JPS5821165A - Measuring method for factor for abnormal blood coagulation - Google Patents

Abstract

PURPOSE:To detect factors for abnormal blood coagulation on account of defficiency of vitamin K in particular by allowing the blood sample which is pretreated by a specific method to react with the immunologically inactive carrier particles sensitized with the II, VII IX or X factors for blood coagulation or the antibodies thereof. CONSTITUTION:A blood sample is added to the suspension prepd. by dispersing fine powder of BaSO4, BaCO3 or aluminum hydroxide[Al(OH)3 gel, etc.]in an aq. buffer soln. and the suspension is treated, then the centrifugally separated supernatant (by which false reaction is prevented) is sampled. One drop of the reagent prepd. by sensitizing (adsorbing or binding) the II, VII, IX or X factors for blood coagulation or the antibodies obtd. by injecting said factors in rabbits, etc. with an immunilogically inactive carrier such as polystyrene latex is added to the two drops of the test liq. which is changed of a dilution magnification of the above-described treated blood sample and is put on a slide plate, then the presence of absence of coagulation is observed. The max. dilution number at which coagulation is admitted is multiplied by 0.2, whereby the concn. of the factors for abnormal blood coagulation is detected. Thus, the simple and quick detection with high sensitivity is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for immunologically measuring a blood coagulation disorder factor in a blood sample, particularly a blood coagulation disorder factor caused by a deficiency in vimemin, and a novel immunoassay reagent for use in the method. Regarding.

The phenomenon of blood coagulation is an enzymatic chain reaction in which activation of the coagulation system is initiated when blood comes into contact with a negatively charged foreign surface.

First, when an inactive enzyme source is activated, the activated enzyme acts on the next enzyme source to make it an active enzyme,
Through a series of reactions as shown below, prothrombin (coagulation factor 2) is activated by thrombin, which acts on the final substrate fibrinogen (factor i) and precipitates as fibrin.

Foreign body side ↓ Factor X Activated factor Factors clearly involved in coagulation, such as macromolecular kininogen, are also known. Some of these coagulation factors are highly active in the hierarchy, including calcium (the first
Although some factors act as cofactors to promote coagulation, such as factor V), factor V, and high-molecular-weight kininogen, all other factors except factor Ⅰ are proteinaceous.

Among these, factors ①, ②, ②, and X are rich in γ-carboxyglutamic acid residues in their protein structures, and activation occurs upon binding of calcium ions to this site and in the presence of phospholipids. Since vitamin K is essential for the normal production of these proteins in the liver, factors ①, ②, ② and X are particularly called vitamin-dependent coagulation factors.

Therefore, in the liver of a patient who is deficient in vitamins or who is receiving vitamin antagonists, these vitamin-dependent coagulation factors are not normally produced, and vitamin-dependent factors with abnormal structures are produced. These abnormally structured vitamin-dependent factors are usually “PIVKA” (proteirr 1ndrb).
ced by vitamin Kabsence
or antagonist), and are also referred to as PIVKA-Ti, ■, ■, and X according to their respective origins (coagulation system ■, ■, ■, and factor X), and are also referred to herein as to be called.

Since P7VKA does not have an r-hydroxyglutamic acid residue in its protein molecular structure, it does not bind to calcium ions, and therefore blood coagulation is inhibited. - For example, PIVKA-R does not produce Cal Deaf Factor) and therefore has a significant effect on the blood coagulation system.

Blood does not clot in normal blood vessels. However, when a blood vessel ruptures and bleeding occurs, the bleeding cannot be stopped unless the coagulation system is constantly activated. On the other hand, if the coagulation system is activated for some reason within a blood vessel, a thrombus is generated, which can lead to serious consequences such as so-called disseminated intravascular coagulation syndrome (hereinafter abbreviated as "DIC"). If the living body is normal, blood that has been coagulated will dissolve due to fibrinolysis (fibrinolysis) as a defense mechanism of the living body, but if abnormally strong fibrinolysis occurs, bleeding will occur. In this way, the coagulation-fibrinolytic system maintains its normal function in a complex equilibrium state of many factors, such as enzymes and their inhibitory substances. Therefore, if a portion of it ruptures, a serious condition such as a blood clot or bleeding will occur. In particular, there are many factors involved in the human coagulation system, which is extremely complex, and unless we can accurately and quickly identify where an abnormality has occurred, we will not be able to take appropriate prevention, treatment, or measures. Become.

Traditionally, for the prevention and treatment of bleeding, transfusion of whole blood or plasma derivatives, administration of vitamins, etc. have been used clinically to act on the coagulation system, and in addition to these, there have been other treatments that affect the coagulation system. The type of drug that works is also used depending on the patient's condition. Conversely, drugs that suppress the coagulation system or platelet function are used to prevent blood clots. Among these, vitamin antagonists, which are widely used as oral anticoagulants, exhibit an anticoagulant effect by lowering coagulation factors ①, ②, ② and factor X, so-called vitamin-dependent factors. be.

However, in actual clinical practice, the use of antagonists for vitamins must be based on a solid diagnosis that they are used specifically to suppress this part of the coagulation system, since vitamins have an abnormal expression of dependent factors. It is not a real drug, but is only used as a comparative therapy for blood clots. In addition, although vitamin antagonists have a remarkable effect on many patients, their use is difficult; for example, excessive administration of vitamin antagonists can lead to γ-carboxylation of vitamin-dependent factors. Since PIVKA is inhibited and has an abnormal structure, the coagulability of the blood is reduced and bleeding occurs.On the other hand, if the dose is insensitive, the effect against blood clots is poor. In such cases, it would be of great clinical significance to know whether PIVKA is present in the blood and its amount. However, there was no conventional method for quantifying this easily and quickly. Therefore, when a vitamin antagonist is administered continuously over a long period of time, the dosage must be determined using the hepaplastin test (hereinafter referred to as "HP").
At present, we rely on so-called trial and error while measuring the blood coagulation ability using methods such as the Thrombotest (hereinafter abbreviated as "TT") and the like.

That is, TT is influenced by PIVKA, and HPT
Since it is said that Inhibition Indegc (7, 1,) is hardly affected by the following formula, if this value is 0.3 to 1.0, it is considered PIVKA positive and indirectly PIVKA I have been seeking the existence of

Inhibition Index (1, 1,)=
HPT (force, ) -, 11T (%) / l1PT (%) However, the tissue thromboplastin used in this method has the problem that the standard curve differs depending on its type, titer, etc., and the measurement procedure is complicated. How to prove it directly,
There are two-dimensional immunoelectrophoresis methods, especially P IVK.
It is possible to prove the existence of A-Il, (G5μtobin9-binPIVKA). However, this method is only qualitative and 1
．． Furthermore, the measurement procedure is complicated and time-consuming, making it unsuitable for routine testing.

Therefore, the present inventors aimed to easily and quickly and accurately measure PIVKA in the blood of patients who are vitamin deficient or who are receiving vitamin antagonists. tactically inactive
- By subjecting the blood sample to an immunological reaction using an immunological measurement reagent such as carrier particles, it is possible to detect a blood coagulation abnormality factor, that is, PIVKA, in a blood sample simply and quickly, and with extremely high sensitivity. Furthermore, in coagulation-fibrinolytic diseases caused by multiple causes, such as DIC, we found that it is difficult to accurately diagnose whether the disease is vitamin-dependent or not. The invention was completed.

Thus, according to the invention, a blood sample pretreated with barium sulfate, barium carbonate or hydrated aluminum oxide is used to prepare an immunologically deficient blood sample sensitized with blood coagulation factors ①, ②, ② or X or antibodies thereof. A blood sample characterized in that it is subjected to an immunological reaction using an immunological measurement reagent made of active carrier particles, and the blood coagulation abnormality factor in the blood sample is determined qualitatively or quantitatively from the result of the reaction. A method for measuring a blood coagulation disorder factor in a sample is provided.

One of the features of the method of the present invention is that the PIVKA-
n, ■, K or X and the corresponding blood coagulation number ■, ■,
(2) Focusing on the fact that they have common antigenicity with factor

Unless otherwise specified, coagulation factor J is separated and purified from human plasma by methods known per se, such as gel filtration, ion exchange chromatography, affinity chromatography, etc., alone or in combination. It can be obtained by
Coagulation, fibrinolysis, kinin,” Chugai Igakusha, 1979].

One-sided liquid coagulation No. 1,
/Unless otherwise specified, "anticoagulant factor antibodies") can also be attacked by methods known per se; The method is to immunize non-human mammals such as rabbits, guinea pigs, goats, and sheep with the coagulation factor K7' r:y obtained as described above, with the addition of Indian adjuvant and other auxiliary agents. The anticoagulant factor antibody can be obtained by collecting the antiserum and separating the anticoagulant factor antibody from the antiserum according to a conventional method, for example, by fractionation using ammonium sulfate precipitation.

Antisera for these human coagulation factors are also commercially available.
You can also use that product.

The coagulation factor and anticoagulation factor antibody to be sensitized according to the present invention correspond to PIVKA in the sample blood to be detected, and although their purity is not strictly limited, they generally contain coagulation factors. It is preferable that the purity is such that it shows a substantially single image by disk electrophoresis,
In addition, the anticoagulant factor antibody should be of such purity that it shows substantially a single image by immunoelectrophoresis in the antigen-antibody reaction with the corresponding coagulation factor in plasma. There is.

In addition, examples of immunologically inert carrier particles used for the sensitization of coagulation factors or anticoagulant antibodies include red blood cells fixed with formalin, polymer latex, bentonite, etc. Any of the carriers commonly used as carriers for conventional immunochemical measurement reagents, such as collodion, silica, and kaolin, can be used. Such carrier particles can generally have an average particle size of about 0.001 to about 20 microns, preferably about 0.05 to about 10 microns.

In the present invention, the term "sensitization" is used in a meaning that includes both cases in which coagulation factors or anticoagulation factor antibodies are physically adsorbed onto carrier particles as described above and cases in which they are chemically bound. .

Thus, sensitization can be carried out by conventional methods, for example, when a coagulation factor or anticoagulant antibody is physically adsorbed onto the carrier particles as described above, the coagulation factor or anticoagulant antibody to be sensitized is to a final concentration of 0.001 to 0.
The carrier is dissolved in a buffer solution (e.g. glycine NaOH buffer) to a concentration of 5% (W/V), preferably -14- or 0.01 to 0.02% (W/V), and is added to the carrier without stirring. (e.g. polystyrene latex),
Sensitization can be carried out by continuing stirring at a temperature of usually about 4 to about 60°C, preferably about 15 to about 40°C, for about 30 to about 180 minutes. In this case, proteins that are immunologically inactive with respect to the coagulation factors and anticoagulant antibodies to be detected, such as albumin and globulin from the serum of animals such as humans, cows, goats, and sheep, are added in a buffer solution [usually 0% 0.01-0.5% (W/V) used once], the carrier particles can be treated before or after sensitization, thereby chemically separating them from non-specific anti-antibodies. Examples include a method in which a carrier particle having a functional group capable of binding on its surface is reacted with a coagulation factor or an anticoagulant antibody to be sensitized in the presence of a suitable binding agent such as carbodiimide, bisdiazobenzidine, geltaraldehyde, etc. . In this case as well, in the same way as above, non-specific reactions can be eliminated by treating (chemically bonding) inactive proteins with carrier particles before or after sensitization to obtain a highly sensitive reagent. can do.

In the above sensitization operation, by appropriately adjusting the sensitizing amount of the coagulation factor or anticoagulant antibody to the carrier particles and/or the degree of treatment with the inert protein, an immunoassay reagent with constant sensitivity can be obtained. can be prepared.

The measurement reagent of the present invention prepared as described above can be obtained from patients.
Collected blood samples, e.g. whole surface, plasma (citrate blood dark,
can be used for the immunological determination of PIVKA in oxalic acid plasma], etc., and the determination utilizes an antigen-antibody reaction known per se, i.e., an immunological agglutination reaction or an immunological agglutination inhibition reaction. You can do it by doing this.

Another feature of the method of the present invention is that the blood sample is prepared in advance!
The point is to perform adsorption treatment with barium A acid, barium carbonate, or water-containing aluminum oxide. This adsorption treatment can prevent false reactions caused by normal coagulation factors that may be present in the blood sample.

Adding to the blood sample an aliquot of a suspension dispersed in barium sulfate, barium carbonate, or hydrated acid (treatment of blood samples with hyaluminum can generally include inert proteins such as sulfated albumin); This can be carried out by shaking continuously or intermittently for a period of time, for example for 5 to 60 minutes, preferably for about 15 minutes, and then removing the -17- fine powder by conventional methods such as centrifugation. The temperature during this treatment is not particularly limited as long as the proteins in the blood sample are not significantly denatured, but it is generally 20 to 40°C.
A temperature within this range, particularly around 37°C, is suitable.

The amount of the fine powder added is not strictly limited,
Although it can be varied widely depending on the quality of the blood sample 1a and/or the type of the fine powder, when barium sulfate and barium carbonate are used, it is generally +Bθ~1 per blood sample 1a.
Blood samples thus pretreated with aluminium hydroxide in an amount of from 000 to 000, preferably from -8-'50 to 60019, can be directly used according to the invention in diluted form with the buffer solution used for said pretreatment. Immunological agglutination or agglutination 1111
-18- It can be used for a termination reaction.

For example, in an immunological agglutination reaction, sliced carrier particles and a measurement reagent are brought into contact with each other. If PIVKA is present in the blood sample, an agglutination reaction will occur;
Can be observed with the naked eye. The degree of PIVKA# in a blood sample can be determined by increasing the dilution factor of the blood sample and determining the double maximum dilution factor at which an agglutination reaction can be confirmed.

In addition, in the immunological aggregation inhibition reaction, as above,
A blood sample diluted to a certain dilution rate and an anticoagulant factor antibody corresponding to PIVKA to be detected are directly placed on a glass slide plate or in a small test tube, and after mixing, the anticoagulant antibody corresponding to the anticoagulant factor according to the present invention is added. A measurement reagent consisting of carrier particles sensitized with the coagulation factor is added and brought into contact, and it is determined whether an agglutination image appears at that time (PIVKA is present at a certain concentration or higher in the nuclear blood sample). Then, the degree of PIVKA# in the blood sample can be determined in the same manner as above based on agglutination 1 (an eye image is observed).

As described above, the method of the present invention requires only a very simple operation of adding a blood sample diluted solution, a reagent, and visually observing the agglutination image on a glass slide plate or in a small test tube. , PIVK', A in a blood sample can be rapidly measured, and clinically, PIVKA can be quantified very easily at the patient's bedside in a few minutes. Such effects of the present invention are completely unexpected from the conventional measurement methods described above, and bring about an epoch-making development in the field of PIVKA measurement.

Next, the present invention will be further explained by examples.

Example 1 (α) Production of prothrombin from human citrate plasma by the method of BttnαrOuB et al.
Thromboa, Diathes, h, ae
morr, 30.

42511973)), i.e. adsorption on notrium citrate, leaching, barium removal with Dowew 5 oW-X-8, rsgphadez G 100 gel p filtration,
Hydroxyapatite, 'DEAE-8ephad
It was purified and separated by exA 50 column chromatography.

(b) Production of anti-prothrombin antibody The prothrombin l- obtained in (α) above was added to 1 ml of physiological saline. Dissolved in 1
．． The mixture was emulsified with the same amount of Freund's adjuvant and injected subcutaneously into rabbits. This injection was performed at 3-week intervals, and after confirming the increase in antibody titer, all mice were collected and suppressed.

After the obtained blood was left at room temperature for 40 minutes.

-21- Serum was separated by centrifugation at room temperature and 3.00 Orpm for 30 minutes. 0.9 in phase with this antiserum 40+d
1/20OA/phosphate buffer (pH?
, 2) and stirred, the saturated M't N? Anti-prothrombin antibody was produced by salting out with ammonium.

(C) Preparation of anti-prothrombin antibody-sensitized polystyrene latex. The anti-glothrombin antibody prepared in 4i-(b) was added to 5'me of 0.24 AI glycine buffer (pH
9,61, and 5 g of 2% IW/V) polystyrene latex suspension (average particle size: 0.109μ) were added thereto and mixed, followed by stirring at room temperature for 2 hours. After this, centrifuge? 8 precipitate 0.3 q6 bovine albumin IB
8.4) Suspended in glycine buffer 1Orne containing
A polystyrene latex sensitized with anti-p-thrombin antibody was obtained.

(d) Coagulation reaction-22- Plasma of a healthy individual was mixed with 0.24 M glycine buffer (pH 9,
6) Dilute plasma 100, 200, 400 and 800 times, drop 2 drops (approximately 100 μt) of each diluted plasma onto the reaction slide plate, and... : At t1, the anti-glothrombin antibody-sensitized polystyrene latex prepared in 1(C) above is added drop by drop. Both were thoroughly mixed, the slide plate was gently rocked, and after 2 minutes, the presence of agglutination was observed with the naked eye, and a clear agglutination+jL image was observed up to the 400-fold diluted plasma.

It was found that the permeability of prothrombin in the plasma of healthy individuals is about 80~/l, and that the tex test that sensitizes anti-prothrombin antibodies from the iJ reaction with pure prothrombin.
Is the electric shock 0.2μ for prothrombin? /me was.

Example 2 (α) Prothrombin sensitized latex lFi & ζ Example 1 (Prothrombin 4 produced in (1) was added to 0,
Dissolved in 24 M glycine buffer (pH 9,6),
2% polystyrene latex 1tffl diameter 0.500μ
) and stirred at room temperature for 1 hour. After stirring 4C, 1
Centrifugation was performed at 0.00 rpm for 20 minutes, and the resulting precipitate was washed with the above buffer solution 10-.

After centrifugation again, the precipitate was suspended in a glycine buffer containing O, flql, and BSA to produce plthrombin-sensitized polystyrene latex.

(6) Aggregation inhibition reaction Blood plasma from healthy individuals was mixed with 0.24 M glycine buffer (pH 9,
6) to 100, 200, 400 and 800 times, 1 drop (approximately 50 μt) of each diluted solution was dropped onto a reaction slide plate, and 400 times of the anti-prothrombin antibody produced in Example 1 (b) was added to the reaction slide plate. One drop of the diluted solution was added dropwise; '□After mixing, the prothrombin-sensitized polystyrene latex suspension prepared in step (a)) was added dropwise. Mix these three ingredients uniformly, gently rock the slide plate,
After 2 minutes, agglutination was observed with the naked eye, and an agglutination inhibition image was observed.
A clear agglutination inhibition reaction was observed up to 400 times diluted plasma. The electric shock measured by this latex reagent is 62μ for professional) on bottles.
It was f/ld.

Example 3 Production of anti-factor ■ antibody sensitized polystyrene latex Anti-factor ■ antibody 10■ prepared from anti-factor ■ antiserum manufactured by Hoechst by ammonium sulfate salting out was added to 0.24 M of 5-
Dissolve in glycine buffer (pH 9,6) and add 2% (
W/V) Polystyrene latex suspension (average particle size:
0.340μ) was added thereto, mixed, and stirred at room temperature for 3 hours. After this, the precipitate obtained by centrifugation was
The suspension was suspended in 10 ml of glycine-containing buffer to obtain polystyrene latex sensitized with anti-factor (■) antibody. Measurement sensitivity is 0.1μ
It was b~.

-25 Example 4 Preparation of anti-factor ■ antibody sensitized polystyrene latex 5 ml of 0.2 mL of anti-factor ■ antibody prepared from anti-factor ■ antiserum manufactured by Hoechst by salting out sulfur-9 ammonium
4M glycine buffer (pH 9.63), knead 2
%tw/v+polystyrene latex suspension (average particle size: 0.220μ) was added and mixed, followed by stirring at room temperature for 2 hours. After this, the precipitate obtained by centrifugation was 0.34B
Suspend in 10 slj of glycine buffer containing SA,
A factor antibody sensitized polystyrene latex was obtained. The measurement sensitivity was 0.4 μ't/ml.

Missing Example 5 Production of anti-factor X antibody sensitized yh u styrene latex D
The method of iaeipi6 et al.
Anti-factor Dissolved in liquid ip&9.63 and added 2% (W
/V) Polystyrene latex suspension (average particle size:
35ml of 0.109μ was added, mixed, and stirred at 37C for 60 minutes. After this, the precipitate fO13%B obtained by centrifugation
The suspension was suspended in SA-containing glycine buffer 10 to obtain anti-factor X antibody sensitized polystyrene latex. The measurement sensitivity is 0.
It was 2μf/at.

Example 6 (α) Production of anti-prothrombin antibody sensitized spheres A 4% af8 solution of formalin-fixed sheep red blood cells (phosphorus #
buffer, pH 7.4) with an equal volume of 0.01% tannin #
After adding the solution and reacting at 56C for 30 minutes, the red blood cells were washed with phosphate buffer.

It was made into an 8-chi suspension. Next, a 0.1% solution of the anti-prothrombin antibody prepared in Example 1(b) was added, and 56C
The mixture was allowed to react for 2 hours. After the reaction, the blood cells were centrifuged and washed with phosphate buffer, and phospho-aM containing 0.2 frBSA'lt was added.
A suspension of 3 blood cells was prepared using a buffer solution.

(6) Hemagglutination reaction Healthy plasma was mixed with 0.24 M glycine buffer (pH 9,
6) at 50.100,200,300.400 and 5
00 times diluted, put 300 μt of each diluted solution into a small test tube, add 50 μt of the suspension of anti-prothrombin antibody-sensitized blood cells prepared in (a) above, mix, and shake vigorously.
The tube was left standing on a stand with a mirror for 2 hours, and the tube bottom image KJ-1 aggregated image was determined.

Shows agglutination images up to 400 times diluted plasma, and measurement sensitivity is 0.2μ
It was t/-.

Example 7 Determination of PIVKA in plasma (clinical application) (a) Test method Barium sulfate (100 η/-) suspended in physiological saline containing 0.2% bovine serum lupulmin (BSA).

To 100 μt of M suspension of barium carbonate + 100 nq/me) or aluminum hydroxide gel + 0.1%) was added 25 μt of test plasma to which citric acid or oxalate had been added, and 37
C for 15 minutes every 2 minutes, then add and mix 375 μt of physiological saline containing 0.2% BSA, and then
Centrifuged for 5 minutes at rpm. This supernatant is a 20-fold dilution of the original plasma.

Furthermore, if necessary, two drops (about iooμt) of the test solution diluted with O2% BsA-containing physiological saline were dropped onto the reaction slide plate, and one drop of anti-prothrombin antibody-sensitized polystyrene latex was added thereto. The slide plate was gently rocked, and the presence or absence of aggregation was observed after 2 minutes. 0.2μ
PIVKAx corresponding to buqthrombin over t/d
Since agglutination will occur if PIVKAu is present, the amount of PIVKAu in the test plasma was calculated by multiplying the highest dilution at which agglutination was observed by 0.2. In addition, in Table 1, PIVK- by the method of the present invention
The 29-An measurement value expressed as 0 indicates that the sensitivity of the PIVKAU mice was less than the measurement sensitivity of this reagent.

(b) Test results (a) Newborn (8-7 days after birth) Cloth I VKAt constant tone μm- 100,0647,0- 20-0,1644,0- 30-1,046,0- 40030,0 - 500,0935,0- 600,3351,0- 700,0451,0- 800,0937,5- 900,1649,2- 1000,30310- 110-0,05410- 1240,2832,0+ 134 0.06 320 +148
0.76 35.0
+-30- Inhibition Index-HP7'(%)-
TT@)/HPT((transformation) HPT: Hepaplasten test, TT: Thrombotest, presence or absence of PIVKAl by DDIEP two-dimensional electrophoresis 11 PT is set as 100 for healthy adults, and newborns have low values as shown in the table. The inhibition index indicates J166.
The presence of PIVKA was suspected in cases 10 and 14, but as a result of measurement using the proposed method, one
InhibitionIndljZ
Then, there are 3 and 1 including Ji12 and 13 that could not be found.
1 indicated the presence of 4 to 8 μ2/− of PIVKA, and this result was consistent with the qualitative findings by DDIEP. Therefore 4
Three cases numbered 12 to 14 were suspected to have abnormalities in vitamin-dependent factors, and were treated with vitamins.

(b) Table 2 of patients with vitamin deficiency 132 1.0 0 + 28-0.19 34.8 + 3 32 1.0 0 +4 3
2 -0.16 12.7 +s 16
-0.34 323 +6 16 -0
．． 55 35.5 +732 1.0 1 18 32 0.65 14.0 + 9 16 0.42 6LO+ Among the 0 patients with 91rid, patients with A2.4.5.6 were:
Despite coagulation abnormalities, conventional Inhi
Although PIVKA was judged to be rare based on the clinical index, all cases of high-grade PIVK were detected using the method of the present application.
The existence of A was revealed, and this result agreed with the DDIEP results, making the treatment policy clear.

(c) Table 3 μ for patients receiving vitamin antagonists? Hawk 116 0.40 44.0 12 32 1.0 0 +3
8 0.28 3
ZO+4 8 0.37 86.0 + 5 2 0.24
38.0 +6 B 0.27 6
3.0 + 7 0 0.37 57.0 --3
3-A, Case 7 Inhibition Intex
This is an example in which the existence of PIVI (A) was not proven using the method of the present application and Dl) IEP, although the diagnosis of PI VKA was overlooked, and the treatment policy became clear from this result.

2) DIC patients Table 4: Method of the present invention Inhibition IiP
PI Index by 'l' DDI
4 EPVKAy constant j
PIt value VKA)
μf/at 1 0 0.47 35.8 -2
0 -0.11 31.4 -3 0
0.28 33.2 -4 0
0.15 39.1-5 0 0.0
2 45.1 -6 0 -0, O?
37.4 -7 0 0.13 3
8.0 -8 0 0.39 26.
9 -9 0 0.08 44.2
-10 0 0.16 47.3 -11
0 -0.13 30, 〇 -1200,
4534,1- 1300,0929,7- 140-0,3031,4- 150-0,2531,4- -34- Showing clear coagulation abnormality, Inhi/J i
In some cases, the existence of PIVKA was suspected based on t 1onIndex, but this method 1 denied the existence of PIVKA on the gold side, and showed that the coagulation abnormality occurred due to a cause other than the abnormality of coagulation factors. , different treatments were given.

-35-

Claims (1)

[Claims] 1. A blood sample pretreated with barium sulfate, barium carbonate, or water-containing aluminum oxide is subjected to blood coagulation tubes ■, ■,
2) Subject to an immunological reaction using an immunoassay reagent consisting of immunologically inert carrier particles sensitized with factor X or its antibody, and determine the blood in the blood sample from the result of the reaction. 1. A method for measuring a blood coagulation disorder factor in a blood sample, the method comprising qualitatively or quantitatively determining the coagulation disorder factor. 2. An immunological measurement reagent for a blood coagulation abnormality factor, characterized by comprising immunologically inactive carrier particles sensitized with blood coagulation factors (1), (2), (2) or X or its antibody.