JPS61502908A - Method for measuring blood clotting time and specific reagents for it - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Coagulation 1m is caused by the conversion of fibrinogen into an insoluble fibrin network structure. You will be able to observe it. Fibrinogen is partially digested by thrombin. This produces fibrin network membranes, which polymerize into fibrin chains, which At the final stage of the reaction system, the chains are intertwined with one another (internal and external), or fibrino They are linked together by ligase (activated by thrombin).

Several methods are known to measure the health of the blood coagulation mechanism. most Important tests include 'prothrombin time 1' 'activated partial thromboplastin time' and “thrombin time.”

(R, Ravel), Clinical Experimental Medicine (C1inical Labo) RatoryMedicine) 71-73 (4th edition, 1984) These trials The following is a brief description of the experiment. ) The above test is not performed on blood itself, but on blood sales or blood. It is carried out regarding Qing.

Determining the endpoint of blood 11i clotting elements by visually observing clot characteristics (Gross, U.S. Pat. Nos. 3 and 4) No. 58, 287). We measured the formation time of the fibrin network M (web). It is possible to measure the clotting time by We discovered that this reticular crotch can be easily observed by using a specific reagent.

The problem with these problems is that they do not work well when used to measure the clotting time of blood itself. It is in. Buttersley (II at Lersley) is US Patent No. 3.492. As stated in issue 096, “Despite great effort and care, The presence of the first blood clot may be missed. be of the same color or physical properties of the surface. The difference is simply a slight increase in viscosity, which makes the initial small clot less likely to become blood clot. It is not easy to tell from yourself. 'Tests with filters that retain blood clots His solution of gradually removing the test tubes is not satisfactory. Throughout the experiment, do you hold the stopwatch in one hand and tilt the test tube with the other? Alternatively, a device that automatically tilts the test tube is required. Our method uses blood It can be successfully applied to the measurement of blood clotting time. .

Furthermore, until now it has been said that operations that prolong blood clotting time are undesirable. It's coming. (Baughman, U.S. Pat. No. 4, 289) , No. 408 Nishinger (Singler), U.S. Patent No. 2.921, 0 No. 00). However, we have shown that prolongation of clotting time is related to clot formation time and fibrin. It has been shown that the reticular membrane formation time can be clearly separated, thereby making it easier to measure the coagulation time. I saw it. Atwood coagulated in U.S. Patent No. 1,614,337. , but his purpose was not to measure clotting time, but rather to measure the coagulation time without solvent. was the production of fibrin from animal blood under the condition.

Various particles can be used as activators of Hagemann factor, which is a related substance in the internal pathway. It has been used. These include diatomaceous earth, bentonite, kaolin, sand and silica. Includes mosquitoes, etc. Lenaham U.S. Patent No. 3,295,210 No. 4,455,377 to Finnerty. latté x particles do not activate Hagemann factor sufficiently.

The early incorporation of latex particles into blood diagnostic tests was made by Singer (Sin ger) and Plotz in the American Medical Journal (Am, J, Me d, ) 2 ts, pp. 88-92 (1956).

A review of the latex particle method is by Llechemy and Michael Shin (Michaelson) is the laboratory manager. agement), pages 27-40 (June issue, 1984) and pages 26-35 (7 Monthly issue, 1984).

Latex particles have been used in agglutination immunoassays. Here, antigen-antibody binding and by fibrin strands to form an unrelated, visible fibrin reticular membrane. The main phenomenon is the bonding of individual latex particles.

Alternatively, agglutination immunoassays use mixtures of latex particles.

For example, Uzgiris U.S. Pat. No. 4,191.73; Cambiaso U.S. Pat. No. 4,184,849, Gia ever) U.S. Pat. No. 4,115.535, and Kampmeyer (Ka+ *pmeyer) See U.S. Pat. No. 4,305,925, but this The literature of It doesn't tell me anything about using child mixtures.

Additionally, some literature contains negative comments about using mixed particle sizes. There are some things. Dorman (Dor+aan) U.S. Patent No. 4,421,896 From column 15, line 15 to column 16, line 3, it states that the particles should be monodisperse. It is described. Toyama (Tasiiyama) U.S. Patent No. 4,321,058 In column 4, lines 20-23, it says, “In order to increase the reproducibility of measurements, It is preferred to use cloth particles.' Pseudo-image coagulation assay for charged substances During the process, fibrinogen was added to change the solubility of fibrinogen and precipitate it. Used against Nogen and its derivatives. Written by Wilner, Pro. Prog, Hemostasis Thro mb, ) Vol. 4, p. 211 (1978).

Becker, in U.S. Pat. No. 4,429.040, incubate the fibrin monomer particles and measure the aggregation of fibrin monomers into the latex. A method for assaying fibrino monomer in biological fluids is described. he, Fibrin monomers are hydrophobic compared to fibrinogen and other plasma proteins. has a high affinity for latex particles. he has his way It is also possible to measure fibrinogen indirectly (fibrinogen can be measured indirectly). (after using enzymes such as thrombin to convert it into fibrin monomers), The use of latex particles for measuring clotting time was not disclosed, but the particle size It is not recognized that it is important for determining the properties of aggregates (floc-like or granular, etc.), and Aggregates obtained using a mixture of particle sizes are better than aggregates obtained using monodisperse specific reagents. , does not take into account that it is easier to observe.

Becker) in column 3, lines 11-19, that he attempted to measure. Emphasis is placed on fibrin monomer and ``soluble fibrin.'' . The term ``soluble fibrin'' refers to fibrin reticular membrane, fibrin-fibrin, contains a nogen conjugate, i.e. it is by definition a polymerized fibril to the point of insolubility. Contains no phosphorus.

Specification Our invention is based on the detection of insoluble fibrin reticular membranes as an indicator of clotting time. ing. Becker said that polymerized fibrin becomes latex particles. It does not state that they have a high affinity.

Beaker uses a high concentration of latex, but we use a low concentration of latex. Concentration is used. Becker uses a very small amount of thrombin. (sufficient to convert fibrinogen into fibrin monomer, but (not enough to convert all fibrin monomer into polymerizable fibrin). I use excessive amounts of thrombin, either directly administered or induced separately. . BeCker's inkjet session lasts about 5 minutes, but Our method takes about 30 to 60 seconds for normal samples. Example of Penker deals with plasma specimens, but we can use the blood itself.

Summary of the invention This invention relates to improvements in the technique of measuring blood clotting time. Allow extra time for blood clotting Extending the clot formation time reduces measurement error and increases clot formation time rather than ambiguous clot formation time. This makes it possible to use the time of formation of the fibrin network as the end point. Also, room In the sense that it can be done at warm temperatures and without special equipment, it can be used to treat blood itself rather than plasma. It is easier to measure the clotting time of a sample of

The improvement of this invention lies in the use of latex particles, especially for fibrin reticular membranes. For convenience, a mixture of latex particles of two different particle sizes is used.

This particular reagent also reduces the effects of blood composition and temperature.

The method of the invention is simple and can be easily carried out using inexpensive equipment. Our invention is biological This is a new method in the sense that latex particles are used to detect and measure polymerization reactions.

The method of the example is applied to the measurement of the formation time of fibrin network membrane during blood coagulation. . By adding latex particles to blood, blood proteins are adsorbed onto the particles. Ru. Adding coagulants (e.g. thrombin, thromboplastin, etc.) One of the adsorbed proteins (fibrinogen) is in phase with the adsorbed latex particles. The reaction product transforms into easily detectable polymerized fibrin by interaction ( fibrin). This makes it easier to measure the formation time of the fibrin reticular membrane. make it easier. This reticular membrane formation time has a simple correlation with the actual clot formation time. . Furthermore, the structure of the latex sufipurine reticular membrane is dependent on fibrinogen concentration. , Therefore, it is also possible to detect the concentration abnormality at the same time.

One object of the present invention is to obtain blood samples from plasma samples, with or without the use of anticoagulants. Rather, it is possible to measure coagulation times induced on samples of blood itself. It is.

Another object of the present invention is to provide a method for testing so that changes in composition do not significantly affect measurements. It is to dilute the liquid.

Another object of the invention is that by using a background that clarifies m recognition, any Changes in the color of the precipitate also allow for visual perception.

Another object of the present invention is to determine the end point of the clotting process by The observer should rely on the precise time of fibrin network formation, rather than the exact time. In order to reduce measurement errors in estimating clotting time, It is to extend blood time.

Another object of the invention is to enable visual observation of polymerization reactions. .

Another object of the present invention is to simultaneously measure blood clotting time and to measure blood clotting time. One purpose is to increase the surface area of a volume of blood by adding particles that increase the surface area of a volume of blood, leading to coagulation of the blood. It is adding a child.

Add particles, thereby providing enough surface area for the maximum rate of coagulation induced. In addition to providing a total surface area that exceeds the total surface area, the resulting surface area is Avoid being a factor in changes in analysis speed. The particles mentioned above do more than just gain surface area. It is intended to be combined with several functions, e.g. to facilitate reaction visualization. This includes laterisomes.

Another object of the invention is to remove particles associated with reactants or products of a coagulation reaction. Add to the reaction and add enough of the particles to components can be easily detected or measured from one or more aspects (e.g. reaction time) and It also detects the properties of the above-mentioned reactions associated with particle reactions of the nature (e.g. condensation or adsorption). For example, fibrinogen in anticoagulated blood can be measured by thromboplastin. When mixed with certain clotting factors (e.g. factor Vll), fibrillation occurs at a time dependent on the concentration. Forms a reticular membrane. A certain amount of specific latex particles (e.g. 0.754 microns) The formation time of the fibrin network film becomes clearer when adding white polystyrene with a diameter of You can see it. When latex particles are introduced into the blood, they are absorbed into the fibril by passive adsorption. Associates with brinogen molecules. As a result of adding thromboplastin, fibrinogen When fibrin polymerizes, it aggregates and the white latex is easily detected. Wear. The time for this latex-mediated, easily detectable phenomenon is the concentration of factor v. It depends on the degree. However, the nature of this phenomenon varies depending on the fibrinogen concentration. It's coming. When the fibrinogen concentration is normal, the fibrin reticular membrane becomes heavily coagulated. It looks like a solid substance, and at a slightly lower concentration it looks like a granular precipitate, and it has a very low concentration. When the concentration of fibrinogen is high, it becomes like a thin Higashi.

Another object of the invention is to add certain substances (e.g. thromboplastin) to of particles (e.g. latex) that associate in the assay reaction caused by The purpose is to provide an easy method for addition. In which the above-mentioned particles are added Regardless of the substances involved in the reaction (e.g. fibrin formation), fibsonogen). Furthermore, in this process, two or more types of particles are used together. adding more particles to the reaction than the addition of the two particles alone at any concentration. For example, prothrombin time is the most easily observed result. As a result, various amounts of white polystyrene with a diameter of 0.277 microns were added. When you do this, small white lumps may suddenly appear. 0.7 of the same substance Larger lumps gradually appear when using 64t chron diameter latex. this When two particles are added in a well-combined amount, a large lump suddenly appears; The degree of response at any concentration is greater than when the two types of particles are added alone. It's also big.

Another object of the present invention is to It is the addition of two or more types of particles that interact with each other to the reaction system, and the above-mentioned reaction the reaction is easily detected or measured (promoted) or more easily detected or measured than when the product is isolated or when only one type of particle is used. Selecting the above-mentioned particle properties (e.g. particle size) to inhibit It is.

Another object of the invention is to introduce particles with different properties (e.g. particle pI) into a reaction system. and certain properties of the reaction (e.g. appearance time) in the absence of particles or are easier to detect or measure than when only one type of particle is present; and other properties of the above-mentioned reactions (e.g. prothrombin clotting time ♂, concentration of compositions such as fibrinogen) and the nature of appearance of latex particles (e.g. High fibrinogen concentrations cause flocculation, while low concentrations form a white film). simply promoting or inhibiting a reaction so that it can be identified .

Another object of the present invention is to start and complete coagulation, #2 granulation, granulation or coagulation. It is to control the time.

Another object of the present invention is to control the size and visibility of aggregate material.

Another object of the present invention is to improve the speed and visibility of the coagulation, flocculation, granulation or flocculation reactions. The goal is to simultaneously control the

Other objects of the invention will be apparent to those skilled in the art after reading these descriptions and claims. would be obvious to a person of ordinary ability.

Brief description of the drawing Figure 1. Particle size and particle dispersion for the addition of monodispersed latex-dispersed coagulation reagents Relationship between product and agglomeration properties Figure 2.2 0, 277 and 0.76 in the addition of dispersed latex-dispersed clotting reagents Relationship of agglomeration properties to relative concentration of 4 micron latex particles Detailed description of preferred examples Some literature in this field distinguishes between fibrin network formation and clot formation. There has been little attempt to separate them. However, very low fibrinogen concentrations In this case, the reticular membrane can be observed without detecting the blood clot that forms later. , it is clear that this distinction is correct. Our test by formation of reticular membrane Without the latex in the kit, there would be an indistinct white glowing thread around the observation chamber. Looking at the polymerization of fibrin monomer into polymerized fibrin that only looks like It can be taken. When the threads are joined together by fipurinoligase, the threads fuse together. It becomes thicker, and other blood elements such as platelets and red blood cells become entangled, creating a more reticular structure. It hardens and becomes a black mass called a true blood clot.

The time from activation of the clotting process to observation of the clot is the clotting time.

This time interval corresponds to the apparent time from activation of the coagulation process to observation of the fibrin reticular membrane. It can be easily related to and calculated with time intervals.

This potential time for the fibrin reticular membrane varies from the first appearance of the fibrin system to the reticular membrane. This should not be confused with the time taken to form a reticular membrane until the formation of a reticular membrane is completed. The latter is fibrin This can be considered an "accurate definition" of reticular membrane formation time.

Blood clotting is a type of "coagulation." Distinguish between the words “coagulation,” “aggregation,” and “agglomeration.” However, the applicant uses these terms interchangeably. ``Condensation'' and “granulation” refers to the nature of agglomeration, with the expected granular nature of the agglomerates. used to describe reactions that result in

(dilution and expansion) Dilution of the blood slows down the aggregation time and leads to the formation of an observable fibrin reticular membrane. Increase the time interval between observable clot formation by several seconds. Preferably, the diluent The volume should be at least 4 times the sample volume.

Additionally, spreading the blood facilitates observation of reticular membrane formation. For example, this invention In the apparatus used during the method, the observation area of 5 x 7 fl in the observation chamber is a few millimeters. Spreads the blood to a thickness of about a meter.

(The total volume of liquid in this chamber is preferably 2.5 ml.) At the beginning of the reaction. The blood-thromboblasten solution is kept in a thin, clean, and clear state in the observation chamber. Blisters can be observed along the edges of the solder layer.

At the same time, this dilution and dilation means that normal blood clotting times are approximately Delay to 60 seconds. This reduces the observer's measurement error by a factor of six. Also, The initial formation of a fibrin reticular membrane and the formation of a true blood clot are physical phenomena that facilitate this extension. Separate for a few seconds. In this method, the observer can detect a more prolonged true clot formation. Rather, it is possible to accurately time the reticular membrane formation.

Blood dilution also reduces the actual change in clotting time associated with its red blood cell volume ratio. . Both dilution and expansion reduce the optical density, allowing adjacent membranes to be more easily observed. do.

Coloring reagents (e.g. pigments and dyes) may also Alternatively, use a colored or fluorescent background to make it easier to see the reticular membrane in the observation chamber. .

(Blood clotting reagent) Some substances can induce blood clotting. Used for blood coagulation measurements These include thrombin, thromboplastin, derivatives, and bothrobus. Thrombin isolated from the venom of Bothrops aLro Leptilase-R (Pentaph-like enzyme) arm Ltd,) 4002 Baste Switzerland, Illinois, USA, 600647. Abbott Laboratory in North Chicago ries) + Enter from the Diagnostic Division. Contains similar substances such as Both routes cause blood clots. Any reagent obtained can be considered a clotting reagent6 (visible by latex particles). (Promotion of awakening) The initial stage observed when fibrin network formation is promoted with latex. The clotting time was measured by an early invention for measuring protropin time. White latex particles of different sizes with dilutions in saline solution used to lengthen It contains a specific amount of a complex aggregate of in a mixed state. This specific substance is used as a blood clotting reagent. Adding it to blood in the presence of A white mass is formed at the same time as the fibulin lock-like membrane is formed. By prothrombin time method is 0.5 ml blood with 50 tag thromboplastin and 2.0 ml diluent. The clotting time for normal human blood is approximately 1 minute. this white lump The fibrin reticular membrane is easier to see than the regular fibrin reticular membrane.

Latex particles in various suspension states can be obtained from three different manufacturers. I can do it. Observation results obtained using particles from one vendor may differ from those obtained using particles from another vendor. This was confirmed when using . Differences between similar particles from different vendors are generally Il suspension medium (e.g. with or without surfactant) or packaging (e.g. This depends on the solid container in which the suspension is placed.

Initial experiments showed that the visualization of solidification was due to some kind of naturally occurring aggregation, or that blood It was designed to be able to determine what fluid is associated with blood clots. The solid material is No blood clotting agents like thrombin or thromboplastin added to diluted blood was not generated. Solid formation time depends on coagulation rather than latex addition. It is related to the time of addition of the agent, and the time of solid matter depends on the clotting time of the blood used. It changed. When observing B with a microscope, latex particles are closely associated with fibrin. It can be seen that this solid substance is caused by a coagulation reaction. interfacial dynamics Interfacial Dynamics Corporation ) (latex particle manufacturer) Dr. Jeffrey Zieman (Kano, Ge Electron microscope photographed and provided kindly by Offrey Seeman The photographs confirmed the microscopic observations.

The onset time of the reaction in the presence of latex is estimated for dilutions with saline only. The value was the same as expected. That is, the apparatus employed in the method of this invention is kept at room temperature. When diluted with 1...1 saline solution, it takes about 30 seconds and 2ml of saline solution. It takes about 60 seconds to form a fibrin reticular membrane when diluted with . gradually of blood It will be different if you dilute it, but the fibrin reticular membrane depends on whether particles are present or not. No significant changes appear in the formation time. In this way, the particle action depends on the start time The goal is not to change it, but to make it easier to see the reticular membrane.

In two types of experiments, chemical condensation is performed prior to coagulation for the setting time measurement test. It has been shown that passive adsorption of substances circulating in the blood is more important than absorption. Ta. (Chemical bonds are also used in this and other tests, although they may be effective.) However). Latex particle suspensions containing surfactants do not contain It's not very effective. Add a small amount of surfactant to prothrombin to coat the hydrophobic surface. When added to the coagulation reaction for a long time, a fibrin network membrane is observed, but the coagulation reaction does not occur. It doesn't bother me. For example, the observation darkness value facilitated by latex is 0.5 ml of blood, 50 IIv of blood, which is the condition that gives the brine reticular membrane formation time. Rhomboplastin and 1. Test of prothrombin time with Oml diluent Table of 8.7% solid polystyrene latex particles with a diameter of 711 microns. by adding 50 μl of saline suspension without surfactant. It will be done.

Unless otherwise noted, all future experiments involving changes in latex particles should be performed using this method. Prothrombin time test conditions are used. Surfactant Tween 20 (Tw When 20 to 40 micromoles of een20) is added, a fibrin network membrane is formed. does not interfere with the process, but the promoting effect of latex is eliminated.

Another observation has shown that passive adsorption is more important than chemical bonding during initial particle mixing. It is shown that something is true. Particles made of polystyrene have an end chain that can be used for chemical bonding. It has sulfonic acid residues. Particles made of other polymers generally have chemical bonding ability. It has terminal functional groups with hydroxyl, carbonyl and amino groups. be The concentration of latex particles required to exhibit a level of promoting effect is determined by It depends primarily on particle size and solids content rather than functional groups. But for a special purpose The use of certain terminal residues may be desirable for users with our In some cases, the terminal amino group has some effectiveness.

The nature of the latex-promoting effect observed in blood coagulation tests depends on particle concentration and diameter. It was found that it varies systematically. For particles with a diameter of 0.1 micron or less , it is difficult to observe the reaction with the naked eye (methods such as Shikashi, Akira Kei, and Kagiri) Such particles can also be used for observation. ) with a diameter of 3.0 microns or more Particles have a small surface area to volume ratio, so to achieve the desired total surface area, , large amounts of expensive particles are required. Particles in the range of 0.1 to 3.0 microns in diameter are collected. It is easy to handle and observe. No inappropriate testing for any particle size or optimum particle concentration It was decided. Regardless of whether the particle concentration increases or decreases from the optimal particle concentration, the reaction proceeds in the following order: Character changes. (1) The amount of coagulation decreases from its maximum value. (2) Many small granules (3) Only many granules are formed. (4) Granules and Blood clot coated white. (5) Only white coated blood clots. (6) Blood clot? A small white fibrin membrane separated from the membrane. (7) Unpromoted fibrin network Membrane formation. By systematically changing the amount of particles of different particle sizes, characteristics of the reaction appear. Ru. Amount variations for different particle sizes yield comparable results as shown in Figure 1. It turns out.

The total surface area per body of a latex particle suspension is determined by the solids content, particle density, and particle size. This calculation for particle suspensions of different particle sizes It was found that a certain total surface area produces each property of latex promotion effect. (for a given sample volume, dilution ratio and fibrinogen concentration).

Certain surface areas of latex particles correspond to specific properties for promoting visualization of clotting time The conclusion is that early latex association in the reaction mediates passive adsorption. This is consistent with the observation that

Ligand adsorption onto latex particles depends on available surface area and ligand concentration . In our case, the total latex surface area is approximately 17-18 cm2 and the dilution The drug and blood volumes were as given previously and the fibrinogen concentration was at standard levels. The fibrinogen molecules are clearly optimally available on the latex in an adsorbs into functional space. The properties of visualization enhancement change with changes in fibrinogen concentration. Ru. If the fibrinogen concentration is normal, it is best adsorbed and It will be a highly condensed view. If fibrinogen levels are lower than normal The amount of adsorption will be reduced, and granules or a white film will be formed. The clot appearance time after the addition of tissue factor, such as It depends on the action of blood factors. Fibrinogen concentration is a latex with a certain structure. Qualitatively indicated by the appearance of gas agglomerates.

The bond between fibrinogen and latex cannot be seen macroscopically. Trombo After adding plastin, fibrinogen degrades and polymerizes at a certain time to form fibrinogen. Form a brine. It is a visually facilitated web formed by associated latex particles. This is polymerization to form a membrane. Determining the appearance time of aggregated particles bound to fibrin molecules. is the addition of thromboplastin or other clotting reagent. This cohesive reaction The reaction is a secondary reaction of fibrinogen being passively adsorbed to latex particles, and blood or start even in the absence of any other processing of the particles. i.e. blood The coagulation test is a two-state latex addition reaction that proceeds through a one-step latex addition reaction. This is a reaction.

Although many molecules have an affinity for latex, coagulation assays Pure coating agents are typically used.

In this method, a pure coating of fibrinogen on latex is produced. rather, the conversion of fibrinogen to fibrin and the fibrinogen on the latex particles because the polymerization of fibrinogen contributes to the desired aggregation. This allows for competitive adsorption of blood proteins involved. Structural changes in aggregate materials are Precursors that can be observed microscopically and macroscopically and during induction reactions (fibrin formation) It is closely related to the initial concentration of protein (fibrinogen). For blood coagulation test Macroscopically, diluted blood is a pink, homogeneous liquid. Clotting substances (e.g. The addition of rhomboplastin) can be predetermined depending on several factors and variables. Inducing fibrin formation during the process. When a certain amount of latex particles are added, the filament If the brinogen concentration is at standard levels (e.g. 4.0 mg/ml), the highest Condensation occurs. The condensed material looks like large white lumps in the red liquid.

The coagulation reaction is at least reduced even when the fibrinogen concentration is above standard levels. . As a result, the fibrinogen concentration decreases to normal levels as the extent of the coagulation reaction decreases. At least a large number of white granules appear instead of lumps. but , the fibrinogen concentration is either sufficiently high (e.g. 7.0/m1) or sufficiently low. (e.g. 1.0 μ/ml) does not result in any type of clotting, but rather clot formation. fibrin reticular membrane and latex grains that give the appearance of a white coating on the surface of Produces an aggregate with children. Conceivably, these structural changes are caused by changes in the reactants. Adding I & other specific proteins that polymerize or react and cause latex agglomeration It can be used to qualitatively measure protein concentration.

(Facilitation of visualization by particle mixture) Mixtures of particles with several sizes are more effective at forming aggregates than monodisperse particles. It turned out to be a mistake. Subsequent experiments show that specific particle mixtures A specific mixture enables a reaction, another inhibits a specific reaction, and different particle sizes The different interactions of mixtures of particles have proven to be a hindrance to clinical practice. Ta.

A compilation of some experimental results is depicted in Figure 2.

Two types of latex particles differing only in particle size of 0.277 and 0.764 microns. used. These were dispersed in saline (containing 8.1% and 8.7% solids, respectively). ) White polystyrene latex particles (interfacial dynamics company Interf) acial Dynamics Corporation) Each axis in the diagram is a grain Compare the liquid volume of the child suspension to the particle suspension for each particle size used. The total surface area of the relevant particles is shown. 1 milliliter of the amount of particles shown in the diagram. of salt solution, and then added 50μ of freeze-dried thromboplastin and carbonate. Mix with Lucilla 7-(Dade). Add 0.5ml to the mixture Anticoagulated (3,8% sodium citrate) blood was added. fibrin-latek The time of aggregation (approximately 30 seconds) and properties of the mixtures were observed for each mixture. For condensation formation The volume and corresponding total surface area for each particle size were determined to be optimal for each particle size.

(For example, for a diameter of 0.277 microns, 100μρ, 17-18 square centimeters) cm, 250 μβ, 17-18 square cells for a 0.764 micron diameter. (inches) If there is a deviation above or below the optimum value, the properties of the aggregate will differ from those at maximum condensation. (e.g., small lumps, granules, white-coated clots, membrane albuginea, or unpromoted fibrin reticular membrane). These are described in more detail in relation to Figure 1. has been done. When particles of a single particle size are added, the properties of the aggregate are is determined for the total surface area of The surface area required to produce maximum agglomeration is At the standard conditions mentioned above, which show a slight variation in the level of maximum condensation with diameter. It was always about 17-1 Bcd.

The total surface area required to produce maximum agglomeration or other specific properties may vary. The particle size varies depending on the particle mixture.

The dotted line in Figure 2 shows the combination of particle mixtures with different particle sizes that yield a constant total surface area of 17- is shown.

The flocculation reaction is more widespread with certain particle size combinations than with a single size particle. can be detected over a large total particle surface area.

For example, when using only particles with a diameter of 0.277 t, the maximum condensation is Occurs at approximately 17c+J, and condensation occurs significantly even if the surface area increases or decreases by 1 cd. decreases, and if the surface area is changed larger than that, condensation will hardly occur. Ru. On the other hand, the surface area of approximately 10 cd consisting of particles with a diameter of 0.764 t is consistent. When adding different amounts of 0.277 micron diameter particles, the total particle table The area is as low as 14cj (10+4) or 25aJ (10+15) or something like that. Condensation occurs very well when the value is greater than or equal to . This observation reflects a wide range of fibrillation This makes clotting time easier to detect and measure across nogen concentrations.

Mixtures of other particle sizes may have a range of total surface area sufficient to cause distinct agglomeration. Decrease. For example, if a particle with a certain number of 0.764 (Kron diameter) has a total surface area of 2- If the total surface area is kept constant, clear condensation will occur at a total surface area of approximately (no condensation occurs with a surface area of 18cj). The value of this discovery is Detection of a wide range of fibrinogen concentrations.

Fibrinogen (or other proteins) that have clinical significance The range of concentrations can be detected by predetermining the particle size mixture and their relative concentrations. selected to do.

There is also a total surface area for mixtures of particles of different size that changes the nature of the reaction. stiffness In blood reactions, it is necessary for the appearance of white-coated blood clots, and below that, the membrane is There is a minimum total particle surface area such that particles are more difficult to detect visually. this The value of discovery is greater when using pre-selected particles of different sizes than when using particles of a single size. It is more accurate to measure fibrinogen at different concentrations using a mixture of This means that it is possible.

A series of other observation results are illustrated in FIG. A kind of outstanding reaction occurs in each grain. This has been shown to occur when judicious combinations of diameter quantities are used.

This outstanding reaction is the most successful reaction observed when using single-sized particles. This seems to be the result of promoting one's best qualities when 0.764 <The most obvious reaction when using only Chron's particle size particles is that the particles gradually become larger. or floc growth (time of 1-2 seconds), while particles of 0.277 micron diameter The most obvious reaction is the sudden appearance of fewer and smaller flocs. Ru. This superior response occurs more rapidly than with any single particle size. It consists of the formation of many large flocs that suddenly appear.

The significance of this is that combinations of particles of different sizes produce more distinct and measurable reactions. It can be used for. For example, the majority of people have a fibrinogen concentration of “ A mixture of large and small particles (e.g. 0.764 mm) is within the normal range. 1007712 for chron diameter, and 75 μl for 0.277 t chron diameter) , a very clear coagulation reaction that is large enough for even beginners to detect and measure. happen. Any difficulty in detection would indicate abnormal fibrinogen levels. cormorant. It could be improved to make it easier to detect and measure other substances. However, particles other than latex could be used as well.

Another advantage of using mixtures is cost savings. Latex is commercially available in liquid volume has been done. Sometimes large particles are more expensive than smaller ones, and their aggregation It has the advantage of being clear. However, when larger particles are used, certain total A larger amount of particles is required to obtain surface area. smaller particles with less volume Adding saves the total cost of particles.

Applicant's invention is listed below as a mechanism for the observed promoting effect of latex particles. Although the requirements should not be overlooked, the explanation is most relevant for the particular application. This is very useful for selecting specific reagents. Promotion between mixtures of particles of different size The mechanism of the protective or inhibitory interaction is thought to be related to the electrostatic properties of the particle. Ru. Latex particles have a negative charge that is directly related to their size. Suspense Particles in the ion are held together by mutual repulsion due to like charges and surface tension. Maintains average distance. These particles are attracted to the particle surface and the distance between them increases. Molecules long enough to bridge the gap form aggregates and bind to each other. match. Many molecules in the blood are attracted to latex particles, but they cannot be detected. Not everything has enough properties to form an aggregate. fiiprino Gen is one of the molecules that is attracted to the surface of latex, but it is difficult to reduce the distance between particles. It cannot be bridged. However, thrombin or (a3< substance (e.g. thromboplastin) is added to Peptides are cleaved from purinogen to produce fibrin monomers, which are further Polymerize. The longer molecular chains create aggregates with spaces between particles. Bridging the gap. Larger particles are farther apart from each other, so they aggregate A longer length of fibrin is required to form the fibrin. But that is -m, the aggregate will be larger than when using small particles. and can be detected more easily. A combination of large and small particles in a specific ratio can be used to make the average distance between particles smaller and the gap It allows the use of initially generated short fibrin molecules to bridge the can form aggregates that can be easily detected as containing larger particles. Wear. For aggregates to form, a sufficient amount of A sufficient amount of fibrinogen must be adsorbed onto the particles. this is available Determined by total surface area and concentration of fipurinogen. However, using two particle sizes When present, some reaction also occurs during polymerization. Usable surface area of one particle is small When available fibrinogen takes up a larger usable area. The degree of aggregation increases as the particles selectively attach to particles. Depends on the surface area of the particle (dominant) with area. Sufficient amount of fibrinogen adsorption onto the dominant particles, and as a result during polymerization the dominant particles (larger surface (having a product), it attracts only an amount of fibrinogen that can cause only weak aggregation. There is a range of relative concentrations of particles beyond which it is no longer possible to Also available Fibrinogen is adsorbed to usable surfaces, resulting in a good fibrinogen concentration per particle. As a result, aggregation occurs due to a larger surface area, corresponding to a larger interparticle distance. There exists a region of relative concentration of particles.

(promotion) We have a large number of small particles (100+++1) and a small number of large particles ( An electron micrograph of the sample (25 ml) was obtained. The photo has a long reaction This indicates that short fibrin chains are preferentially used.

Is the ligand of a certain length bridging the interparticle gap? If possible, the average distance from each other such that agglomeration will result The hypothesized mechanism is it makes sense.

Because of the importance of "charge" in this hypothesized mechanism, other charged particles are included in these measurements. child is considered valid. However, latex particles are more effective.

Several theoretical studies on the stability of latex dispersions have been reported. O OtLewill and Shaw, members of the Faraday Society. Discnsions of Faraday 5ociety 154-163 (1966) discusses the stability of monodisperse polystyrene particles. ing. Theoretically and experimentally, coagulation concentration is independent of particle size, or at most There is only a weak dependency. See pages 159-160 of the same report. Two contradictory particle sizes Tests of mutual coagulation of charged particles show that some particles may be affected by changes in pH stability ρ. It is said that Hogg established a legal theory that the effect due to the difference in diameter can be ignored. gg) etc. in the same magazine, pp. 1638-1651 (June 1966). It was.

Wise and Mealy, Vol. 66, pp. 490-499. (1970) found that for monodisperse systems, the stability of the dispersion system depends on the particle size. It is proposed that it be done. And they are talking about a two-dispersion system! It's a hit.

Of course, these theoretical analyzes are for uncoated particles. It is. The interaction between the coatings clearly affects the stability of the dispersion. It will make an impact.

(Origin of reagent) Thromboplastin is produced by a reaction with latex particles.

Liquid thromboplastin was mixed with latex suspension.

The mixture can be prepared by using them individually or by using lyophilized thromboplastin. It is easier to measure and handle than when using a fine powder alone.

The lyophilized mixture is used as a dry reagent in the device, where it is dissolved in a diluent. blood is added to the blood in the observation chamber. latex liquid suspension in the same way as when thromboplastin or alone is lyophilized. Formation of a reticular membrane by rhomboplastin and aggregation by latex also occur.

The lyophilized reagents are described in US Pat. No. 3,918.708 to Mayer. No. 3,497,319 to Altschul and and Lenahan, U.S. Pat. No. 3,395.210. However, these references do not discuss the freeze-drying of latex particles with coagulation reagents. It is not mentioned.

Other reagents are attached to the latex particles and the latex and reagent are simultaneously delivered to a specific point. added to the reaction to ensure transport.

(Control of surface area) Clotting time depends, up to an asymptotic level, on exposed surface area.

It is useful to spread the blood over a large surface in the observation chamber. latex The addition of gas increases the amount of exposed surface to blood, and maximum exposure Ensure uniformity.

(promoting additional visualization) Particles may be treated to further facilitate detection of reactions, e.g. using attached particles, against a red blood solution for easy detection when aggregated. Use colors that stand out. (e.g. yellow).

Fluorescent particles are also observed under ultraviolet light, and the reaction of this particle is more easily It is now possible to easily detect fibrinogen at low concentrations.

(Further modifications and applications) When two or more particles are added to a reaction, each of them contributes to a different composition or product of the reaction. As a result, the reaction is easier to detect than if no particles were present. different compositions and reaction products can be easily detected and measured.

Different reagents can be attached to particles of different sizes. Detection and measurement made easier The particles used for dextran, dextran, etc.) may be adsorbed, and these substances may the substance of interest in the solution or the substance that mediates the reaction to be detected; binds to the reaction products generated during the test, thereby further increasing the ease of detection and measurement. It may be promoted.

Two or more of the substances mentioned above may be combined in one species to further facilitate ease of detection or measurement. The particles probably adhere to one or more types of particles rather than the other particles.

Other devices adsorb substances and, by the introduction of certain reagents, cause the presence of the substances mentioned above. It is used to guide (e.g. by magnetic force) and collect the particles to be tested.

Can adsorb or react with other proteins or latex particles, or with each other. Other substances that can react with the fibrinogen concentration detection and It was tested for its concentration in a manner similar to the Especially if the reaction product is When the molecules have molecules that are significantly different from the substances, the gap between the particles, similar to fibrin polymers, can bridge the gap. In this way, the specific reagent of the present invention has the potential for polymerization. It turns out that it has a special use in promoting dimerization reactions.

Here, the latex specific reagent refers to aggregates formed by polymerization of fibrin. used to facilitate visualization. See, for example, the polymerization of Anne, Additionally, particles other than latex (e.g., silica, cells, or liposomes) may method or with latex particles.

Although the present invention has particular applicability to the testing of blood per se, it is possible with suitable modifications. In addition, it can also be used for plasma specimens. The context is consistent and there is no other way to say otherwise. In some places, the word "blood" has a meaning of 1!46 and 4. riX Nine, Niga Oei□1゜FIG, 1 Pure book (no changes in content) 0m2+o7a4+FIG, 2 garden Procedural amendment (formality) %formula% 1. Indication of case: PCT/US851004982, title of invention: Measurement of blood clotting time Standard method and specific reagents for it 3. Person making corrections Relationship to the case: Applicant Name: International Health Services 5, date of amendment order: 1937 International search report dated May 27, 2017 11-^mc11-1・pc? /+τc1. lζl＾chu◇.

Claims (36)

[Claims] 1. (a) dilute whole blood with a diluent containing a clotting reagent, and (b) the optical density of that solution. In order to reduce the (c) Clotting time of whole blood, consisting of observing the formation time of fibrin reticular membrane. How to measure visually. 2. (c) Before step, treat the blood with a staining method that facilitates visualization of the fibrin reticular membrane. The method according to claim 1, wherein the method is managed. 3. A method of observing the formation of a fibrin reticular membrane against a background that makes it stand out in contrast. The method described in Scope 1. 4. A clotting reagent consisting of a blood clotting reagent and a plurality of negatively charged particles. 5. 5. The reagent according to claim 4, wherein the particles are latex particles. 6. Blood clotting reagents include thrombin, thromboplastin, partial thromboplastin, and and activated partial thromboplastin. Reagent according to item 4. 7. The particles are selected from the group consisting of latex particles, ribosomes and red blood cells. The reagent according to claim 4, wherein the reagent is used. 8. A reagent according to claim 5, which is used in lyophilized form. 9. Claims where the average diameter of the particles is between 0.1 microns and 3.0 microns. A reagent according to scope item 5. 10. The above-mentioned treatments promote or inhibit the aggregation response of the substance to which the particles adhere. The reagent according to claim 4, which is applied to the particles. 11. As a result, predetermined Contacting blood with a clotting reagent in the presence of a specific reagent of particles of defined size and concentration A method for measuring blood clotting time, including: 12. The method according to claim 11, wherein the specific reagent comprises a plurality of latex particles. . 13. Claims that particle size and concentration were selected to provide desirable agglomeration properties. The method according to item 12. 14. 14. The method of claim 13, wherein the desired property of agglomeration is maximum agglomeration. . 15. 14. The method of claim 13, wherein the desired property of agglomeration is maximum granulation. . 16. Particle size and concentration are selected to reduce the time required to form a fibrin network. 13. The method according to claim 12, which selects. 17. Particle size and concentration were chosen to maximize visualization of the fibrin reticular membrane. 13. The method according to claim 12. 18. The particles are comprised of at least two different predetermined particles at a predetermined concentration. 13. The method according to claim 12, wherein the method has a size. 19. The above sizes and concentrations are chosen to minimize the formation time of the fibrin network and to 13. The method of claim 12, wherein the method is selected to maximize visualization of the membrane. 20. The desired properties of aggregation are achieved over the desired fibrinogen concentration range. 14. The method of claim 13, wherein: 21. Adjust the particle size and concentration to a predetermined number of particles per unit volume of diluted sample. 13. The method of claim 12, wherein the total surface area is given as: 22. The first particle size is 0.764 microns, the second particle size is 0.277 microns, Then, the proportion of particles of both particle sizes falls into the “good” or “excellent” region in Figure 2. 19. The method of claim 18, wherein the method is selected such that: 23. The proportion of both particle sizes should be settled in the "excellent" region in Figure 2. 23. The method according to claim 22, wherein: 24. In order to form a fibrin network membrane, a certain amount of Mixing a certain amount of blood with a coagulation reagent and incorporating the above-mentioned specific reagent into the net. Measuring the clotting time, which consists of observing the formation time of the fibrin reticular membrane and How to do it. 25. Including adjustments for dilution rate and solids content, represented by the dotted line in Figure 1. relative to the suspension volume expressed in microliters. A volume of latex particle suspension, with particle size D in microns, A method of measuring blood clotting time comprising contacting a blood sample with a blood sample. 26. Mixing the dispersed particles and monomer in the presence of a polymerization reagent and producing Time elapses until coagulation of polymer or oligomer and latex particles is observed. It consists of measuring the time until the monomer becomes a polymer or oligomer. How to measure time in. 27. 27. The method of claim 26, wherein the monomer is a fibrin monomer. 28. Fibrin monomer is obtained by the conversion of fibrinogen to fibrin. 28. The method according to claim 27. 29. 28. The method of claim 27, wherein the monomer is actin. 30. The particles are of at least two different sizes and have a predetermined size and concentration. 27. The method of claim 26, wherein: 31. When fibrinogen is normal, the particle size and concentration are reduced to form clots. Select and characterize latex of predetermined particle size and concentration in the presence of coagulation reagents. Mixing the fixed reagent with the blood sample and determining the time and onset of fibrin network formation. , clotting time, which consists of observing the properties of aggregation of latex particles and fibrin. and at the same time detect abnormal fibrinogen levels in the blood. 32. Claim 1 comprising a coagulation reagent and a plurality of types of latex particles. The method described in section. 33. 33. The method of claim 32, wherein the particle size of the latex particles is varied. 34. The method according to claim 33, wherein particles of each particle size are present in a predetermined concentration. Law. 35. Claim 3, wherein the particles are treated to facilitate their detection. The method described in Section 2. 36. Claim 32, wherein the coagulation reagent comprises an aggregation inhibitor or accelerator. Method.