EP-3961211-B1 - METHOD FOR MEASURING TITER OF COAGULATION FACTOR INHIBITOR

Inventors

• KAWABE, TOSHIKI
• ODA, YUKIO
• EMMI, MARI
• SHIMA, MIDORI
• NOGAMI, KEIJI
• OGIWARA, Kenichi
• SHIMONISHI, Naruto

Dates

Publication Date

20260506

Application Date

20200423

Claims (6)

1. A method for measuring a titer of a blood coagulation factor VIII inhibitor, comprising: (a) preparing a mixed specimen by mixing a subject blood specimen and a normal blood specimen; (b) heating the mixed specimen and acquiring a coagulation reaction curve for the heated mixed specimen; (c) acquiring a coagulation reaction curve for a mixed specimen that has not been heated; (d) calculating a parameter related to the coagulation reaction curve of the mixed specimen that has not been heated as a first parameter; (e) calculating a parameter related to the coagulation reaction curve of the heated mixed specimen as a second parameter; and (f) calculating a titer of a blood coagulation factor VIII inhibitor in the subject blood specimen on the basis of the ratio or the difference between the first parameter and the second parameter, wherein a titer of a blood coagulation factor VIII inhibitor in the subject blood specimen is calculated on the basis of a calibration curve from the ratio or the difference between the first parameter and the second parameter, wherein the parameter related to the coagulation reaction curve is at least one parameter selected from the group consisting of a parameter related to a weighted average point in a predetermined area of a primary differential curve of the coagulation reaction curve, and a parameter related to a weighted average point in a predetermined area of a secondary differential curve of the coagulation reaction curve, wherein either (i) the weighted average point is a weighted average point of the primary differential curve represented by coordinates (vT, vH) defined by a weighted average time vT and a weighted average height vH, wherein, when the primary differential curve is defined as F(t) (t represents time) and a time when F(t) is a predetermined value X is defined as t1, t2 (t1 < t2), the vT and the vH are represented by the following formulas, respectively: v T = M ∑ i = t 1 t 2 F i v H = M ∑ i = t 1 t 2 i wherein M = ∑ i = t 1 t 2 i × F i and the parameter related to the weighted average point includes one or more parameters selected from the group consisting of the vT, the vH, a peak width vB, a weighted average peak width vW, a B flattening vAB, a B time rate vTB, a W flattening vAW, a W time rate vTW, an average time vTa, an average height vHa, vTm, vABa, and vAWa, wherein the peak width vB is a length of time satisfying F(t) ≥ X from the t1 to t2, the weighted average peak width vW is a length of time satisfying F(t) ≥ vH from the t1 to t2, the vAB represents a ratio between the vH and the vB, the vTB represents a ratio between the vT and the vB, the vAW represents a ratio between the vH and the vW, the vTW represents a ratio between the vT and the vW, when F(t), t1 and t2 have the same definitions as described above, and the number of data points from F(t1) to F(t2) is defined as n, the vTa, the vHa, and the vTm are represented by the following formulas, respectively: vTa = ∑ i = t 1 t 2 i n vHa = ∑ i = t 1 t 2 F i n vTm = t 1 + t 2 2 the vABa represents a ratio between the vHa and the vB, and the vAWa represents a ratio between the vHa and the vW; or (ii) the weighted average point is a weighted average point of a plus peak of the secondary differential curve represented by coordinates (pT, pH) defined by a weighted average time pT and a weighted average height pH, wherein, when the secondary differential curve is defined as F'(t) (t represents time) and a time when F'(t) is a predetermined value X is defined as t1, t2 (t1 < t2), the pT and the pH are represented by the following formulas, respectively: p T = M ∑ i = t 1 t 2 F ′ i p H = M ∑ i = t 1 t 2 i wherein M = ∑ i = t 1 t 2 i × F ′ i and the parameter related to the weighted average point includes one or more parameters selected from the group consisting of the pT, the pH, a peak width pB, a weighted average peak width pW, a B flattening pAB, a B time rate pTB, a W flattening pAW, and a W time rate pTW, wherein the peak width pB is a length of time satisfying F'(t) ≥ X from the t1 to t2, the weighted average peak width pW is a length of time satisfying F'(t) ≥ pH from the t1 to t2, the pAB represents a ratio between the pH and the pB, the pTB represents a ratio between the pT and the pB, the pAW represents a ratio between the pH and the pW, and the pTW represents a ratio between the pT and the pW, or (iii) the weighted average point is a weighted average point of a minus peak of the secondary differential curve represented by coordinates (mT, mH) defined by a weighted average time mT and a weighted average height mH, wherein, when the secondary differential curve is defined as F'(t) (t represents time) and a time when F'(t) is a predetermined value X is defined as t1, t2 (t1 < t2), the mT and the mH are represented by the following formulas, respectively: m T = M ∑ i = t 1 t 2 F ′ i m H = M ∑ i = t 1 t 2 i wherein M = ∑ i = t 1 t 2 i × F ′ i and the parameter related to the weighted average point includes one or more parameters selected from the group consisting of the mT, the mH, a peak width mB, a weighted average peak width mW, a B flattening mAB, a B time rate mTB, a W flattening mAW, and a W time rate mTW, wherein the peak width mB is a length of time satisfying F'(t) ≤ X from the t1 to t2, the weighted average peak width mW is a length of time satisfying F'(t) ≤ mH from the t1 to t2, the mAB represents a ratio between the mH and the mB, the mTB represents a ratio between the mT and the mB, the mAW represents a ratio between the mH and the mW, and the mTW represents a ratio between the mT and the mW.
2. The method according to claim 1, wherein the mixed specimen contains the subject blood specimen and the normal blood specimen at a volume ratio of 1 : 9 to 9 : 1.
3. The method according to claim 2, further comprising diluting the subject blood specimen before mixing with the normal blood specimen, or diluting the mixed specimen prepared.
4. The method according to any one of claims 1 to 3, wherein the heating of the mixed specimen is performed at 30°C or more and 40°C or less for 2 to 30 minutes.
5. The method according to any one of claims 1 to 4, wherein the subject blood specimen is a blood specimen showing prolongation of APTT due to the presence of the blood coagulation factor VIII inhibitor.
6. The method according to any one of claims 1 to 5, wherein a titer of the blood coagulation factor VIII inhibitor is calculated in Bethesda units.

Description

Field of the Invention The present invention relates to a method for measuring a titer of a coagulation factor inhibitor of a blood specimen. Background of the Invention The blood coagulation test is a test for diagnosing a blood coagulation ability of a patient with the addition of a predetermined reagent to a blood specimen of the patient and measuring, for example, the blood coagulation time. By the blood coagulation test, the state of a hemostatic ability or fibrinolytic capacity of the patient can be grasped. Typical examples of the blood coagulation time include a prothrombin time (PT), an activated partial thromboplastin time (APTT), and a thrombin time. In recent years, an automatic analyzer that automatically measures a blood coagulation test has been widely used, and the blood coagulation test can be easily performed. Examples of the cause of prolongation of blood coagulation time include influence of a coagulation inhibitor, reduction of components involved in coagulation, congenital deficiency of blood coagulation factors, and acquired autoantibodies that inhibit coagulation reaction. For example, in a case where prolongation of APTT is observed, a cross-mixing test is generally performed, and it is determined by which of a coagulation factor inhibitor (anticoagulation factor), lupus anticoagulant (LA), or deficiency of a coagulation factor such as hemophilia, the prolongation of APTT is caused. In the cross-mixing test, the APTT (immediate reaction) immediately after mixture, and the APTT (delayed reaction) after incubation at 37°C for 2 hours, of a normal plasma, a plasma to be tested, and mixed plasmas containing a plasma to be tested and a normal plasma at various volume ratios are measured. The measured values are graphed by putting the measured values (seconds) of APTT on the vertical axis and the volume ratios of the plasma to be tested to the normal plasma on the horizontal axis. Each of the created graphs of the immediate reaction and delayed reaction shows a pattern of "downward convex", "straight line", or "upward convex" depending on the APTT prolongation factor. On the basis of such patterns of the immediate reaction and delayed reaction, the APTT prolongation factor is determined. In a case where the APTT prolongation is determined to be caused by a coagulation factor inhibitor, the inhibitor titer is generally measured by a Bethesda assay. In the Bethesda assay, a sample obtained by mixing a dilution series of a plasma to be tested with a normal plasma is heated at 37°C for 2 hours, and then the residual activity of the coagulation factor in the sample is measured, and the titer of an inhibitor of the coagulation factor is measured from the measured value on the basis of the calibration curve. The Bethesda assay is currently a standard assay for the inhibitor titer against a coagulation factor VIII (FVIII) and a coagulation factor IX (FIX). In the blood coagulation test, a coagulation reaction curve can be obtained by measuring the amount of blood coagulation reaction over time after addition of a reagent to a blood specimen. The coagulation reaction curve has a different shape depending on the type of the abnormality in the blood coagulation system (Non Patent Literature 1). For this reason, a method for determining the abnormality in the blood coagulation system on the basis of a coagulation reaction curve has been disclosed. For example, Patent Literatures 1 to 3 disclose a method for evaluating the presence or absence of abnormalities of coagulation factors in a patient, on the basis of the parameters related to primary and secondary differential curves of a coagulation reaction curve for the blood of the patient, for example, the maximum coagulation rate, the maximum coagulation acceleration, and the maximum coagulation deceleration. Patent Literature 4 discloses a method for determining the severity of hemophilia on the basis of the average rate of the change in the coagulation rate during the time when the coagulation reaction of a patient reaches the maximum coagulation rate or the maximum coagulation acceleration. Patent Literature 5 discloses a method for determining the presence of a FVIII inhibitor on the basis of the ratio of the slope of the straight line representing the coagulation time for the dilution ratio of patient plasma to the slope of the straight line representing the coagulation time for the dilution ratio of control plasma. Patent Literature 1:JP 2016-194426 APatent Literature 2:JP 2016-118442 APatent Literature 3:JP 2017-106925 APatent Literature 4:JP 2018-017619 APatent Literature 5:JP 2018-517150 A Non Patent Literature Non Patent Literature 1: British Journal of Haematology, 1997, 98: 68-73 EP 3 882 628, which is a document under Article 54(3) EPC, discloses a further method for measuring a titer of a coagulation factor inhibitor. US 2015/111235 A1 discloses a method for the determination of a titer of coagulation inhibitors including factor VIII, co