinically relevant concentration ranges.
    The difference in concentration of blood or blood plasma in the reactionmixtures when using the Quick type PT method may be about 1.5 to 5 times,1.5 to 4 times, 1.5 to 3 times or 1.5 to 2 times. ln spite of the difference in blood or blood plasma concentration in thereaction mixture, all the two or more different PT methods of the assay arecalibrated to give, with relevant reference blood or blood plasma samples thatlack the anticoagulant of interest, the same or approximately the same PTresult. This PT is expressed in such a way that this is possible. Expression inregular time units will not do as these will vary. A reaction mixture with a lowconcentration of blood or blood plasma shows long PT:s and vice versa. Anatural way to express PT is in INR (internationalized normalized ratio), butother expressions, various synthetic time-like units, or ratios of such, arepossible. Important is that a given relevant sample lacking anticoagulant(s),analyzed by the two or more PT methods, will yield as closely as possible thesame result. A natural way to compare the results from one PT method toanother is by the average PT-result and by the CV (coefficient of variation) ofthe comparison. The calibration is such that the average PT-result for severalrelevant samples that lack the anticoagulant should be as nearly the same aspossible with all of the two or more PT-methods, and the CV of thecomparisons should be as low as possible.
    The at least two PT methods may be calibrated such that the PTmethods give the same or approximately the same PT result when used to analyze reference blood or blood plasma from a normal individual anddilutions of such reference blood or blood plasma. With blood or blood plasmafrom a normal individual is here meant blood or blood plasma from one orseveral normal person(s) or a pool of normal individuals under noanticoagulation treatment.
    The at least two PT methods may be calibrated to give the same orapproximately the same PT results when used to analyze blood or bloodplasma (from a single or many individual(s) or pool of samples from differentindividuals) which lack anticoagulants of interest for the present assay andwhich blood or blood plasma has an above normal PT value determined byone or several established PT methods. ln step c) of the assay a difference in PT between PT measurements iscalculated. The terms “significant” and “insignificant” are here given theirconventional statistical definition. A significant difference is then a relativedifference of 2 times CV or more, an insignificant difference less than 2 timesCV. lf the difference in PT calculated in step c) is significant and the identityof the anticoagulant is known, a concentration of the anticoagulant in theblood or blood plasma sample may be computed. lf the difference in PT calculated in step c) is non-significant and theidentity of the anticoagulant is known, the level above which the anticoagulantis not present in said blood or blood plasma sample may be assigned. lf the difference in PT calculated in step c) is significant, an estimatedPT of the blood or blood plasma sample in the absence of anticoagulants maybe computable.
    Computation of anticoagulant concentration in a blood or blood plasmasample can only be performed if the identity of the anticoagulant is known orassumed. lf analyses with two PT methods have been performed it may beexpedient to use the difference in PT results and the PT-result from theanalysis with the highest sample dilution for this calculation. The PT-result atthe highest dilution will be closest to the PT-result should there be noanticoagulant present. Using the INR-formalism, this imagined PT-result is designated lNRo. lNRo may be obtained by subtracting a fraction of thedifference, or subtracting a function of the difference, depending on if thedose-response to the anticoagulant level is linear or not. Since the dose-response may depend on this lNRo and on the temperature at which thedeterminations have been made, the conversion of the size of the differenceinto anticoagulant concentration may require a mu|titude of so called standardcurves. Alternatively a multidimensional function may be employed tocompute lNRo and the anticoagulant level. lf analyses with more than two PT methods have been performed inpracticing the assay of the invention, more than one difference can becomputed, and these several differences, and the PT-value obtained at thehighest sample dilution, can be used to compute the lNRo and the possibleanticoagulant levels depending on the identity of the anticoagulant. lt is alsopossible to favor one of the differences as being most useable as it is afavorable range for the determinations.
    The liquid reagent may comprise thromboplastin, fibrinogen andcoagulation factor V.
    Any kind of PT-reagent may be used in practicing the invention, but aliquid reagent of the Owren type, a reagent containing thromboplastin,fibrinogen and FV, is preferred because it allows a heftier final dilution of thesample than when a Quick type PT liquid reagent is used. This is a propertyvalue in POC-applications because a smaller sample volume can be used,e.g. the volume size that is readily obtained from a finger prick, i.e. bloodvolumes in the range 2 to 50 micro-liters.
    The PT measurements may be performed at an ambient temperaturein the range of 17 °C to 45 °C, preferably in the range of 18 °C to 30 °C, morepreferably in the range of 21 °C to 30 °C and most preferably in the range of25 °C to 30 °C.
    The anticoagulants which may be determined in the present assay aremembers of a group comprising direct acting inhibitors of activatedcoagulation factors lla and Xa, which may be selected from a groupcomprising dabigatran, apixaban, rivaroxaban or hirudin, or are members of agroup comprising indirect acting inhibitors of activated coagulation factors lla and Xa, which may be selected from a group comprising fractionated orunfractionated heparins, lupus anticoagulants or antibodies interfering withthe activity of FVIII.
    The first volume of the liquid reagent in the first reaction mixture maybe equal to the second volume of the liquid reagent in the second reactionmixture.
    The volume of blood or blood plasma diluted in the liquid reagent maybe in the range of 1 to 20 uL.
    The volume of blood or blood plasma may be added to the liquidreagent with an end-to-end capillary.
    Brief Description of the Drawinqs Fig. 1 is a graph showing apixaban content measured with the present assayin normal blood plasma.
    Fig. 2 is a magnification of a portion of the graph in Fig. 1.
    Fig. 3 is a graph showing apixaban content measured with the present assayin normal blood plasma (primary data: PT in seconds).
    Fig. 4 is a magnification of a portion of the graph in Fig. 3.
    Fig. 5 is a graph showing apixaban content measured with the present assayin normal blood.
    Fig. 6 is a magnification of a portion of the graph in Fig. 5 Fig. 7 is a graph showing dabigatran content measured with the presentassay in normal blood.
    Fig. 8 is a magnification of a portion of the graph in Fig. 7.
    Fig. 9 is a graph showing heparin content measured with the present assay innormal blood plasma.
    Fig. 10 is a magnification ofa portion of the graph in Fig. 9.
    Detailed DescriptionExamples below are given, not to limit the scope of the invention, but to further explain and describe the invention, and to stimulate development inthis important medical diagnostic area. Experimental work was performedwith the commercially available ambient room temperature coagulation 11 instrument, Simple Simon PT Zafena AB, Borensberg, Sweden, with functionsdescribed in EP 1 636 595 B2. One instrument or reader was used for eachPT-method. Alternatively, the same instrument could be used for all PT-methods. The ambient room temperature in the laboratory where the workwas done varied between 21°C and 26°C. Such a temperature interval, lowerthan but not distant from 30 °C, is favored since the temperature hardlyaffects the PT, thereby improving the precision of the provided PT analysis. lnaddition, a lower temperature increases the assay sensitivity foranticoagulants in the examples below. The assay is, however, possible toperform at an ambient temperature in the range of 17-45 °C.
    The PT-reagent was the one delivered together with lot N223M of theSimple Simon PT product, a reagent of Owren-type that thus containseffective amounts of thromboplastin, fibrinogen and FV (the thromboplastin ofrabbit brain origin and the fibrinogen and the FV of bovine blood plasmaorigin). Other Owren type reagents could also be used.
    The reagent was in portions of 200 uL to which sample, citrated bloodor citrated blood plasma, was added and mixed. The addition was by an end-to-end plastic capillary mounted at one end of a tubular body with adisplacement mechanism in the other end, this to allow convenient mixing ofsample and reagent, such Mixxocaps with 10 uL capillaries are supplied withthe Simple Simon PT product, to perform the experiments described, someMixxocaps were fitted with 20 uL capillaries, or 5 uL capillaries instead of the10 uL capillaries with which they were supplied by themanufacturer.Alternatively, standard pipettes or similar may be used to mixthe blood or blood plasma sample with the liquid reagent.
    The ratios between the volume of blood or blood plasma to the volumeof liquid reagent used in the experiments were 1:11, 1:21 and 1:41. Otherratios lying within the range of 1:5 and 1:100 could be used.
    The difference in concentration of blood or blood plasma between thedifferent reaction mixtures used in the assays in the experiments wasbetween 1.5 to 5 times. The difference in concentration could, however, beabout 1.5 times to 100 times. 12 Anticoagulated normal plasma was the NKP product GHI-163 lot10188, MediRox AB, Nyköping, Sweden. Stock solutions of dabigatran(Pradaxa, Boehringer-Ingelheim) and apixaban (Eliquis, Bristol-Myers Squibb)containing 100 mg/L were kind gifts of Professor Tomas Lindahl of theDepartment of Experimental Medicine of Linköping University. Unfractionatedheparin was Heparin Leo lot A6888B, Leo Pharma A/S, Ballerup, Denmark.
    Example 1Apixaban is a direct acting inhibitor of activated coagulation factor X (FXa). It is the active compound of the antithrombotic drug Eliquis, Bristol-Myers Squibb. lt is of clinical interest to measure apixaban in blood plasma inthe range 50 to 1000 ug/L. To prepare suitable samples, small volumes of thestock solution of apixaban was added to normal plasma, the control plasmaNKP, to give normal plasma with apixaban content in the range 0 to 1000ug/L. These normal plasmas with apixaban, including the unadulteratednormal plasma (NKP) and the same diluted 1:2 in 9 g/L NaCl (NKP 1:2) wereall analyzed by Simple Simon PT at sample additions of 20 uL, 10 uL and 5uL in 200 uL reagents, thus with three PT-methods with different samplecontent in the reaction mixtures, according to the invention. It was furtherrelevant that all three PT-methods were calibrated, according to the invention,to show INR 1.000 for unadulterated NKP, and INR 1.357 for the NKP 1:2. Allprimary INR values, the results that appeared on the instrument screens (oneinstrument or reader was used for each PT-method), were thereforetransformed to the corrected/calibrated lNRc by the formula a*lNRexp(b)where a and b were selected to given NKP and NKP 1:2 their desired values,1.000 and 1.357, respectively. The INR-value of the NKP 1:2 was calculatedby the formula of Lindahl et al for the INR of corresponding to PT-activity of50% (half of the 100% that equates to INR 1.000). The data treatment isshown in table 1. Above the INR calibrated columns (lNRc) there is an uppernumber and a lower number, these are the above mentioned a and b,respectively. 13 Two measurements of the apixanban content of the normal plasma areshown, a more sensitive measure, the difference in lNRc given by PT-methods with 20uL sample and 5uL sample, and a less sensitive measure,the lNRc-difference between 10uL and with 5 uL. These lNRc-differences areplotted against the apixaban content of the normal plasmas as shown in thegraph in Fig. 1. The square symbols are the difference in lNRc between theresults of 20uL method and the 5uL method, and the round symbols thedifference in lNRc between the 10uL method and the 5uL. lt is pointed out that it is possibility to estimate the INR of the normalplasma in the absence of anticoagulant, lNRo. ln the above this is done in aless sophisticated way. A portion (56%) of the lesser lNRc-difference issubtracted from the lNRc-results of the 5uL sample method, the resultobviously closest to lNRo. These simple lNRo-estimates are surprisinglygood, only the one at top apixaban content (1000 ug/L) is off by more than atenth of an INR-unit.
    Fig. 1 and 2 show graphs in which the rate of increase in the measuresof apixaban content increases with the content. However, for those many thatappreciate/favors linear dose-response relationships, such relationships arehere displayed by apixaban measures that are below one INR-unit as seen inthe graph in Fig. 2, where Fig. 2 shows a magnification of a portion of thegraph in Fig. 1.
    The data treatment showed in Fig. 1 and 2 could be confusing to thoseunfamiliar to ambient room temperature determination of INR. lnstead of a 14 primary INR-result which has been corrected for temperature effect by thecomputations by the instrument/reader, we could instead consider the PT inseconds. This is done to increase the clarity in describing the invention inspite of introducing an error due to the temperature not being strictly constantduring the time period in which the assays were performed (temperaturesvaried from 22.3 °C to 24.2 °C). ln spite of this bias the results are still goodenough to be convincing, and explains more clearly because itallows adata-treatment familiar to all with knowledge in the art, i.e. computation of INR bydividing the PT (in seconds) by a normal PT and powering the ratio with anormalizing constant equivalent to the lSl.ln table 2 the results of the assay on normal plasma (NKP) with added apixaban is shown, but the primary data is here in PT, in seconds, and thedata is converted into lNRc by division with the PT of a normal plasma andsubjecting the ratio to the power of an lSl, upper and lower number,respectively, shown above the lNRc-columns. As reamed relevant the uppernumber is selected to make NKP 1:2 to show lNRc 1.357. . .êíiïiššs_ lßpL-Epl.3,37 5.7.E- ' ln table 2 data is biased by a temperature gradient running from 22.3°C at the top to 24.2 °C at the bottom, this however does little to disturb thegeneral picture. The PT-methods used, ambient temperature Owrensmethods, are more sensitive to inhibitors than others, still it is obvious, that asingle of the selected PT-methods would not give much useful information atlower levels of apixaban. lmagine that you employ the most apixaban sensitive of the three, the one with 20 uL of sample, and you obtain a PT of35.2 seconds. This is more than that of a normal, 32.8 seconds, but is thisdifference due to anticoagulants or is it just due to the sample having aslightly elevated PT (the PT of NKP 1:2 is 36.9 seconds) How would youknow Practice of the invention, performing two or more PT-methods withdifferent sample content in the reaction mixture gives a c|earer picture, sincethe methods are calibrated to show the same INR if the PT-vary for reasonsother than anticoagulant content, there would be little or no difference, nosignificant difference, between the lNRc-results. ln the present example it isknown that the samples contain the anticoagulant apixaban because this hasbeen added, and a difference in lNRc-result appears. ln the example therewere three PT-methods used according to the invention, and there are threedifferences in lNRc-results that can be examined (two of the three are shownin table 2), and for the assay of normal plasma with 50 ug/L of apixaban, allthree differences indicate the presence of anticoagulant. lf the identity of theanticoagulant is known its content can be estimated. Also the expected lNRcfor the sample in absence of inhibitor, lNRo, can be estimated. Table 2showing the PT:s in seconds, clarifies the utility of practicing the invention.The temperature bias, only muddles to a minor extent, the of this the highestINR values, those at the top, are exaggerated by about 10%, otherwise thedata is near identical to those shown in the previous table, including the lNRoestimates. ln the graphs in Figs 3 and 4 the data from table 2 has been inserted.lNRc-differences are plotted against the apixaban content of the normalplasmas (difference in lNRc given by PT-methods with 20uL sample and 5uLsample (squares), and lNRc-difference between 10uL and with 5 uL (dots)).The graph in Fig. 4 is a magnification of a portion of the graph in Fig. 3.
    Example 2 Apixaban in the range 0 to 1000 ug/L was added to a normal citratedblood. These blood samples and a blood sample with an INR 2.3 wereanalyzed by Simple Simon PT using sample volumes of 20uL, 10 uL and 5uL 16 in 200 uL reagents. The primary results in INR, and after relevant calibration,lNRc, are displayed in the table below, table 3: 8,37 1.01 ___________________________________________________________________________________________ .ënšasšælzšm INR i INR .Itëäfšßë . lwšf: AINBQ êlNBsy ëfli-il-.tzlßí flflßf-.šzšsš usxfL fiuI-.hïsl V Stil-hårt ßfluL-äxll- V lflæll--Ssfl- A i. ..'. t' 'i ° "l i' “3 1 8 ' Table 3 Here it was decided, for all three PT-methods, that the normal bloodwithout addition of apixaban was to show lNRc 1.00, and a citrated blood,reported by the central hospital laboratory to show INR 2.3 was here to showlNRc 2.30. As in previous examples the difference in lNRc-results are viewedas measures of anticoagulant content, here apixaban content, and two of thethree possible such differences are plotted against the known apixanbancontent and displayed in Figs 5 and 6. The difference in lNRc given by PT-methods with 20uL sample and 5uL sample is shown as squares and lNRc-difference between 10uL and with 5 uL as dots. The graph in Fig. 6 is amagnification of a portion of the graph in Fig. 5.
    The dose-responses of the anticoagulant method of the invention showgood positive dose-response characteristics over a clinical range of interest ofapixaban in blood. The dose-response is again linear for differences in lNRcof less than unity, see Fig. 6.
    Example 3Dabigatran is the active compound formed in vivo when the pharmceutical preparation containing dabigatran-etexilate, Pradaxa,Boehringer-lngelheim, is administered per os, i.e. orally.s. Clinical levels ofinterest range from 50 to1000 ug/L in blood plasma. Such samples werecreated by adding small volumes of stock solution of dabigatran to normal 17 blood plasma, NKP. Results and data-treatment, similar to that in Example 1 are shown in table 4.
    Table 4 ln comparing table 4 with table 1 in Example 1, it appears that thedisplayed embodiment of the invention is less sensitive for dabigatran than forapixaban in normal plasma. The impression is strengthened when comparingthe graphs in Figs 7 and 8, with the corresponding figures of Example 1.
    The more sensitive lNRc-difference (20uL minus 5uL, shown assquares in Figs 7 and 8) in the linear part of the graph (see Fig. 8, amagnification of a portion of the graph in Fig. 7) reaches 0.5 at about 200ug/L of dabigatran, whereas the same is reached at about 80 ug/L ofapixaban, see Fig. 2, i.e. a more than twofold difference in sensitivity. Yet,dabigatran levels of 50 ug/L of dabigatran appear to be detectable.
    Obviously, the assay to determine anticoagulants shows differentsensitivities for different anticoagulants, and in addition different sensitivitiesare expected with different reagents, different relevant calibrations, anddifferent temperature at which the Pt analyses are performed.
    Example 4Heparin is an anticoagulant that is tedious to determine at point of care sites, such as at surgical wards. More convenient methods are wanted. Theutility of the present invention is exemplified below. The example is withunfractionated heparin, often used as antithrombotic agent in extensivesurgery. 18 Samples were prepared by adding small volumes of a stock solution ofheparin to normal plasma, NKP. ln the selected calibration INR 1.000 andINR 1.357 were assigned to NKP and NKP 1:2, respectively, which was thesame relevant calibration as selected in Examples 1 and 3 above.Experimental details are the same as example 1 and 3, and the results anddata-treatment are shown in the Table 5. “v13ia, ašïåiiå-.fål-ëif ____ .êiïiiå-fålsiåf ........ ..... ._ass ma om:så sms oss_________ .......... :mn :mn Lßo Plotting the measure of anticoagulant content in the normal plasma,the difference in lNRc for two of possible three differences against the heparincontent gives the graphs in Figs 9 and 10. As seen from table 5 and Figs 9and 10, the heparin may be detected at about 0.2 U/mL and determined in theconcentration range 0.2 to 5 U/mL, which is of clinical relevance.
    权利要求:
    Claims (10)
    [1] 1. An assay to determine anticoagulants in a blood or bloodplasma sample, wherein said assay comprises analyses with at least two wetchemistry prothrombine time (PT) methods, the assay comprising the stepsof: a) in a first PT analysis with a first PT method measure PT in a firstreaction mixture comprising a first volume of blood or blood plasmadiluted in a first volume of a liquid reagent; b) in a second PT analysis with a second PT method measure PT in asecond reaction mixture comprising a second volume of said bloodor blood plasma diluted in a second volume of said liquid reagent,wherein the concentration of blood or blood plasma in the secondreaction mixture differs from the concentration of blood or bloodplasma in the first reaction mixture; characterized inthat said at least two PT methods are calibrated to:- give the same or approximately the same PT results when usedto analyze reference blood or blood plasma which lackanticoagulants of interest for said assay; and wherein the assay further comprises the step of: c) calculating a difference in PT from the measurements in step a)and b), wherein if said difference in PT is1) significant, this is indicative of a presence of anticoagulants inthe blood or blood plasma sample; or 2) non-significant, this is indicative of an absence of anti-coagulants above detectable level in the blood or bloodplasma sample.
    [2] 2. The assay according to claim 1, wherein if the difference in PTcalculated in step c) is significant and the identity of the anticoagulant isknown, a concentration of the anticoagulant in said blood or blood plasmasample is computable.
    [3] 3. The assay according to claim 1, wherein if the difference in PTcalculated in step c) is non-significant and the identity of the anticoagulant isknown, the level above which the anticoagulant is not present in said blood orblood plasma sample is assignable.
    [4] 4. The assay according to any of claims 1 or 2, wherein if thedifference in PT calculated in step c) is significant, an estimated PT of theblood or blood plasma sample in the absence of anticoagulants iscomputable.
    [5] 5. The assay according to any of the preceding claims, whereinthe liquid reagent comprises thromboplastin, fibrinogen and coagulation factorV.
    [6] 6. The assay according to any of the preceding claims, wherein thePT analyses are performed at an ambient temperature in the range of 17 °Cto 45 °C, preferably in the range of 18 °C to 30 °C, more preferably in therange of 21 °C to 30 °C and most preferably in the range of 25 °C to 30 °C.
    [7] 7. The assay according to any of the preceding claims, whereinthe anticoagulants determined in said assay are members of a groupcomprising direct acting inhibitors of activated coagulation factors lla and Xa,which are selected from a group comprising dabigatran, apixaban,rivaroxaban or hirudin or a group comprising indirect acting inhibitors of 21 activated coagulation factors lla and Xa, selected from a group comprisingfractionated or unfractionated heparins, lupus anticoagulants or antibodiesinterfering with the activity of FVIII.
    [8] 8. The assay according to any of the preceding claims, whereinthe first volume of the liquid reagent in the first reaction mixture is equal to the second volume of the liquid reagent in the second reaction mixture.
    [9] 9. The assay according to any of the preceding claims, wherein thevolume of blood or blood plasma diluted in the liquid reagent is in the range of1 to 20 uL.
    [10] 10.volume of blood or blood plasma is added to the liquid reagent with an end-to- The assay according to any of the preceding claims, wherein the end capillary.
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    同族专利:
    公开号 | 公开日
    PL3146347T3|2020-03-31|
    WO2015177293A1|2015-11-26|
    US20170089932A1|2017-03-30|
    EA036308B1|2020-10-23|
    SE540132C2|2018-04-10|
    US10613104B2|2020-04-07|
    CN106415275A|2017-02-15|
    EA201650035A1|2017-05-31|
    CN106415275B|2019-05-17|
    ZA201607732B|2020-05-27|
    EP3146347A1|2017-03-29|
    ES2750350T3|2020-03-25|
    EP3146347B1|2019-07-31|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    SE1450612A|SE540132C2|2014-05-22|2014-05-22|Method of analysis for the determination of anticoagulants in blood or blood plasma|SE1450612A| SE540132C2|2014-05-22|2014-05-22|Method of analysis for the determination of anticoagulants in blood or blood plasma|
    EP15726897.0A| EP3146347B1|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    EA201650035A| EA036308B1|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    PCT/EP2015/061292| WO2015177293A1|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    ES15726897T| ES2750350T3|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    CN201580027220.6A| CN106415275B|2014-05-22|2015-05-21|Determine the measurement of the anti-coagulants in blood or blood plasma|
    US15/311,255| US10613104B2|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    PL15726897T| PL3146347T3|2014-05-22|2015-05-21|Assay to determine anticoagulants in blood or blood plasma|
    ZA2016/07732A| ZA201607732B|2014-05-22|2016-11-09|Assay to determine anticoagulants in blood or blood plasma|
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