Patent Application
20150111235
The present invention relates to a method for determining a cause of the prolongation of blood coagulation time in a blood sample.
A blood coagulation test is carried out for screening of the presence or absence of any abnormality in the blood coagulation reaction system, or for measurement of the activity of individual factors, by measuring the time period starting from the time point at which an activator and/or Ca2+ and the like are added into a specimen or a specimen mixture, until the time point at which detectable fibrin clots are formed (blood coagulation time; hereinafter, occasionally also referred to simply as coagulation time). Typical examples of the blood coagulation time include prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time.
The PT is the time taken from the addition of a mixed liquid of tissue thromboplastin and Ca2+ to a test plasma until the coagulation of the blood plasma, and the PT is intended for a comprehensive examination of the coagulation activities of factor VII, factor X, factor V, prothrombin, fibrinogen and the like, which are related to the extrinsic pathway of coagulation. The APTT is the time taken from the addition of a sufficient amount of phospholipids and an activating agent (kaolin, silicic anhydride, ellagic acid, or the like) and an appropriate amount of Ca2+ to a test plasma until the coagulation of the blood plasma, and the APTT is intended for a comprehensive examination of the coagulation activities of factor XII, factor XI, prekalikrein, high molecular weight kininogen, factor IX, factor VIII, factor X, factor V, prothrombin, fibrinogen and the like, which are related to the intrinsic pathway of coagulation. In general, what is referred to as abnormality in these blood coagulation tests is the prolongation of the coagulation time. Abnormality in the blood coagulation reaction system reflects the signs of the tendency to hemorrhage or the tendency to thrombosis (tendency to blood coagulation) in the body.
Causes of these abnormalities that can be considered include: 1) a deficiency or decrease in the blood coagulation factors, 2) the presence of an antibody to a blood component that constitutes the blood coagulation system, 3) the presence of an antibody to a component in the reagent for measuring the blood coagulation time, 4) the presence of an antibody to a composite between a blood component that constitutes the blood coagulation system and a component in the reagent for measuring the blood coagulation time, and 5) administration of a drug that inhibits the blood coagulation reaction.
However, simply performing the measurement of the blood coagulation time does not enable differentiation of whether the cause is a decrease in the activity due to simple deficiency of coagulation factors, or a decrease in the activity due to the inhibition of the coagulation reaction by an antibody (inhibitor) to a component or the like in a component that constitutes the blood coagulation system or a component in the reagent for measuring the blood coagulation time. Furthermore, since the therapeutic policy varies with the difference in the causes, differentiation of the cause is important. Thus, there has been carried out a blood coagulation correction test (mixing test) in which normal plasma is added to a test plasma, and the extent of the blood coagulation time of the test plasma being corrected (normalized) is plotted to determine the cause (Non-Patent Document 1).
The mixing test has been traditionally carried out, for example, in the manner described below.
A sample is prepared by adding normal plasma to test plasma and mixing them such that the proportion of the normal plasma is 0, 20, 50, 80 or 100%, and the APTT is measured. The results are plotted into a graph (horizontal axis: proportion of the normal plasma mixed or the proportion of the test plasma(s), vertical axis: coagulation time (seconds)), and the cause is visually determined from the shape of the graph. For example, in the case of a deficiency of a coagulation factor, the addition of a small amount of normal plasma (20% in
When a coagulation factor inhibitor is present, the inhibitor inactivates the coagulation factor in the normal plasma, even though the proportion of the normal plasma added is increased. Therefore, the extent of an improvement in the coagulation time due to the addition of normal plasma is low, and a convex curve is shown above the straight line (
However, a graph with a shape similar to the dotted line (
Furthermore, since the mixing test involves making a determination by visual inspection, there is no unified method for quantifying the extent of correction, and the final determination is entrusted to the judge. Therefore, there is another problem that it is possible to have the result of determination varied depending on the degree of proficiency of the judge.
Moreover, there is also a possibility that the result of determination may vary depending on the difference in the sensitivity of the reagent for measurement to the coagulation factor inhibitor. Particularly, lupus anticoagulant (hereinafter, LA) is known as a coagulation factor inhibitor that depends on the sensitivity of the reagent. The LA is not an inhibitor to individual coagulation factors, but is an immunoglobulin that inhibits the phospholipid-dependent coagulation reaction. Since the presence of phospholipids is essential to the coagulation reaction, usually, many of the reagents for measuring blood coagulation are rich in phospholipids. The LA reacts with the phospholipids in the reagent, thereby consuming these phospholipids, and consequently inhibits the coagulation reaction. Therefore, coagulation tests such as PT and APTT are often found to be abnormal. However, since the LA results in reaction intensities that vary with the type of the phospholipids (origin, phospholipid composition, and the like), it is known to obtain different results of determination based on the reagent used.
There are also known methods for determination as follows, which do not depend on the degree of proficiency of the judge and facilitate the determination (Non-Patent Document 2). When the coagulation time for a test plasma is designated as a, the coagulation time for a mixture of a test plasma and a normal plasma at a mixing ratio of 5:5 is designated as b, and the coagulation time for a normal plasma is designated as c,
1) (a+c)/2≦b, 2) (b−c)/a≧x, 3) b−c≧x, 4) b/c≧x
The method for determination (1) merely expresses in numerical values of whether the graph is convex above the straight line, or the graph is convex below the straight line, and thus the determination by visual inspection and the results do not change. The method for determination (2) is also known as Rosner Index, and is considered as a useful method for determination in determining the LA in particular. In this method, a value of 0.15 is considered appropriate for x, but this value is a value set up for the kaolin coagulation time (KCT). Actually, each reagent used in various facilities requires appropriate setting for x, but the methods for such setting are not clearly known (Non-Patent Document 3). The methods for determination (3) and (4) also require setting of x for various facilities, but the methods for such setting are not clearly known, and cannot be said to be satisfactory as methods for determination.
As discussed above, the conventional methods lack clarity in the technique of setting the cut-off value, and have a risk that the coagulation factor inhibitors miss weakly positive specimens. Actually, in the review carried out by the inventors of the present invention, there has been acknowledged an example in which the graph obtained by a mixing test in the case of a LA weakly positive specimen shows a convex curve below the straight line. Furthermore, the mixing test is largely dependent on the difference in the sensitivity to the coagulation factor inhibitor of the reagents for measurement, and there is a possibility that the result of measurement may vary with the reagent. Moreover, the method of determining by visual inspection greatly depends on the experience of the judge as described above, and has a risk that the result of determination may vary with the difference in the level of proficiency.
Therefore, there is a strong demand for the development of a method for determination which does not depend on the intensity of the sensitivity to a coagulation factor inhibitor in a reagent or on the difference in the level of proficiency of the judge, and which is intended to obtain consistent results of determination in regard to whether the prolongation of the blood coagulation time is caused by the presence of a coagulation factor inhibitor or by a deficiency of a coagulation factor.
The inventors of the present invention made a thorough investigation, and as a result, they found that the problems described above can be solved by a method for determination as shown below, in which the results of a mixing test are not by determined by visual inspection but by a comparison of the area ratio in the graph, and the setting of the cut-off value is also clearly defined. Thus, the inventors completed the present invention.
Specifically, the present invention provides the following inventions.
1. A method of determining a cause of prolongation of blood coagulation time in test plasma, the method including:
(1) measuring blood coagulation times of samples including (a) the test plasma only, (b) normal plasma only, and (c) the test plasma and the normal plasma mixed at least at a mixing ratio;
(2) drawing a polygonal line graph by plotting the measurement results of the samples (a), (b) and (c), with a vertical axis representing the blood coagulation time or a prolongation ratio of blood coagulation time and a horizontal axis representing the mixing ratio or mixing proportion of the test plasma or the normal plasma, and thereby determining an area A under the polygonal line and an area B under a line segment that connects the plotted measurement results of the samples (a) and (b);
(3) calculating a ratio of the area (A−B) obtained by subtracting the area B from the area A, with respect to the area B, ((A−B)/(B)):area ratio X);
(4) performing the steps of (1) and (2) for a coagulation factor inhibitor-positive plasma, and thereby determining in advance a standard area ratio Y; and
(5) comparing the area ratio X and the standard area ratio Y, and determining the test plasma as a coagulation factor inhibitor type in the case where Y≦X, or as a coagulation factor deficient type in the case where Y>X.
2. The method of 1, wherein the standard area ratio Y for the coagulation factor inhibitor-positive plasma is a value determined with respect to a coagulation factor inhibitor-positive plasma exhibiting a blood coagulation time that is larger than the upper limit of a standard range which has been obtained by statistically processing the blood coagulation time of a plurality of healthy persons' plasmas, and is not greater than a value of the upper limit+50%.
3. The method of 2, wherein the standard range for the blood coagulation time of healthy persons' plasmas was obtained by statistically processing the blood coagulation time of a plurality of healthy persons' plasmas.
4. The method of 1, wherein when the vertical axis is used to represent the blood coagulation time in (2) of 1, values obtained by subtracting a constant number therefrom are used for the area A and the area B.
5. The method of 4, wherein the constant number is obtained by drawing an auxiliary line parallel to the horizontal axis in the plot of the blood coagulation time of the normal plasma drawn in the form of a polygonal line graph, and calculating the area under the auxiliary line.
6. The method of any one of 1 to 5, wherein the blood coagulation time is at least one selected from PT (Prothrombin time), APTT (activated partial thromboplastin time), dPT (dilute PT), dAPTT (dilute APTT), KCT (kaolin coagulation time), and dRVVT (dilute Russell's viper venom time).
7. The method of any one of 1 to 6, wherein the coagulation factor inhibitor is at least one selected from Lupus anticoagulant (LA), a factor VIII inhibitor, a factor IX inhibitor, and a factor V inhibitor.
According to the method of the present invention, the determination of plasma in which the coagulation factor inhibitor is weakly positive, for which determination has been traditionally difficult, can be more accurately carried out. Furthermore, according to the method of the present invention, accurate determination can be easily carried out, without being affected by the degree of proficiency of the judge. Therefore, according to the method of the present invention, a proper therapeutic policy for a patient with prolonged coagulation time can be determined by a blood coagulation time test.
As the test plasma to be used in the present invention, it is preferable to use blood plasma, rather than to use the blood directly, and it is more preferable to use platelet-poor plasma. This is because the platelet-derived phospholipids remaining in the blood plasma can eliminate the possibility of a phospholipid-dependent coagulation factor inhibitor such as LA becoming negative.
Normal blood plasma that is used in the mixing with the test plasma may be self-prepared or commercially available normal plasma, and preferably, normal plasma which has been subjected to platelet removal is preferable. Known techniques may be used for the method of removing platelets as described above, and for example, centrifugation or platelet removal filter treatment can be used. Known examples of commercially available products include Pooled Normal Plasma (manufactured by Precision Biologic, Inc.), Platelet Poor Plasma (Technoclone GmbH), and LAtrol (Trade Mark) Normal Control (American Diagnostica, Inc.).
Known examples of the coagulation factor inhibitor include, but not limited to, a factor VIII inhibitor, a factor IX inhibitor and a factor V inhibitor, in addition to the LA. The method of the present invention can be applied, when a reagent for measuring the blood coagulation time which shows sensitivity to the subject coagulation factor inhibitor is used.
For example, when the LA is used as the subject, any reagent for measuring the time of phospholipid-dependent blood coagulation, which reagent exhibits sensitivity to the LA, may be used, and reagents that measure the PT, APTT, dAPTT, dPT, KCT and dRVVT are in use. Furthermore, when the factor VIII inhibitor or the factor IX inhibitor is used as the subject, an APTT measuring reagent or the like is used, and when the factor V inhibitor is used as the subject, an APTT measuring reagent, a PT measuring reagent or the like is used. Commercially available products can be used for all of these reagents. For instance, examples of commercially available PT measuring reagents include Coagpia (registered trademark) PT-S (manufactured by Sekisui Medical Co., Ltd.), ThromboCheck PT Plus (manufactured by Sysmex Corp.), and STA Reagent Series PT (manufactured by F. Hoffmann-La Roche, Ltd.). Examples of commercially available APTT measuring reagents include Coagpia (registered trademark) APTT-S (manufactured by Sekisui Medical Co., Ltd.), ThromboCheck APTT-SLA (manufactured by Sysmex Corp.), and APTT Liquid RD (manufactured by F. Hoffmann-La Roche, Ltd.).
On the other hand, examples of a coagulation factor, which is suspected to be deficient or reduced due to an abnormality of the blood coagulation test, include fibrinogen, prothrombin, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, prekalikrein, high molecular weight kininogen, and von Willebrand factor (vWF).
In step (1) of the method for determination of the invention, the blood coagulation time of samples including (a) test plasma only, (b) normal plasma only, and (c) a mixed sample obtained as described above, are measured.
In the measurement of the blood coagulation time (mixing test) using a sample prepared by mixing a test plasma and a normal plasma at least at one mixing ratio, the plot number may be 3 points or higher including the test plasma and the normal plasma themselves, but in order to clarify the variation pattern of the coagulation time that accompanies the variation of the mixing ratio between the test plasma and the normal plasma, the plot number is preferably 4 points or higher, and more preferably 5 points or higher. The upper limit of the plot number is not particularly limited, but when the economic efficiency or measurement time is considered, the plot number is preferably 10 points or lower, and more preferably 7 points or lower.
The prolongation ratio of the coagulation time is a relative ratio defined when the difference between the coagulation time of normal plasma and the coagulation time of test plasma only is taken as 1. As a matter of fact, the prolongation ratio may also be represented by the relative percentage defined by taking the difference in the coagulation time as 100.
Step (2) is a step carried out by drawing a polygonal line graph using the results of measurement of (a) the coagulation time of the sample containing the test plasma only, (b) the coagulation time of the sample containing a normal plasma only, and (c) the coagulation time of the sample containing a mixed liquid thereof, and plotting the results of measurement of the items (a), (b) and (c), with the vertical axis representing the blood coagulation time or the prolongation ratio of blood coagulation time and the horizontal axis representing the mixing ratio or mixing proportion of the test plasma or the normal plasma, so as to determine the area A under the polygonal line and the area B under a line segment that connects the plotted results of measurement of the items (a) and (b). For example,
Step (3) is a step of calculating the ratio of the area (A−B), which is the value obtainable by subtracting the area (B) of a right-angled triangle having the diagonal line connecting the blood coagulation time or the prolongation ratio of the coagulation time of the sample containing the test plasma only and the sample containing the normal plasma only, from the area (A) of a polygon formed by the polygonal line graph, the horizontal axis and the vertical axis, with respect to the area of the right-angled triangle, ((A−B)/(B)):area ratio X). The area ratio may also be expressed in percentage.
In the case of
Step (4) is a step of setting the cut-off value.
It is desirable to use, as the sample for setting the cut-off value, a coagulation factor inhibitor-positive plasma exhibiting a coagulation time that is close to the coagulation time for a healthy person's plasma, in order to increase the detection sensitivity of the coagulation factor inhibitor. For example, the blood coagulation time of a sample for setting the cut-off value is larger than the upper limit of a standard range for healthy persons, which has been obtained by statistically processing the blood coagulation time of plural healthy persons' plasmas, and the blood coagulation time of the sample for setting the cut-off value is preferably not greater than the value of the upper limit+50%, more preferably not greater than the value of the upper limit+20%, and even more preferably not greater than the value of upper limit+10%.
Furthermore, any coagulation factor inhibitor-positive plasma falling in the above range may be used, and weakly positive plasma may be used, or a series of stepwise dilutions of strongly positive plasma in normal plasma may also be used.
Commercially available products can also be used as the known coagulation factor inhibitor-positive plasma mentioned above. Known examples of the commercially available products of LA-positive plasma include LAtrol (Trade Mark) (American Diagnostica, Inc.), Lupus Positive Control (manufactured by Precision Biologic, Inc.), and Human Plasma Lupus Anticoagulant (George King Bio-Medical, Inc.). Known examples of the commercially available products of factor VIII inhibitor include Human Plasma FVIII with Inhibitor (George King Bio-Medical, Inc.) and Factor VIII Inhibitor Plasma (Technoclone GmbH).
However, the cut-off value (standard area ratio Y) may be appropriately set up for each facility with which the mixing test is performed, in consideration of the differences in the reagent used or the measuring instrument.
In addition, an identical product is used for the reagent for measuring the blood coagulation time of a diluted sample and the reagent for measuring the blood coagulation time of a healthy person. As an example, the examples of APTT in the case of using Coagpia (Registered Trademark) APTT-S (manufactured by Sekisui Medical Co., Ltd.; hereinafter, reagent A) and ThromboCheck APTT-SLA (manufactured by Sysmex Corp.; hereinafter, reagent B) are presented in Table 1.
In the Table 1, in the case of the reagent A, the 8-fold diluted sample exhibiting an APTT value of 39.9 seconds, which is larger than the standard range of healthy persons, 36.6 seconds, serves as the sample for setting the cut-off value. In the case of the reagent B, the 8-fold diluted sample exhibiting an APTT value of 35.8 seconds, which is larger than the standard range of healthy persons, 35.7 seconds, serves as the sample for setting the cut-off value. Here, the standard range of healthy persons is preferably obtained by statistically processing the blood coagulation time of plural healthy persons' plasmas, as will be described below.
Step (5) is a step of determining whether the test plasma is of the coagulation factor inhibitor type or of the coagulation factor deficient type. The area ratio X is compared with the standard area ratio Y, and in the case where Y≦X, the test plasma can be determined as the coagulation factor inhibitor type, while in the case where Y>X, the test plasma can be determined as the coagulation factor deficient type. Since the determination is based on a comparison of numerical values, the determination is clear and does not require proficiency. Here, the coagulation factor inhibitor type implies that the prolongation of the blood coagulation time is caused by the coagulation factor inhibitors discussed above. The coagulation factor deficient type implies that the prolongation of the blood coagulation time is caused by the deficiency of the coagulation factors.
The area or area ratio as discussed above may be calculated by actually plotting a graph, or the like, but as long as the same calculation results are obtained, the method of calculating the area or area ratio is not limited. That is, the instrument for measuring the coagulation time may be imparted with a function such as one capable of obtaining the relevant calculation results, so that the calculation is automatically carried out from the results of sample analysis.
Hereinafter, the present invention will be described in more detail by way of Examples, but the present invention is not intended to be limited to these.
(1) Determination of sample for setting cut-off value
50 μL each of specimens prepared by diluting a LA-positive plasma with a normal plasma to 4 to 16 times were provided, and 50 μL each of a reagent for measuring APTT was added thereto. The mixtures were heated to 37° C. for 3 minutes, and then 50 μL each of a calcium chloride liquid was added to the mixtures. The coagulation time was measured using a fully automated blood coagulation analyzer, Coapresta (registered trademark) 2000 (sold by Sekisui Medical Co., Ltd.).
Lupus Positive Control was used as the LA-positive plasma, and Pooled Normal Plasma was used as the normal plasma (all manufactured by Precision Biologic, Inc.). Furthermore, Coagpia (registered trademark) APTT-S (manufactured by Sekisui Medical Co., Ltd.; hereinafter, reagent A) and ThromboCheck APTT-SLA (manufactured by Sysmex Corp.; hereinafter, reagent B) were used as the reagents for measuring the APTT. The normal standard ranges of the reagents were set up based on the measurement values measured in 48 healthy persons by using each of the reagents, and were set as the mean value±2SD of the coagulation time.
As shown in the Table 1, in the case of the reagent A, since the 10-fold diluted specimen resulted in an APTT value of 36.0 seconds, which was within the normal standard range (26.5 to 36.6 seconds), it was decided to use the 8-fold diluted specimen for the setting of the cut-off value. Likewise, in the case of the reagent B, since the 10-fold diluted specimen resulted in an APTT value of 33.4 seconds, which was within the normal standard range (24.6 to 35.7 seconds), it was decided to use the 8-fold diluted specimen for the setting of the cut-off value.
(2) Performing of Mixing Test
A mixing test was performed according to the procedure shown below, using the 8-fold diluted specimen used in section (1), as well as LA-positive plasma samples (specimen Nos. 1 to 5), coagulation factor deficient plasma samples (specimen Nos. 6 and 7) and warfarin-administered patient's plasma samples (specimen Nos. 8 to 10), such as shown in Table 2.
Samples prepared by mixing a test plasma and a normal plasma at ratios of 0, 20, 50 and 100%, were provided, and the coagulation time was measured using the reagent for measuring APTT described in section (1).
Subsequently, the prolongation ratios of coagulation time with respect to a sample containing 0% of a test plasma (containing normal plasma only) were calculated from the respective coagulation times measured, and a polygonal line graph was produced by plotting the data, while using the vertical axis to represent the prolongation ratio of coagulation time and the horizontal axis to represent the proportion of the test plasma.
The area of a polygon formed by a diagonal line connecting the prolongation ratio of coagulation time of the sample containing 100% of a test plasma and the prolongation ratio of coagulation time of the sample containing 0% of a test plasma (containing normal plasma only) and by the polygonal line graph (
The area ratios (%) calculated based on the data of the 8-fold diluted specimens were set as the cut-off values of the respective reagents, and when the area ratio of a sample was equal to or higher than the cut-off value, the sample was determined as the coagulation factor inhibitor type (hereinafter, may be indicated simply as “inhibitor type”), while when the area ratio of a sample was lower than the cut-off value, the sample was determined as the coagulation factor deficient type (hereinafter, may be indicated simply as “factor deficient type”).
This time, the area ratio (%) was calculated by two methods, such as one method in which 3 points were used for the measurement points (proportions of test plasma: 0, 50 and 100%) and one method in which 4 points were used for the measurement points (Proportions of test plasma: 0, 20, 50 and 100%), and the area ratio values thus obtained are indicated in the table as “Invention-3 point” and “Invention-4 point,” respectively (Tables 2 and 3).
An analysis was performed on the specimens such as described above (specimen Nos. 1 to 5) according to an enclosed document, using commercially available reagents for measuring the LA, LA Test “Gradipore” (manufactured by Institute of Medical Biology) and Staclot LA (manufactured by F. Hoffmann-La Roche GmbH).
In regard to the LA Test “Gradipore,” the “ratio of coagulation time” (coagulation time of test plasma/coagulation time of normal plasma) was calculated, and a sample having a value of the ratio of 1.3 or higher was determined as LA-positive. In regard to the Staclot LA, the “difference of coagulation time” (coagulation time of test plasma-coagulation time of normal plasma) was calculated, and a sample having a value of the difference of 8 seconds or greater was determined as LA-positive.
The results measured in section (2) were used and applied into the following expressions to perform calculations.
Conventional method 1: (a+c)/2−b
Conventional method 2 (Rosner Index): (b−c)/a
a: Coagulation time of test plasma (seconds)
b: Coagulation time of sample containing 50% of test plasma (seconds)
c: Coagulation time sample containing 0% of test plasma (seconds)
In the conventional method 1, a sample having a value of 0 or less was determined as “inhibitor type,” and in the conventional method 2, a sample having a value equal to or greater than the calculated value of the 8-fold diluted specimen used in section (1) of Example 1 as described above, was determined as “inhibitor type.”
A comparison of the results of the Example and the Comparative examples are presented in Table 3. Strongly LA-positive specimens (specimen Nos. 1 to 3: positive for both Gradipore and Staclot) were analyzed with the reagent A, and as a result, the specimens were determined as the “inhibitor type,” regardless of whether the determination was made by using the method of the present invention or any one of the conventional methods. However, in the case of weakly positive specimens (specimen Nos. 4 and 5: positive for any one of Gradipore and Staclot), the convention method 1 was not able to detect the “inhibitor type.” Furthermore, the same test was performed on LA-negative specimens, and coagulation factor deficient specimens (specimen Nos. 6 and 7) were determined as the “factor deficient type” by all of the calculation methods. However, in the case of warfarin-administered specimens (specimen Nos. 8 to 10), pseudo-positivity was recognized in the conventional method 2 such that the specimen No. 9 was determined as the “inhibitor type,” while the methods of the present invention (3 point and 4 point methods) were able to determine the specimen as the “factor deficient type,” similarly for both Gradipore and Staclot. From these results, it was found that the methods of the present invention are capable of specifically detecting the “inhibitor type.”
Similarly, the results obtained by an analysis using the reagent B are presented in Tables 4 and 5. The results for the strongly LA-positive specimens (specimen Nos. 1 to 3) were determined as the “inhibitor type,” regardless of whether the determination was made by using the method of the present invention or any one of the conventional methods. However, a weakly positive specimen (specimen No. 4), which had been determined as LA-negative with Gradipore, was determined as the “factor deficient type” according to the 3-point method and the conventional method 1. However, when the number of measurement points was increased to 4 points, the same specimen was determined as the “inhibitor type,” and thus, it was found that the degree of accuracy of determination was enhanced.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-159144 | Jun 2008 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12999527 | Dec 2010 | US |
| Child | 14579362 | US |
