Method for estimating abnormal blood coagulation factor

Abstract

Provided is a method for estimating a factor of coagulation abnormality in a blood sample. The method comprises the following steps: 1) calculating a parameter Ps related to a coagulation reaction of a specimen S and a parameter Pm related to a coagulation reaction of a specimen M; 2) inferring, on the basis of Ps, whether the specimen S is positive for a coagulation factor inhibitor; and 3) inferring, on the basis of Pm, or Ps and Pm, that a lupus anticoagulant is positive or a coagulation factor is deficient, from the specimen (S) for which the coagulation factor inhibitor is not inferred to be positive in step 2). The specimen S is a blood specimen to be tested having a prolonged coagulation time, the specimen M is a mixed specimen of the specimen S and the specimen N, and the specimen N is a normal blood specimen. Ps is calculated on the basis of the measurement point or time at which the coagulation reaction curve or the coagulation velocity curve of the specimen S reaches a prescribed value, and Pm is calculated on the basis of the measurement point or time at which the coagulation reaction curve or the coagulation velocity curve of the specimen M reaches a prescribed value.

Description

Methods for inferring factors of coagulation disorders Technical Field

[0001] This invention relates to a method for inferring factors of coagulation disorders. Background Technology

[0002] Coagulation tests are used to diagnose a patient's coagulation ability by adding prescribed reagents to a blood sample and measuring clotting time. Typical examples of clotting time include prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time. Generally, abnormalities in coagulation ability lead to prolonged clotting time. Factors contributing to abnormal coagulation ability include the effects of anticoagulants, a reduction in coagulation-related components, the influence of autoantibodies on phospholipids that contribute to the coagulation reaction (lupus anticoagulants), congenital deficiencies in coagulation factors (e.g., congenital hemophilia), and the presence of autoantibodies against coagulation factors (inhibitors). As an example of coagulation abnormalities caused by inhibitors, in patients with congenital hemophilia A, antibodies against FVIII (FVIII inhibitors) appear due to the administration of factor VIII (FVIII) preparations. Another example is acquired hemophilia A (AHA). In AHA, autoantibodies against FVIII are present.

[0003] Previously, when prolonged clotting time was found in coagulation tests, a cross-mixing test was generally performed to infer the cause of the prolonged clotting time, provided there was no suspicion of anticoagulant use. In a cross-mixing test, a mixture of the tested sample and normal samples was prepared. The APTT (immediate response time) of the tested sample, normal samples, and the sample immediately after mixing was measured, as well as the APTT (delayed response time) of each sample after incubation at 37°C for 2 hours. Based on the APTT profiles of the immediate and delayed responses, it was determined whether the prolonged APTT was caused by a coagulation factor inhibitor (anticoagulant), lupus anticoagulant (LA), or a deficiency of a coagulation factor such as hemophilia. However, as mentioned above, cross-mixing tests suffer from drawbacks such as requiring a large number of samples, being extremely cumbersome and time-consuming, and lacking the ability to provide quantitative analysis.

[0004] Patent Document 1 describes a method for obtaining information related to the cause of prolonged coagulation time, comprising: obtaining first, second, and third coagulation times of a tested blood sample, a normal blood sample, and a mixed sample thereof; obtaining fourth, fifth, and sixth coagulation times of a sample obtained by incubating the tested blood sample, normal blood sample, and mixed sample at a specified temperature for 45 minutes to 4 hours; obtaining a first quantitative indicator based on the first, second, and third coagulation times; obtaining a second quantitative indicator based on the fourth, fifth, and sixth coagulation times; and obtaining a value of the ratio or difference between the value of the first quantitative indicator and the value of the second quantitative indicator, or a value obtained by combining the value of the ratio and the value of the difference, wherein the value is information indicating whether the tested blood sample is suspected of containing a coagulation factor inhibitor.

[0005] Patent Document 2 describes a method for determining a blood sample, comprising: a step of preparing a mixed plasma by mixing the plasma to be tested and normal plasma; a step of obtaining parameters related to the differential of the coagulation waveform of the mixed plasma, namely, the maximum coagulation rate, the maximum coagulation acceleration, and the maximum coagulation deceleration; and a step of determining, based on the values ​​of the obtained parameters, whether the plasma to be tested is suspected to be coagulation factor deficient plasma or suspected to be plasma containing lupus anticoagulant (LA) or coagulation factor inhibitor.

[0006] Patent document 3 describes a blood analysis method comprising the following steps: preparing a mixed sample consisting of a blood sample to be tested and a normal blood sample; heating the mixed sample; obtaining coagulation reaction data for the heated mixed sample and the unheated mixed sample; obtaining a differential curve of the coagulation reaction data; calculating a parameter related to the centroid of the differential curve for the unheated mixed sample as a first parameter; calculating a parameter related to the centroid of the differential curve for the heated mixed sample as a second parameter; and evaluating the coagulation characteristics of the blood sample to be tested based on the ratio or difference between the first parameter and the second parameter.

[0007] Patent document 4 describes a method for inferring the coagulation time extension factor, which includes the following steps: detecting the coagulation reaction endpoint Pe in the coagulation reaction curve of the tested blood sample with prolonged coagulation time; calculating the curve T(X) representing the measurement point or time when the coagulation reaction curve reaches Pe by X%; and inferring the coagulation time extension factor of the tested blood sample based on the shape of T(X).

[0008] Patent document 5 describes an analytical method for coagulation reaction, which includes the following steps: determining points pk and qk that are specified values ​​before and after the coagulation rate curve V(i) of the tested blood sample reaches its maximum value Vmax; and using the parameters calculated using pk and qk as a benchmark to infer the coagulation abnormality factors of the blood sample.

[0009] Patent document 6 describes the following: LA-positive specimens and coagulation factor inhibitor-positive specimens can be distinguished based on the maximum coagulation velocity, maximum coagulation acceleration, or maximum coagulation deceleration calculated from the coagulation waveform of the blood specimen.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent No. 6696963

[0013] Patent Document 2: Japanese Patent No. 6871673

[0014] Patent Document 3: International Publication No. 2021 / 229556

[0015] Patent Document 4: International Publication No. 2022 / 186381

[0016] Patent Document 5: International Publication No. 2022 / 054819

[0017] Patent Document 6: Japanese Patent Application Publication No. 2016-118442 Summary of the Invention

[0018] This invention provides a method for inferring factors causing coagulation abnormalities in blood samples.

[0019] That is, the present invention provides the following solution.

[0020] [1] A method for inferring factors of abnormal coagulation in blood samples, comprising the following steps:

[0021] 1) Calculate the parameter Ps related to the coagulation reaction of specimen S and the parameter Pm related to the coagulation reaction of specimen M.

[0022] here,

[0023] Specimen S is a blood sample with prolonged clotting time.

[0024] Specimen M is a mixture of specimens S and N.

[0025] Specimen N is a normal blood sample.

[0026] Ps is calculated based on Ts(X), or vT1s(X) and vT2s(X).

[0027] Pm is calculated based on the first-order differential curve of the coagulation reaction curve of Tm(X), vT2m(X), vT1m(X), and vT2m(X), or the coagulation reaction curve of the specimen M.

[0028] here,

[0029] Ts(X) represents the measurement point or time at which the coagulation reaction curve of sample S reaches X% of Es, where Es is the coagulation reaction endpoint in the coagulation reaction curve of sample S.

[0030] vT1s(X) represents the measurement point or time at which the coagulation rate curve of sample S reaches its maximum value by X percent.

[0031] vT2s(X) represents the measurement point or time at which the coagulation rate curve of sample S reaches its maximum value, which is X% of the maximum value.

[0032] Tm(X) represents the measurement point or time at which the coagulation reaction curve of specimen M reaches X% of Em, where Em is the coagulation reaction endpoint in the coagulation reaction curve of specimen M.

[0033] vT1m(X) represents the measurement point or time at which the coagulation rate curve of specimen M reaches its maximum value by X percent.

[0034] vT2m(X) represents the measurement point or time at which the coagulation rate curve of specimen M reaches its maximum value, which is X% of the maximum value.

[0035] 0 < X ​​≤ 100;

[0036] 2) Inferring whether specimen S is positive for coagulation factor inhibitors based on Ps; and

[0037] 3) Specimens S that were not inferred to be positive for coagulation factor inhibitors in 2) are inferred to be positive for lupus anticoagulant or lacking in coagulation factor based on Pm, or based on Ps and Pm.

[0038] [2] According to the method described in [1], wherein the Ps is selected from the following (a) to (j):

[0039] (a)rTs(X1)

[0040] (Here, rTs(X1) = Ts(X1) / Ts(As) × Qs (IIa')

[0041] X1=35~95, As=3~75, X1>As, Qs>0);

[0042] (b) {rTs(X2)-rTs(X1)} / (X2-X1),

[0043] (Here, rTs(X1) and rTs(X2) are defined by the above equation (IIa'), where X1 = 1 to 85, X2 = 25 to 95, (X2 - X1) = 5 to 94, As = 5 to 95, and Qs > 0).

[0044] (c) rSLs[X1:X2]

[0045] (here,

[0046]

[0047] UX is the average value from X1 to X2.

[0048] UrTs is the average value from rTs(X1) to rTs(X2).

[0049] rTs(X2) is defined by the above equation (IIa'),

[0050] X1=1~85, X2=25~100, (X2-X1)=5~99, As=5~95, Qs>0);

[0051] (d) wXs[X1:X2]

[0052] (here,

[0053]

[0054] X1=1~75, X2=30~100, (X2-X1)=10~99, Qs>0);

[0055] (e)vT2s(X1) / vT1s(X2)

[0056] (Here, X1 = 3 to 58, X2 = 13 to 98);

[0057] (f)vT2s(X1) / vT2s(X2)

[0058] (Here, X1 = 13-63, X2 = 58-83, X2 > X1);

[0059] (g) (vT2s (X1) - vT1s (X2)) / vT2s (Bs)

[0060] (Here, X1 = 3~58, X2 = 13~98, Bs = 3~100);

[0061] (h) (vT2s (X1) - vT1s (X2)) / vT1s (Bs)

[0062] (Here, X1 = 3~58, X2 = 3~93, Bs = 3~100);

[0063] (i) (vT2s(X1)-vT2s(X2)) / vT2s(Bs)

[0064] (Here, X1 = 8~58, X2 = 48~83, X2 > X1, Bs = 3~100);

[0065] (j) (vT2s (X1) - vT2s (X2)) / vT1s (Bs)

[0066] (Here, X1 = 8~58, X2 = 48~98, X2 > X1, Bs = 3~100).

[0067] Furthermore, the Pm is selected from the following (a') to (p'):

[0068] (a')Tm(X1) (where X1 = 90~100);

[0069] (b')rTm(X1)

[0070] (Here, rTm(X1) = Tm(X1) / Tm(Am) × Qm (IIb')

[0071] X1=30~95, Am=1~35, X1-Am=10~94, Qm>0);

[0072] (c')aTm(X1,X2)

[0073] (here,

[0074] aTm (X1, X2) = {Tm (X2) - Tm (X1)} / (X2 - X1) × Qm,

[0075] X1=1~90, X2=25~100, X2-X1=5~99, Qm>0);

[0076] (d')SLm[X1:X2]

[0077] (here,

[0078]

[0079] UX is the average value from X1 to X2.

[0080] UTm is the average value from Tm(X1) to Tm(X2).

[0081] X1=1~95, X2=30~100, (X2-X1)=5~99, Qm>0);

[0082] (e')rSLm[X1:X2]

[0083] (here,

[0084]

[0085] rTm(X) is defined by the above equation (IIb').

[0086] UX is the average value of X from X1 to X2.

[0087] UrTm is the average value of rTm(X) from rTm(X1) to rTm(X2).

[0088] X1=1~70, X2=25~100, (X2-X1)=5~99, Am=5~95, Qm>0);

[0089] (f')wXm[X1:X2]

[0090] (here,

[0091]

[0092] X1=1~45, X2=30~100, (X2-X1)=10~99, Qm>0);

[0093] (g')wTm[X1:X2]

[0094] (here,

[0095]

[0096] X1=75~95, X2=95~100, (X2-X1)=5~25, Qm>0);

[0097] (h')wrTm[X1:X2]

[0098] (here,

[0099]

[0100] rTm(X) is defined by the above equation (IIb').

[0101] X1=1~90, X2=30~100, (X2-X1)=5~99, Am=3~34, Qm>0);

[0102] (i') vT2m (X1) / vT1m (X2)

[0103] (Here, X1 = 3 to 93, X2 = 3 to 88);

[0104] (j') vT2m (X1) / vT2m (X2)

[0105] (Here, X1 = 38-63, X2 = 63-93, X2 > X1);

[0106] (k') (vT2m (X1) - vT1m (X2)) / vT2s (Bs)

[0107] (Here, X1 = 3~93, X2 = 3~88, Bm = 3~100);

[0108] (l') (vT2m (X1) - vT2m (X2)) / vT2s (Bs)

[0109] (Here, X1 = 28-63, X2 = 68-93, X2 > X1, Bm = 3-100);

[0110] (m') (vT2m (X1) - vT1m (X2)) / vT1s (Bs)

[0111] (Here, X1 = 3~93, X2 = 3~93, Bm = 3~100);

[0112] (n') (vT2m (X1) - vT2m (X2)) / vT1s (Bs)

[0113] (Here, X1 = 23~63, X2 = 63~93, X2 > X1, Bm = 3~100);

[0114] (o')iCmax_m

[0115] (Here, iCmax_m is the reciprocal of Cmax_m,)

[0116] Cmax_m is the maximum value of Cm(i).

[0117] Cm(i)=Vm(i)×(Q / Em) (XIb)

[0118] In equation (XIb), Vm(i) is the first differential curve of the coagulation reaction curve of the specimen M, i is the time or the number of measurement points, and Q > 0).

[0119] (p')iwCm[X1:X2]

[0120] (here,

[0121]

[0122] Cm(i) is defined by the above equation (XIb).

[0123] X1 and X2 satisfy Cm(X1) = Cm(X2) = Vm(i) × [Q / (Em×S%)], where X1 < VmaxT_m < X2, Qm > 0.

[0124] S = 1 ~ 100,

[0125] VmaxT_m is the time or measurement point at which the first-order differential curve of the coagulation reaction curve of the specimen M reaches its maximum value.

[0126] [3] The method according to [1] or [2], wherein 2) above includes the following steps:

[0127] If the above Ps is at or above the threshold of Ps, the above specimen S is inferred to be positive for coagulation factor inhibitor.

[0128] The above 3) includes the following steps:

[0129] If the above-mentioned Pm is at or above the threshold of Pm, the above-mentioned specimen S is inferred to be positive for lupus anticoagulant; or if the above-mentioned Pm is at or below the threshold of Pm, the above-mentioned specimen S is inferred to be deficient in coagulation factor.

[0130] [4] The method according to any one of [1] to [3], wherein, as described in 2) and 3) above, includes the following steps:

[0131] Ps and Pm of the above specimen S are plotted on a two-dimensional map. Based on the position of the plotted data on the two-dimensional map, the specimen S is inferred to be positive for coagulation factor inhibitors, positive for lupus anticoagulants, or lacking coagulation factors.

[0132] [5] The method according to any one of [1] to [4], wherein the method comprises the following steps:

[0133] Calculate the parameter Pd for specimen S that is inferred to be positive for lupus anticoagulant, and

[0134] Based on Pd, it can be inferred whether the specimen S is suspected to have come from a patient with acquired hemophilia A.

[0135] here,

[0136] Pd is calculated based on Td(X).

[0137] Td(X)=Ts(X)-Tm(X),

[0138] 0 < X ​​≤ 100.

[0139] [6] The method according to any one of [5], wherein the Pd is selected from the following (a'') to (e''):

[0140] (a'')Td(X1) (where X1 = 43~100);

[0141] (b'')Td(X2)-Td(X1) (where X1 = 1~80, X2 = 15~100, and X2-X1 = 5~99);

[0142] (c'')SLd[X1:X2]

[0143] (here,

[0144]

[0145] X1 = 1~95, X2 = 25~100, X2 - X1 = 5~99, UX is the average value of X from X1 to X2, UTd is the average value of Td(X) from Td(X1) to Td(X2), Qd > 0).

[0146] (d'')wXd[X1:X2]

[0147] (here,

[0148]

[0149] X1 = 1, X2 = 50-90, or X1 = 15-25, X2 = 25-45, and X2 - X1 = 5-30, Qd > 0);

[0150] (e'')wTd[X1:X2]

[0151] (here,

[0152]

[0153] X1=1~95, X2=45~100, X2-X1=5~99, Qd>0).

[0154] [7] The method according to [5] or [6], wherein the method includes the following steps:

[0155] If Pd is at or above the threshold of Pd, the specimen S is inferred to be suspected to be from a patient with acquired hemophilia A.

[0156] [8] A procedure for implementing the method described in any one of [1] to [7].

[0157] [9] An apparatus for carrying out the method described in any one of [1] to [7].

[0158] According to the method of the present invention, factors contributing to coagulation abnormalities in blood samples can be quantitatively inferred without the need for creating APTT profiles of immediate and delayed responses for each sample, as is done in conventional cross-mixing tests. The method of the present invention allows for a faster and simpler inference of factors contributing to coagulation abnormalities in blood samples. Attached Figure Description

[0159] Figure 1 is an example of a solidification reaction curve.

[0160] Figure 2A illustrates T(X). The moments when the solidification reaction reaches 10%, 50%, and 90% of the solidification endpoint E on the solidification reaction curve are represented as T(10), T(50), and T(90), respectively. Figure 2B shows the changes in T(X) and vT(X) relative to X. vT(X) is represented by vT1(X) and vT2(X).

[0161] Figure 3 shows the T(X) of the specimen type at an APTT of approximately 100 seconds. A: Ts(X), B: Tm(X) with a 1:1 mixture ratio. Solid line: LA, dashed line: dFVIII, dotted line: iFVIII. C: iFVIII (APTT: 107 seconds), D: dFVIII (APTT: 101 seconds), E: LA (APTT: 108 seconds). Solid line: Ts, dashed line: Tm.

[0162] Figure 4 is a conceptual diagram showing the configuration of an automatic analysis device for the method of inferring factors of solidification anomalies according to the present invention.

[0163] Figure 5 shows the rTs (X1) of the tested specimens. LA: LA-positive specimen. Def: Coagulation factor deficient specimen, 8, 9, and 5 represent FVIII, FIX, and FV, respectively. Inh: Coagulation factor inhibitor positive specimen, 8 and 9 represent FVIII and FIX, respectively.

[0164] Figure 6 shows the difference between the minimum value of rTs(X1) of group Inh and the maximum value of rTs(X1) of groups LA and Def. Gray bars indicate that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, × is used.

[0165] Figure 7 shows the rTs(X2) - rTs(X1) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0166] Figure 8 shows the difference between the minimum value of rTs(X2) - rTs(X1) for group Inh and the maximum value of rTs(X2) - rTs(X1) for groups LA and Def. Gray bars indicate that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, an × is used.

[0167] Figure 9 shows the arTs (X1, X2) of the tested specimens. The labels on the horizontal axis are the same as in Figure 5.

[0168] Figure 10 shows the difference between the minimum value of arTs(X1, X2) of group Inh and the maximum value of arTs(X2, X1) of groups LA and Def. As = 5. Gray bars indicate that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, an × is used.

[0169] Figure 11 is the same as Figure 10. As = 50.

[0170] Figure 12 is the same as Figure 10. As = 95.

[0171] Figure 13 shows the rSLs [X1:X2] of the tested specimens. The labels on the horizontal axis are the same as in Figure 5.

[0172] Figure 14 shows the difference between the minimum value of rSLs[X1:X2] for group Inh and the maximum value of rSLs[X1:X2] for groups LA and Def. As = 5. Gray columns indicate that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, an × is used.

[0173] Figure 15 is the same as Figure 14. As = 50.

[0174] Figure 16 is the same as Figure 14. As = 95.

[0175] Figure 17 shows the wXs[X1:X2] of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0176] Figure 18 shows the difference between the minimum value of wXs[X1:X2] of group Inh and the maximum value of wXs[X1:X2] of groups LA and Def. Gray bars indicate that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, an × is used.

[0177] Figure 19 shows the vT2s(X1) / vT1s(X2) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0178] Figure 20 shows the difference between the minimum value of the Inh group and the maximum value of the LA and Def groups for vT2s(X1) / vT1s(X2) and vT2s(X1) / vT2s(X2). Gray columns indicate that the minimum value of the Inh group is greater than the maximum value of the LA and Def groups; otherwise, an × is used.

[0179] Figure 21 shows the relative values ​​of the differences in vTs(X) between the tested specimens. The labels on the horizontal axis are the same as in Figure 5.

[0180] Figure 22 shows the difference between the minimum value of the Inh group and the maximum value of the LA and Def groups in terms of the relative values ​​of the difference of vTs(X). Gray columns indicate that the minimum value of the Inh group is greater than the maximum value of the LA and Def groups; otherwise, × is used.

[0181] Figure 23 is the same as Figure 22.

[0182] Figure 24 is the same as Figure 22.

[0183] Figure 25 shows the Tm (X1) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0184] Figure 26 shows the rTm (X1) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0185] Figure 27 shows the difference between the minimum value of rTm(X1) of group LA and the maximum value of rTm(X1) of group Def. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × sign is used.

[0186] Figure 28 shows the SLm[X1:X2] of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0187] Figure 29 shows the difference between the minimum value of SLm[X1:X2] for group LA and the maximum value of SLm[X1:X2] for group Def. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × sign is used.

[0188] Figure 30 shows the rSLm[X1:X2] of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0189] Figure 31 shows the difference between the minimum value of rSLm[X1:X2] for group LA and the maximum value of rSLm[X1:X2] for group Def. Am = 5. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × is used.

[0190] Figure 32 is the same as Figure 31. Am = 50.

[0191] Figure 33 is the same as Figure 31. Am = 95.

[0192] Figure 34 shows the samples A: wXm [X1:X2], B: wTm [X1:X2], and C: wrTm [X1:X2]. The labels on the horizontal axis are the same as in Figure 5.

[0193] Figure 35 shows the difference between the minimum value of wXm[X1:X2] in group LA and the maximum value of wXm[X1:X2] in group Def. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × is used.

[0194] Figure 36 shows the difference between the minimum value of wTm[X1:X2] for group LA and the maximum value of wTm[X1:X2] for group Def. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × is used.

[0195] Figure 37A shows the difference between the minimum value of wrTm[X1:X2] for group LA and the maximum value of wrTm[X1:X2] for group Def. Am = 3. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × is used.

[0196] Figure 37B is the same as Figure 37A. Am = 10.

[0197] Figure 37C is the same as Figure 37A. Am = 34.

[0198] Figure 38 shows the aTm(X1, X2) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0199] Figure 39 shows the difference between the minimum value of aTm(X1, X2) in group LA and the maximum value of aTm(X1, X2) in group Def. Gray bars indicate that the minimum value of group LA is greater than the maximum value of group Def; otherwise, an × is used.

[0200] Figure 40A shows the ratio of vTm(X) to vTm(X) of the tested specimen. The labels on the horizontal axis are the same as in Figure 5.

[0201] Figure 40B shows the difference between the minimum value of vT2m(X1) / vT1m(X2) and the maximum value of vT2m(X1) / vT2m(X2) in the LA group and the maximum value in the Def group. Gray columns indicate that the minimum value of the LA group is greater than the maximum value of the Def group; if this is not the case, × is used.

[0202] Figure 41 shows the relative values ​​of the differences in vTm(X) between the tested specimens. The labels on the horizontal axis are the same as in Figure 5.

[0203] Figure 42 shows the difference between the minimum value in the LA group and the maximum value in the Def group regarding the relative values ​​of the difference of vTm(X). Gray columns indicate that the minimum value in the LA group is greater than the maximum value in the Def group; otherwise, × is used.

[0204] Figure 43 is the same as Figure 42.

[0205] Figure 44 is the same as Figure 42.

[0206] Figure 45 shows the iCmax_m and iwCm[X] of the tested specimen.

[0207] Figure 46 is a two-dimensional graph plotting (Ps, Pm) of the tested sample. In the figure, 〇 represents LA, △ represents iFVIII, □ represents iFIX, × represents dFVIII, + represents dFIX, and * represents dFV. The dashed line parallel to the Y-axis represents the threshold Ls, and the dashed line parallel to the X-axis represents the threshold Lm. The diagonal line represents the threshold line Lc.

[0208] Figure 47 shows the results for the tested specimens: A: Ts (50), B: Vmax, C: (1 / Vmax) × 100, and D: VmaxT. The labels on the horizontal axis are the same as in Figure 5.

[0209] Figure 48 shows the results for mixed samples: A: Tm (50), B: Vmax, C: (1 / Vmax) × 100, and D: VmaxT. The labels on the horizontal axis are the same as in Figure 5.

[0210] Figure 49A is a two-dimensional plot of (Ps, Pm) of subjects using a mixed sample (subject:NPP) at a mixing ratio of 1:1. dFVIII, dFIX, and dFV represent FVIII-deficient, FIX-deficient, and FV-deficient samples, respectively; iFVIII and iFIX represent FVIII inhibitor-positive and FIX inhibitor-positive samples, respectively. The dashed line parallel to the Y-axis represents the threshold Ls, and the dashed line parallel to the X-axis represents the threshold Lm.

[0211] Figure 49B is a two-dimensional graph plotted using Pm of a mixed sample with a mixing ratio (sample:NPP) of 9:1, showing (Ps, Pm). The representations and dashed lines in the figure are the same as in Figure 49A.

[0212] Figure 49C is a two-dimensional graph plotted using Pm of a mixed sample with a mixing ratio (sample: NPP) of 1:9, showing (Ps, Pm) of the sample. The representations and dashed lines in the figure are the same as in Figure 49A.

[0213] Figure 50 is a two-dimensional plot of (Ps, Pm) for AHA and LA. The dashed line parallel to the Y-axis represents the threshold Ls, and the dashed line parallel to the X-axis represents the threshold Lm. The mixing ratios (subject: NPP) of the mixed samples used were 9:1 (A), 1:1 (B), and 1:9 (C).

[0214] Figure 51 compares the T(X) of AHA-a (inferred as LA), AHA-b (inferred as Inh), and LA in the AHA group. Solid line: AHA-a, dashed line: AHA-b, dotted line: LA. A: Ts(X), B: Tm(X) of mixed specimens with a mixing ratio (subject: NPP) of 1:1, C: Td(X).

[0215] Figure 52 shows the Td(X) of AHA-a, AHA-b, and LA under different mixing ratios of specimen S to NPP. Specimen S:NPP ratios are 9:1 (A), 1:1 (B), and 1:9 (C).

[0216] Figure 53A shows the Pd = Td(X2) - Td(X1) values ​​for the AHA and LA groups. The horizontal axis labels are: LA: LA-positive specimen; AHA: Acquired hemophilia A specimen. B: The difference between the maximum and minimum Pd (= Td(X2) - Td(X1)) values ​​in the LA group and the AHA group. Gray columns indicate that the minimum value in the AHA group is greater than the maximum value in the LA group; if this is not the case, an × sign is used.

[0217] Figure 54A shows Pd = SLd [X1:X2] for the AHA and LA groups. Figure 54B shows the difference between the maximum value and the minimum value of Pd (=SLd [X1:X2]) for the LA group and the AHA group. The labels on the horizontal axis of Figure A and the descriptions in Figure B are the same as those in Figure 53.

[0218] Figure 55A shows Pd = wXd[X1:X2] for the AHA and LA groups. Figure 55B shows the difference between the maximum value and the minimum value of Pd (= wXd[X1:X2]) for the LA group and the AHA group. The labels on the horizontal axis of Figure A and the descriptions in Figure B are the same as those in Figure 53.

[0219] Figure 56A shows Pd = wTd[X1:X2] for the AHA and LA groups. Figure 56B shows the difference between the maximum value and the minimum value of Pd (= wTd[X1:X2]) for the LA group and the AHA group. The labels on the horizontal axis of Figure A and the descriptions in Figure B are the same as those in Figure 53.

[0220] Figure 57 is a two-dimensional plot of (Pd, Pm) for AHA and LA. The dashed line parallel to the Y-axis represents the threshold Ld, and the dashed line parallel to the X-axis represents the threshold Lm. For the mixing ratio (subject: NPP) of the mixed samples used, Figure A is 9:1, Figure B is 1:1, and Figure C is 1:9.

[0221] Figure 58 is a two-dimensional spectrum obtained by converting the two-dimensional spectrum of Figure 49A into index ratios (Ps ratio, Pm ratio).

[0222] Figure 59 shows the relationship between factor activity and Ps ratio (A: FVIII, B: FIX, C: FV). D: Relationship between potency and Pm ratio (△: iFVIII, □: iFIX).

[0223] Figure 60 is a two-dimensional graph plotting the (Ps ratio, Pm ratio) of LA, iFVIII, and AHA.

[0224] Figure 61 is a two-dimensional graph of LA and AHA. A is a graph plotted with (Ps ratio, Pm ratio), and B is a graph plotted with (Pd ratio, Pm ratio).

[0225] Figure 62 is a flowchart for inferring factors of coagulation abnormalities in specimens with known prolonged APTT.

[0226] Figure 63 shows the flowchart for APTT determination and inference of factors causing coagulation abnormalities in specimens with unknown APTT.

[0227] Figure 64 shows the flowchart for inferring factors causing abnormal coagulation of the specimen after APTT prolongation is discovered.

[0228] Figure 65 shows the detailed process of parameter comparison and inference of factors contributing to solidification anomalies.

[0229] Figure 66 is the inference flow of factors incorporating the solidification anomaly inferred by Inh-X. Detailed Implementation

[0230] In coagulation tests, prescribed reagents are added to the blood sample being tested, and the subsequent coagulation reaction is measured. The clotting time is determined based on this reaction. In routine samples with coagulation abnormalities, the clotting time is often prolonged compared to normal samples. In this instruction manual, coagulation reaction, clotting time, coagulation abnormality, and blood sample are sometimes referred to simply as "coagulation reaction," "clotting time," "coagulation abnormality," and "sample," respectively. A prolonged clotting time is an indicator of the presence or absence of a coagulation abnormality. On the other hand, factors causing coagulation abnormalities (such as factors leading to prolonged clotting time) cannot be inferred from clotting time alone.

[0231] In coagulation tests, if a prolonged clotting time, such as APTT, is found in the sample, the conventional procedure would, if no anticoagulant such as heparin was present in the sample, involve a cross-mixing test to determine the factors contributing to the prolonged clotting time (hereinafter referred to as "prolongation factors"). Specifically, the cross-mixing test determines whether the prolonged APTT is caused by a deficiency of coagulation factor inhibitors (anticoagulants), lupus anticoagulants (LA), or coagulation factors such as hemophilia. In the cross-mixing test, the APTT of a normal sample, the sample being tested, and a mixture of the sample and the normal sample is measured immediately after preparation (immediate reaction) and after incubation at 37°C for 2 hours (delayed reaction). The APTT prolongation factors are determined based on the APTT profiles of these immediate and delayed reactions. In conventional methods, without a separate cross-mixing test in addition to clotting time measurement, it is impossible to determine the prolongation factors in the sample. However, cross-mixed tests require a large number of samples to generate APTT profiles for both immediate and delayed responses in each sample, which is not only very cumbersome but also takes a long time until the results are obtained.

[0232] In this invention, there is no need for further preparation of the specimens subjected to long-term incubation treatment as a pretreatment for the delayed reaction determination in the aforementioned conventional cross-mixing test. This allows for the inference of factors causing abnormal coagulation of the specimen using a smaller quantity of specimen. Furthermore, a series of analytical steps can be easily automated.

[0233] [Methods for inferring factors of solidification anomalies]

[0234] This invention provides a method for inferring factors of abnormal coagulation in blood samples. In the method for inferring factors of abnormal coagulation in blood samples of this invention (hereinafter also referred to as the method of this invention), blood samples with prolonged coagulation time are used as the tested blood samples (hereinafter also referred to as the tested samples) to infer the factors of abnormal coagulation in the tested samples (i.e., factors of prolonged coagulation time).

[0235] 1. Specimen

[0236] Plasma is preferred as the blood sample described above. An anticoagulant, commonly used in coagulation tests, can be added to this sample. For example, plasma can be obtained by centrifuging blood collected using a blood collection tube containing an aqueous solution of sodium citrate.

[0237] In routine coagulation tests, time-series data of the coagulation reaction of a specimen (e.g., a coagulation reaction curve) are obtained, and the coagulation time is calculated based on this data. Specimens with coagulation times longer than normal values ​​(e.g., a baseline value determined based on the coagulation time of a normal specimen group) can be selected as specimens with prolonged coagulation time and used as subjects in the method of the present invention. Examples of the calculated coagulation times include prothrombin time (PT) and activated partial thromboplastin time (APTT), but are not limited to these.

[0238] In the method of this invention, the coagulation reaction curves of the test sample and a mixture of the test sample and a normal sample are used. The normal sample refers to a normal blood sample that does not exhibit prolonged coagulation time. As the normal sample, blood samples from individuals without coagulation abnormalities, for example, blood samples taken from healthy individuals, preferably plasma, mixtures thereof, commercially available normal plasma, etc., can be used. In the preparation of the mixture, the test sample and the normal sample are mixed at a predetermined ratio. The mixing ratio of the test sample to the normal sample is only required to be in the range of 1:9 to 9:1 in volumetric proportions.

[0239] In this instruction manual, the tested specimen is sometimes referred to as specimen S, the normal specimen as specimen N, and the mixed specimen as specimen M. In addition, the tested specimen and the mixed specimen, or the tested specimen, the normal specimen and the mixed specimen, are sometimes collectively referred to as "scrutiny".

[0240] 2. Measurement of solidification reaction

[0241] The coagulation reaction of the tested specimens and mixed specimens is measured. Coagulation reaction curves for each specimen can be obtained based on the time-series data of the coagulation reaction obtained in this measurement. This coagulation reaction measurement can be performed following the usual procedures for coagulation reaction measurements such as prothrombin time (PT), activated partial thromboplastin time (APTT), or fibrinogen concentration (Fbg). In the following description, the method of the present invention is primarily based on coagulation reaction measurement using APTT. Those skilled in the art can modify the method of the present invention to other measurement methods (e.g., coagulation reaction measurement using PT).

[0242] In the measurement of coagulation reaction, a coagulation time assay reagent is added to the above-mentioned sample to induce a coagulation reaction. The coagulation reaction of the mixture (reaction solution) after reagent addition can be measured. The reagent used can be arbitrarily selected according to the purpose of the measurement. Reagents for measuring various coagulation times are commercially available (e.g., APTT reagent Coagpia APTT-N; manufactured by Sekisui Medical Co., Ltd.). The measurement of coagulation reaction can be performed using conventional methods, such as optical methods that measure the amount of scattered light, transmittance, absorbance, etc., or mechanical methods that measure plasma viscosity. In the following specification, the method of the present invention will be described using the measurement of coagulation reaction based on the amount of scattered light as an example.

[0243] The start time for coagulation reaction measurement is typically the moment when the reagent is mixed with the sample and the coagulation reaction is initiated, but other time points can also be specified as the start time for coagulation reaction measurement. The duration of continuous measurement of the coagulation reaction can be, for example, from tens of seconds to approximately 10 minutes from the moment the sample and reagent are mixed. This measurement time can be any fixed time, or it can be until the end of the coagulation reaction of each sample is detected. The progress of the coagulation reaction can be measured repeatedly at specified intervals during this measurement time (photometry in the case of optical detection). For example, measurements can be taken at 0.1-second intervals. The temperature of the mixture used in the measurement is the usual condition for coagulation reaction, for example, 30°C to 40°C, preferably 35°C to 39°C. Furthermore, various measurement conditions can be appropriately set according to the sample, reagent, measurement method, etc.

[0244] Unlike existing methods such as cross-mixing tests or those described in Patent Document 1, the method of the present invention does not require further preparation of specimens subjected to prolonged incubation. For example, in the present invention, during coagulation reaction measurement, the prolonged incubation required in the delayed reaction of conventional cross-mixing tests is not required before the addition of the coagulation time measuring reagent (e.g., the first reagent for APTT measurement). In this specification, "prolonged incubation" of the specimen refers to an incubation at a temperature preferably 15°C to 40°C, more preferably 30°C to 40°C, and preferably for 2 minutes or more, more preferably 5 minutes or more. Furthermore, in this specification, "incubation" of the specimen refers to active temperature control performed by a heater, cooler, thermostat, etc., and does not include passive temperature changes or maintenance such as placement at room temperature.

[0245] On the other hand, in this invention, the specimen for coagulation reaction measurement can undergo a preheating treatment, typically used for coagulation reaction measurement, before the addition of the coagulation time determination reagent, such as a heating treatment at 30°C to 40°C for less than 1 minute. Therefore, the specimen for coagulation reaction measurement in this invention is one that has not undergone an incubation treatment at 15°C to 40°C for more than 5 minutes before the addition of the coagulation time determination reagent, preferably one that has not undergone an incubation treatment at 15°C to 40°C for more than 2 minutes, and more preferably a sample that has undergone an incubation treatment at 15°C to 40°C for less than 1 minute. The specimen for coagulation reaction measurement in this invention can undergo a heating process, typically used in coagulation reaction measurement, after the addition of the coagulation time determination reagent (prepared as a reaction solution), such as maintaining the reaction solution at 30°C to 40°C.

[0246] The series of operations in the coagulation reaction measurement described above can be performed using an automated analytical device. An example of such an automated analytical device is the CP3000 coagulation automated analyzer (manufactured by Sekisui Medical Co., Ltd.). Alternatively, some operations can be performed manually. For example, the sample can be prepared manually, with subsequent operations performed using an automated analytical device.

[0247] 3. Obtaining the solidification reaction curve

[0248] The coagulation reaction curve R(i) of the specimen is obtained based on the above coagulation reaction measurement. Here, i represents the time or number of measurement points from the start of the coagulation reaction measurement of the specimen (hereinafter, they are also referred to as "time" or "number of measurement points," respectively). For example, if the measurement (photometric) interval is 0.1 seconds, it is represented by time = 0.1 × number of measurement points. That is, R(i) can be a function of the number of measurement points or a function of time. In the following specification, R(i) is sometimes abbreviated as R. Generally speaking, the coagulation reaction curve R is obtained by using known methods to remove noise or smooth the measured values ​​of the coagulation reaction measurement, or by zero-point adjustment or relative value conversion of the curve for adjusting the initial value of the coagulation reaction measurement as needed. An example of the coagulation reaction curve R is shown in Figure 1. The horizontal axis of Figure 1 represents time, and the vertical axis represents the amount of scattered light. The coagulation reaction curve R rises due to the increase in the amount of scattered light as the coagulation reaction of the reaction liquid progresses over time, and reaches a plateau as the coagulation reaction nears its end, with the entire process exhibiting an S-shape.

[0249] The derivative, such as the first-order derivative curve, of the solidification reaction curve R can be obtained from the calculated solidification reaction curve R. The derivative of the solidification reaction curve can be performed using any method, for example, by calculating the average slope value over the interval. In this specification, the first-order derivative curve of the solidification reaction curve R is also referred to as the "solidification rate curve," denoted by V(i) (sometimes abbreviated as V).

[0250] 4. Calculation of setting time

[0251] The clotting time of the specimen can be calculated based on R(i) or V(i). The clotting time can be calculated using any method. Examples of methods for calculating solidification time include: calculating the time when R(i) reaches N% of the solidification reaction endpoint E (described later); calculating the time when V(i) reaches N% of its maximum value; using the time when the ratio of the cumulative values ​​of R(i) within a small time period reaches a predetermined value as the starting point Te and calculating the time when R(i) reaches N% of R(Te) as the solidification time (Japanese Patent Application Publication No. 6-249855); calculating the solidification time based on the time-varying cumulative value of R(i) within a small time period (refer to WO2021 / 132552); calculating the solidification time based on the weighted average time of V(i) (refer to WO2021 / 177452); and using the time when V(i) reaches its maximum value and then reaches a predetermined value as the starting point Te and calculating the time when R(i) reaches N% of R(Te) as the solidification time (refer to WO2021 / 206107), etc.

[0252] When the calculated coagulation time of a specimen is longer than the normal value (e.g., a standard value determined based on the coagulation time of a normal specimen group), it is determined that the coagulation time of the specimen is prolonged. The specimen with prolonged coagulation time can be used as the test specimen (specimen S) in the method of the present invention. In the method of the present invention, when it is necessary to infer the factors (or prolongation factors) of the coagulation abnormality of the test specimen with prolonged coagulation time, the test specimen is used as specimen S. A mixed specimen (specimen M) can be prepared from specimen S and a normal specimen (specimen N), and the coagulation reaction curves of specimen S and specimen M are obtained.

[0253] 5. Obtaining T(X)

[0254] 5-1) End point of solidification reaction E

[0255] In one embodiment of the method of the present invention, a measurement point or time T(X) representing X% of the coagulation reaction curve R(i) of the specimen reaching the coagulation reaction endpoint E is obtained. The coagulation reaction endpoint E in the coagulation reaction curve R of the specimen is detected. The coagulation reaction endpoint E can be detected based on any criterion such as the time when R(i) reaches a plateau, the time when V(i) decreases to 0 or a certain value after reaching a peak (see WO2021 / 206107), or the earliest point where the ratio of the cumulative values ​​of R(i) within a small time period is less than a threshold (e.g., 1.001) (see WO2021 / 132552 or the embodiments described later). The detection of the coagulation reaction endpoint E can be performed after obtaining R up to a predetermined measurement time, or the detection of E can be performed in parallel with the acquisition of R and the acquisition of R can end at the time E is detected. For example, the steps described in WO2021 / 206107 or WO2021 / 132552 can be performed in parallel with the acquisition of R (so-called real-time E detection).

[0256] As an embodiment of the method of the present invention, an example of the detection step of the solidification reaction endpoint E based on the method described in WO2021 / 132552 will be described in detail. The ratio of the cumulative values ​​of R(i) within a small time period is defined as the cumulative ratio Z(i), which is calculated using the following formula:

[0257] Z(i)={R(i+1)+R(i+2)+…+R(i+m-1)+R(i+m)} / {R(i-m)+R(i-m+1)+…+R(i-2)+R(i-1)}

[0258] In the above formula, i represents the measurement point number, and m can be appropriately set according to the measurement conditions of the solidification reaction, the analytical items, etc. For example, when the measurement interval is 0.1 seconds, m = 10 to 30. The earliest measurement point or time at which Z(i) is detected to be less than the threshold Zs is taken as the end point E of the solidification reaction. Zs can be appropriately set according to the analytical items, and is greater than 1 and less than 1.100. For example, in the case of APTT measurement, it is preferably less than 1.050, and more preferably Zs is in the range of 1.010 to 1.001. In order to prevent false detection of E due to abnormalities in the initial reaction, Z(i) is preferably calculated after i reaches the specified calculation start point and R(i) becomes above the specified value. In this step, R(i) can be obtained and Z(i) can be calculated in parallel while measuring the solidification reaction to detect E.

[0259] In the method of this invention, the coagulation reaction endpoint E in the coagulation reaction curves of specimen S and specimen M can be obtained respectively. Hereinafter, the coagulation reaction endpoint in the coagulation reaction curve of specimen S will be referred to as Es, and the coagulation reaction endpoint in the coagulation reaction curve of specimen M will be referred to as Em.

[0260] 5-2) Ts(X) and Tm(X)

[0261] T(X) can be obtained for specimens S and M respectively. In this specification, R(i) and T(X) for specimen S are referred to as Rs(i) (or simply Rs) and Ts(X), respectively, and R(i) and T(X) for specimen M are referred to as Rm(i) (or simply Rm) and Tm(X), respectively. More specifically, Ts(X) represents the measurement point or time at which the coagulation reaction curve Rs of specimen S reaches X% of Es, and Tm(X) represents the measurement point or time at which the coagulation reaction curve Rm of specimen M reaches X% of Em. On the other hand, T(X) in this specification is used as a general term that includes Ts(X) and Tm(X), and the description of T(X) can also be applied to Ts(X) and Tm(X) unless otherwise specified. In this specification and accompanying drawings, T(X), Ts(X) and Tm(X) are sometimes represented by parentheses of X, or sometimes the parentheses are removed and written with X as "TX", "TsX" and "TmX" respectively, or sometimes they are written as "T", "Ts" and "Tm" respectively.

[0262] More specifically, T(X) is the number of measurement points or time from the start of the solidification reaction measurement (time 0) to when R reaches X% of E. That is, for T(X), the following equation (I) holds.

[0263] R(T(X))=E×X%(I)

[0264] Therefore, for Ts(X) and Tm(X), the following equations (Ia) and (Ib) hold true respectively.

[0265] Rs(Ts(X))=Es×X%(Ia)

[0266] Rm(Tm(X))=Em×X%(Ib)

[0267] Here, Rs and Rm are the coagulation reaction curves of specimen S and specimen M, respectively, and 0 < X ​​≤ 100. Therefore, T(X) can be expressed as a function of the variable X. In the method of the present invention, X is preferably 1 to 100. For example, each X can be a value that differs by more than 0.1 (i.e., the increment of X is ≥ 0.1), or it can be a value that differs by more than 5 (i.e., the increment of X is ≥ 5). In one example, X is a variable that varies in increment 1 within the range of 1 to 100 (i.e., X = {1, 2, 3, ..., 97, 98, 99, 100}). In another example, X is a variable that varies in increment 3 within the range of 3 to 99 (i.e., X = {3, 6, 9, ..., 96, 99}). In yet another example, X is a variable that varies in increment 3 within the range of 1 to 97 (i.e., X = {1, 4, 7, ..., 94, 97}). In another example, X is a variable ranging from 5 to 100 and changing in increments of 5 (i.e., X = {5, 10, 15, ..., 90, 95, 100}). In yet another example, X can be a variable ranging from 0.5 to 100 and changing in increments of 0.5 (i.e., X = {0.5, 1.0, 1.5, 2.0, ..., 98.5, 99.0, 99.5, 100.0}).

[0268] Alternatively, T(X) can be expressed as the average value from T(X-K) to T(X+K). In this case, K is preferably less than the increment of X mentioned above. For example, if K=2, then T(X) can be expressed as the average value from T(8) to T(12) when X=10, or as the average value from T(13) to T(17) when X=15. Or, T(X) can be expressed as the average value from T(X-K+1) to T(X). For example, if K=5, then T(X) can be expressed as the average value from T(6) to T(10) when X=10, or as the average value from T(91) to T(95) when X=95.

[0269] Figure 2A is a diagram illustrating T(X). The figure shows the solidification reaction curves representing the points from 10% to 90% of E, with the times when 10%, 50%, and 90% of E are represented as T(10), T(50), and T(90), respectively. The time tE of E corresponds to T(100).

[0270] To calculate the parameters used to infer the solidification anomalies described later, Ts(X) and Tm(X) can be further relativeized. When the relativeized Ts(X) and Tm(X) are expressed as rTs(X) and rTm(X) respectively, rTs(X) and rTm(X) are preferably defined by the following equations (IIa) and (IIb).

[0271] rTs(X)=Ts(X) / Ts(As)×Qs(IIa)

[0272] rTm(X)=Tm(X) / Tm(Am)×Qm(IIb)

[0273] Here, As and Am only need to be independently within the range of the values ​​of X mentioned above, preferably 3 to 97, more preferably 40 to 60 (e.g., 50). Qs > 0, Qm > 0. Qs and Qm are coefficients used to adjust the size of the parameters, and can be selected from the range of 1 to 100, for example. In addition, Qs and Qm do not need to be the same value.

[0274] The method of the present invention may include the following steps: calculating the above-mentioned Ts(X) or rTs(X) and Tm(X) or rTm(X) based on the coagulation reaction of specimen S and specimen M. Alternatively, the above-mentioned Ts(X), Tm(X), rTs(X) or rTm(X) used in the method of the present invention may be pre-calculated or stored data.

[0275] 6. Obtaining the function of V(i)

[0276] 6-1) vT1(X) and vT2(X)

[0277] In one embodiment of the method of the present invention, the point at which the coagulation rate curve V(i) of the specimen reaches a predetermined value is obtained. This predetermined value can be determined as X% (0 < X ​​≤ 100) of the maximum value Vmax of V(i). Since V(i) is a curve with Vmax as its peak value, the points at which V(i) reaches this predetermined value are detected before and after the peak value. In this specification, the measurement point or time at which V(i) reaches Vmax is referred to as VmaxT. The measurement point or time at which V(i) reaches X% of Vmax before VmaxT is defined as vT1(X), and the measurement point or time at which V(i) reaches X% of Vmax after VmaxT is defined as vT2(X). Therefore, vT1(X) ≤ vT2(X), where, when X = 100, vT1(X) = vT2(X) = VmaxT. In this specification, vT1(X) and vT2(X) are sometimes combined and represented as vT(X). Figure 2B is a graph showing the changes in T(X) and vT(X) relative to X. In the figure, vT(X) is represented by vT1(X) and vT2(X).

[0278] In the method of this invention, vT1(X) and vT2(X) can be obtained from specimen S and specimen M, respectively. In this specification, V(i), Vmax, VmaxT, vT1(X), and vT2(X) of specimen S are referred to as Vs(i), Vmax_s, VmaxT_s, vT1s(X), and vT2s(X), respectively; and V(i), Vmax, VmaxT, vT1(X), and vT2(X) of specimen M are referred to as Vm(i), Vmax_m, VmaxT_m, vT1m(X), and vT2m(X), respectively. Additionally, sometimes vT1s(X) and vT2s(X) are collectively referred to as vTs(X), and vT1m(X) and vT2m(X) are collectively represented as vTm(X). In addition, in this specification and the accompanying drawings, vT1s(X), vT2s(X), vT1m(X) and vT2m(X) are sometimes written as “vT1sX”, “vT2sX”, “vT1mX” and “vT2mX” respectively, with the parentheses of X removed; and sometimes the parentheses and X are removed and written as “vT1s”, “vT2s”, “vT1m” and “vT2m” respectively.

[0279] Therefore, for vT1s(X), vT2s(X), vT1m(X), and vT2m(X), the following equations (Xa) to (Xd) hold respectively.

[0280] Vs (vT1s (X)) = Vmax_s × X% (Xa)

[0281] (where vT1s(X)≤VmaxT_s)

[0282] Vs (vT2s (X)) = Vmax_s × X% (Xb)

[0283] (where vT2s(X)≥VmaxT_s)

[0284] Vm (vT1m (X)) = Vmax_m × X% (Xc)

[0285] (where vT1m(X)≤VmaxT_m)

[0286] Vm (vT2m (X)) = Vmax_m × X% (Xd)

[0287] (where vT2m(X)≥VmaxT_m)

[0288] That is, vT1s(X) and vT2s(X) represent the measurement points or times at which Vs(i) reaches X% of Vmax_s before and after VmaxT_s, respectively, and vT1m(X) and vT2m(X) represent the measurement points or times at which Vm(i) reaches X% of Vmax_m before and after VmaxT_m, respectively. In equations (Xa) to (Xd), 0 < X ​​≤ 100.

[0289] In the calculation of vT1(X) and vT2(X) above, when V(i) exhibits a bimodal characteristic, sometimes multiple points where V(i) is Vmax×X% are detected after VmaxT. In this case, the largest point among the detected points can be selected as vT2(X). Similarly, when multiple points where (i) is Vmax×X% are detected before VmaxT, the smallest point among the detected points (excluding points in the initial noise) can be selected as vT1(X). Alternatively, sometimes V(i) < (Vmax×X%) < V(i+1) or V(i-1) < (Vmax×X%) < V(i). In this case, i can be selected as the number of measurement points or time that satisfies V(i) = Vmax×X%.

[0290] 6-2)C(i)

[0291] Sometimes V(i) is corrected based on the solidification reaction endpoint E. In this specification, the V(i) after this correction is called the corrected V(i), denoted as C(i). For example, the corrected Vs(i) and corrected Vm(i) are denoted as Cs(i) and Cm(i) respectively, and can be calculated according to the following formula.

[0292] Cs(i)=Vs(i)×(Q / Es) (XIa)

[0293] Cm(i)=Vm(i)×(Q / Em) (XIb)

[0294] (In each formula, Q > 0)

[0295] It should be noted that in the calculation of vT1s(X), vT2s(X), vT1m(X) and vT2m(X) above, these C(i) (Cs(i) and Cm(i)) can be used instead of Vs(i) and Vm(i).

[0296] In this specification, the maximum values ​​of Cs(i) and Cm(i) are represented as Cmax_s and Cmax_m, respectively. Furthermore, the reciprocals of Cs(i) and Cm(i) are represented as iCs(i) and iCm(i), respectively, iCs(X) = 1 / Cs(X) and iCm(X) = 1 / Cm(X). Similarly, the reciprocals of Cmax_s and Cmax_m are represented as iCmax_s and iCmax_m, respectively, iCmax_s = 1 / Cmax_s and iCmax_m = 1 / Cmax_m.

[0297] 7. Calculation of parameters Ps and Pm

[0298] In the method of the present invention, parameters Ps related to the coagulation reaction of specimen S and parameters Pm related to the coagulation reaction of specimen M are calculated.

[0299] In one embodiment, Ps is a parameter calculated based on Ts(X), preferably based on rTs(X). In another embodiment, Ps is a parameter calculated based on vTs(X) (vT1s(X) and / or vT2s(X)). Preferably, at least two Ts(X) or vTs(X) under at least two X (e.g., X1 and As, or X1 and X2) are used in the calculation of Ps.

[0300] In one embodiment, Pm is a parameter calculated based on Tm(X), preferably based on rTm(X). In another embodiment, Pm is a parameter calculated based on vTm(X) (vT1m(X) and / or vT2m(X)). In yet another embodiment, Pm is a parameter calculated based on vT2m(X). Preferably, at least two Tm(X) or vTm(X) under at least two X (e.g., X1 and Am, or X1 and X2) are used in the calculation of Pm.

[0301] 7-1) Ps

[0302] In one implementation, Ps is rTs(X1) as defined by the following formula (IIa').

[0303] rTs(X1)=Ts(X1) / Ts(As)×Qs(IIa')

[0304] Here, it is preferred that X1 = 35 to 95, As = 3 to 75, where X1 > As, and preferably (X1 - As) = 5 to 92, and Qs as described above.

[0305] In one implementation, Ps is rTs(X2) - rTs(X1) as defined by the following formula (IIIa').

[0306] rTs(X2) - rTs(X1)

[0307] (={Ts(X2)-Ts(X1)} / Ts(As)×Qs) (IIIa')

[0308] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 2 to 80, X2 = 50 to 95, and (X2 - X1) = 5 to 93. As and Qs are as described above.

[0309] In one implementation, Ps is the ratio of the difference between rTs(X1) and rTs(X2) to the difference between X1 and X2, as defined by the following formula (IVa').

[0310] arTs(X1, X2)

[0311] ={rTs(X2)-rTs(X1)} / (X2-X1)

[0312] =[{Ts(X2)-Ts(X1)} / Ts(As)×Qs] / (X2-X1) (IVa')

[0313] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2. Preferably, X1 = 1 to 85, X2 = 25 to 95, (X2 - X1) = 5 to 94, and As = 5 to 95. More preferably, X1 = 4 to 80, X2 = 55 to 95, and X2 - X1 = 5 to 91. Qs is as described above.

[0314] In one implementation, Ps is the slope of the regression line of rTs(X) within a specified interval of X. The slope of the regression line of rTs(X) within the interval from X1 to X2 is denoted as rSLs[X1:X2], and is calculated using the following formula (Va').

[0315]

[0316] Here, UX is the average value of X from X1 to X2, and UrTs is the average value of rTs(X) from rTs(X1) to rTs(X2). As used to calculate rTs is 5 to 95. X1 and X2 only need to be independently within the range of the aforementioned X values, where X1 < X2. Preferably, X1 = 1 to 85, X2 = 25 to 100, and (X2 - X1) = 5 to 99. More preferably, X1 = 1 to 80, X2 = 55 to 100, and (X2 - X1) = 5 to 99. Qs is as described above.

[0317] In one implementation, Ps is the weighted average of X over a specified interval of Ts(X). The weighted average of X over the interval from Ts(X1) to Ts(X2) is denoted as wXs[X1:X2] and is calculated using the following formula (VIa).

[0318]

[0319] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 1 to 75, X2 = 30 to 100, and (X2 - X1) = 10 to 99. Qs is as described above.

[0320] In one embodiment, Ps is the ratio of vT(X), which is vT2s(X1) / vT1s(X2). Here, it is preferred that X1 = 3 to 58 and X2 = 13 to 98. In another embodiment, Ps is vT2s(X1) / vT2s(X2). Here, it is preferred that X1 = 13 to 63 and X2 = 58 to 83 (where X2 > X1).

[0321] In one embodiment, Ps is the ratio of the difference with respect to vT(X), which is (vT2s(X1) - vT1s(X2)) / vT2s(Bs). Here, preferably X1 = 3 to 58, X2 = 13 to 98, and Bs = 3 to 100. More preferably, X1 = 13 to 63 and X2 = 58 to 83.

[0322] In another embodiment, Ps is (vT2s(X1) - vT2s(X2)) / vT2s(Bs). Here, it is preferred that X1 = 8 to 58, X2 = 48 to 83, where X2 > X1, and Bs = 3 to 100. More preferably, X1 = 8 to 58, X2 = 68 to 83.

[0323] In another embodiment, Ps is (vT2s(X1) - vT2s(X2)) / vT1s(Bs). Here, it is preferred that X1 = 8 to 58, X2 = 48 to 98, where X2 > X1, and Bs = 3 to 100. More preferably, X2 = 38 to 83.

[0324] In another embodiment, Ps is (vT2s(X1) - vT1s(X2)) / vT1s(Bs). Here, it is preferred that X1 = 3 to 58, X2 = 3 to 93, and Bs = 3 to 100. More preferably, X2 = 78 to 83.

[0325] 7-2) Pm

[0326] In one implementation, Pm is Tm(X1), where X1 only needs to be within the range of the values ​​of X mentioned above, for example, X1 = 90 to 100.

[0327] In one implementation, Pm is the ratio of Tm(X) and is defined by the following formula (IIb') rTm(X1).

[0328] rTm (X1) = Tm (X1) / Tm (Am) × Qm (IIb')

[0329] Here, it is preferred that X1 = 30 to 95, Am = 1 to 35, where X1 > Am, and preferably (X1 - Am) = 10 to 94, and Qm as described above.

[0330] In one implementation, Pm is the ratio of the difference between Tm(X1) and Tm(X2) to the difference between X1 and X2, which is aTm(X1, X2) as defined by the following formula (IVb).

[0331] aTm(X1, X2)

[0332] ={Tm(X2)-Tm(X1)} / (X2-X1)× Qm(IVb)

[0333] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 1 to 90, X2 = 25 to 100, and X2 - X1 = 5 to 99. Qm is as described above.

[0334] In one implementation, Pm is the slope of the regression line of Tm(X) within a specified interval of X. The slope of the regression line of Tm(X) within the interval from X1 to X2 is denoted as SLm[X1:X2] and is calculated using the following formula (Vb).

[0335]

[0336] Here, UX is the average value of X from X1 to X2, and UTm is the average value of Tm(X) from Tm(X1) to Tm(X2). X1 and X2 only need to be independently within the range of the above-mentioned values ​​of X, where X1 < X2, preferably X1 = 1 to 95, X2 = 30 to 100, and (X2 - X1) = 5 to 99. Qm is as described above.

[0337] In one implementation, Pm is the slope of the regression line of rTm(X) within a specified interval of X. The slope of the regression line of rTm(X) within the interval from X1 to X2 is denoted as rSLm[X1:X2], and is calculated using the following formula (Vb').

[0338]

[0339] Here, UX is the average value of X from X1 to X2, and UrTm is the average value of rTm(X) from rTm(X1) to rTm(X2). X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 1 to 70, X2 = 25 to 100, (X2 - X1) = 5 to 99, more preferably X1 = 1 to 55, X2 = 25 to 95, (X2 - X1) = 5 to 94. rTm is as described above, and Am used to calculate rTm is as described above, preferably Am = 5 to 95. Qm is as described above.

[0340] In one implementation, Pm is the weighted average of X over a specified interval of Tm(X). The weighted average of X over the interval from Tm(X1) to Tm(X2) is denoted as wXm[X1:X2] and is calculated using the following formula (VIb).

[0341]

[0342] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 1 to 45, X2 = 30 to 100, and (X2 - X1) = 10 to 99. Qm is as described above.

[0343] In one implementation, Pm is the weighted average of Tm(X) over a specified interval of X. The weighted average of Tm(X) over the interval from X1 to X2 is denoted as wTm[X1:X2], and is calculated using the following equation (VIIb).

[0344]

[0345] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 75~95, X2 = 95~100, and (X2 - X1) = 5~25. Qm is as described above.

[0346] In one implementation, Pm is the weighted average of rTm(X) over a specified interval of X. The weighted average of rTm(X) over the interval from X1 to X2 is denoted as wrTm[X1:X2] and is calculated using the following formula (VIIb').

[0347]

[0348] Here, X1 and X2 only need to be independently within the range of the values ​​of X mentioned above, where X1 < X2, preferably X1 = 1 to 90, X2 = 30 to 100, (X2 - X1) = 5 to 99, more preferably X1 = 15 to 75, X2 = 50 to 85, (X2 - X1) = 5 to 65. rTm is as described above, preferably Am = 3 to 34, and Qm is as described above.

[0349] In one embodiment, Pm is vT2m(X1). Here, X1 is preferably 3 to 18. In one embodiment, Pm is vT2m(X1) / vT1m(X2). Here, X1 is preferably 3 to 93, and X2 is preferably 3 to 88. In one embodiment, Pm is vT2m(X1) / vT2m(X2). Here, X1 is preferably 38 to 63, X2 is preferably 63 to 93, and X2 > X1.

[0350] In one embodiment, Pm is the ratio of the difference between Pm and vT(X), which is (vT2m(X1) - vT1m(X2)) / vT2m(Bm). Here, it is preferred that X1 = 3 to 93, X2 = 3 to 88, and Bm = 3 to 100. More preferably, X1 = 18 to 93 and X2 = 3 to 78.

[0351] In another embodiment, Pm is (vT2m(X1) - vT2m(X2)) / vT2m(Bm). Here, it is preferred that X1 = 28 to 63, X2 = 68 to 93, where X2 > X1, and Bm = 3 to 100. More preferably, X1 = 43 to 58, X2 = 63 to 68.

[0352] In another embodiment, Pm is (vT2m(X1) - vT1m(X2)) / vT1m(Bm). Here, it is preferred that X1 = 3 to 93, X2 = 3 to 93, and Bm = 3 to 100. More preferably, X1 = 18 to 93 and X2 = 3 to 78.

[0353] In another embodiment, Pm is (vT2m(X1) - vT2m(X2)) / vT1m(Bm). Here, preferably X1 = 23 to 63, X2 = 63 to 93, where X2 > X1, and Bm = 3 to 100. More preferably, X1 = 43 to 58, X2 = 63 to 68.

[0354] In one embodiment, Pm is the reciprocal of Cm(X1) iCm(X1), where X1 = 100. Therefore, a preferred example of iCm(X1) used in Pm is iCmax_m. In another embodiment, Pm is the reciprocal of the weighted average of Cm(X) in the interval X = X1 to X2, denoted as iwCm[X1:X2], and calculated using the following formula (XIIb).

[0355]

[0356] Here, X1 and X2 satisfy Cm(X1) = Cm(X2) = Vm(i) × [Q / (Em × S%)] (S = 3 ~ 100), X1 < VmaxT_m < X2. Qm is as described above. iwCm[X1:X2] is sometimes represented as iwCm[S].

[0357] 8. Inference of factors contributing to solidification anomalies using Ps and Pm

[0358] In the method of the present invention, parameters concerning specimens S and M, namely Ps and Pm as described above, are used to infer factors (or prolongation factors) of coagulation abnormality of specimen S.

[0359] Factors indicating coagulation abnormalities that can be inferred by the method of the present invention include coagulation factor inhibitor positivity (Inh), lupus anticoagulant positivity (LA), and coagulation factor deficiency (Def). Coagulation factor deficiency (Def) includes deficiency of coagulation factor V (FV), coagulation factor VIII (FVIII), coagulation factor IX (FIX), coagulation factor X (FX), coagulation factor XI (FXI), and coagulation factor XII (FXII). Preferably, coagulation factor deficiency refers to a state where the activity value (IU / dL) of a coagulation factor (e.g., FVIII) is less than about 40% of the normal value. Examples of coagulation factor inhibitors include FVIII inhibitors and FIX inhibitors. Preferably, inhibitor positivity refers to an inhibitor titer of 0.6 BU / mL or higher. It should be noted that in this specification, inhibitor titer is sometimes simply referred to as "potency," and the inhibitor titer in this specification is expressed in Bethesda units (BU / mL).

[0360] As illustrated in the examples described later, Ts(X) can exhibit different characteristics depending on factors contributing to abnormal coagulation of the specimen (the type of specimen). Figure 3A shows Ts(X) for each specimen type at an APTT of approximately 100 seconds. The solid line represents LA, the dashed line represents dFVIII (FVIII activity: less than 1%) as an example of Def, and the dotted line represents iFVIII (FVIII inhibitor positive, titer: 0.8) as an example of Inh. APTT is Ts(X) at X = 50, showing that all three specimen types are around 100 seconds. When comparing the three specimen types relatively, LA is higher when X < 50, and LA, dFVIII, and iFVIII increase sequentially when X > 50. The slope in the range of X = approximately 10 to approximately 90 also shows that LA, dFVIII, and iFVIII increase sequentially. Based on these characteristics, it is shown that even for specimens with the same degree of APTT prolongation (approximately 100 seconds), Ts(X) varies significantly depending on the specimen type. The slope of Ts(X) is the largest for iFVIII, and the difference in Ts(X) increases with X > 50, reaching its maximum at X = 100 for iFVIII. That is, the Ts(X) of Inh indicates that the coagulation reaction of Inh is weaker than that of LA and Def. Therefore, based on the Ts(X) of specimen S showing coagulation abnormalities (prolonged coagulation time), it can be inferred whether the coagulation abnormality of specimen S is due to Inh or other factors (LA or Def). As an example, as shown in Figure 3A, with X as the horizontal axis and Ts(X) as the vertical axis, since the slope of Ts(X) for Inh is greater than that for LA and Def, Inh can be distinguished by using the index Ps of Ts(X) within a specified range of X, which quantifies the characteristics of such Ts(X), as a parameter.

[0361] Furthermore, Tm(X) can also exhibit different characteristics depending on the factors causing abnormal coagulation of the specimen. Figure 3B shows the Tm(X) for each specimen type in a 1:1 mixture of the tested specimen and normal specimen (NP), with the solid, dashed, and dotted lines being the same as in Figure 3A. The Tm(X) for the mixture of dFVIII (Def) and iFVIII (Inh) specimens shows a smaller increase with increasing X because the coagulation reaction is closer to that of normal specimens due to the correction effect from the coagulation factor from the normal specimen. On the other hand, in the mixture from LA, the phospholipids from the NP mixed with specimen S for preparing the mixture immediately bind to the autoantibodies against phospholipids contained in specimen S, thus inhibiting the coagulation reaction. As a result, Tm(X) increases significantly with increasing X. That is, the Tm(X) for LA contains data showing a greater intensity of inhibition of the coagulation reaction of LA than that of Def and Inh. However, it is important to note that even with high-valence Inh, the solidification reaction is suppressed, causing Tm(X) to increase. However, if Inh can be distinguished based on Ts as described above, then it is only necessary to distinguish between Def and LA. Therefore, Tm(X) can be used to distinguish between LA and Def. As an example, as shown in Figure 3B, with X as the horizontal axis and Tm(X) as the vertical axis, the slope of Tm(X) for LA is greater than that for Def. Therefore, by using the index Pm of Tm(X) within a specified range based on X, which quantifies the characteristics of such Tm(X), as a parameter, LA can be distinguished.

[0362] As described above, in this invention, the presence or absence of a specimen S is first inferred based on its Ts(X). Next, specimens S that are not inferred to be Inh can be inferred to be LA or Def based on Tm(X).

[0363] More specifically, in this invention, the aforementioned Ps and Pm are used to infer factors contributing to the coagulation abnormalities of the specimen S. More specifically, firstly, it is inferred whether the specimen S is Inh based on Ps, and then specimens S not inferred as Inh are inferred as LA or Def based on Pm, or Ps and Pm.

[0364] In one implementation, Ps is the above-mentioned rTs(X1), rTs(X2)-rTs(X1), arTs(X1, X2), rSLs[X1:X2], wXs[X1:X2], vT2s(X1) / vT1s(X2), vT2s(X1) / vT2s(X2), (vT2s(X1)-vT1s(X2)) / vT2s(Bs), (vT2s(X1)-vT1s(X2)) / vT2s(Bs), (vT2s(X1)-vT1s(X2)) / vT2s(Bs), (vT2s(X1)-vT1s(X2)) / vT1s(Bs), (vT2s(X1)-vT1s(X2)) / vT2s(Bs), or (vT2s(X1)-vT2s(X2)) / vT1s(Bs). If Ps is a threshold or greater than the threshold, then the specimen S is inferred to be Inh. In this specification, the threshold for Ps is set as Ls. The threshold Ls can be predetermined before the method of the present invention is implemented. Ls can be determined based on Ps calculated from a population of specimens for which factors of coagulation anomalies are known. Ls can be set for each Ps, namely rTs(X1), rTs(X2) - rTs(X1), arTs(X1, X2), rSLs[X1:X2], wXs[X1:X2], vT2s(X1) / vT1s(X2), vT2s(X1) / vT2s(X2), (vT2s(X1) - vT1s(X2)) / vT2s(Bs), (vT2s(X1) - vT1s(X2)) / vT1s(Bs), (vT2s(X1) - vT2s(X2)) / vT2s(Bs), and (vT2s(X1) - vT2s(X2)) / vT1s(Bs). On the other hand, when the Ps of the specimen S is Ls or not greater than it, the specimen S is not inferred to be Inh, but is inferred to be LA or Def in the inference process based on Pm described later.

[0365] Two thresholds, Ls1 and Ls2, related to Ps can be set as needed, where Ls1 > Ls2. For example, if the Ps of specimen S is Ls1 or greater, specimen S is inferred as Inh. On the other hand, if the Ps of specimen S is Ls2 or less, specimen S is not inferred as Inh, and in the Pm-based inference process described later, it is inferred as LA or Def.

[0366] In one example, the Ps of each specimen are calculated from the Inh specimen population, and Ls are set based on their statistical values ​​(e.g., mean - [k × standard deviation], k preferably 2 to 5). In another example, the Ps of each specimen are calculated from the Inh specimen population, and their minimum value × k (k preferably 0 < k < 100%) is set as Ls. In yet another example, the Ps of Inh specimens with potency exceeding the cutoff value (0.6 BU / mL) but near 1 BU / mL are calculated, and this value × k (k preferably 0 < k ≤ 100%) is set as Ls. It should be noted that these Ls can be set as Ls1 when thresholds Ls1 and Ls2 are set.

[0367] In another example, Ps for each specimen is calculated from a population of specimens with a non-Inh prolongation factor (e.g., a population of LA and / or Def specimens), and Ls is set based on their statistical values ​​(e.g., mean + [k × standard deviation], k preferably 2 to 5). In another example, Ps for each specimen is calculated from a population of specimens with a non-Inh prolongation factor (e.g., a population of LA and Def specimens), and Ls is set based on their maximum value × k (k preferably 1 < k). In another example, Ps for each specimen is calculated from a population of normal specimens (NP), and Ls is set based on their statistical values ​​(e.g., mean + [k × standard deviation], k preferably 2 to 5). It should be noted that these Ls can be set as Ls2 when thresholds Ls1 and Ls2 are set. Preferably, Ls2 is set based on the Ps of each specimen in the LA and Def or NP specimen population according to the above criteria. In another example, Ls is set between the maximum value of Ps from the specimen populations from LA and Def and the minimum value of Ps from the specimen population from Inh.

[0368] In one implementation, Pm is the aforementioned Tm(X1), rTm(X1), aTm(X1, X2), SLm[X1:X2], rSLm[X1:X2], wXm[X1:X2], wTm[X1:X2], wrTm[X1:X2], vT2m(X1) / vT1m(X2), vT2m(X1) / vT2m(X2), ( vT2m (X1) - vT1m (X2)) / vT2s (Bs), (vT2m (X1) - vT2m (X2)) / vT2s (Bs), (vT2m (X1) - vT1m(X2)) / vT1s(Bs), (vT2m(X1)-vT2m(X2)) / vT1s(Bs), iCm(X1) or iwCm[X1:X2].

[0369] In one implementation, if Pm is a threshold or greater than the threshold, the specimen S is inferred to be LA; otherwise, the specimen S is inferred to be Def. In this specification, the threshold for Pm is set as Lm. The threshold Lm can be predetermined before the method of the present invention is implemented. Lm can be determined based on Pm calculated from a population of specimens for which factors of coagulation abnormality are known. Lm can be applied to each Pm, i.e., Tm(X1), rTm(X1), aTm(X1, X2), SLm[X1:X2], rSLm[X1:X2], wXm[X1:X2], wTm[X1:X2], wrTm[X1:X2], vT2m(X1) / vT1m(X2), vT2m(X1) / vT2m(X2), (vT2m) / vT2m(X2), (vT2m) / vT1 ...1m / vT1m(X2), (vT1m / vT1m(X2), (vT1m / vT2m(X2), (vT1m / vT1m(X2), (vT1m / vT1m(X2), (vT1m / vT1m(X2), (vT1m / vT1m(X2), (vT1 (X1) - vT1m(X2)) / vT2s(Bs), (vT2m(X1) - vT2m(X2)) / vT2s(Bs), (vT2m(X1) - vT1m(X2)) / vT1s(Bs), (vT2m(X1) - vT2m(X2)) / vT1s(Bs), iCm(X1) and iwCm[X1:X2] are set respectively.

[0370] Two thresholds, Lm1 and Lm2, related to Pm can be set as needed, where Lm1 > Lm2. For example, if the Pm of specimen S is Lm1 or greater, specimen S is inferred to be LA; on the other hand, if the Pm of specimen S is Lm2 or less, specimen S is inferred to be Def.

[0371] In one example, Pm is calculated for each specimen from the LA specimen population, and Lm is set based on their statistical values ​​(e.g., mean - [k × standard deviation], k preferably 2 to 5). In another example, Pm is calculated for each specimen from the LA specimen population, and Lm is set as their minimum value × k (k preferably 0 < k < 100%). It should be noted that these Lm values ​​can be set as Lm1 when thresholds Lm1 and Lm2 are set.

[0372] In another example, Pm is calculated for each specimen from the Def specimen population, and Lm is set based on their statistical values ​​(e.g., mean + [k × standard deviation], k preferably 2 to 5). In another example, Pm is calculated for each specimen from the Def specimen population, and Lm is set as the maximum value × k (k preferably 1 < k). In another example, Lm is set between the maximum value of Pm from the Def specimen population and the minimum value of Pm from the LA specimen population. In another example, Pm is calculated for each specimen from the normal specimen (NP) population, and Lm is set based on their statistical values ​​(e.g., mean + [k × standard deviation], k preferably 2 to 5). It should be noted that these Lm values ​​can be set to Lm2 when thresholds Lm1 and Lm2 are set.

[0373] In another implementation, a threshold line Lc separating LA and Def is set based on Pm and Ps. Ps is set as the horizontal axis (X-axis), and Pm as the vertical axis (Y-axis), and the threshold line (Y = aX + b) is obtained by following these steps.

[0374] 1) Find the slope SL and intercept IC of the regression line for the LA group.

[0375] 2) Calculate Pm and Py for each group of LA (Py = SL × Ps + IC).

[0376] 3) Calculate the standard deviation SD of (Pm - Py) for the LA group.

[0377] 4) Set the slope a and intercept b of the threshold line as a = SL, b = IC - k × SD (k is preferably 2 to 3).

[0378] In one implementation, by performing a two-dimensional mapping of Ps and Pm of the specimen S, the factors contributing to coagulation abnormalities in the specimen S can be visually displayed. Specifically, Ps and Pm of the specimen S are plotted on a two-dimensional map with Ps on one axis and Pm on the other. For example, on a two-dimensional map where Ps is represented by the X-axis and Pm by the Y-axis, the specimen S is plotted at (Ps, Pm). The relationship between Ps and Pm and the coagulation reaction can be interpreted as follows: Ps is an indicator of the weakness of the coagulation reaction of the specimen S, and Pm is an indicator of the strength of the inhibition of the coagulation reaction. Therefore, when comparing the relative positions of Inh, Def, and LA on the two-dimensional map, the Ps of Inh (which has a coagulation factor inhibitor) is larger than that of Def and LA, and the Pm of LA (which has an autoantibody against phospholipids that contribute to the coagulation reaction) is larger than that of Def. Even in Inh, since higher titers result in stronger inhibition of the coagulation reaction, the Pm of Inh is affected by the titer, and Pm increases according to the titer. That is, in Inh, the Pm of Inh-A, which has a relatively higher potency, is greater than the Pm of Inh-B, which has a relatively lower potency. Furthermore, it can be inferred that the reason for the prolonged APTT is that the (Ps, Pm) of the specimen, which combines both the weakness of the coagulation reaction and the strength of the coagulation reaction inhibition, is located on the two-dimensional spectrum corresponding to the degree of influence of these two factors. Because Ps and Pm have such characteristics, the specimen S can be inferred to be Inh, LA, or Def based on its plotted position on the two-dimensional spectrum.

[0379] On the aforementioned two-dimensional graph, lines (threshold lines or dividing lines) representing Ls and Lm can be shown respectively. For example, the two-dimensional graph is divided into four regions by the lines representing Ls and Lm. The factors contributing to the coagulation abnormality of specimen S can be intuitively inferred from which of these four regions the curve of specimen S lies. For example, on a two-dimensional graph where the X-axis represents Ps and the Y-axis represents Pm, when specimen S is Inh, its plotted (Ps, Pm) is located in the region to the right of the line representing Ls (also called region HX). If this is not the case (located in the region to the left of the line representing Ls, also called region LX), specimen S is LA or Def. Specifically, when the plotted (Ps, Pm) is located above the line representing Lm (also called region LH), specimen S is LA; and when this is not the case (located below the line representing Lm, also called region LL), specimen S is Def.

[0380] Alternatively, lines representing Ls and Lc can be shown separately on the aforementioned two-dimensional graph. Specimen S can be inferred as Inh, LA, or Def based on Ls, just as described above. Among specimens inferred as LA or Def, if its plot is located above the line representing Lc, specimen S is LA; otherwise, specimen S is Def.

[0381] 9. Further inference from specimens with an inferred result of LA

[0382] While extremely rare, specimens showing coagulation abnormalities can be found in patients with acquired hemophilia A (AHA). AHA is a disease characterized by hemophilia symptoms caused by an acquired autoimmune disorder resulting in autoantibodies against coagulation factors (mostly FVIII). AHA is characterized by the presence of residual activity of coagulation factor inhibitors despite a positive PT (prolonged PT) test, prolonged APTT (prolonged acute-to-thickness test), and increased inhibitor titer with decreased coagulation factor activity. AHA requires rapid and accurate diagnosis and treatment. Diagnosis of AHA typically requires cross-mixing tests and inhibitor titer assays. However, facilities capable of performing inhibitor titer testing are very limited, and many facilities rely on external testing. Furthermore, cross-mixing tests are hardly a widely available procedure, and are not always performed even in large facilities where they are necessary. In other words, only a limited number of medical institutions can perform the tests required for AHA diagnosis, and only a very small percentage of facilities can provide results on the same day of blood collection. On the other hand, AHA requires immediate and appropriate treatment (hemostatic therapy and immunosuppressive therapy) after rapid diagnosis. Because the treatment methods differ from other coagulation abnormalities, it takes time to obtain test results, which poses a risk of delay in initiating appropriate treatment for AHA patients.

[0383] When investigating the plotting location of specimens from AHA patients (hereinafter also referred to as "AHA") on the Ps-Pm two-dimensional map based on the method of the present invention described above, even if the coagulation factor inhibitor is positive, it sometimes does not fall into the Inh region HX (see Figure 50). Since the onset of AHA is very rare, it is assumed that its probability of occurrence is also very small. Such AHAs can cause significant problems to the inference results of the coagulation abnormality factors of the present invention. For example, if the AHA is plotted in the LA region (e.g., the LH region described above) on the above two-dimensional map, the AHA will be incorrectly inferred as LA. However, the treatment for LA is different from the treatment for AHA, so incorrect inference as LA poses a risk to AHA patients. Therefore, it is clinically significant to investigate the possibility that specimens inferred as LA in the method of the present invention described above are suspected AHA specimens. In addition, it is beneficial to be able to quickly confirm the possibility of suspected AHA in the examined specimens.

[0384] Therefore, in another embodiment of the method of the present invention, for a specimen inferred to be LA based on Ps and Pm calculated from the coagulation reaction in the above-mentioned APTT determination, the LA status of the specimen is re-inferred based on the parameter Pd. In this embodiment, the parameter Pd is calculated for the specimen inferred to be LA based on Ps and Pm. Pd is calculated based on Td(X), where Td(X) = Ts(X) - Tm(X) (0 < X ​​≤ 100). In this specification and drawings, Td(X) is sometimes referred to as "TdX" or abbreviated as "Td".

[0385] As examples of Pd, as shown in the embodiments described later (Figures 52-56), Td(X1), Td(X2)-Td(X1), SLd[X1:X2], wXd[X1:X2], and wTd[X1:X2] can be given by the following formulas. It should be noted that in the following formulas, Qd is a coefficient used to adjust the size of Pd, Qd>0, and can be selected from the range of 1 to 100, for example.

[0386] ·Td(X1) (where X1 is preferably 43 to 100);

[0387] ·Td(X2)-Td(X1) (where X1=1~80, X2=15~100, and X2-X1=5~99);

[0388]

[0389] (In the formula, X1 = 1 to 95, X2 = 25 to 100, X2 - X1 = 5 to 99, UX is the average value of X from X1 to X2, and UTd is the average value of Td(X) from Td(X1) to Td(X2).)

[0390]

[0391] (In the formula, X1 = 1, X2 = 50~90, or X1 = 15~25, X2 = 25~45, and X2 - X1 = 5~30);

[0392]

[0393] (In the formula, X1 = 1 to 95, X2 = 45 to 100, X2 - X1 = 5 to 99);

[0394] The calculated Pd is used to infer whether the inferred LA specimen is a suspected AHA specimen (hereinafter also referred to as Inh-X). As shown in the embodiments described later, although it is actually AHA, the Pd of AHA located in region LH on the Pd-Pm two-dimensional map tends to be greater than that of the true LA (see Figure 57). Therefore, if the Pd of the specimen is greater than the threshold Ld, the specimen is inferred to be Inh-X. For example, when the region where Pd > Ld and Pm > Lm on the Pd-Pm two-dimensional map is set as region dHH, the specimen plotted (Ps, Pm) located in region dHH is inferred to be Inh-X. The threshold Ld can be predetermined before the method of the present invention is implemented. For example, Ld can be determined based on Pd calculated from the AHA specimen population or as a true LA specimen population or a normal specimen population. Ld can be set separately for each Pd. On the other hand, if the Pd of the specimen is Ld or smaller than it, the specimen is inferred to be LA.

[0395] Two thresholds, Ld1 and Ld2, related to Pd can be set as needed, where Ld1 > Ld2. For example, if the Pd of a specimen located in region LH on the Ps-Pm two-dimensional spectrum is Ld1 or greater, the specimen is inferred to be Inh-X. On the other hand, if the Pd of the specimen is Ld2 or less, the specimen is inferred to be LA.

[0396] In one example, Pd is calculated for each specimen from the population of specimens that constitute true LA, and Ld is set based on their statistical values ​​(e.g., mean + [k × standard deviation], k preferably 2 to 5). In another example, Pd is calculated for each specimen from the population of specimens that constitute true LA, and Ld is set as the maximum value × k (k preferably 1 < k). It should be noted that these Lds can be set as Ld2 if thresholds Ld1 and Ld2 are set.

[0397] In another example, the Pd for each specimen is calculated from the AHA specimen population, and Ld is set based on their statistical values ​​(e.g., mean - [k × standard deviation], k preferably 2 to 5). In yet another example, the Pd for each specimen is calculated from the AHA specimen population, and Ld is set as the minimum value × k (k preferably 0 < k < 100%). It should be noted that these Lds can be set as Ld1 if thresholds Ld1 and Ld2 are set.

[0398] In another example, Ld is set between the maximum value of Pd from the specimen population that is the true LA and the minimum value of Pd from the specimen population that is the AHA.

[0399] In another example, Pd for each specimen is calculated from the normal specimen population, and Ld is set based on their statistical values ​​(e.g., mean + [k × standard deviation], where k is preferably 2 to 5).

[0400] 10. Inference Process

[0401] An illustrative flow diagram of the method of the present invention for inferring factors of coagulation abnormalities in a specimen is shown in Figures 62-66. As one embodiment, Figure 62 shows the flow diagram for inferring factors of coagulation abnormalities in a specimen (specimen S) with a known prolonged APTT. The detailed steps of the flow diagram are described below.

[0402] S1: Obtain the Rs of specimen S. Perform APTT on a mixed specimen (specimen M) containing specimen S and normal specimens to obtain Rm.

[0403] S2: Calculate Ts from Rs, and calculate Tm from Rm.

[0404] S3: Calculate parameter Ps from Ts, and calculate parameter Pm from Tm.

[0405] S4: Compare Ps with Ls, and compare Pm with Lm.

[0406] S5: Based on the comparison in S4, the inference result of the extension factor is determined to be any one of the following four.

[0407] When Ps > Ls and Pm > Lm, the specimen is inferred to be Inh-A.

[0408] ※Inh-A is a sample containing a relatively strong inhibitory effect against coagulation.

[0409] When Ps > Ls and Pm ≤ Lm, the specimen to be examined is inferred to be Inh-B.

[0410] ※Inh-B is a relatively weak inhibitor of the coagulation reaction.

[0411] When Ps≤Ls and Pm>Lm, the specimen to be examined is inferred to be LA.

[0412] In the case that Ps≤Ls and Pm≤Lm, the examined specimen is inferred to be Def.

[0413] S6: Output information related to the inference results of the extension factor.

[0414] * Ps and Pm can be output to a two-dimensional graph as needed.

[0415] As one implementation, Figure 63 illustrates the process for APTT determination and inference of factors causing coagulation abnormalities in specimens with unknown APTT. The detailed steps of the process are described below.

[0416] S1: APTT determination (Rs acquisition) of the test subject (test subject S candidate).

[0417] S2: Calculate APTT from Rs (calculation of Ts).

[0418] S3: Determine whether APTT is extended.

[0419] If the condition is determined to be extended, proceed to S4.

[0420] If it is not determined to be an extension, proceed to S20.

[0421] S4: Calculate parameter Ps from Ts.

[0422] S5: APTT determination (Rm acquisition) of mixed specimens (specimen M) of the tested specimen and normal specimen.

[0423] S6: Calculate Tm from Rm.

[0424] S7: Calculate parameter Pm from Tm.

[0425] S8: Compare Ps with Ls, and compare Pm with Lm.

[0426] S9: Based on the comparison in S8, the inference result of the extension factor is determined to be any one of the following four.

[0427] When Ps > Ls and Pm > Lm, the specimen is inferred to be Inh-A.

[0428] ※Inh-A is a sample containing a relatively strong inhibitory effect against coagulation.

[0429] When Ps > Ls and Pm ≤ Lm, the specimen to be examined is inferred to be Inh-B.

[0430] ※Inh-B is a relatively weak inhibitor of the coagulation reaction.

[0431] When Ps≤Ls and Pm>Lm, the specimen to be examined is inferred to be LA.

[0432] In the case that Ps≤Ls and Pm≤Lm, the examined specimen is inferred to be Def.

[0433] S10: Output information related to the inference results of APTT and the extension factor.

[0434] * Ps and Pm can be output on a two-dimensional graph as needed.

[0435] S20: Output APTT.

[0436] As one implementation, Figure 64 shows the procedure for inferring factors causing coagulation abnormalities in a specimen (specimen S) after an APTT prolongation specimen (specimen S) is found in a standard APTT assay. The detailed steps of the procedure are described below.

[0437] S1: Rs is obtained based on the APTT test data of specimen S.

[0438] S2: Calculate Ts from Rs.

[0439] S3: Calculate parameter Ps from Ts.

[0440] S4: Compare Ps with Ls.

[0441] If Ps > Ls, proceed to S10.

[0442] If Ps≤Ls, proceed to S5.

[0443] S5: APTT determination (Rm acquisition) of a mixed specimen (specimen M) of specimen S and normal specimen.

[0444] S6: Calculate Tm from Rm.

[0445] S7: Calculate parameter Pm from Tm.

[0446] S8: Compare Pm with Lm.

[0447] If Pm > Lm, proceed to S20.

[0448] Enter S30 if Pm≤Lm.

[0449] S10: The specimen to be examined is inferred to be Inh.

[0450] S20: The specimen to be examined is inferred to be LA.

[0451] S30: The specimen to be examined is inferred to be Def.

[0452] ※In S10~S30, APTT can be output as needed. In addition, Ps and Pm can be output to the two-dimensional graph.

[0453] As one implementation, a detailed process for comparing parameters in S4-S5 of Figure 62 or S8-S9 of Figure 63 and inferring factors of solidification anomalies is shown in Figure 65.

[0454] As one implementation, a flow chart in FIG66 is shown that further includes an Inh-X deduction step using Pd, which is also included in the flow chart of FIG65. The flow chart of FIG66 is the same as that of FIG65 except for the steps after S30. The detailed steps of the flow chart after S30 are described below.

[0455] S30: When Ps≤Ls and Pm>Lm:

[0456] Calculate Td from Ts and Tm, and then calculate the parameter Pd.

[0457] S31: Compare Pd with Ld.

[0458] If Pd≤Ld, proceed to S32.

[0459] If Pd > Ld, proceed to S33.

[0460] S32: The specimen to be examined is inferred to be LA.

[0461] S33: The specimen to be examined is inferred to be Inh-X.

[0462] It should be noted that, in order to identify each region, region symbols (HH, HL, LH, LL, and dHH) can be assigned separately, and the region information using these region symbols can be represented as the inference result. Additionally, this region information can be output as the inference result.

[0463] 11. Applications of other solidification reaction measurement methods

[0464] The above description uses the example of coagulation reaction measurement based on the amount of scattered light to illustrate the method for inferring factors of coagulation anomalies according to the present invention. However, anyone skilled in the art can apply the above-described method based on the amount of scattered light to methods using other coagulation reaction measurement methods (e.g., coagulation reaction measurement methods based on transmittance, absorbance, viscosity, etc.), and therefore, such applications are also included within the scope of the present invention.

[0465] 12. Other

[0466] For those skilled in the art, it will be understood that in the above-described steps for inferring factors of solidification anomalies, as long as it is possible to distinguish between factors inferring different solidification anomalies, "above the threshold" can be used instead of "greater than the threshold," or "below the threshold" can be used instead of "less than the threshold." Furthermore, for those skilled in the art, it will be understood that when using two-dimensional graphs for inference, as long as it is possible to distinguish between factors inferring different solidification anomalies, the "region to the right (upper) of the line representing the threshold" can be defined as a region that includes or excludes a portion of that line.

[0467] The inference results of the factors causing coagulation abnormalities in the tested specimen obtained through the above steps can be output in any form. For example, they can be output as electronic data, printed data, or displayed on a monitor. Preferably, the inference results include information that the tested specimen is any one of Inh, LA, and Def. Alternatively, if no prolonged coagulation time is found in the tested specimen, information that the tested specimen is normal or has no coagulation abnormalities (or prolonged coagulation time) can be output. The coagulation time of the tested specimen, as well as R(i), Ts(X), Tm(X), vT1s(X) and vT2s(X), vT1m(X) and vT2m(X), Ps, Pm, Pd, etc., of the tested specimen can be output along with the inference results as needed. In addition, Ps, Pm, and Pd can also be output as a relative comparison of the statistical values ​​of the specimen group relative to the normal specimen (NP) (when the mean of Ps, Pm, and / or Pd of the NP group is set as M and the standard deviation is set as SD, the denominator of the comparison = M + k × SD, k = 2 to 3). Alternatively, the output can be the relative ratios of Ps, Pm, and Pd based on the selected NP (for example, mixed plasma from multiple normal samples, or normal plasma for crossmixing tests). (When the values ​​of Ps, Pm, and / or Pd for the NP are set to N, the denominator of the relative ratio = N × k, where k = 100–200%). Furthermore, as information corresponding to the values ​​of each relative ratio, for example, as indicators of the magnitude of the values, + and ++ can be output respectively. Additionally, the output can also be the region information (e.g., HH, HL, LH, LL, or dHH) where the plotted (Ps, Pm) or (Pd, Pm) of the tested sample is located on the two-dimensional plot.

[0468] 13. Procedures and apparatus

[0469] The method for inferring factors of solidification anomalies according to the present invention described above can be performed automatically using a computer program. Therefore, one aspect of the present invention is a program for performing the method for inferring factors of solidification anomalies according to the present invention. In one embodiment, the program of the present invention is a program for performing the following processes: calculating the aforementioned Ps and Pm; inferring whether the specimen S is Inh based on the calculated Ps; and inferring that the specimen S not inferred as Inh is LA or Def based on the calculated Pm. In one embodiment, the program of the present invention further performs a process of calculating Ts(X), or vT1s(X) and vT2s(X), and Tm(X), or vT1m(X) and vT2m(X), before the aforementioned Ps and Pm calculation process. In one embodiment, the program of the present invention further performs a process of calculating R(i) or V(i) for specimen S and specimen M. The obtained R(i) or V(i) can be used for the calculation of Ts(X), etc., and the calculation of the solidification time of specimen S. In one embodiment, the program of the present invention further executes a process of calculating the coagulation time of the specimen S. In one embodiment, the program of the present invention executes a process of calculating the aforementioned Ts(X), or vT1s(X) and vT2s(X), and Tm(X), or vT1m(X) and vT2m(X), if the calculated coagulation time of the specimen S is prolonged. In one embodiment, the program of the present invention further executes a process of outputting the inference results obtained in the above-mentioned process of inferring the factors (Inh, LA, or Def) of coagulation abnormality of the specimen S.

[0470] In one embodiment, the program of the present invention further executes a process of calculating Td(X) and Pd of the specimen S inferred to be LA. In one embodiment, the program of the present invention further executes a process of inferring whether the specimen S is Inh-X based on the calculated Pd. In one embodiment, the program of the present invention further executes a process of outputting the inference results (Inh, LA, Def, or Inh-X) of the factors of coagulation abnormality of the specimen S obtained in the above inference process.

[0471] In a preferred embodiment, the program of the present invention is a program for implementing the processes of Figures 62-66 above.

[0472] One aspect of the present invention is an apparatus for performing the method for inferring factors of coagulation abnormalities according to the present invention described above. The apparatus includes a computer for executing the procedure of the present invention to infer factors of coagulation abnormalities in a test specimen. Depending on the need, the apparatus includes a measuring device for measuring the coagulation reaction of the specimen or an output device for outputting the inferred results of the factors of coagulation abnormalities.

[0473] The following describes one embodiment of the device of the present invention. One embodiment of the device of the present invention is the automatic analysis device 1 shown in FIG4. The automatic analysis device 1 includes: a control unit 10, an operation unit 20, a measurement unit 30, and an output unit 40.

[0474] The control unit 10 controls the overall operation of the automatic analysis device 1. The control unit 10 may be, for example, a computer (personal computer, etc.). The control unit 10 includes a CPU, memory, storage, communication interface (I / F), etc., and performs tasks such as processing instructions from the operation unit 20, controlling the operation of the measurement unit 30, saving or analyzing measurement data received from the measurement unit 30, saving analysis results, and controlling the output based on the measurement data or analysis results from the output unit 40. Furthermore, the control unit 10 may also be connected to external media, a host computer, or other devices. It should be noted that in the control unit 10, the computer controlling the operation of the measurement unit 30 and the computer performing measurement data analysis may be the same or different.

[0475] The operation unit 20 receives input from the operator and transmits the input information to the control unit 10. For example, the operation unit 20 may have a user interface (UI) such as a keyboard or touch panel. Under the control of the control unit 10, the output unit 40 outputs the measurement data from the measurement unit 30, the solidification reaction curve R based on the measurement data, the solidification rate curve V, the solidification reaction endpoint E, the solidification time, T(X), vT1(X), vT2(X), Ps, Pm, Pd, inferences about factors causing abnormal solidification of the specimen (e.g., Inh, LA, or Def), a two-dimensional map showing the specimen's plot (Ps, Pm), information about the region where the specimen's plot (Ps, Pm) is located on the two-dimensional map (e.g., HH, HL, LH, LL, or dHH), and information related to the inference results (e.g., labels). For example, the output unit 40 may have a display device such as a monitor.

[0476] The measuring unit 30 performs a series of operations for coagulation testing to obtain measurement data on the coagulation reaction of a sample containing a blood specimen. The measuring unit 30 includes: various instruments or analytical modules required for coagulation testing, such as a specimen container for holding the blood specimen, a reagent container for holding the reagents used in the test, a reaction vessel for reacting the specimen with the reagents, a probe for dispensing the specimen, diluent, reagents, etc., into the reaction vessel, a light source, a detector for detecting scattered or transmitted light from the sample in the reaction vessel, a data processing circuit for sending data from the detector to the control unit 10, and a control circuit for receiving instructions from the control unit 10 to control the operation of the measuring unit 30, etc.

[0477] The control unit 10 infers factors contributing to abnormal coagulation of the specimen based on data measured by the measurement unit 30. This analysis may include the calculation of Ps and Pm, as well as the inference of factors contributing to abnormal coagulation of the specimen using Ps and Pm. Furthermore, the control unit 10 can obtain the coagulation reaction curve R, coagulation rate curve V, coagulation time, coagulation reaction endpoint E, T(X), vT1(X), vT2(X), etc. Alternatively, the coagulation reaction curves R and E may be obtained by the control unit 10 based on measurement data from the measurement unit 30, or they may be obtained by other devices, such as the measurement unit 30, and sent to the control unit 10. The control unit 10 may store the specimen's coagulation time, T(X), V, vT1(X), vT2(X), and various parameters used to calculate Ps, Pm, Pd, etc. (e.g., formulas for calculating Ps, Pm, and Pd, etc.). Additionally, the control unit 10 may also store parameters (e.g., the aforementioned thresholds Ls, Lm, Ld, etc.) used to infer coagulation abnormalities in the specimen. Alternatively, the control unit 10 may also retrieve these parameters or reference values ​​stored on an external device or network during parsing.

[0478] The above analysis can be implemented by the program described above for carrying out the method of the present invention. Therefore, the control unit 10 may be equipped with a program for inferring factors of solidification anomalies according to the present invention.

[0479] The analysis results from the control unit 10 are sent to the output unit 40 and output. The output can take any form, such as display on a screen, transmission to the host computer, or printing. The output information from the output unit may include the coagulation reaction curve R, the coagulation rate curve V, the coagulation reaction endpoint E, the coagulation time, T(X), vT1(X), vT2(X), Ps, Pm, Pd, and inferences about factors contributing to abnormal coagulation of the sample. The types of output information from the output unit can be controlled by the program of this invention.

[0480] Example

[0481] The present invention will be described in more detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0482] Example 1: Inference of Factors Contributing to Solidification Abnormalities

[0483] 1. Method

[0484] 1) Specimen

[0485] The following specimens with prolonged coagulation time will be displayed as the test specimens.

[0486] • Positive coagulation factor inhibitors (Inh): Plasma from patients diagnosed as positive for FVIII inhibitors (N=11, titer 0.8–260 BU / mL) and positive for FIX inhibitors (N=4, titer 0.6–3.2 BU / mL) (total N=15).

[0487] • LA-positive specimen (LA): Plasma from patients diagnosed with LA positivity (N=11).

[0488] • Coagulation factor deficiency specimens (Def): Plasma from patients diagnosed with FVIII deficiency (N=37), FIX deficiency (N=23) and FV deficiency (N=5) as shown in Table 1 below, and congenital factor V deficiency plasma from George King Bio-Medical, Inc. with confirmed FV activity values ​​less than 1% (N=1) (total N=65).

[0489] [Table 1]

[0490]

[0491] Patient blood was collected in blood collection tubes containing 3.2% sodium citrate solution at a blood-to-sodium citrate aqueous solution ratio of 9:1. The blood collection tubes were then centrifuged (1500g for 15 minutes), and the separated plasma was collected and stored in a preservation container at -80°C. The frozen plasma was thawed at 37°C before testing and used as the test sample. For the preparation of the mixed sample, normal plasma (NPP) was prepared from 20 normal individuals without confirmed coagulation abnormalities. The mixed sample was prepared by mixing the test sample with NPP at a mixing ratio (test sample:NPP volume ratio) of 9:1, 1:1, and 1:9.

[0492] 2) Measurement of solidification reaction

[0493] For the APTT assay, Coagpia APTT-N (manufactured by Sekisui Medical Co., Ltd.) was used as the first reagent, and Coagpia APTT-N calcium chloride solution (25mM calcium chloride solution, manufactured by Sekisui Medical Co., Ltd.) was used as the second reagent. Coagulation reaction was measured using a CP3000 automated coagulation analyzer (manufactured by Sekisui Medical Co., Ltd.). 50 μL of the sample was heated in a cuvette at 37°C for 45 seconds, followed by the addition of 50 μL of the first reagent at approximately 37°C. After a further 171 seconds, 50 μL of the second reagent was added to initiate the coagulation reaction. The reaction was carried out at 37°C. The coagulation reaction was measured as follows: the cuvette was illuminated with light of 660 nm wavelength using an LED as the light source, and the amount of scattered light from a 90-degree side scattering was measured at 0.1-second intervals. The measurement time was 600 seconds.

[0494] 3) Obtaining the solidification reaction curve

[0495] After smoothing the photometric data of each specimen, including noise removal, the coagulation reaction curve R(i) is generated by zero-point adjustment with the amount of scattered light at the start of photometric measurement being 0.

[0496] 4) Detection of the solidification reaction endpoint E and determination of APTT

[0497] The earliest time at which the cumulative ratio Z(i) of R(i) (refer to WO2021 / 132552) is less than the cumulative ratio threshold Zs is taken as the solidification reaction endpoint E. The cumulative ratio threshold Zs is 1.001. The time T(50) at which R(i) reaches 50% of E is calculated and determined as APTT. The cumulative ratio Z(i) at time i is calculated as follows:

[0498] Cumulative ratio Z(i) = Rb(i) / Ra(i)

[0499] Ra(i)=sum from R(i-20) to R(i-1)

[0500] Rb(i) = the sum of R(i+1) to R(i+20)

[0501] 5) Calculation of T(X)

[0502] For each sample, the time T(X) for R(i) to reach X% of the E detected in 4) is calculated based on the following formula. Specifically, based on the following formula, for the tested sample and the mixed sample, R(i), E, ​​and T(X) are calculated as Rm(i), Em, and Tm(X) for the tested sample and R(i), E, ​​and T(X) for the mixed sample. X is set from 1 to 100 in intervals of 1, and T(1) to T(100) are calculated. APTT is defined as T(50) when X = 50.

[0503] Rs(Ts(X))=Es×X%(Ia)

[0504] Rm(Tm(X))=Em×X%(Ib)

[0505] 6) Calculation of the function of V(i)

[0506] Take the first derivative of Rs(i) and Rm(i) to calculate the solidification rate curves Vs(i) and Vm(i), respectively. The maximum values ​​of Vs(i) and Vm(i) are obtained as Vmax_s and Vmax_m. Based on the following equations (Xa) to (Xd), calculate vT1s(X), vT2s(X), vT1m(X), and vT2m(X). X is set from 3 to 98 in increments of 5, and then to 100.

[0507] Vs (vT1s (X)) = Vmax_s × X% (Xa)

[0508] (where vT1s(X)≤VmaxT_s)

[0509] Vs (vT2s (X)) = Vmax_s × X% (Xb)

[0510] (where vT2s(X)≥VmaxT_s)

[0511] Vm (vT1m (X)) = Vmax_m × X% (Xc)

[0512] (where vT1m(X)≤VmaxT_m)

[0513] Vm (vT2m (X)) = Vmax_m × X% (Xd)

[0514] (where vT2m(X)≤VmaxT_m)

[0515] 2. Coagulation reaction of the specimen

[0516] 1) T(X)

[0517] Figure 3 shows the T(X) (X represents time (seconds)) of specimens selected from the LA, Def, and Inh groups with the same APTT (approximately 100 seconds). The specimens selected from the Inh group were positive for FVIII inhibitors with a potency of 0.8 BU / mL (iFVIII). Additionally, the specimens selected from the Def group were FVIII-deficient specimens with factor activity less than 1% (dFVIII). Figure 3A shows the Ts(X) of each specimen, and Figure 3B shows the Tm(X) of a mixed specimen with a 1:1 ratio (subject:NPP). In Figures 3A and 3B, solid lines represent LA, dashed lines represent dFVIII (Def), and dotted lines represent iFVIII (Inh). Figures 3C–E show the Ts (solid lines) and Tm (dashed lines) for iFVIII (Inh), dFVIII (Def), and LA, respectively.

[0518] As shown in Figure 3A, even when Ts(X) is at the same level (APTT is approximately 100 seconds) when X = 50, the slope of Ts(X) for Inh is significantly larger. When X > 50, the difference between Ts(X) for Inh and Ts(X) for LA and Def increases with increasing X. Therefore, this indicates that it is possible to distinguish and infer Inh from specimens showing prolonged coagulation time based on Ts(X).

[0519] Furthermore, as shown in Figure 3B, the Tm(X) of the mixed specimen from Def increases only slightly with increasing X. This is presumably because the coagulation reaction is closer to that of a normal specimen due to the action of the coagulation factor from the NPP mixed with specimen S for preparing the mixed specimen. On the other hand, the Tm(X) of the mixed specimen from LA increases with increasing X. This is presumably because the phospholipids from the NPP mixed with specimen S for preparing the mixed specimen immediately bind to the autoantibodies against the phospholipids contained in specimen S, thereby inhibiting the coagulation reaction.

[0520] 3. Inference of Inh based on Ps

[0521] 1) rTs(X1)

[0522] As Ps, rTs(X1) of each specimen is calculated according to the following formula (IIa').

[0523] rTs(X1)=Ts(X1) / Ts(As)×Qs(IIa')

[0524] (X1 = 10, 60, 95 or 100, As = 50, Qs = 1)

[0525] The calculated rTs(X1) for each specimen is shown in Figure 5. In the figure, the solid line represents the minimum rTs(X1) for the Inh group, and the dashed line represents the maximum rTs(X1) for the LA and Def groups. When X1 > As, it was found that the rTs(X1) for Inh tends to be larger compared to LA and Def. Therefore, it shows that a threshold that can distinguish Inh from LA and Def can be set based on rTs(X1), and specimens with rTs(X1) greater than this threshold when X1 > As can be inferred to be Inh.

[0526] Figure 6 shows the difference between the minimum value of rTs(X1) of the Inh group and the maximum value of rTs(X1) of the LA and Def groups when various changes were made to X1 and As (X1 > As). In the figure, when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, it is indicated by a gray bar; otherwise, it is indicated by ×. When X1 > As, it can be confirmed that the minimum value of rTs(X1) of the Inh group increases compared to the maximum value of rTs(X1) of the LA and Def groups. This trend is not observed when As ≥ 80. Based on this result, it is indicated that rTs(X1) in the range of X1 = 35–95, As = 3–75, and X1 - As = 5–92 are preferred for inferring Inh.

[0527] 2) rTs(X2) - rTs(X1)

[0528] As Ps, rTs(X2) - rTs(X1) of each specimen is calculated according to the following formula (IIIa').

[0529] rTs(X2) - rTs(X1)

[0530] =(Ts(X2)-Ts(X1)) / Ts(As)×Qs(IIIa')

[0531] (X1 = 20 and X2 = 95, As = 50, Qs = 1)

[0532] The calculated rTs(X2) - rTs(X1) for each specimen is shown in Figure 7. In the figure, the solid line represents the minimum value of rTs(X2) - rTs(X1) for the Inh group, and the dashed line represents the maximum value of rTs(X2) - rTs(X1) for the LA and Def groups. When X2 > X1, it is found that the rTs(X2) - rTs(X1) of Inh tends to be larger compared with LA and Def. Therefore, it shows that a threshold that can distinguish Inh from LA and Def can be set based on rTs(X2) - rTs(X1), and specimens with rTs(X2) - rTs(X1) greater than this threshold can be inferred to be Inh.

[0533] For As = 50 and with various changes to X1 and X2, the difference between the minimum value of the Inh group and the maximum value of the LA and Def groups is shown in Figure 8. In the figure, when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, it is indicated by a gray bar; otherwise, it is indicated by ×. When X2 > X1, there is a trend that the minimum value of the Inh group's rTs(X2) - rTs(X1) increases compared to the maximum value of the LA and Def groups. However, this trend is not observed when X1 ≥ 85, X2 ≤ 45, or X2 = 100. Based on this result, it is indicated that the rTs(X2) - rTs(X1) within the ranges of X1 = 2–80, X2 = 50–95, and X2 - X1 = 5–93 are preferred for inferring Inh.

[0534] 3) arTs(X1, X2)

[0535] As Ps, the ratio arTs(X1, X2) of the difference between rTs(X1) and rTs(X2) of each specimen relative to the difference between X1 and X2 is calculated according to the following formula (IVa').

[0536] arTs(X1, X2)

[0537] ={rTs(X2)-rTs(X1)} / (X2-X1)

[0538] =[{Ts(X2)-Ts(X1)} / Ts(As)×Qs] / (X2-X1) (IVa')

[0539] (X1 = 50, X2 = 95, As = 5, 50 or 95, Qs = 1)

[0540] The calculated arTs(X1, X2) for each specimen are shown in Figures 9A-C. In the figures, the solid line represents the minimum arTs(X1, X2) for the Inh group, and the dashed line represents the maximum arTs(X1, X2) for the LA and Def groups. A trend was observed that the arTs(X1, X2) for Inh is larger compared to LA and Def. Therefore, it is shown that a threshold can be set based on arTs(X1, X2) to distinguish Inh from LA and Def, and specimens with arTs(X1, X2) greater than this threshold can be inferred to be Inh.

[0541] For arTs(X1, X2) with various changes to X1 and X2 when As = 5, 50, and 95, the difference between the minimum value of the Inh group and the maximum value of the LA and Def groups is shown in Figures 10-12. In the figures, when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, it is indicated by a gray bar; otherwise, it is indicated by an ×. It can be confirmed that the minimum value of arTs(X1, X2) of the Inh group is greater than the maximum value of arTs(X1, X2) of the LA and Def groups. Based on this result, it is shown that in order to deduce Inh, arTs(X1:X2) in the range of As = 5-95, X1 = 1-85, X2 = 25-95, and X2-X1 = 5-94 can be used, preferably in the range of X1 = 4-80, X2 = 55-95, and X2-X1 = 5-91.

[0542] 4) rSLs[X1:X2]

[0543] As Ps, rSLs[X1:X2], which is the slope of the regression line of rTs(X) for each specimen, is calculated according to the following formula (Va').

[0544]

[0545] Where UX is the average value of X from X1 to X2, and UrTs is the average value of rTs(X) from rTs(X1) to rTs(X2).

[0546] (X1 = 10, X2 = 90, As = 5, 50 or 95, Qs = 100)

[0547] The calculated rSLs[X1:X2] for each specimen are shown in Figure 13. In the figure, the solid line represents the minimum rSLs[X1:X2] for the Inh group, and the dashed line represents the maximum rSLs[X1:X2] for the LA and Def groups. Examples can be identified where the rSLs[X1:X2] for Inh is larger compared to LA and Def. Therefore, it shows that a threshold can be set based on rSLs[X1:X2] to distinguish Inh from LA and Def, and specimens with rSLs[X1:X2] greater than this threshold can be inferred to be Inh.

[0548] The differences between the minimum value of rSLs[X1:X2] of group Inh and the maximum value of rSLs[X1:X2] of groups LA and Def are shown in Figures 14-16 for As = 5, 50, and 95, with various changes to X1 and X2. In the figures, a gray bar indicates that the minimum value of group Inh is greater than the maximum value of groups LA and Def; otherwise, an × is used. There is a trend that the minimum value of rSLs[X1:X2] of group Inh is greater than the maximum value of rSLs[X1:X2] of groups LA and Def. This trend is not observed when X2 < 40, and also when As is large, this trend is not observed if the difference between X1 and X2 is small. Based on this result, it is indicated that in order to infer Inh, rSLs[X1:X2] in the range of As=5~95, X1=1~85, X2=25~100, X2-X1=5~99 can be used, and irSLs[X1:X2] in the range of X1=1~80, X2=55~100, X2-X1=5~99 can be preferred.

[0549] 5) wXs[X1:X2]

[0550] As Ps, the weighted average value wXs[X1:X2] of X in the interval from Ts(X1) to Ts(X2) for each specimen is calculated according to the following formula (VIa).

[0551]

[0552] (X1=1, X2=100, Qs=1)

[0553] The calculated wXs[X1:X2] values ​​for each specimen are shown in Figure 17. In the figure, the solid line represents the minimum wXs[X1:X2] value for the Inh group, and the dashed line represents the maximum wXs[X1:X2] value for the LA and Def groups. Examples can be identified where the wXs[X1:X2] value for Inh is larger compared to LA and Def. Therefore, it shows that a threshold can be set based on wXs[X1:X2] to distinguish Inh from LA and Def, and specimens with wXs[X1:X2] values ​​greater than this threshold can be inferred to be Inh.

[0554] Figure 18 shows the difference between the minimum value of wXs[X1:X2] of the Inh group and the maximum value of wXs[X1:X2] of the LA and Def groups when X1 and X2 are modified in various ways. In the figure, when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, it is indicated by a gray bar, and when it is not, it is indicated by ×. It can be confirmed that the minimum value of wXs[X1:X2] of the Inh group is greater than the maximum value of wXs[X1:X2] of the LA and Def groups. Based on this result, it is indicated that in order to deduce Inh, wXs[X1:X2] is preferably in the range of X1 = 1 to 75, X2 = 30 to 100, and X2 - X1 = 10 to 99.

[0555] 6) The ratio of vTs(X)

[0556] An example of vT2s(X1) / vT1s(X2) (X1 = 8 and X2 = 78) for each specimen is shown in Figure 19. In the figure, the solid line represents the minimum value of the Inh group, and the dashed line represents the maximum value of the LA and Def groups. The minimum value of Inh is greater than the maximum value of LA and Def. Figure 20 shows the difference between the minimum value of vT2s(X1) / vT1s(X2) and vT2s(X1) / vT2s(X2) in the Inh group and the maximum value in the LA and Def groups when various changes are made to X1 and X2. In the figure, when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, it is indicated by a gray bar; otherwise, it is indicated by an ×. Based on this result, it is indicated that vT2s(X1) / vT1s(X2) in the range of X1=3~58 and X2=13~98 and vT2s(X1) / vT2s(X2) in the range of X1=13~63 and X2=58~83 (where X1<X2) are preferred as Ps for inferring Inh.

[0557] 7) The relative value of the difference between vTs(X)

[0558] The relative values ​​of the differences in vTs(X) for each specimen are shown in Figure 21. Figure 21A represents (vT2s(X1) - vT2s(X2)) / vT1s(Bs) (X1 = 53 and X2 = 63, Bs = 3). Figure 21B represents (vT2s(X1) - vT1s(X2)) / vT2s(Bs) for each specimen (X1 = 8 and X2 = 83, Bs = 3). In the figures, the solid line represents the minimum value of the Inh group, and the dashed line represents the maximum value of the LA and Def groups. It can be confirmed that the minimum value of Inh is greater than the maximum value of LA and Def.

[0559] Figure 22 shows the difference between the minimum value of (vT2s(X1) - vT1s(X2)) / vT2s(Bs) and (vT2s(X1) - vT2s(X2)) / vT2s(Bs) (Bs = 3) in the Inh group and the maximum value in the LA and Def groups. Figure 23 shows the difference between the minimum value of (vT2s(X1) - vT1s(X2)) / vT2s(Bs) and (vT2s(X1) - vT2s(X2)) / vT2s(Bs) (Bs = 100) in the Inh group and the maximum value in the LA and Def groups. Figure 24 shows the difference between the minimum value of (vT2s(X1) - vT1s(X2)) / vT1s(Bs) and (vT2s(X1) - vT2s(X2)) / vT1s(Bs) (Bs = 3) in the Inh group and the maximum value in the LA and Def groups. In Figures 22-24, a gray bar is used when the minimum value of the Inh group is greater than the maximum value of the LA and Def groups, and × is used when this is not the case. Based on this result, it is shown that the relative value of the difference of vTs(X) is preferably used as Ps for inferring Inh.

[0560] 4. Inference of LA and Def based on Pm

[0561] 1) Tm(X1)

[0562] As Pm, Tm(X1) of each specimen is calculated according to the following formula (X1 = 1, 25, 50, 75, 100).

[0563] The Tm(X1) values ​​for each specimen are shown in Figure 25. In the figure, the solid line represents the maximum value of Tm(X1) for the Def group, and the dashed line represents the minimum value of Tm(X1) for the LA group. A trend was observed that the Tm(X1) for LA increased compared to Def. Therefore, it is shown that a threshold for distinguishing LA from Def can be set based on Tm(X1), and specimens with Tm(X1) greater than this threshold can be inferred as LA from specimens excluding Inh. As the Pm used to infer LA, Tm(X1) is preferably in the range of X1 = 90–100.

[0564] 2) rTm(X1)

[0565] As Pm, rTm(X1) of each specimen is calculated according to the following formula (IIb').

[0566] rTm (X1) = Tm (X1) / Tm (Am) × Qm (IIb')

[0567] (X1=90, Am=3, Qm=1)

[0568] The calculated rTm(X1) values ​​for each specimen are shown in Figure 26. In the figure, the solid line represents the maximum value of rTm(X1) for the Def group, and the dashed line represents the minimum value of rTm(X1) for the LA group. Examples can be identified where the rTm(X1) for LA is larger than that for Def. Therefore, it shows that a threshold for distinguishing LA from Def can be set based on rTm(X1), and specimens with rTm(X1) greater than this threshold can be inferred to be LA from specimens excluding Inh.

[0569] Figure 27 shows the difference between the minimum value of rTm(X1) of the LA group and the maximum value of rTm(X1) of the Def group when various changes were made to X1 and Am (X1 > Am). In the figure, when the minimum value of the LA group is greater than the maximum value of the Def group, it is indicated by a gray bar, and when it is not, it is indicated by ×. According to the results, it is shown that rTm(X1) in the range of Am = 1 to 35, X1 = 30 to 95, and X1 - Am = 10 to 94 is preferred for inferring LA and Def.

[0570] 3) SLm[X1:X2]

[0571] As Pm, SLm[X1:X2], which is the slope of the regression line of Tm(X) for each specimen, is calculated according to the following formula (Vb).

[0572]

[0573] Where UX is the average value of X from X1 to X2, and UTm is the average value of Tm(X) from Tm(X1) to Tm(X2).

[0574] (X1=5, X2=100, Qm=1)

[0575] The calculated SLm[X1:X2] values ​​for each specimen are shown in Figure 28. In the figure, the solid line represents the maximum value of SLm[X1:X2] for the Def group, and the dashed line represents the minimum value of SLm[X1:X2] for the LA group. Examples can be identified where the SLm[X1:X2] of LA is larger than that of Def. Therefore, it shows that a threshold for distinguishing LA from Def can be set based on SLm[X1:X2], and specimens with SLm[X1:X2] greater than this threshold can be inferred to be LA from specimens excluding Inh.

[0576] Figure 29 shows the difference between the minimum value of SLm[X1:X2] of the LA group and the maximum value of SLm[X1:X2] of the Def group when X1 and X2 are modified in various ways. In the figure, when the minimum value of the LA group is greater than the maximum value of the Def group, it is indicated by a gray bar, and when it is not, it is indicated by ×. Based on this result, it is shown that SLm[X1:X2] in the range of X1 = 1 to 95, X2 = 30 to 100, and X2 - X1 = 5 to 99 is preferred for inferring LA and Def.

[0577] 4) rSLm[X1:X2]

[0578] As Pm, rSLm[X1:X2], which is the slope of the regression line of rTm(X) for each specimen, is calculated according to the following formula (Vb').

[0579]

[0580] Where rTm(X) is rTm(X) = Tm(X) / Tm(Am) × Qm, UX is the average value of X from X1 to X2, and UrTm is the average value of Tm(X) from rTm(X1) to rTm(X2).

[0581] (X1 = 20, X2 = 40, As = 5, 50 or 95, Qm = 1)

[0582] The calculated rSLm[X1:X2] values ​​for each specimen are shown in Figure 30. In the figure, the solid line represents the maximum value of rSLm[X1:X2] for the Def group, and the dashed line represents the minimum value of rSLm[X1:X2] for the LA group. Examples can be identified where the rSLm[X1:X2] value for LA is larger than that for Def. Therefore, it shows that a threshold can be set based on rSLm[X1:X2] to distinguish between LA and Def, and specimens with rSLm[X1:X2] greater than this threshold can be inferred to be LA from specimens excluding Inh.

[0583] The difference between the minimum value of rSLm[X1:X2] of the LA group and the maximum value of rSLm[X1:X2] of the Def group when X1 and X2 are modified in various ways is shown in Figures 31-33. In the figures, when the minimum value of the LA group is greater than the maximum value of the Def group, it is indicated by a gray bar, and when it is not, it is indicated by ×. According to the results, it is shown that in order to infer LA and Def, rSLm[X1:X2] in the range of Am = 5 to 95, X1 = 1 to 70, X2 = 25 to 100, and X2 - X1 = 5 to 99 can be used, preferably in the range of X1 = 1 to 55, X2 = 25 to 95, and X2 - X1 = 5 to 94.

[0584] 5) Weighted average

[0585] As Pm, wXm[X1:X2], which is the weighted average of X in the interval from Tm(X1) to Tm(X2) for each specimen, is calculated according to the following formula (VIb). Similarly, wTm[X1:X2], the weighted average of Tm(X) in the interval from X1 to X2 for each specimen, is calculated according to the following formula (VIIb). Similarly, wrTm[X1:X2], the weighted average of rTm(X) in the interval from X1 to X2 for each specimen, is calculated according to the following formula (VIIb').

[0586]

[0587] The calculated wXm[X1:X2], wTm[X1:X2], and wrTm[X1:X2] for each specimen are shown in Figures 34A-C. In the figures, the solid line represents the maximum value of the Def group, and the dashed line represents the minimum value of the LA group. The parameters of LA are larger than those of Def. On the other hand, it is impossible to distinguish Inh from LA or Def based on these parameters. Therefore, it shows that a threshold that can distinguish LA from Def can be set based on wXm[X1:X2], wTm[X1:X2], and wrTm[X1:X2], and specimens with parameters greater than this threshold can be inferred to be LA from specimens that exclude Inh.

[0588] The differences between the minimum values ​​of wXm[X1:X2], wTm[X1:X2], and wrTm[X1:X2] of the LA group and the maximum values ​​of the same parameters of the Def group when X1 and X2 are modified in various ways are shown in Figures 35, 36, and 37A-C. In the figures, when the minimum value of the LA group is greater than the maximum value of the Def group, a gray bar is used; otherwise, an × is used. Based on this result, it is shown that wXm[X1:X2] in the range of X1 = 1-45, X2 = 30-100, and X2-X1 = 10-99 is preferably used to infer LA and Def. In addition, it is shown that as the range for inferring LA and Def based on wTm[X1:X2], X1 = 75-95, X2 = 95-100, and X2-X1 = 5-25 are preferred. Furthermore, it is indicated that as the range for inferring LA and Def based on wrTm[X1:X2], X1 = 1 to 90, X2 = 30 to 100, X2 - X1 = 5 to 99 are preferred, and X1 = 15 to 75, X2 = 50 to 85, X2 - X1 = 5 to 65, Am = 3 to 34 are more preferred.

[0589] 6) aTm(X1, X2)

[0590] As Pm, aTm(X1, X2) is calculated according to the following formula (IVb).

[0591] aTm(X1, X2)

[0592] ={Tm(X2)-Tm(X1)} / (X2-X1) (IVb)

[0593] (X1 = 85, X2 = 90)

[0594] The calculated aTm(X1, X2) values ​​for each specimen are shown in Figure 38. In the figure, the solid line represents the maximum value of aTm(X1, X2) for the Def group, and the dashed line represents the minimum value of aTm(X1, X2) for the LA group. Examples can be identified where aTm(X1, X2) for LA is larger than that for Def. On the other hand, it is not possible to distinguish Inh from LA or Def based on aTm(X1, X2). Therefore, it shows that a threshold can be set based on aTm(X1, X2) to distinguish LA from Def, and specimens with aTm(X1, X2) greater than this threshold can be inferred to be LA from specimens excluding Inh.

[0595] Figure 39 shows the difference between the minimum value of aTm(X1, X2) in the LA group and the maximum value in the Def group when X1 and X2 are modified for various reasons. In the figure, when the minimum value of the LA group is greater than the maximum value of the Def group, it is indicated by a gray bar, and when it is not, it is indicated by ×. Based on this result, it is shown that aTm(X1, X2) in the ranges of X1 = 1 to 90, X2 = 25 to 100, and X2 - X1 = 5 to 99 is preferred for inferring LA and Def.

[0596] 7) vTm(X)

[0597] The vT2m(X1) (X1=3) of each specimen, serving as an example of vTm(X), and the vT2m(X1) / vT1m(X2) (X1=18, X2=23) and vT2m(X1) / vT2m(X2) (X1=43, X2=88) of each specimen, serving as examples of vTm(X), are shown in A-C of Figure 40A. In the figure, the solid line represents the maximum value of the Def group, and the dashed line represents the minimum value of the LA group. The value of LA is larger than that of Def. Therefore, it shows that LA and Def can be inferred from the specimens excluding Inh based on vTm(X) or its ratio. It should be noted that the preferred range for vT2m(X1) is X1=3 to 18.

[0598] Figure 40B shows the results of investigating the vTm ratio by making various changes to X1 and X2. In the figure, when the minimum value of the LA group is greater than the maximum value of the Def group, a gray bar is used; otherwise, an × is used. The preferred ranges for vT2m(X1) / vT1m(X2) are X1 = 3–93 and X2 = 3–88, and the preferred ranges for vT2m(X1) / vT2m(X2) are X1 = 38–63 and X2 = 63–93. Therefore, it is shown that the vTm ratio can be used to infer LA and Def.

[0599] 8) The relative value of the difference between vTm(X)

[0600] Figure 41A shows the (vT2m(X1) - vT1m(X2)) / vT1m(Bm) for each specimen (X1 = 43 and X2 = 78, Bm = 3), and Figure 41B shows the (vT2m(X1) - vT1m(X2)) / vT2m(Bm) for each specimen (X1 = 43 and X2 = 78, Bm = 3). In the figures, the solid line represents the maximum value of the Def group, and the dashed line represents the minimum value of the LA group. The value of LA is larger than that of Def.

[0601] Figures 42 and 43 show the difference between the minimum and maximum relative values ​​of the difference of vTm(X) in the LA group and the Def group. In the figures, a gray bar indicates that the minimum value in the LA group is greater than the maximum value in the Def group; otherwise, an × is used. Figure 42 shows the results for (vT2m(X1) - vT1m(X2)) / vT1m(Bm) and (vT2m(X1) - vT2m(X2)) / vT1m(Bm), and Figure 43 shows the results for (vT2m(X1) - vT1m(X2)) / vT2m(Bm) and (vT2m(X1) - vT2m(X2)) / vT2m(Bm). Figure 44 shows the results when Bm = 100. Based on these results, it is shown that the relative value of the difference of vTm(X) is preferably used to infer LA or Def. Furthermore, based on these results, it is indicated that in (vT2m(X1) - vT1m(X2)) / vT1m(Bm), the preferred values ​​are Bm = 3~100, X1 = 3~93, and X2 = 3~93. It is also indicated that in (vT2m(X1) - vT1m(X2)) / vT2m(Bm), the preferred values ​​are Bm = 3~100, X1 = 3~93, and X2 = 3~88. Furthermore, it is indicated that in (vT2m(X1) - vT2m(X2)) / vT1m(Bm), the preferred values ​​are Bm = 3~100, X1 = 23~63, and X2 = 63~93. Finally, it is indicated that in (vT2m(X1) - vT2m(X2)) / vT2m(Bm), the preferred values ​​are Bm = 3~100, X1 = 28~63, and X2 = 68~93.

[0602] 9) Cm(i)

[0603] As a correction parameter for Vm(i), Cm(i) is calculated (Q=1) according to the above equation (XIb). The maximum value of Cm(i), Cmax_m, is found, and its reciprocal, iCmax_m, is calculated. In addition, iwCm[X1:X2] is calculated according to the above equation (XIIb). In equation (XIIb), X1 and X2 are the points that satisfy Cm(X1) = Cm(X2) = {the maximum value of Cm(i) × S%} (S=3 or 98, X1 < Vmax_m < X2). Figures 45A, B, and C show iCmax_m and iwCm[X1:X2] when S=3 and 98.

[0604] 5. Inference of factors contributing to solidification anomalies using two-dimensional spectra

[0605] As shown in Figures 46A and 46B, using a 1:1 (subject: NPP) mixed sample Pm, the (Ps, Pm) values ​​for each subject were plotted on a two-dimensional Ps-Pm graph with Ps on the X-axis and Pm on the Y-axis. Ps is rSLs[X1:X2] when As = 50, and Pm is SLm[X1:X2]. Both Ps and Pm have X1 = 10 and X2 = 90. The plots of Inh, LA, and Def are distributed in different regions on the two-dimensional graph. In Figures 46A and 46B, the dashed line parallel to the Y-axis represents the threshold Ls (average Ps of the Def group + 3SD), and the plots of Inh are all located to the right of this line (region HX). In Figure 46A, the dashed line parallel to the X-axis represents the threshold Lm (average Ps of the Def group + 4SD), and the distribution areas of the plots of LA and Def are separated by this dashed line. In Figure 46B, the solid line (slanted line) represents the threshold line Lc. The steps for calculating the threshold line Lc (Y = aX + b) with Ps as the X-axis and Pm as the Y-axis are as follows: First, calculate the slope SL and intercept IC of the regression line for the LA group, and calculate Py (Py = SL × X + IC). Next, calculate the standard deviation SD of (Pm - Py) for the LA group. Calculate Lc (Y = aX + b) with slope a = SL and intercept b = IC - 3 × SD. The plotted distribution areas of LA and Def are separated by this threshold line Lc. This indicates that by plotting Ps and Pm of the tested specimen on a two-dimensional graph, it is possible to visually show which of Inh, LA, and Def is responsible for the coagulation abnormality of the tested specimen.

[0606] 6. Comparative Examples

[0607] 1) Setting time

[0608] T(50) is the moment when R(i) reaches 50% of E, which has traditionally been calculated in the form of solidification time. As shown in Figure 47A, it is not possible to distinguish Inh, LA and Def based on Ts(50) of the specimen.

[0609] 2) Parameters related to the solidification rate curve

[0610] The coagulation reaction curve R(i) of the specimen is differentiated to obtain the coagulation rate curve V(i). The maximum value (maximum reaction rate) Vmax of V(i), its reciprocal 1 / Vmax, and the time VmaxT for V(i) to reach Vmax are calculated. As shown in Figures 47B-D, it is impossible to distinguish Inh, LA, and Def based on any one of Vmax, 1 / Vmax, and VmaxT. For example, in the case of Vmax shown in Figure 47B, although there is a trend that Vmax decreases in the order of LA, Def, and Inh, the distribution areas of the specimen types partially overlap.

[0611] For mixed samples, Tm(50), Vmax, 1 / Vmax, and VmaxT were also calculated. As shown in Figures 48A-D, it is impossible to distinguish Inh, LA, and Def based on any one of Tm(50), Vmax, 1 / Vmax, and VmaxT.

[0612] 7. The effect of mixing ratio in mixed samples

[0613] Following the same steps as in step 5 above, but preparing a mixed specimen with a mixing ratio of 1:9 or 9:1 (subject:NPP), the Pm of the resulting mixed specimen is determined. Figure 49 shows a two-dimensional map plotting the specimen types (Ps, Pm) for each subject. Ps, Pm, Ls, and Lm are the same as in Figure 46A. Figure 49A shows the Pm of a 1:1 (subject:NPP) mixed specimen, Figure 49B shows the Pm of a 1:9 (subject:NPP) mixed specimen, and Figure 49C shows the Pm of a 9:1 (subject:NPP) mixed specimen. In the figures, dFVIII, dFIX, and dFV represent FVIII deficiency, FIX deficiency, and FV deficiency, respectively, while iFVIII and iFIX represent FVIII inhibitor positivity and FIX inhibitor positivity, respectively. The dashed line parallel to the Y-axis represents the threshold Ls, and the dashed line parallel to the X-axis represents the threshold Lm. Regardless of the mixing ratio of the mixed samples, the distribution trends of Inh and Def on the two-dimensional spectrum are roughly the same, with Pm for Def being below Lm. The results indicate that regardless of the mixing ratio of the mixed samples, Pm can be used to infer LA and Def.

[0614] Example 2: Inference of AHA

[0615] Acquired hemophilia A (AHA) is a specific pathological condition in which factor VIII activity remains despite the presence of factor VIII inhibitors. Therefore, the location of (Ps, Pm) in the samples of AHA patients on a two-dimensional map was investigated.

[0616] For samples from AHA patients (AHA; N=6), Ps and Pm were calculated using the same procedure as in Example 1. Ps is {Ts(X2) - Ts(X1)} / Ts(As) × Qs in formula (IIIa'). Pm is SLm[X1:X2] in formula (Vb). For LA-positive samples (LA; N=11) used in Example 1, Ps and Pm were also calculated in the same way. Mixed samples with mixing ratios of 1:9, 1:1, and 9:1 (subject: NPP) were used in the calculation of Pm. Figure 50 is a two-dimensional plot of (Ps, Pm) for each subject. In the figure, the dashed line parallel to the Y-axis represents the threshold Ls (where the mean Ps of the Def group is set to M, and the standard deviation is set to SD, Ls = M + 3SD), and the dashed line parallel to the X-axis represents the threshold Lm (where the mean Pm of the Def group is set to M, and the standard deviation is set to SD, Lm = M + K × SD, where K is 3, 3.5, or 4). Although there is a trend for Ps of AHA to be greater than LA, some AHAs are plotted in regions inferred as LA. This indicates that in inferences using coagulation abnormality factors such as Ps and Pm as in Example 1, there is a possibility that AHAs may be incorrectly inferred as LAs. Since the onset of AHA is very rare, the probability of such incorrect inference is very low, but considering that the treatments for LA and AHA are completely different, such incorrect inferences pose a significant risk to AHA patients.

[0617] Figure 51 shows the Ts (Figure A), Tm (Figure B), and Td (Figure C) of the following three specimens. AHA-a (solid line) is the AHA located in the region LH inferred as LA in the two-dimensional plot of Figure 50 (Ps, Pm). AHA-b (dashed line) is the AHA with Pm of the same degree as AHA-a and a larger Ps. LA (dotted line) is the LA with Ps closest to AHA-a. In Figure 50, the Ps of AHA-a and LA are of the same degree because the slope of Ts of AHA-a in Figure 51A is approximately parallel to the Ts of LA. In addition, the Pm of AHA-a and LA are of the same degree because the Tm of AHA-a is approximately the same as the Tm of LA in Figure 51B. On the other hand, as shown in Figure 51C, the Td of both AHA-a and AHA-b increases with increasing X, but the Td of LA shifts near zero. The difference between Td(X) in such AHA and LA indicates that the AHA group containing (Ps, Pm) located in the region LH inferred to be LA can be distinguished from the LA group.

[0618] The Pd values ​​of the specimens from the AHA and LA groups used in Example 1 are shown in Figures 52-56. Figures 52A-C show the Td(X) of the mixed specimens when the mixing ratio (specimen S:NPP) is 9:1 (A), 1:1 (B), and 1:9 (C), respectively. In Figures 52A-C, AHA-a (solid lines) represents the AHAs (Ps, Pm) located in the region LH (i.e., the region with the smallest Ps) inferred as LA in the two-dimensional plots of Figures 50A-C. In Figures 52A-C, AHA-b (dashed lines) represents the AHA specimens with the second smallest Ps after AHA-a in the two-dimensional plots of Figures 50A-C. In Figures 52A-C, LA (dotted lines) represents the LAs in the two-dimensional plots of Figures 50A-C where Ps is closest to AHA-a. In Figures 52A-C, within the range where X is greater than approximately 40, the Td(X) of AHA-a and AHA-b is greater than that of LA. Based on this result, it can be shown that Td(X) within the specified interval of X can be used as Pd.

[0619] Figures 53-56 show the Td(X2)-Td(X1), SLd[X1:X2], wXd[X1:X2], and wTd[X1:X2] for each specimen. In the tables of Figures 53-56, gray columns indicate that the minimum value of the AHA group is greater than the maximum value of the LA group; if this is not the case, × is used.

[0620] Figure 57 is a two-dimensional plot of (Pd, Pm) for each subject in AHA and LA. The mixing ratios (subject: NPP) of the mixed samples in Figures A, B, and C are 9:1, 1:1, and 1:9, respectively. Pd is SLd[X1:X2], and Pm is SLm[X1:X2], with X1 = 10 and X2 = 90 for both Pd and Pm. In the figure, the dashed line parallel to the Y-axis represents the threshold Ld (with the mean of Pd in ​​the LA group set as Md, the standard deviation as SDd, and the coefficient as kd, Ld = Md + kd × SDd). Additionally, the dashed line parallel to the X-axis represents the threshold Lm (with the mean of Pm in the Def group set as Mm, the standard deviation as SDm, and the coefficient as km, Lm = Mm + km × SDm). In Figures 57A, B, and C, kd is 4, 4, and 3, respectively, and km is 3.5, 4, and 3, respectively. Using thresholds Ld and Lm, all samples with AHA can be distinguished from LA. This demonstrates that potential AHAs in samples inferred as LA from the steps of Example 1 can be distinguished from LA based on Pd.

[0621] Example 3 shows the inference results using the ratio of indicators.

[0622] Normal plasma from healthy individuals was used as the specimen (N=10). Coagulation reaction measurements, coagulation reaction curve acquisition, detection of the coagulation reaction endpoint E, determination of APTT, and calculation of T(X) were performed following the same procedures as in Example 1.

[0623] Calculation of index ratio

[0624] Index ratio = (Ps, Pm, or Pd of the tested sample) / (Control index value)

[0625] Control index value = average value of Ps, Pm, or Pd in ​​the normal sample group + [2 × standard deviation]

[0626] Both the horizontal axis Ps and the vertical axis Pm are calculated based on the same indicators as in Example 1, and a two-dimensional graph is created. Examples of units for the indicator ratios include ratios and unitsless ratios. By using the indicator ratios, it is possible to relatively show how much Ps and Pm of the specimen S compare to the normal specimen group.

[0627] Figure 58 shows the two-dimensional map obtained by converting the two-dimensional map of Figure 49A into index ratios (Ps ratio, Pm ratio). The control index values ​​for the index ratios were calculated using the mean + [2 × standard deviation] of each Ps and Pm from the normal plasma group (N=10). The Ps and Pm used were the same as in Figure 49A. Figure 58 shows the distribution of all specimens by specimen type on a two-dimensional map. Figures 58A, B, and C show dFVIII, dFIX, and dFV for Def; Figures 58D and E show iFVIII and iFIX for Inh; and Figure 58F shows LA. The Ps ratios for LA, Def, and Inh range from 0.9 to 1.6, 0.6 to 2.0, and 2.1 to 3.3, respectively, and the Pm ratios range from 1.5 to 6.5, 0.9 to 1.3, and 1.0 to 9.3, respectively. Based on these results, specimens with a Ps ratio greater than 2.0 can be classified as Inh, and specimens with a Pm ratio greater than 1.3 can be classified as LA. Additionally, specimens with a Ps ratio below 2.0 and a Pm ratio below 1.3 can be classified as Def.

[0628] 1) Relationship between Ps ratio and factor activity

[0629] Figure 59 shows the relationship between the Ps ratio and coagulation factor activity, with factor activity (IU / dL) on the horizontal axis (logarithmic scale) and the Ps ratio on the vertical axis. The Ps ratio was calculated in the same way as in Figure 58. Figure 59A represents dFVIII, Figure 59B represents dFIX, and Figure 59C represents dFV. In Figures 59A and C, specimens from severely ill patients with factor activity less than 1% are plotted at the horizontal axis scale mark 1. Additionally, Inh is plotted at the horizontal axis scale mark 0.1 in a manner that allows for comparison with coagulation factor inhibitors (iFVIII in Figure 59A and iFIX in Figure 59B). Figures 59A–C show a negative correlation between the Ps ratio and factor activity. Furthermore, it is evident that specimens with a Ps ratio greater than 2.0 can be distinguished from Inh, and specimens with a Ps ratio less than 2.0 can be distinguished from Def.

[0630] 2) Relationship between Pm ratio and inhibitor potency

[0631] Figure 59D shows the relationship between the Pm ratio and inhibitor potency for iFVIII (△) and iFIX (□), with inhibitor potency (BU / mL) on the x-axis (logarithmic scale) and Pm ratio on the y-axis. The Pm ratio was calculated in the same manner as in Figure 58. Figure 59D indicates a correlation between the Pm ratio and potency when the potency of iFVIII is above approximately 5. Based on this correlation, since it covers samples with a potency up to 260 (Pm ratio approximately 9), it suggests that the Pm ratio can infer the potency level.

[0632] 3) Advantages of index ratio

[0633] For index ratios, by measuring the normal plasma group used for the control index value (denominator) with the same device and the same batch of reagents as the test subject (specimen S), it can be expected that the effects of batch-to-batch differences in reagents and device-to-device differences in automated analysis devices can be avoided. Therefore, it can be said to be a practical display method.

[0634] Figure 60 shows the differences in the distribution positions of various specimen types of LA (〇), iFVIII (△), and AHA (◇) in the two-dimensional Ps ratio and Pm ratio map. The (Ps ratio, Pm ratio) of the 6 AHA cases were divided into the following three positions: 3 cases between the LA group and the iFVIII group; 2 cases between the LA group and the iFVIII group and close to the iFVIII group; and 1 case between the LA group and the iFVIII group and close to the LA group. The reason why all AHA cases are distributed in the position between the LA group and the iFVIII group can be explained by the fact that although AHA contains a factor VIII inhibitor, factor VIII activity remains. That is, compared with iFVIII where factor VIII activity is almost gone, Ps is smaller, but because it is a high-titer specimen with an inhibitor potency greater than 5 BU / mL, Pm is larger. In summary, the distribution position (coordinates) of Ps and Pm in the two-dimensional spectrum shows that Ps reflects the activity (weakness) of the factor, while Pm reflects the inhibition (strength) of the solidification reaction.

[0635] 4) Relationship between AHA specimens and index ratios

[0636] The positional relationships between the specimen types in a two-dimensional graph using LA and AHA as specimen types and index ratios on the horizontal and vertical axes were investigated. The two-dimensional graph is shown in Figure 61. In the figure, 〇 represents LA and ◇ represents AHA. For the index ratios on the horizontal and vertical axes, Figure 61A shows (Ps ratio, Pm ratio), and Figure 61B shows (Pd ratio, Pm ratio). Ps is rSLs[X1:X2] when As=50, Pm is SLm[X1:X2], and Pd is SLd[X1:X2]. Ps, Pm, and Pd all use X1=10 and X2=90. The control index values ​​used to calculate the Ps ratio and Pm ratio are the mean of the normal specimen group + [2×standard deviation], and the control index values ​​used to calculate the Pd ratio are the mean of the LA group + [3×standard deviation]. In addition, the mixing ratio (subject:NPP) of the mixed specimens is 1:1. In the AHA group, the specimen with the minimum Ps ratio (1.4) had a Pd ratio of 2.0. This specimen is AHA-a whose (Ps, Pm) position on the 2D graph is located in the region LH inferred as LA. On the other hand, the specimen with the maximum Ps ratio (1.6) in LA had a Pd ratio of 0.6. Based on this result, it can be shown that AHA-a whose (Ps, Pm) position on the 2D graph is located in the region LH inferred as LA can be distinguished from LA using Pd. As an example, when the Pd ratio threshold Ld = 1.5, if the Pd ratio of a specimen whose (Ps, Pm) position on the 2D graph is located in the region LH inferred as LA is > Ld, the specimen can be inferred to be a specimen that is not LA or is suspected to be AHA.

Claims

1. A method for inferring factors of abnormal coagulation in a blood sample, comprising the following steps: 1) Calculating a parameter Ps related to the coagulation reaction of sample S and a parameter Pm related to the coagulation reaction of sample M, where sample S is a blood sample with prolonged coagulation time, sample M is a mixed sample of sample S and sample N, sample N is a normal blood sample, Ps is calculated based on Ts(X), or vT1s(X) and vT2s(X), and Pm is calculated based on Tm(X), vT2m(X), vT1m(X) and vT2m(X), or the first derivative curve of the coagulation reaction curve of sample M, where Ts(X) represents the measurement point or time at which the coagulation reaction curve of sample S reaches X% of Es, Es is the coagulation reaction endpoint in the coagulation reaction curve of sample S, and vT1s(X) represents the time before the coagulation rate curve of sample S reaches its maximum value. The measurement point or time at which the maximum value is X%, vT2s(X) represents the measurement point or time at which the coagulation rate curve of specimen S reaches the maximum value after reaching the maximum value, Tm(X) represents the measurement point or time at which the coagulation reaction curve of specimen M reaches Em, where Em is the coagulation reaction endpoint in the coagulation reaction curve of specimen M, vT1m(X) represents the measurement point or time at which the coagulation rate curve of specimen M reaches the maximum value before reaching the maximum value, vT2m(X) represents the measurement point or time at which the coagulation rate curve of specimen M reaches the maximum value after reaching the maximum value, 0 < X ​​≤ 100; 2) inferring whether specimen S is positive for coagulation factor inhibitor based on Ps; and 3) inferring that specimen S not inferred to be positive for coagulation factor inhibitor in 2) is positive for lupus anticoagulant or lacking coagulation factor based on Pm, or based on Ps and Pm.

2. The method according to claim 1, wherein, The Ps are selected from the following (a) to (j): (a) rTs(X1) where rTs(X1) = Ts(X1) / Ts(As) × Qs (IIa') X1 = 35 ~ 95, As = 3 ~ 75, X1 > As, Qs > 0; (b) {rTs(X2) - rTs(X1)} / (X2 - X1), where rTs(X1) and rTs(X2) are defined by the above formula (IIa'), where X1 = 1 ~ 85, X2 = 25 ~ 95, (X2 - X1) = 5 ~ 94, As = 5 ~ 95, Qs > 0; (c) rSLs[X1:X2] where, UX is the average value from X1 to X2, UrTs is the average value from rTs(X1) to rTs(X2), rTs(X2) is defined by the above equation (IIa'), X1 = 1~85, X2 = 25~100, (X2-X1) = 5~99, As = 5~95, Qs>0; (d) wXs[X1:X2] Here, X1 = 1~75, X2 = 30~100, (X2 - X1) = 10~99, Qs > 0; (e) vT2s(X1) / vT1s(X2) Here, X1 = 3~58, X2 = 13~98; (f) vT2s(X1) / vT2s(X2) Here, X1 = 13~63, X2 = 58~83, X2 > X1; (g) (vT2s(X1) - vT1s(X2)) / vT2s(Bs) Here, X1 = 3~58, X2 = 13~98, Bs = 3~100; (h) (vT2s(X1) - vT1s(X2)) / vT1s(Bs) Here, X1 = 3~58, X 2 = 3~93, Bs = 3~100; (i) (vT2s(X1) - vT2s(X2)) / vT2s(Bs) Here, X1 = 8~58, X2 = 48~83, X2 > X1, Bs = 3~100; (j) (vT2s(X1) - vT2s(X2)) / vT1s(Bs) Here, X1 = 8~58, X2 = 48~98, X2 > X1, Bs = 3~100, and, the Pm is selected from the following (a')~(p'): (a') Tm(X1), where X1 = 90~100; (b') rTm(X1) Here, rTm(X1) = Tm(X1) / Tm(Am) × Qm (IIb')X1=30~95, Am=1~35, Qm, X1=1~90, X2=25~100, UX is the average value from X1 to X2, UTm is the average value from Tm(X1) to Tm(X2), X1 = 1 to 95, X2 = 30 to 100, (X2 - X1) = 5 to 99, Qm > 0; (e')rSLm[X1:X2] Here, rTm(X) is defined by the above equation (IIb'), where UX is the average value of X from X1 to X2, UrTm is the average value of rTm(X) from rTm(X1) to rTm(X2), X1 = 1~70, X2 = 25~100, (X2-X1) = 5~99, Am = 5~95, Qm>0; (f') wXm[X1:X2] Here, X1 = 1–45, X2 = 30–100, (X2–X1) = 10–99, Qm > 0; (g')wTm[X1:X2] Here, X1 = 75–95, X2 = 95–100, (X2–X1) = 5–25, Qm > 0; (h')wrTm[X1:X2] Here, rTm(X) is defined by the above formula (IIb'), X1 = 1~90, X2 = 30~100, (X2-X1) = 5~99, Am = 3~34, Qm>0; (i') vT2m(X1) / vT1m(X2) Here, X1 = 3~93, X2 = 3~88; (j') vT2m(X1) / vT2m(X2) Here, X1 = 38~63, X2 = 63~93, X2>X1; (k') (vT2m(X1)-vT1m(X2)) / vT2s(Bs) Here, X1 = 3~93, X2 = 3~88, Bm = 3~100; (l') (vT2m(X1)-vT2m(X2)) / vT2s(Bs) Here, X1 = 3~93, X2 = 3~88, Bm = 3~100; 2)) / vT2s(Bs) Here, X1 = 28~63, X2 = 68~93, X2 > X1, Bm = 3~100; (m')(vT2m(X1) - vT1m(X2)) / vT1s(Bs) Here, X1 = 3~93, X2 = 3~93, Bm = 3~100; (n')(vT2m(X1) - vT2m(X2)) / vT1s(Bs) Here, X1 = 23~63, X2 = 63~93, X2 > X1, Bm = 3~100; (o')iCmax_m Here, iCmax_m is the reciprocal of Cmax_m, Cmax_m is the maximum value of Cm(i), Cm(i) = Vm(i)×(Q / Em) (XIb) In equation (XIb), Vm(i) is the first differential curve of the coagulation reaction curve of the specimen M, i is the time or the number of measurement points, and Q>0; (p')iwCm[X1:X2] Here, Cm(i) is defined by the above formula (XIb), where X1 and X2 satisfy Cm(X1) = Cm(X2) = Vm(i) × [Q / (Em × S%)], where X1 < VmaxT_m < X2, Qm > 0, S = 1 to 100, and VmaxT_m is the time or measurement point at which the first differential curve of the coagulation reaction curve of the specimen M reaches its maximum value.

3. The method according to claim 1, wherein, 2) includes the following steps: if Ps is at or above the threshold of Ps, the specimen S is inferred to be positive for coagulation factor inhibitor; 3) includes the following steps: if Pm is at or above the threshold of Pm, the specimen S is inferred to be positive for lupus anticoagulant, or if Pm is at or below the threshold of Pm, the specimen S is inferred to be deficient in coagulation factor.

4. The method according to claim 1, wherein, As described in 2) and 3), the steps include: plotting the Ps and Pm of the specimen S on a two-dimensional map, and inferring the specimen S as positive for coagulation factor inhibitors, positive for lupus anticoagulants, or lacking coagulation factors based on the position of the plotted data on the two-dimensional map.

5. The method according to claim 1, wherein, The process includes the following steps: calculating the parameter Pd for a specimen S that is presumed to be positive for lupus anticoagulant, and inferring whether the specimen S is suspected to be from a patient with acquired hemophilia A based on Pd. Here, Pd is calculated based on Td(X), where Td(X) = Ts(X) - Tm(X), and 0 < X ​​≤ 100.

6. The method according to claim 5, wherein, The Pd is selected from the following (a'') to (e''): (a'') Td(X1), where X1 = 43 to 100; (b'') Td(X2) - Td(X1), where X1 = 1 to 80, X2 = 15 to 100, and X2 - X1 = 5 to 99; (c'') SLd[X1:X2] where, X1 = 1~95, X2 = 25~100, X2 - X1 = 5~99, UX is the average value of X from X1 to X2, UTd is the average value of Td(X) from Td(X1) to Td(X2), Qd > 0; (d'')wXd[X1:X2] Here, X1 = 1, X2 = 50–90, or X1 = 15–25, X2 = 25–45, and X2 – X1 = 5–30, Qd > 0; (e'')wTd[X1:X2] Here, X1=1~95,X2=45~100,X2-X1=5~99,Qd>0。 7. The method according to claim 5, wherein, The procedure includes the following steps: if Pd is at or above the threshold of Pd, the specimen S is inferred to be suspected to be from a patient with acquired hemophilia A.

8. A procedure for implementing the method according to any one of claims 1 to 7.

9. An apparatus for carrying out the method according to any one of claims 1 to 7.

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