Patent Application
20100235103
1. Field of the Invention
This invention relates to analyzing blood for carrying out coagulation studies and other chemistry procedures, including determining the presence of prothrombotic abnormalities such as conditions of hypercoagulability, and monitoring oral anticoagulant therapy to take into account the platelet count in determining prothrombin times (PT), and a new Anticoagulant Therapy Factor (nATF).
2. Description of the Prior Art
Testing of blood and other body fluids is commonly done in hospitals, labs, clinics and other medical facilities. For example, to prevent excessive bleeding or deleterious blood clots, a patient may receive oral anticoagulant therapy before, during and after surgery. Oral anticoagulant therapy generally involves the use of oral anticoagulants—a class of drugs which inhibit blood clotting. To assure that the oral anticoagulant therapy is properly administered, strict monitoring is accomplished and is more fully described in various medical technical literature, such as the articles entitled “PTs, PR, ISIs and INRs: A Primer on Prothrombin Time Reporting Parts I and II” respectively published November, 1993 and December, 1993 issues of Clinical Hemostasis Review, and herein incorporated by reference.
These technical articles disclose anticoagulant therapy monitoring that takes into account three parameters which are: International Normalized Ratio (INR), International Sensitivity Index (ISI) and prothrombin time (PT), reported in seconds. The prothrombin time (PT) indicates the level of prothrombin and blood factors V, VII, and X in a plasma sample and is a measure of the coagulation response of a patient. Also affecting this response may be plasma coagulation inhibitors, such as, for example, protein C and protein S. Some individuals have deficiencies of protein C and protein S. The INR and ISI parameters are needed so as to take into account various differences in instrumentation, methodologies and in thromboplastins' (Tps) sensitivities, used in anticoagulant therapy. In general, thromboplastins (Tps) used in North America are derived from rabbit brain, those previously used in Great Britain from human brain, and those used in Europe from either rabbit brain or bovine brain. The INR and ISI parameters take into account all of these various factors, such as the differences in thromboplastins (Tps), to provide a standardized system for monitoring oral anticoagulant therapy to reduce serious problems related to prior, during and after surgery, such as excessive bleeding or the formation of blood clots.
The ISI itself according to the WHO 1999 guidelines, Publication no. 889-1999, have coefficients of variation ranging from 1.7% to 8.1%. Therefore, if the ISI is used exponentially to determine the INR of a patient, then the coefficients of variation for the INR's must be even greater than those for the ISI range.
As reported in Part I (Calibration of Thromboplastin Reagents and Principles of Prothrombin Time Report) of the above technical article of the Clinical Hemostasis Review, the determination of the INR and ISI parameters are quite involved, and as reported in Part II (Limitation of INR Reporting) of the above technical article of the Clinical Hemostasis Review, the error yielded by the INR and ISI parameters is quite high, such as about up to 10%. The complexity of the interrelationship between the International Normalized Ratio (INR), the International Sensitivity Index (ISI) and the patient's prothrombin time (PT) may be given by the below expression (A),
wherein the quantity
is commonly referred to as prothrombin ratio (PR):
The possible error involved with the use of International Normalized Ratio (INR) is also discussed in the technical article entitled “Reliability and Clinical Impact of the Normalization of the Prothrombin Times in Oral Anticoagulant Control” of E. A. Loeliger et al., published in Thrombosis and Hemostasis 1985; 53: 148-154, and herein incorporated by reference. As can be seen in the above expression (B), ISI is an exponent of INR which leads to the possible error involved in the use of INR to be about 10% or possibly even more. A procedure related to the calibration of the ISI is described in a technical article entitled “Failure of the International Normalized Ratio to Generate Consistent Results within a Local Medical Community” of V. L. Ng et al., published in Am. J. Clin. Pathol. 1993; 99: 689-694, and herein incorporated by reference.
The unwanted INR deviations are further discussed in the technical article entitled “Minimum Lyophilized Plasma Requirement for ISI Calibration” of L. Poller et al. published in Am. J. Clin. Pathol. February 1998, Vol. 109, No. 2, 196-204, and herein incorporated by reference. As discussed in this article, the INR deviations became prominent when the number of abnormal samples being tested therein was reduced to fewer than 20 which leads to keeping the population of the samples to at least 20. The paper of L. Poller et al. also discusses the usage of 20 high lyophilized INR plasmas and 7 normal lyophilized plasmas to calibrate the INR. Further, in this article, a deviation of +/−10% from means was discussed as being an acceptable limit of INR deviation. Further still, this article discusses the evaluation techniques of taking into account the prothrombin ratio (PR) and the mean normal prothrombin time (MNPT), i.e., the geometric mean of normal plasma samples.
The discrepancies related to the use of the INR are further studied and described in the technical article of V. L. NG et al., entitled, “Highly Sensitive Thromboplastins Do Not Improve INR Precision,” published in Am. J. Clin. Pathol., 1998; 109, No. 3, 338-346 and herein incorporated by reference. In this article, the clinical significance of INR discordance is examined with the results being tabulated in Table 4 therein and which are analyzed to conclude that the level of discordance for paired values of individual specimens tested with different thromboplastins disadvantageously range from 17% to 29%.
U.S. Pat. No. 5,981,285 issued on Nov. 9, 1999 to Wallace E. Carroll et al., which discloses a “Method and Apparatus for Determining Anticoagulant Therapy Factors” provides an accurate method for taking into account varying prothrombin times (PT) caused by different sensitivities of various thromboplastin formed from rabbit brain, bovine brain or other sources used for anticoagulant therapy. This method does not suffer from the relatively high (10%) error sometimes occurring because of the use of the INR and ISI parameters with the exponents used in their determination.
The lack of existing methods to provide reliable results for physicians to utilize in treatment of patients has been discussed, including in a paper by Davis, Kent D., Danielson, Constance F. M., May, Lawrence S., and Han, Zi-Qin, “Use of Different Thromboplastin Reagents Causes Greater Variability in International Normalized Ratio Results Than Prolonged Room Temperature Storage of Specimens,” Archives of Pathol. and Lab. Medicine, November 1998. The authors observed that a change in the thromboplastin reagent can result in statistically and clinically significant differences in the INR.
Considering the current methods for determining anticoagulant therapy factors, there are numerous opportunities for error. For example, it has been reported that patient deaths have occurred at St. Agnes Hospital in Philadelphia, Pa. There the problem did not appear to be the thromboplastin reagent, but rather, was apparently due to a failure to enter the correct ISI in the instrument, used to carry out the prothrombin times when the reagent was changed. This resulted in the incorrect INR's being reported. Doses of coumadin were given to already overanticoagulated patients based on the faulty INR error, and it is apparent that patient deaths were caused by excessive bleeding due to coumadin overdoses.
But even in addition to errors where a value is not input correctly, the known methods for determining anticoagulant therapy factors still may be prone to errors, even when the procedure is carried out in accordance with the reagent manufacturer's ISI data. One can see this in that current methods have reported that reagents used to calculate prothrombin times, may, for healthy (i.e., presumed normal) subjects, give rise to results ranging from 9.7 to 12.3 seconds at the 95th % reference interval for a particular reagent, and 10.6 to 12.4 for another. The wide ranges for normal patients illustrates the mean normal prothrombin time differences. When the manufacturer reference data ranges are considered, if indeed 20 presumed normal patients' data may be reported within a broad range, then there is the potential for introduction of this range into the current anticoagulation therapy factor determinations, since they rely on the data for 20 presumed normal patients. Considering the reagent manufacturer expected ranges for expected normal prothrombin times, INR units may vary up to 30%. This error is apparently what physicians must work with when treating patients. A way to remove the potential for this type of error is needed.
This invention relates to the inventions disclosed in U.S. Pat. Nos. 3,905,769 ('769) of Sep. 16, 1975; 5,197,017 ('017) dated Mar. 23, 1993; and 5,502,651 ('651) dated Mar. 26, 1996, all issued to Wallace E. Carroll and R. David Jackson, and all of which are incorporated herein by reference. The present invention provides apparatus and methods for monitoring anticoagulant therapy and conditions relating to prothrombotic abnormalities, such as, for example, a hypercoagulation condition.
The blood and blood components of individual beings are often measured to evaluate levels of particular substances, including exogenous as well as endogenous molecules and compounds. Blood may be evaluated for blood abnormalities which relate to clotting (or the inability to clot). For example, blood and blood components may be measured in conjunction with blood clotting evaluations and analyses for determining treatment levels for the administration of anticoagulants, such as oral anticoagulant therapy, referred to above. For example, patients being treated or cared for, for certain heart or blood disorders, may receive blood thinning agents as a course of therapy. Some individuals exhibit what is clinically average or normal coagulation, whereas in others, the ability of their blood to coagulate may be referred to as a hypercoagulable condition, where clotting of the blood is considered to occur more rapidly than that of the clinically average individual. Conversely, another clinical condition is hypocoagulability, where the blood clotting requires additional time than that of the clinically average individual.
Hypercoagulability is a state of a person which involves an increased clotting function of the blood relative to what is considered to be presumed normal coagulation. Individuals presenting with hypercoagulable states have the potential to develop arterial or venous thromboembolism (VTE). Components considered to be responsible for effecting clot formation include fibrinogen, Factor VIII, von Willebrand Factor (VWF) and Factor XIII. Factor VIII is considered not to participate in the Prothrombin Time (time to the first clot formation). VWF concerns platelets. Platelets may be removed by centrifugation, such as where a plasma sample of the blood components is separated from the platelets. It is considered that thrombin is the component in blood responsible for the clotting to occur. The presence' of too much free thrombin is considered to be a condition hypercoagulability, and the lack or inactivity of thrombin results in the condition of hypocoagulability. Both, hypercoagulability and hypocoagulability, are conditions or states which may be brought about by various pathological conditions.
It is clinically important to know the state of an individual's clotting function, that is, in particular, where the individual is hypercoagulable, since treatments may be altered to account for this condition. In many cases, the presence of, or suspicion of, hypercoagulability is used to drive further treatments or testing of a patient, which may be very costly. Currently, the determination of hypercoagulability for an individual may take as long as about thirty minutes. See e.g., J. L. Curnow, et al., J. Thrombosis and Haemostasis, 5, 528-534 (2006). During the time it takes to make the determination, many things may happen, and, in many instances, the administration of treatment agents to an individual may be required prior to the time of completion of the hypercoagulability determination.
A prior method is the von Clauss fibrinogen method, which is based on the consideration that the greater the amount of fibrin present, the less the time for the thrombin clot time. However, the prior methods for determining hypercoagulability as a state of a person's blood, including the von Clauss method, have generally involved lengthy durations. Another example of a prior reported attempt to clinically determine hypercoagulable states is discussed in “Reduced fibrinolysis and increased fibrin generation can be detected in hypercoagulable patients using the overall haemostatic potential assay,” J. L. Curnow, et al., J. Thrombosis and Haemostasis, 5, 528-534 (2006). However, the Curnow determination proceeded over a course of minutes, where maximum optical density (OD) was not attained until after about 50 minutes, and where the first detection response appears to be after 5 minutes. (Id. at 530) Given the immediacy with which, in many situations, hypercoagulation must be resolved, or treatment option's for a patient considered, the time duration of thirty minutes, provided by prior methods, or even on the order of magnitude of minutes for prior determinations, may place many patients at a disadvantage or at an increased risk, including any of the risks associated with the condition of hypercoagulability. Often, further costly tests are given to patients who present with symptoms that may be clinically associated with hypercoagulable conditions. In some cases, these tests are unnecessary, adding further costs to patient care, and subjecting the patient to further waiting or discomfort. A need exists for a method and apparatus that may facilitate a determination of a hypercoagulable condition with speed and accuracy, and in an economical manner.
Methods and apparatus useful for processing coagulation studies, and other chemistry procedures involving blood and blood components. The apparatus and methods may be used to determine anticoagulant therapy factors which are designated herein, in particular, to determine new Anticoagulant Therapy Factors (nATF's) which preferably may replace International Normalized Ratio (INR) in anticoagulation therapy management. Previously, anticoagulation therapy involved the use of International Normalized Ratios (INR's). The International Normalized Ratio (INR) was utilized in order to arrive at an anticoagulant therapy factor (ATF). The INR based ATF was dependent on the prothrombin time (PT), the prothrombin ratio (PR), a fibrinogen transformation rate (FTR), and a maximum acceleration point (MAP) having an associated time to maximum acceleration (TMA).
Methods and apparatus are disclosed for determining a new anticoagulant therapy factor (nATF) for monitoring oral anticoagulant therapy to help prevent excessive bleeding or deleterious blood clots that might otherwise occur before, during or after surgery. In one embodiment, a new anticoagulant therapy factor (nATF) is based upon a determination of the fibrinogen transformation rate (FTR) which, in turn, is dependent on a maximum acceleration point (MAP) for fibrinogen (FBG) conversion. The nATF quantity is also based upon the time to maximum acceleration from the time of reagent injection (TX) into a plasma sample, but does not require the difficulty of obtaining prior art International Normalized Ratio (INR) and International Sensitivity Index (ISI) parameters. The International Normalized Ratio (INR) was created to relate all species' clotting material to human clotting material, and nATF can replace INR in anticoagulant therapy management.
In accordance with other embodiments, methods and apparatus are provided for determining an anticoagulation therapy factor, which do not require the use of a mean normal prothrombin time (MNPT) and ISI data. In other words, the need to obtain and calculate the prothrombin time of 20 presumed normal patients, is not required to determine an anticoagulant therapy factor.
In accordance with the present invention, there is provided apparatus and methods for carrying out coagulation studies and other chemical procedures and analyses.
According to embodiments of the invention, there is provided a method for determining a hypercoagulability condition in an individual. The method may include monitoring a sample of an individual's blood and/or blood components for changes associated with fibrinogen to fibrin formation.
An apparatus for determining a hypercoagulable condition involving monitoring of a sample of an individual's blood and/or blood components for changes associated with fibrinogen to fibrin formation also is provided by the invention.
The method and apparatus may be used for determining the presence of a hypercoagulable state of a patient in an effective and efficient manner. According to preferred embodiments of the invention, the method and apparatus may facilitate making a determination of hypercoagulability within seconds.
Embodiments of the method and apparatus may include regulating further screening, testing and/or therapy programs by evaluating for a potential hypercoagulable state of a patient. A further object and advantage of the invention is to prevent the ordering of extensive laboratory tests. Preventing unnecessary testing has a benefit of convenience and comfort to a patient, as well as the economic value and benefit of costs savings to patients and healthcare insurers.
According to embodiments of the invention relating to the determination of a hypercoagulable condition, the method and apparatus further may include monitoring a voltage signal of a spectrophotometer to determine fibrinogen to fibrin formation in conjunction with or association with the readings taken of the sample to evaluate the passage and/or absorption of particular wavelengths or a spectral range.
According to preferred embodiments, the method and apparatus may be used to determine hypercoagulable conditions in an individual which are due to one or more or numerous conditions causing the condition. In other words, preferred embodiments may determine the presence of a hypercoagulable condition occurring from a different cause.
The methods and apparatus of the present invention are designed to provide an effective way to detect a hypercoagulability condition in a human, and within times of as short as about thirty seconds, as opposed to prior determinations which were on the order of thirty minutes.
Referring to the drawings, wherein the same reference numbers indicate the same elements throughout, there is shown in
Wiper 24 is illustrated placed at a position to give a suitable output and is not varied during the running of the test. The output between line 18 and wiper 24 is delivered to an A/D converter 26 and digital recorder 28. As is known, the A/D converter 26 and the digital recorder 28 may be combined into one piece of equipment and may, for example, be a device sold commercially by National Instrument of Austin, Tex. as their type Lab-PC+. The signal across variable resistor 22 is an analog signal and hence the portion of the signal between leads 18 and wiper 24, which is applied to the A/D converter 26 and digital recorder 28, is also analog. A computer 30 is connected to the output of the A/D converter 26, is preferably IBM compatible, and is programmed in a manner described hereinafter.
For example, preferably, the detector cell 10 is positioned adjacent an opposite wall of the sample container 8, and the emitter light source 4 positioned adjacent on opposite wall, so the light 6 emitted from the light source 4 passes through the container 8. The light source 4 is preferably selected to produce light 6 which can be absorbed by one or more components which are to be measured.
The apparatus can be used to Carry out coagulation studies in accordance with the invention. In accordance with a preferred embodiment of the present invention, the light source 4 may, for example, comprise a light emitting diode (LED) emitting a predetermined wavelength, such as for example, a wavelength of 660 nm, and the detector cell 10 may, for example, comprise a silicon photovoltaic cell detector. Optionally, though not shown, a bar code reader may also be provided to read bar code labels placed on the sample container 8. The bar code reader may produce a signal which can be read by the computer 30 to associate a set of data with a particular sample container 8.
To carry out a coagulation study on blood plasma, the citrated blood is separated from the red blood cell component of the blood. Conventional methods of separation, which include centrifugation, may be employed. Also, the use of a container device such as that disclosed in our issued U.S. Pat. No. 6,706,536, may also be used, and the method disclosed therein for reading the plasma volume relative to the sample volume may also be employed.
Illustrative of an apparatus and method according to one embodiment is a coagulation study which can be carried out therewith. A reagent, such as, for example, Thromboplastin-Calcium (Tp-Ca), is added to the plasma sample which is maintained at about 37° C. by any suitable temperature control device, such as a heated sleeve or compartment (not shown). The reagent addition is done by dispensing an appropriate amount of the reagent into the plasma portion of the blood. The plasma portion may be obtained by any suitable separation technique, such as for example, centrifugation. In one embodiment illustrated herein, the container 8 is vented when reagent is added. The reagent for example, may comprise thromboplastin, which is added in an amount equal to twice the volume of the plasma. The reagent is mixed with the plasma. It is preferable to minimize air bubbles so as not to interfere with the results. The plasma sample to which the reagent has been added is heated to maintain a 37° C. temperature, which, for example, may be done by placing the container holding the plasma and reagent in a heating chamber (not shown).
Readings are taken of the optical activity of the components in the sample container 8.
Reaction kinematics may be studied by observing changes in the optical density of the plasma layer. For example, an amount of reagent, such as Thromboplastin-Calcium (Tp-Ca), may be added to the plasma sample in the container. The plasma sample in the container may comprise a known amount of volume. Alternately, the plasma volume may be ascertained through the method and apparatus described in our U.S. Pat. No. 6,706,536. A controlled amount of Tp-Ca reagent is added to the plasma sample. The amount of reagent added corresponds to the amount of plasma volume. The detector cell 10 and emitter light source 4 are preferably positioned so the absorbance of the plasma sample may be read, including when the reagent is added and the sample volume is thereby increased.
With the detection elements, such as the cell 10 and emitter 4, positioned to read the plasma sample and the reagents added thereto, the reaction analysis of the extended prothrombin time curve can be followed.
The coagulation study of the type described above is used to ascertain the results shown in the graph plotted on
Prior patents for obtaining an anticoagulant therapy factor (ATF) relied on the International Normalized Ratio (INR) system which was derived in order to improve the consistency of results from one laboratory to another. The INR system utilized the calculation of INR from the equation:
INR=(PTpatient/PTgeometric mean)ISI
wherein the PTpatient is the prothrombin time (PT) as an absolute value in seconds for a patient, PTgeometric mean is the mean, a presumed number of normal patients. The International Sensitivity Index (ISI) is an equalizing number which a reagent manufacturer of thromboplastin specifies. The ISI is a value which is obtained through calibration against a World Health Organization primary reference thromboplastin standard. Local ISI (LSI) values have also been used to provide a further refinement of the manufacturer-assigned ISI of the referenced thromboplastin in order to provide local calibration of the ISI value.
For illustration, the present invention can be employed for accurate determination of a new Anticoagulant Therapy Factor (nATF) from a human blood sample, for use during the monitoring of oral anticoagulant therapy, without the need for an ISI or LSI value, and without the need for an INR value. As is known in the art, blood clotting Factors I, II, V, VII, VIII, IX and X are associated with platelets (Bounameaux, 1957); and, among these, Factors II, VII, IX and X are less firmly attached, since they are readily removed from the platelets by washing (Betterle, Fabris et al, 1977). The role of these platelet-involved clotting factors in blood coagulation is not, however, defined. The present invention provides a method and apparatus for a new Anticoagulant Therapy Factor (nATF) which may be used for anticoagulant therapy monitoring without the need for INR.
The International Normalized Ratio (INR) is previously discussed in already incorporated reference technical articles entitled “PTs, PRs, ISIs and INRs: A Primer on Prothrombin Time Reporting Part I and II respectively,” published in November, 1993 and December, 1993 issues of Clinical Hemostasis Review. The illustrative example of an analysis which is carried out employing the present invention relies upon the maximum acceleration point (MAP) at which fibrinogen conversion achieves a maximum and from there decelerates, the time to reach the MAP (TX), and the mean normal time to MAP (MNTX), and a fibrinogen transformation rate (FTR), that is, the thrombin activity in which fibrinogen (FBG) is converted to fibrin to cause clotting in blood plasma.
More particularly, during the clotting steps used to determine the clotting process of a plasma specimen of a patient under observation, a thromboplastin (Tp) activates factor VII which, activates factor X, which, in turn, under catalytic action of factor V, activates factor II (sometimes referred to as prothrombin) to cause factor IIa (sometimes referred to as thrombin) that converts fibrinogen (FBG) to fibrin with resultant turbidity activity which is measured, in a manner as to be described hereinafter, when the reaction is undergoing simulated zero-order kinetics.
From the above, it should be noted that the thromboplastin (Tp) does not take part in the reaction where factor IIa (thrombin) converts fibrinogen (FBG) to fibrin which is deterministic of the clotting of the plasma of the patient under consideration. The thromboplastin (Tp) only acts to activate factor VII to start the whole cascade rolling. Note also that differing thromboplastins (Tps) have differing rates of effect on factor VII, so the rates of enzyme factor reactions up to II-IIa (the PT) will vary.
Therefore, the prothrombin times (PTs) vary with the different thromboplastins (Tps) which may have been a factor that mislead authorities to the need of taking into account the International Normalized Ratio (INR) and the International Sensitivity Index (ISI) to compensate for the use of different types of thromboplastins (Tps) during the monitoring of oral anticoagulant therapy. It is further noted, that thromboplastins (Tps) have nothing directly to do with factor IIa converting fibrinogen (FBG) to fibrin, so it does not matter which thromboplastin is used when the fibrinogen transformation is a primary factor.
The thromboplastin (Tp) is needed therefore only to start the reactions that give factor IIa. Once the factor IIa is obtained, fibrinogen (FBG) to fibrin conversion goes on its own independent of the thromboplastin (Tp) used.
In one embodiment, the present method and apparatus has use, for example, in coagulation studies where fibrinogen (FBG) standard solutions and a control solution are employed, wherein the fibrinogen standard solutions act as dormant references to which solutions analyzed with the present invention are compared, whereas the control solution acts as a reagent that is used to control a reaction. The fibrinogen standards include both high and low solutions, whereas the control solution is particularly used to control clotting times and fibrinogens of blood samples. It is only necessary to use fibrinogen standards when PT-derived fibrinogens (FBG's) are determined. In connection with other embodiments of the invention, fibrinogen (FBG) standards are not necessary for the INR determination (such as for example INRz described herein).
Another embodiment provides a method and apparatus for determining an anticoagulation therapy factor which does not require the use of fibrinogen standard solutions. In this embodiment, the apparatus and method may be carried out without the need to ascertain the mean normal prothrombin time (MNPT) of 20 presumed normal patients.
Where a fibrinogen standard solution is utilized, a fibrinogen (FBG) solution of about 10 g/l may be prepared from a cryoprecipitate. The cryoprecipitate may be prepared by freezing plasma, letting the plasma thaw in a refrigerator and then, as known in the art, expressing off the plasma so as to leave behind the residue cryoprecipitate. The gathered cryoprecipitate should contain a substantial amount of both desired fibrinogen (FBG) and factor VIII (antihemophilic globulin), along with other elements that are not of particular concern to the present invention. The 10 g/l fibrinogen (FBG) solution, after further treatment, serves as the source for the high fibrinogen (FBG) standard. A 0.5 g/l fibrinogen (FBG) solution may then be prepared by a 1:20 (10 g/l/20=0.5 g/l) dilution of some of the gathered cryoprecipitate to which may be added an Owren's Veronal Buffer (pH 7.35) (known in the art) or normal saline solution and which, after further treatment, may serve as a source of the low fibrinogen (FBG) standard.
The fibrinogen standard can be created by adding fibrinogen to normal plasma in an empty container. Preferably, the fibrinogen standard is formed from a 1:1 fibrinogen to normal plasma solution. For example, 0.5 ml of fibrinogen and 0.5 ml of plasma can be added together in an empty container. Thromboplastin calcium is then added to the fibrinogen standard. Preferably, twice the amount by volume of thromboplastin is added into the container per volume amount of fibrinogen standard which is present in the container. The reaction is watched with the apparatus 10.
Then, 1 ml of each of the high (10 g/l) and low (0.5 g/l) sources of the fibrinogen standards may be added to 1 ml of normal human plasma (so the cryoprecipitate plasma solution can clot). Through analysis, high and low fibrinogen (FBG) standards are obtained. Preferably, a chemical method to determine fibrinogen (FBG) is used, such as, the Ware method to clot, collect and wash the fibrin clot and the Ratnoff method to dissolve the clot and measure the fibrinogen (FBG) by its tyrosine content. The Ware method is used to obtain the clot and generally involves collecting blood using citrate, oxalate or disodium ethylenediaminetetraacetate as anticoagulant, typically adding 1.0 ml to about 30 ml 0.85% or 0.90% sodium chloride (NaCl) in a flask containing 1 ml M/5 phosphate buffer and 0.5 ml 1% calcium chloride CaCl2, and then adding 0.2 ml (100 units) of a thrombin solution. Preferably, the solution is mixed and allowed to stand at room temperature for fifteen minutes, the fibrin forming in less than one minute forming a solid gel if the fibrinogen concentration is normal. A glass rod may be introduced into the solution and the clot wound around the rod. See Richard J. Henry, M.D., et al., Clinical Chemistry: Principals and Techniques (2nd Edition) 1974, Harper and Row, pp. 458-459, the disclosure of which is incorporated herein by reference. Once the clot is obtained, preferably the Ratnoff method may be utilized to dissolve the clot and measure the fibrinogen (FBG) by its tyrosine content. See “A New Method for the Determination of Fibrinogen in Small Samples of Plasma”, Oscar D. Ratnoff, M. D. et al., J. Lab. Clin. Med., 1951: V. 37 pp. 316-320, the complete disclosure of which is incorporated herein by reference. The Ratnoff method relies on the optical density of the developed color being proportional to the concentration of fibrinogen or tyrosine and sets forth a calibration curve for determining the relationship between optical density and concentration of fibrinogen. The addition of a fibrinogen standard preferably is added to the plasma sample based on the volume of the plasma.
As is known, the addition of the reagent Thromboplastin C serves as a coagulant to cause clotting to occur within a sample of citrated blood under test which may be contained in a container 8. As clotting occurs, the A/D converter 26 of
The study which measures the concentration of the fibrinogen (FBG) in the plasma that contributes to the clotting of the plasma and uses an instrument, such as, for example, the potentiophotometer apparatus illustrated in
As seen in
An anticoagulant therapy factor (nATF) is determined. The optical density of a quantity c1 directly corresponds to a specified minimum amount of fibrinogen (FBG) that must be present for a measuring system, such as the circuit arrangement of
Considering the clotting curve of
As shown in
The deceleration of fibrinogen (FBG) to fibrin conversion continues until a quantity cEOT is reached at a time tEOT. The time tEOT is the point where the deceleration of the fibrinogen (FBG) to fibrin conversion corresponds to a value which is less than the required amount of fibrinogen (FBG) that was present in order to start the fibrinogen (FBG) to fibrin conversion process. Thus, because the desired fibrinogen (FBG) to fibrin conversion is no longer in existence, the time tEOT represents the ending point of the fibrinogen (FBG) to fibrin conversion in accordance with the coagulation study exemplified herein, which may be referred to as the end of the test (EOT). The fibrinogen (FBG) to fibrin conversion has a starting point of t1 and an ending point of tEOT. The differential of these times, t1 and tEOT, define a second delta (IUT).
The “clot slope” method that gathers typical data as shown in
One embodiment of the method and apparatus is illustrated in accordance with the clotting curve shown in
nATFz=XR
(2−nFTR) (1)
This embodiment utilizes a zero order line (L) to obtain a first delta, in particular IUXz, which is a first differential taken along the simulated zero order kinetic line (L), and preferably along the segment between the start of the simulated zero order kinetic (T2S) to the last highest absorbance value (T2) (i.e., preferably, the last highest absorbance value of a simulated zero order kinetic). As previously discussed, the acceleration of the fibrinogen conversion proceeds from a first time, here time (T1) and continues, eventually reaching a time where the last highest delta absorbance value or maximum acceleration point (T2) having a corresponding quantity cT2 is reached. The values for “T” correspond with times, and the values for “c” correspond with quantity, which may be measured in instrument units based on optical density readings (also referred to as optical density or o.d.). The time T2, as well as the quantity cT2, is the point of maximum acceleration of the fibrinogen (FBG) to fibrin conversion and is also the point where deceleration of fibrinogen (FBG) to fibrin conversion begins. In this embodiment, IUXz is the change in optical density preferably from the beginning of the at the time T2S at which the simulated zero order kinetic begins to the optical density at time T2 which is the maximum acceleration point or the last highest delta absorbance value of a simulated zero order kinetic.
The (IUXz) represents the fibrinogen (FBG) converted between time T2S and T2. The (IUTz) represents fibrinogen converted from the time T2S to the end of the test or T3.
The maximum acceleration ratio (XR) for this embodiment is calculated to arrive at the new alternate anticoagulation therapy factor (nATFz). The maximum acceleration ratio (XR) is defined as the time to maximum acceleration from reagent injection (TX) divided by the mean normal TX value of a number of presumed normal specimens (MNTX). For example, the mean normal TX value may be derived based on the value of 20 or more presumed normal specimens. The maximum acceleration ratio (XR) may be expressed through the following formula:
XR=TX/MNTX (2)
The clotting curve of
nATFz=XR
(2−nFTR) (3)
with FTR being IUXz/IUTz.
The preferred IBM-compatible computer 30 of
(b) the computer 30 may be programmed to look for a digital quantity representative of a critical quantity c1, and when such occurs, record its instant time T1. (The time span between To and T1 is the prothrombin time (PT), and has an normal duration of about 12 seconds, but may be greater than 30 seconds);
nFTRz=IUXz/IUTz (4)
XR=TX/MNTX (2)
The MNTX value may be ascertained and stored in the computer for reference.
nATFz or INRz=XR(2−nFTR) (3)
where, in the exponent, the value 2 is the logarithm of the total fibrinogen, which, as expressed in terms of the optical density, is 100% transmittance, the log of 100 being 2.
The new anticoagulation therapy factor (nATFz) does not require an ISI value, as was previously used to determine anticoagulation therapy factors. The new anticoagulation therapy factor (nATFz) uses for its ascertainment the values extracted from the clotting curve (see
The apparatus and method for obtaining a new anticoagulant therapy factor, (nATFz), may be accomplished without encountering the complications involved with obtaining the prior art quantities International Normalized Ratio (INR) and International Sensitivity Index (ISI).
The new anticoagulant therapy factor (nATFz or ATF) preferably is a replacement for the International Normalized Ratio (INR), hence it may be referred to as INRz. Existing medical literature, instrumentation, and methodologies are closely linked to the International Normalized Ratio (INR). The nATFz was compared for correlation with the INR by comparative testing, to INR quantities, even with the understanding that the INR determination may have an error of about ten (10) % which needs to be taken into account to explain certain inconsistencies.
Table 2, below, includes anticoagulant therapy factors obtained from patients at two different hospitals. The ATFz values were obtained, with GATFz representing one geographic location where patients were located and MATFz being another location. The ATFz was obtained as the new anticoagulant therapy factor, and as illustrated in Tables 4 and 5, below, compares favorably to results obtained for INR determinations.
Another alternate embodiment for determining a new anticoagulant therapy factor (ATFt) is provided. The alternate embodiment for determining ATFt eliminates the need for determining a mean normal prothrombin time (MNPT) (or MNXT) and ISI, saving considerable time and costs, and removing potential sources of error, as the MNPT (the expected value of MNPT's depending on the varying 20 presumed normals population) and ISI (generally provided by the manufacturer of the reagent—such as, for example, the thromboplastin, etc.) are not required for the determination of the ATFt. An alternate embodiment for determining ATFt is illustrated in accordance with the clotting curve shown in
nATFt=Value 1*Value 2 (4)
The alternate embodiment utilizes the zero order line (L) to obtain a first delta, in particular IUXz, which is a first differential taken along the simulated zero order kinetic line (L), and preferably along the segment between the start of the simulated zero order kinetic (T2S) to the last highest absorbance value (T2) (i.e., preferably, the last highest absorbance value of a simulated zero order kinetic). As previously discussed, the acceleration of the fibrinogen conversion proceeds from a first time, here time (T1) and continues, eventually reaching a time where the last highest delta absorbance value or maximum acceleration point (T2) having a corresponding quantity cT2 is reached. The time T2, as well as the quantity cT2, is the point of maximum acceleration of the fibrinogen (FBG) to fibrin conversion and also is the point where deceleration of fibrinogen (FBG) to fibrin conversion begins. As illustrated on the clotting chart in
The (IUXz) represents the fibrinogen (FBG) converted between time T2S and T2. The (IUTz) represents fibrinogen converted from the time T2S to the end of the test or T3.
The first value V1 corresponds to the value determined for the theoretical end of test (TEOT), which, as illustrated in the clotting curve representation in
V1=TEOT=ZTM/IUXz*IUTz (5)
where ZTM is the time between Tmap (i.e., T2 shown on
ZTM=T
2
−T
2
S (6)
A second value, V2, also referred to as a multiplier, is determined based on the value T2S. In the expression for the ATFt, the second value, V2, may be obtained by taking the value of the time (T2S) corresponding to a second time (t2) or the maximum acceleration point (Tmap), and scaling this value. It is illustrated in this embodiment that the multiplier is derived from the natural log base “e”, which is 2.71828, scaled to provide an appropriately decimaled value. The scaling number used in the example set forth for this embodiment is 100. The second value (V2) may be expressed by the following relationship:
V2=T2S/100e (7)
where T2S is the maximum acceleration point for the sample, and 100e is the value 100 multiplied by the natural log base “e” (2.71828) or 271.828. The new anticoagulation therapy factor according to the alternate embodiment may be expressed as follows:
nATFt=[(T2−T2S)/IUXz*IUTz]*[T2S/M] (8)
where M represents a multiplier. In the present example, the multiplier M, corresponds to the value 271.828 (which is 100 times the natural log base “e”).
An alternate embodiment of an anticoagulant therapy factor, ATFt2, which does not require the ascertainment of a mean normal prothrombin time (MNPT) or use of an ISI value, is derived using the expression (5), wherein the IUTz is replaced by the expression (IUTz+IULz). In this alternate expression the method is carried out to ascertain the values for Value1 and Value2, in the manner described herein, with Value 1 being obtained through expression (5.1):
V1=TEOT=ZTM/IUXz*(IUTz+IULz) (5.1)
where IULz is time to convert the lag phase fibrinogen (FBG) measured along the ordinate between T1 and T2S. In expression 5.1, the theoretical end of test (TEOT) is set to include the time to convert the fibrinogen (FBG) in the lag phase of the clotting curve.
nATFt2=[(T2−T2S)/IUXz*(IUTz+IULz)]*[T2S/M] (8.1)
The apparatus may comprise a computer which is programmed to record, store and process data. The zero order rate may be determined by ascertaining data from analyzing the sample, and optical density properties. One example of how this may be accomplished is using two arrays, a data array and a sub array. A data array may be ascertained by collecting data over a time interval. In one embodiment, for example, the data array may comprise a sequential list of optical densities, taken of a sample by an optical analytical instrument, such as, for example, a spectrophotometer, for a frequency of time. In the example, the frequency of sample data is taken every 100th of a second. In this embodiment, a computer is programmed to record the optical density of the sample, every 100th of a second. Two values, NOW and THEN, for the data array are provided for ascertaining the Prothrombin Time (PT) (which is the time point T1), maximum acceleration point (MAP), and end of test point (EOT). Two time definitions may be specified, one being the interval between NOW and THEN on the clotting curve, which may be 2.72 seconds ( 272/100th of a second), the second being the size of the filter used for signal averaging. NOW is the sum of the last 20 optical densities and THEN is the sum of the 10 prior data points 2.72 seconds prior to NOW. A graphical illustration is provided in
The sub array may be defined as a sequential list of delta absorbance units. This may begin at T1, the prothrombin time (PT), and continue until the last highest delta absorbance (delta A) has been detected, then continues an additional five (5) seconds to insure the last delta A has been found. A determination of T2S may be accomplished by locating within the sub array, the first occurrence of when the sub array delta value is greater than or equal to 80% of the highest delta absorbance units. The first derivative is ascertained by computing the difference between (NOW) and (THEN). The PT is ascertained by determining the point prior to the positive difference between AVERAGE(THEN) and AVERAGE(NOW) for a period of 2.72 seconds or 272 ticks. The MAP is the point where the last highest difference between SUM(THEN) and SUM(NOW) has occurred. The computer may be programmed to store this delta A value in the sub array. The EOT may be ascertained by determining the point prior to where the difference between SUM(THEN) and SUM(NOW) is less than one.
Table 2 illustrates examples of samples, identified by ID numbers, along with corresponding data which compares the ATF values obtained for an ATF determined through the prior method, using ISI and INR values (represented as ATFa), an ATF determined through the use of a zero order kinetic reaction using the MNTX (nATFz), and an ATF determined without using the MNXT or ISI (nATFt). The data in table 2 represents universal laboratory data from combined locations for the patients listed. The data is based on analysis of absorbance data, storage of the data by the computer, such as, for example, with a storage device, like a hard drive, and retrieving the data and processing the data. The data, in the example represented in Table 2 was processed using the definitions and NOW and THEN intervals.
A statistical comparison of the above data from Table 2 is presented below in Tables 4 and 5. The value AINR in Table 2 represents the INR value obtained pursuant to the World Health Organization (WHO), using expressions (A) and (B) above. GINR and MINR correspond to INR values used to determine the comparison data set forth in Tables 4 and 5.
The determination of the new anticoagulant therapy factor (ATFt) may be carried out with a computer. According to one example, the gathering, storing, and manipulation of the data generally illustrated in
In accordance with one embodiment, the IBM-compatible computer 30 of
TEOT=ZTM/IUXz*IUTz (5)
M=100e (9)
VALUE 2=T2S/100e (7)
The data may be ascertained and stored in the computer for reference.
nATFt=VALUE 1*VALUE 2 (4)
The computer 30 may be used to manipulate and derive the quantities of expression (4) to determine a new anticoagulant therapy factor nATFt utilizing known programming routines and techniques. The data collected by a computer 30 may be used to manipulate and derive the new anticoagulant therapy factor (nATFt) of expression (4). Similarly, one skilled in the art, using known mathematical techniques may derive the theoretical end of test TEOT of expression (5) and the second value VALUE 2 of expression (7) which, in turn, are used to determine the new anticoagulant therapy (nATFt) of expression (4). In the nATFt determination, the determination is based on the patient's own sample, and does not rely on the determination of normal prothrombin times for the reagent used (e.g., thromboplastin, innovin or the like). With the nATFt, no longer does the accuracy of the quantities determined depend, in whole or part, on the number of specimens used, that is, the number of stable (or presumed stable) patients.
The new anticoagulation therapy factor (nATFt) does not require an ISI value, as was previously used to determine anticoagulation therapy factors. The new anticoagulation therapy factor (nATFt) uses for its ascertainment the values extracted from the clotting curve (see
It should now be appreciated that the present invention provides an apparatus and method for obtaining a new anticoagulant therapy factor (nATF) without encountering the complications involved with obtaining the prior art quantities International Normalized Ratio (INR) and International Sensitivity Index (ISI).
The new anticoagulant therapy factor (nATFt) preferably is a replacement for the International Normalized Ratio (INR). Existing medical literature, instrumentation, and methodologies are closely linked to the International Normalized Ratio (INR). The nATFt was compared for correlation with the INR by comparative testing, to INR quantities, even with the understanding that the INR determination may have an error of about +/−15%, at a 95% confidence interval, which needs to be taken into account to explain certain inconsistencies.
The hereinbefore description of the new anticoagulant therapy factor (nATFt) does correlate at least as well as, and preferably better than, studies carried out using the International Normalized Ratio (INR). For some comparisons, see the tables below, and in particular Table 4 and Table 5.
Table 3 (Part A) and Table 3 (Part B) provide corresponding data for a coagulation study. In Table 3 (Part A and B), the following references are used:
Comparative Results of nATFt's and nATFz's
Results between patients in two different geographic locations (i.e., two different hospitals) were compared for correlation with each other. This comparison is expressed in Table 4 below, and includes a comparison of INR values calculated by the WHO method for each respective location, with GInr representing one location for these traditionally WHO determined values, and MInr representing values based on data obtained at the other location. The values identified as ATFz and ATFt, such as, GATFt and MATFt, and GATFz and MATFz, represent anticoagulant therapy factors derived from the expressions (1) through (9) above.
The ATFa represents an anticoagulation therapy factor derived from our method and apparatus for the expression ATFa=XR(2−nFTR) wherein a maximum acceleration point is obtained, and nFTR=IUX/IUT, where IUX is the change in optical density from a time prior to the MAP time (t<MAP which is tMAP minus some time from MAP) to the optical density at a time after the MAP time (t>MAP which is tMAP plus some time from MAP); and wherein IUT=the change in optical density at the time t1 to the optical density measured at time tEOT, where time tEOT is the end of the test (EOT). The (IUX) represents the fibrinogen (FBG) for MAP (−a number of seconds) to MAP (+a number of seconds) (that is the fibrinogen (FBG) converted from t<MAP to t>MAP on
A comparison of combined location data is shown in Table 5, below. The sample size was 217.
Comparative results were also calculated for the ATFt which includes the lag phase fibrinogen, in accordance with the IULz, using the expression (5.1) for the TEOT value. Table 6 below provides the values for the ATFz, ATFt, and the ATFt2 (which is obtained from expression 5.1 using the IULz).
Table 7 represents a comparison of the data from Table 6.
Table 8 provides comparative data for the anticoagulant therapy factors, similar to Table 2, but using the ATFt2 method from expressions (4) and (5.1) for corresponding GINRt2 and MINRt2 values.
Table 9 provides comparative data for the ATFa, ATFz and ATFt2 and INR values calculated by the WHO method for each respective location, with GInr representing one location for these traditionally WHO determined values, and MInr representing values based on data obtained at the other location. The values identified as ATFz and ATFt2, such as, GATFt2 and MATFt2, and GATFz and MATFz, represent anticoagulant therapy factors derived from the expressions (1) through (9) above, inclusive of expressions (5.1) and (8.1).
Further comparative results are provided in Table 10 to illustrate the effect of prothrombin time (PT) on INR values. Table 10 provides a comparison based on data from Table 3, and provides INR values for PT's of PT=PT (under the heading “INR”), PT=PT+0.5 (under the heading “+0.5”), PT=PT+1.0 (under the heading “+1.0”), PT=PT+1.5 (under the heading “+1.5”), and PT=+2.0 (under the heading “+2.0”). The new anticoagulation therapy factor (ATFt2) was compared with the WHO method for determining ATF. The WHO method utilizes the mean prothrombin time of 20 presumed normal patients. The thromboplastin reagents list MNPT “expected ranges” listed in the accompanying thromboplastin-reagent (Tp) brochures. These brochures acknowledge that MNPT differences are inevitable because of variations in the 20 “normal donor” populations. Geometric, rather than arithmetic mean calculation limits MNPT variation somewhat, but simulated 0.5 second incremented increases over a total 2.5 second range, show ever-increasing INR differences notably at higher INR levels. To exemplify this, Table 10 shows these changes with Thromboplastin C Plus (which has a manufacturer's reported ISI=1.74 and MNPT=9.89 seconds) in POTENS+.
Since the in-house determined MNPT would continue with that Tp lot, intralaboratory results would be relatively unaffected. However, between laboratory INR agreements, or interlab results, are compromised. As a denominator, considering the expression used to derive the MNPT, such as expression (B), above, MNPT is, of course, less problematic for INRs than the exponent, ISI. Comparative results, showing interlab results, are provided in Table 11. ATFt is seen to be numerically equal to WHO/INRs determined in both analytical instruments, namely, the MDA-Electra 9000C and the POTENS+. Identical computer bits derived in POTENS+from the absorbances creating the thrombin-fibrinogen-fibrin clotting curve are used for the POTENS+WHO/INR and ATFt (NO ISI, NO MNPT) determinations. MNPT is, of course, still necessary for the WHO method. For ATFt, Zero Order Kinetics Line's slope is extended in both directions to intersect with the Tp-plasma baseline and the absorbance at total fibrin formation. The sum of this interval and the time from the Tp injection to the beginning of Zero Order Kinetics (T2S) is Value 1. Value 2 is T2S/100e. “e” is the Natural Logarithm, base 2.71828. ATFt=(Value 1)*(Value 2), in accordance with expression (4) herein (and the expression (8.1) for ATFt2).
Table 11 provides statistical comparisons for results obtained using two POTENS+coagulometers (one designated as GINR and another designated as MINR), and using a Bio Merieux MDA-180 coagulometer (designated as AINR). The POTENS+, WHO/INRs, INRzs, and ATFts and the MDA-180 (AINR) WHO/INRs are compared. Statistical data and Bland-Altman plot data demonstrate that the new anticoagulant therapy factor ATFt may replace WHO/INR and provide results which are within the parameters of traditional therapeutic or reference ranges.
The linear regression analysis expression y=mx+b, when solved for the slope, m, is expressed as (y−b)/x. This is biased, so the expression is y/x is when b is equal to zero. The comparison in Table 11, above, provides comparative data for mean y (mY) and mean x (mX) values, including the slope mY/mX. The use of mY/mX is used to provide comparative results.
In another embodiment, an article may be provided to derive an anticoagulant therapy factor (ATF). The article may comprise stored instructions on a storage media which can be read and processed with a processor. For example, the computer may be provided with a stored set of instructions, or chip, which is programmed to determine a new ATF for the spectral data obtained from the coagulation activity of a sample. For example, the computer chip may be preprogrammed with a set of instructions for cooperating with the output of a photodetection device, such as, the device shown and described in
According to alternate embodiments of the invention, the method and apparatus may involve the detection of additional prothrombotic abnormalities (or disorders) in a patient. According to preferred embodiments of the invention, methods and apparatus for conducting a determination relating to a hypercoagulation condition are provided. According to preferred embodiments of the invention, the prothrombotic abnormality evaluated is hypercoagulability. In other words, the hypercoagulable condition in an individual may be evaluated in a relatively short duration of time. The method and apparatus include analyzing a blood sample as a clotting activator, such as thromboplastin, is added, to track the change in the optical density corresponding to a blood component, such as fibrin formation based on the fibrinogen content of the blood sample. The absorbance curve develops when optically-clear fibrinogen is converted into turbid fibrin after the clotting agent. Preferred embodiments of the method and apparatus evaluate fibrinogen conversion activity.
The method involves taking readings and using the readings, such as, for example, by recording the readings over a particular time interval or duration. The absorbance value (which may be measured in instrument units) is plotted against time (which may be measured in seconds or fractions of seconds). The method involves the collection and processing of the data. The data may be represented in a clotting curve plot. A plot is formed which has a slope where the maximum acceleration of the rate of conversion of fibrinogen to fibrin occurs. According to preferred embodiments, the method and apparatus utilize the tangent of the slope to derive a zero order kinetic. According to preferred embodiments, a portion of the zero order slope line (such as the zero order line L shown in
Referring to
The derivation of a hypercoagulability indicator may be carried out by evaluating trigonometric values associated with the slope. According to a preferred embodiment of the method, an angle may be derived as an indicator of potential prothrombotic abnormality.
tangent θ°=Tm/IUT (10)
where Tm=TEOT−T2S, and wherein IUT=cT2S−cEOT.
Though the actual absorbance values themselves would not be less than zero, it is conceivable that the line representing t=T2S may be extended below the abscissa (the line t=0), and the additive absolute value of c determined (e.g., where c=a negative ordinate) adding the absolute value of the ordinate C1=|−X| with the value CT2S. The value of the sum of C1 and CT2S may be determined to yield a trigonometric function value, an adjacent leg (AL). The opposite leg (OL) may be determined by T(CI/L)−T2S, where T(CI/L) is the time where L crosses (i.e., intersects with) the line c=C1. According to preferred embodiments, the determination of a hypercoagulable condition may be made using the slope of the clotting curve and a reference, namely T=T2S. In other words determining absorbance values for a sample during a coagulation reaction at time T2S and T2 may be utilized by the present method and apparatus to determine the presence of a hypercoagulable condition.
The method involves reacting a sample of a patient's blood or blood components with a clotting agent, such as thrombin. The clotting curve in
The invention also includes an apparatus useful for carrying out analyses on a sample of a patient's blood or blood components. According to a preferred embodiment, the apparatus may include a light source and a photocell for detecting the light emitted from the light source. The light source may be provided to generate energy of a particular wavelength, such as, for example, 660 nm, or other useful wavelength or spectral range, which is capable of detecting the formation of fibrin. The sample may be placed between the path of the light source and a photocell detector. According to preferred embodiments, the wavelength selected is capable of distinguishing the fibrin formation. The apparatus also may include or be linked with a processor, such as a computer, which may record absorbance values over a time interval, where the absorbance values represent the fibrin level in the sample at the corresponding given time values. According to the examples shown in
It is noted that the prothrombin times (PT's) for a blood sample of a patient with a hypercoagulable condition may be in the shorter range, as compared with the mean or average PT, (that is a PT that is presumed to correspond with that of a patient considered to have normal coagulation). The shorter PT may be an indicator of the possibility of a hypercoagulable condition, but the PT being short does not necessarily mean that the patient has a hypercoagulability state.
The hypercoagulability condition is illustrated in relation to clotting curves showing both the presumed normal coagulation patients and those exhibiting the characteristics of a hypercoagulable condition. As discussed herein, from the results of the absorbance values, expressed in units, and the corresponding time values of the clotting reaction, a zero order kinetic slope is obtained. The indicator value is derived from the zero order line. Examples of the slopes obtained are illustrated in the clotting plots of
As illustrated in
According to an alternate embodiment of the invention, in order to provide even more rapid results, the method, upon ascertaining the clotting curve data to form the line L, may utilize an implied end of test line, IEOT, which corresponds with c=cIEOT. The implied end of test line IEOT may be selected from the values cIEOT=x, where x is: 0<x<cT2S. The time differential between a first differential reference (DIFF1) tIEOT/L (which is at the intersection of cIEOT and L) and a second differential reference (DIFF2) (which is at the intersection of cIEOT and t=T2S) may be used to determine an opposite leg (OL) of the tangent of the angle θ° to be determined.
Opposite leg (OL)=DIFF1−DIFF2
An adjacent leg (AL) is also determined. A unit differential (expressed in instrument units) forms an adjacent leg for determining the tangent of the angle θ°. The unit differential for the adjacent leg (AL) may be determined as the vertical distance (in instrument units) between c=x and cT2S, that is cT2S less x (where c=x).
Adjacent leg (AL)=CT2S−x (12)
Accordingly, tangent θ°=(DIFF1−DIFF2)/(CT2Sx) (13)
Alternately, the method may derive an angle θ° using the opposite and adjacent legs formed from a horizontal line segment c=X, where c lies between cT2S and zero, and c=X is The expression may be represented as:
tangent θ°=Tval/IU (11)
where Tval is a time differential between time T2S and a point TcX, where TcX is the time corresponding with the intersection of c=X and L. IU represents the instrument units between cT2S (which is an absorbance value at time T2S) and c=x (which is an absorbance value at time TcX).
Conversely, the line L may be extended above the clotting curve to intersect with the vertical line t=T1 forming the vertex of an angle, with the vertical line t=T2S forming an adjacent leg for determining the tangent of the angle θ1° (see
The slope of the clotting curve may be derived by equations described herein and represents the increase in the rate of fibrinogen conversion. The tangent line L also may be determined using the methods and apparatus described herein. Through the use of the clotting curve data, the indicator values indicative of and/or corresponding with a hypercoagulable condition in an individual may be determined.
One or more reference parameters may be established to serve as a measure against which patient samples may be compared. A patient's blood sample is used to react with a clotting activator, and clotting curve data is collected. The methods described herein for reacting the blood sample with a clotting agent that activates the fibrinogen conversion, such as, for example, thromboplastin C Plus, BTP or Innovin may be employed in conjunction with the hypercoagulability determination. The methods and apparatus used to obtain a clotting curve for a sample may be applied in order to facilitate the hypercoagulable determination. The hypercoagulability condition is assigned by the evaluation of an indicative expression ascertained through the sample data.
Referring to
According to embodiments of the method and apparatus, the angle deviation in its simplest expression may be considered to be a statistically significant deviation from the angle determined for the clotting data: of blood samples of persons presumed to have a normal coagulation condition. These presumed normal coagulation condition patients may be patients who do not exhibit hypercoagulable or hypocoagulable conditions, as may have been previously determined through more expensive and time consuming tests, or by presenting a symptom confirming the condition. The angle deviation may be circumscribed to be a percent deviation from a mean normal angle value (e.g., that obtained for samples from presumed normal coagulation condition persons). The angle deviation, according to some embodiments, may be a measure of standard deviations from the mean normal angle value. If one standard deviation is within the sampling or instrumentation error or range, then a different value may be selected. For example, two standard deviations may be used to categorize test results for angles which are considered to be discrepant or considered an angle deviation (indicative of a hypercoagulable condition for that sample). According to alternate embodiments, angles equal to or greater than three standard deviations may be used to categorize test results that correspond with a condition considered to be a hypercoagulable condition.
According to preferred embodiments, a standard may be established. The blood or blood component samples from presumed normal persons may be run through the test in order to derive a standard for the angle. The standardization angle data (SAD) may be stored. A computer with, or operating linked to, suitable storage means, such as a hard drive, or other suitable apparatus, including those described herein, may store the standardization angle data (SAD). In addition, a standardized sample solution, which may be a blood sample, or other sample containing fibrinogen, may be provided as an instrument calibration standard. The standard may be used to provide a standard angle for a particular instrument. The standard angle may be used as a reference measurement against which angle deviations of patient samples undergoing the hypercoagulability test may be determined. Alternately, or in conjunction with the calibration standard samples, the standard data may be stored on a device, such as a computer memory element. The data may be stored in electronic or other digital form. Though the method may be carried out through a physical or manual manipulation and comparison of the clot slope data, including the expression of that data in the form of a clotting curve. A module may be provided that contains data and may include software with instructions for instructing a processor to record and compare data from a sample analysis. For example, the method for determining a hypercoagulable condition may be carried out using the clotting curve determination, as described herein.
Alternately, an angle deviation from a presumed normal coagulation sample may be derived through the use of high standard samples which correspond with the elevated levels of one or more blood components consistent with a hypercoagulable condition. For example, high standard solutions containing elevated levels of fibrinogen and Factor VIII may be used. Fibrinogen activity conversion data may be recorded for the high standard. The angle θ° may be determined using the methods described herein. The angle ascertained for the high reference standards may serve as a reference against which to make hypercoagulability determinations for blood samples of individuals (i.e., those being tested) by comparing the angles. An angle θ° correspondence with a high standard angle (θ°HS) which is derived from the clotting data for a test run with the sample of a person may be used as a positive indicator of a hypercoagulable condition for that person. Correspondence and deviations from a test sample specimen data, in particular the angle, with the high standard data, may be circumscribed to assign a sample (and essentially the individual of that sample) within or without a classification or category of a prothrombotic disorder, and, in particular, a hypercoagulable condition.
Examples are set forth wherein high standard solutions were prepared and compared with samples of persons having presumed normal coagulation. According to the following examples, the presence of hypercoagulable conditions was determined for patient samples. Samples were prepared as follows. High standard samples were prepared which contained elevated levels of fibrinogen and Factor VIII. Cryoprecipitate was used in conjunction with the sample preparation. Cryoprecipitate is a blood product prepared from plasma and contains concentrations of proteins, including von Willebrand factor, fibrinogen, factor VIII and fibronectin. Cryoprecipitate may be obtained, for example, by a slow thawing of fresh frozen plasma at a low temperature, such as at about 4° C., and centrifuging at a low temperature to precipitate the aforementioned proteins, including fibrinogen and Factor VIII. Cryoprecipitate may be quantified in units, with each unit being defined as that amount or portion obtained from 250 ml plasma (which essentially is the amount of one single fresh frozen plasma (or FFP)). One unit of cryoprecipitate (CPP) contains about at least 80 IU (international units) Factor VIII and about 250 mg of fibrinogen. According to some measurements, for example, each 15 ml unit of cryoprecipitate may contain about 100 IU of factor VIII and about 350 mg of fibrinogen, von Willebrand factor, factor XIII, and fibronectin.
The cryoprecipitate is concentrated from the original plasma volume to a volume of about 10 to 15 ml. The cryoprecipitate may be stored, preferably at a temperature of about 0 C. After storage, the cryoprecipitate is reconstituted. In this example, about 10 ml of a saline solution was used to make up the cryoprecipitate concentrate to 25 ml total volume. The concentration of Factor VIII and of fibrinogen in the 25 ml sample was:
80 IU/25 ml=3.2 IU Factor VIII/ml CPP
250 mg/25 ml=10 mg FBG/ml CPP
A high standard solution was prepared using normal plasma and CPP. The high standard contained Factor VIII in an amount greater than the plasma of a person with presumed normal coagulation (that is neither hypocoagulable nor hypercoagulable). Normal plasma has about 1 IU Factor VIII per 1 ml. A high standard (HS) was prepared based on mixing CPP in a 1:1 ratio by volume with normal plasma, which results in the following:
(3.2 IU Factor VIII+1.0 IU Factor VIII)/2=2.1 IU Factor VIII/ml in the high standard.
The high standard (HS) contained an amount of Factor VIII which was about 210% greater than the Factor VIII content in plasma of a person with presumed normal coagulation. The high standard (HS) also contained an amount of fibrinogen which was greater than the fibrinogen of the plasma of a person with presumed normal coagulation. The fibrinogen content of the high standard was calculated as follows:
The CPP, as considered above, contains about 10 mg/ml fibrinogen (or 1000 mg/di). Plasma of a person with presumed normal coagulation contains about 300 mg/dl of fibrinogen. Considering the fibrinogen content in the high standard:
((1000+300) mg/dl)/2=650 mg/dl FBG in the high standard (HS) or 650/300=2.17 or, expressed in other terms, 217% greater than the FBG content of presumed normal plasma.
The high standard prepared contained about 200% greater levels of Factor VIII found in the plasma of persons with presumed normal coagulation. Factor VIII is associated with a shortened prothrombin time (PT), which is the period of time calculated from the addition of a reagent used to activate the clotting process (e.g., thromboplastin-calcium) to a point where the conversion of fibrinogen to fibrin begins (i.e., the formation of the first clot).
In accordance with the clotting curves illustrated in
The testing of samples was carried out, and time and absorbance data, including the PT and XT times, were recorded. The test included twenty-two samples. High standards were prepared to contain an amount of a clotting factor at a level which is greater than that contained in the blood or blood component of a person considered to have a clinically normal coagulation. According to the example, two high standards containing the higher amounts of a clotting factor (here containing higher amounts of Factor VIII) were also analyzed. The twenty-two samples were run with three different clotting agents, and two high standard samples were prepared and run for each of the three clotting agents. The data from the analysis is presented in Table 12. The twenty-two samples were from the blood of persons presumed to have normal coagulation. The time to maximum acceleration (XT), the point at which the angle θ° is completed, ranged, for the samples evaluated, from 12.2 seconds to 14.6 seconds (for the Dade TPC coagulation agent). The information utilized to determine an indicator for hypercoagulable condition may be obtained within about 14 seconds. Accordingly, a determination of hypercoagulability, may be completed within about thirty seconds. In accordance with the evaluation, two high standard samples, HSx1 and HSx2 were included for each clotting agent. HSx1 and HSx2 represent high standard samples and are included on the results in Table 12 as respective references, HS
Analyses were conducted using three different clotting agents. One was TPC (Dade thromboplastin C Plus, which is a thromboplastin with calcium). Each of the twenty-two samples also was run with this clotting agent added (see the results identified on Table 12 as “Tp 1”). Another clotting agent was used, namely, BTP, or bovine thrombin, which is obtained from bovine plasma and is a clotting enzyme that facilitates the formation of fibrin clots from fibrinogen. BTP is a serine protease and functions by cleaving Arginine-Glycine bonds in fibrinogen. Fibrin and fibrinpeptide A and B result from the cleaving. Each of the twenty-two samples was run using the BTP clotting agent (identified on Table 12 as “Tp 2”). A third clotting agent, Innovin, also was used (see Table 12, “Tp 3”).
The angle obtained for the samples in the analysis was determined by using the slope data to obtain a value corresponding to a trigonometric function. According to a preferred embodiment, the measurements were used to correspond with the tangent of the angle. The tangent was determined using the clot slope data obtained from the clotting analysis. The following expressions were used in conjunction with a tangent determination.
tc=XT−T2S (14)
Tm=TEOT−T2S (15)
tan θ=Tm/IUT (16)
In accordance with the expressions (14) (15) and (16), tc/IUX and Tm/IUT are equivalent (see
An apparatus according to the invention may be constructed to include spectrophotometric means for spectroscopically analyzing a sample. For example, a spectrophotometer as described herein may be used to record changes in the absorbance values for fibrinogen during the sample analysis. The clotting curves illustrated in
According to the method, angle indicator values were determined for a sampling of individuals. Samples were obtained from individuals and run with three different clotting agents, including Dade Thromboplastin C, Dade Innovin and Biopool TP. Each sample was placed into a cuvette and placed into a spectrophotometer. Readings were taken of absorbance values throughout the test. The clotting agent was added to the sample contents as the absorbance values were being recorded. A wavelength of about 660 nm was used for the absorbance analysis. The data was collected and stored for each sample. Twenty-two samples were run and are represented in the analysis. A computer was programmed to manipulate the data to determine angles for each corresponding sample. According to the plot on
Referring to
The data for the twenty-two individual samples and the high standards is reported in Table 12.
Table 12 lists two high standard values HSx1 and HSx2 for each clotting agent used. A total of six high standard values are reported in Table 12 for three different clotting agents. The high standard values are listed as HSx1 and HSx2, where x corresponds to the clotting agent, e.g., x=TPC for Dade Thromboplastin C Plus, x=INN for Dade Innovin, and x=BPT for BioPool Thromboplastin. Each of the high standards (HSx) shows significantly smaller (or more acute) angles θ when compared with the angles (θ) for the presumed normal coagulation condition patient samples. Turning to the sample run for the first clotting agent Dade thromboplastin C Plus, the smallest angle θ of the twenty two samples run, was sample ID 19, whose corresponding angle θ° was 77.32°. Considering the high standards (HS
There also may be provided a normal coagulation standard, where individuals having presumed normal coagulation are sampled and their angle values compared as a reference. Conversely, reference angles for samples of individuals known to have a hypercoagulable condition may be determined and used as a reference against which to compare angles derived from the testing of samples from other individuals.
The angle determinations discussed herein in their broadest sense provide a relationship of a clotting condition. More particularly, the relationship is one which may be determinative of a prothrombotic abnormality, such as, for example, a hypercoagulable condition. The present method and apparatus enable the use of clotting agents with a blood sample or blood component sample to derive a indicator of a hypercoagulable condition.
While the invention has been described with reference to specific embodiments, the description is illustrative and is not to be construed as limiting the scope of the invention. The sample container used to contain the sample may comprise a vial, or cuvette, including, for example, the sample container disclosed in our U.S. Pat. No. 6,706,536. For example, although described in connection with body fluids of a human, the present invention has applicability to veterinary procedures, as well, where fluids are to be measured or analyzed. Various modifications and changes may occur to those skilled in the art without departing from the spirit and scope of the invention described herein and as defined by the appended claims.
This application is a continuation-in-part of U.S. application Ser. No. 11/359,667, filed on Feb. 22, 2006, issued as U.S. Pat. No. 7,276,377 on Oct. 2, 2007, which is a continuation-in-part of U.S. application Ser. No. 10/662,043, filed on Sep. 12, 2003, which is a continuation of U.S. application Ser. No. 10/428,708 filed on May 2, 2003; the application also claims priority to U.S. Provisional application Ser. No. 60/679,423, filed on May 10, 2005, the disclosures of which are herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60679423 | May 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10428708 | May 2003 | US |
| Child | 10662043 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11359667 | Feb 2006 | US |
| Child | 11906325 | US | |
| Parent | 10662043 | Sep 2003 | US |
| Child | 11359667 | US |
