Patent Application
20240402197
The present invention relates to a method for analyzing blood coagulation reaction.
A blood coagulation test is a test for diagnosing the blood coagulation ability of a subject by examining the coagulation reaction of the subject's blood specimen. In the blood coagulation test, generally, after adding a predetermined reagent to a blood specimen, the coagulation reaction is measured over time, and the blood coagulation time is measured based on the time series data of the obtained coagulation reaction. The blood coagulation time is used as an indicator for detecting an anomaly in blood coagulation ability. In recent years, automatic analysis devices for measuring the blood coagulation time have been widely used.
When measuring the blood coagulation time with an automatic analysis device, a specimen and a reagent are put into a cell, and the coagulation reaction in the cell is measured over time with an optical method and the like. Since the measurement of the coagulation reaction is performed in a measurement port of the device, a cell is supplied to and removed from the measurement port for each measurement. A common automatic analysis device includes a plurality of measurement ports for measuring the coagulation reaction in a cell, thereby being able to measure the coagulation reaction in a plurality of cells in parallel. A specimen is dispensed into a cell supplied to the measurement port, and thereafter the coagulation reaction in the cell is measured after a reagent is added to the cell until the cell is removed from the measurement port. In normal automatic analysis devices, the measurement time is set in advance, and is the same between cells (specimens).
Patent Literatures 1 and 2 describe a blood coagulation analysis device comprising a drive mechanism which rotates and stops a table in a reciprocating direction, the table including a plurality of measurement unit modules for measuring the coagulation time of a cell, the blood coagulation analysis device capable of moving the measurement unit modules to an attachment/detachment position of a cell, a specimen dispensing position, and a reagent dispensing position, and stopping the measurement unit modules. Patent Literature 3 describes a blood coagulation analysis device comprising a fixed table including a plurality of light measurement ports for measuring the coagulation time of cuvette (cell), and a cell transfer arm which supplies a cell including a specimen to the light measurement ports on the table.
In conventional blood coagulation tests, it is common to measure the coagulation reaction for the time sufficient for the coagulation reaction to complete, and then to calculate the coagulation time based on the obtained data. However, an abnormal specimen which contains an anomaly in a blood coagulation reaction often shows a delay in the initiation and progress of the coagulation reaction, and as a result, it tends to take a long time for the coagulation reaction to complete. Therefore, when measuring the coagulation reaction of a large number of blood specimens with a conventionally common automatic analysis device, in order to prevent measurement termination midway through the coagulation reaction, it has been necessary to set a long measurement time in consideration of existence of an abnormal specimen with prolonged coagulation time. On the other hand, since most specimens to be measured are normal samples, the longer the measurement time is set, the lower the overall analysis efficiency becomes.
The present invention relates to appropriately adjusting the measurement time of each blood specimen in blood coagulation reaction analysis by an automatic analysis device, and improving the analysis efficiency.
The present invention provides the following.
[1] A method for analyzing a blood coagulation reaction, comprising,
[2] The method according to [1], wherein the reaction table has a disk-like shape.
[3] The method according to [1] or [2], wherein the reaction table performs an intermittent rotation operation, and a time for which the reaction table completes one full round of rotation is from 320 to 380 seconds.
[4] The method according to [3], wherein an interval of the intermittent rotation operation is α seconds, and a number of the measurement port on the reaction table is β, where α=from 8 to 20, β=from 16 to 47, and [α×β]=from 320 to 380 seconds, and
[5] The method according to any one of [1] to [4],
[6] The method according to [5], wherein when the measurement port of the reaction table reaches a predetermined position A, the cell is supplied to the measurement port, when the measurement port reaches a predetermined position B, the subject blood specimen is dispensed to the cell, when the measurement port reaches a predetermined position C or a predetermined position D, the reagent is dispensed to the cell, and thereafter, when the measurement port reaches the predetermined position A or a predetermined position E, the cell is removed from the measurement port.
[7] The method according to [6], wherein the position A is disposed on a location shifted from the position E in a rotation direction by one intermittent rotation operation of the reaction table.
[8] The method according to [6] or [7], wherein when the cell supplied to the measurement port reaches the position E on the reaction table, if it has been determined that the coagulation reaction of the sample solution in the cell is completed, measurement of the coagulation reaction of the sample solution is terminated, and then, at the position E, or when the cell reaches the position A, the cell is removed from the measurement port.
[9] The method according to any one of [6] to [8], wherein when the cell supplied to the measurement port reaches the position E on the reaction table, if it has not been determined that the coagulation reaction of the sample solution in the cell is completed, the cell is rotated on the reaction table without being removed from the measurement port, and measurement of the coagulation reaction of the sample solution in the cell is continued.
[10] The method according to any one of [6] to [9], wherein after dispensing the subject blood specimen to the cell supplied to the measurement port at the position B, when the measurement port reaches the position C or the position D for the first time, the reagent is dispensed to the cell.
[11] The method according to any one of [6] to [9], wherein after dispensing the subject blood specimen to the cell at the position B, before dispensing the reagent to the cell at the position C or the position D, the reaction table is rotated one or more times, during which heating of the subject blood specimen is continued.
According to the present invention, the efficiency of analysis of the blood coagulation reaction by an automatic analysis device can be improved without complicating the configuration and operation of the device.
In analysis of a blood coagulation reaction, a predetermined reagent is added to a blood specimen to prepare a sample solution, and the blood coagulation reaction thereof is measured. The blood coagulation reaction is generally represented by a coagulation reaction curve which indicates changes over time in the amount of coagulation reaction. In the present specification, a blood specimen, a blood coagulation reaction, and a blood coagulation time may be simply called a sample, a coagulation reaction, and a coagulation time, respectively.
Conventional blood coagulation analysis devices typically measure the coagulation reactions of a large number of sample solutions in parallel. The measurement time of the coagulation reactions is set in advance, and is basically constant between the sample solutions. Therefore, in analysis by blood coagulation analysis devices, it is necessary to set a longer measurement time in consideration of the possibility that an abnormal sample with prolonged coagulation time exists in a sample group to be tested. Although the aforementioned Patent Literatures 1 and 2 indicate the possibility that the measurement time can be changed for each sample (for example,
Meanwhile, in a method for analyzing a blood coagulation reaction of the present invention (hereinafter also referred to as the method of the present invention), in parallel with the measurement of the coagulation reaction of a sample solution in a blood coagulation analysis device, it is determined whether or not the coagulation reaction of the sample solution is completed. When it is determined that the coagulation reaction is completed, the measurement of the coagulation reaction of the sample solution is terminated, and otherwise, the measurement is basically continued until the coagulation reaction is completed. Accordingly, in the method of the present invention, the measurement time of a coagulation reaction may be changed for each sample solution. The method of the present invention can optimize the measurement time of a coagulation reaction for each sample solution, thereby enabling reduction of overall measurement time, so to speak, improvement of analysis efficiency.
On the other hand, a blood coagulation analysis device generally performs complicated operations, such as supplying a cell to a measurement port, dispensing of a sample and a reagent to the cell, movement of the measurement port, and removal of the cell from the measurement port. However, complication of the operations of the device easily results in blurring and noise in measurement values, which in turn leads to a decrease in measurement accuracy. For example, in cases of the devices described in Patent Literatures 1 and 2, a measurement port is moved to a predetermined position by rotating a table on which the measurement port is mounted in a reciprocating direction. Although such reciprocating operations make it possible to immediately move a cell to a target position with the minimum moving distance, since the cell is swung and the sample solution in the cell is shaken with the reciprocating operations of the table, noise is mixed into a coagulation reaction curve (measurement data), which will have a significant negative influence on measurement values (coagulation times). It is desired to prevent blurring and noise in measurement values by simplifying the operations of the device.
The method for analyzing a blood coagulation reaction of the present invention comprises the following:
The blood coagulation analysis device used in the method of the present invention comprises a reaction table including a plurality of equally spaced measurement ports to which a cell can be attached. A cell into which a sample solution is to be put can be supplied to each one of the measurement ports. At the time of analysis, the coagulation reactions of the sample solution in the cells supplied to the measurement ports are measured. Although it is preferable that the coagulation reactions are optically measured, well-known methods may be used, and the methods are not particularly limited. In the blood coagulation analysis device used in the method of the present invention, each measurement port is moved to an attachment/detachment position of a cell and to a dispensing position of a sample and a reagent by rotating the reaction table in one direction. Such a mechanism simplifies the configuration and operation of the device, and reduces blurring and noise in measurement values due to the operation of the device.
The data of the measured coagulation reaction of the sample solution is successively sent to a data processing unit, and is accumulated as the time series data of the coagulation reaction. The data processing unit determines whether or not the coagulation reaction of the sample solution is completed from the time series data of the accumulated coagulation reaction. When it is determined that the coagulation reaction is completed, the measurement of the coagulation reaction of the sample solution is terminated. The cell containing the sample solution whose coagulation reaction is completed is removed from the measurement port, and is discarded.
Hereinafter, referring to the drawings, an embodiment of a method for analyzing a blood coagulation reaction according to the present invention will be described. However, the embodiment of the present invention is not limited to the following description. Additionally, each embodiment can be combined with other embodiments or conventional technologies.
The control unit controls the operation of the entire system. The control unit may be constituted by, for example, one or more computers. The control unit includes a CPU, a memory, a storage, a communication interface (I/F), and the like, and performs processing of a command from the operation unit, control of the operation of the measurement unit, saving of measurement data and analysis results received from the measurement unit, control of output of the measurement data and the analysis results by the output unit, and the like. Further, the control unit may be connected to other equipment, such as an external media and a host computer. The control of the system by the control unit may be performed by a program.
The operation unit obtains an input from an operator, and transmits the obtained input to the control unit. For example, the operation unit includes a user interface (UI), such as a keyboard and a touch panel. The output unit outputs measurement data and analysis results of the measurement unit under control of the control unit.
The measurement unit is mainly constituted by an analysis module 100, which will be described later, and obtains measurement data of a coagulation reaction of a sample solution including a subject specimen. In the measurement unit, n subject specimens are analyzed in parallel in n reaction units (for example, a measurement port 2, which will be described later). The obtained measurement data is analyzed in the data processing unit, and it is determined whether or not the coagulation reaction is completed. The data processing unit can also perform arithmetic processing of coagulation time calculation and the like. Measurement and analysis of data are controlled by the measurement control unit. Alternatively, measurement and analysis of data may be controlled by the control unit via the measurement control unit.
In the embodiment illustrated in
An analysis result in the data processing unit is sent to the output unit under control of the control unit. An output from the output unit can take any forms, such as display on a screen, transmission to a host computer, and printing. Output information from the output unit can include the data of a coagulation reaction curve, a coagulation time, and the like.
The analysis module 100 includes a reaction table 1, the measurement port 2, a cell transfer unit 3, a sample dispensing unit 4, a first reagent dispensing unit 5, and a second reagent dispensing unit 6. Further, in the analysis module 100 illustrated in
Additionally, although not illustrated in
The analysis module 100 may also include a control unit 12 for controlling the operation of the drive mechanism, the measurement by the measurement port 2, or the analysis of measurement data. The analysis module 100 may also include a data processing unit 13 for analyzing the measured coagulation reaction data, determining whether or not the coagulation reaction of the sample solution is completed, and calculating the coagulation time of a subject specimen. Alternatively, the control unit 12 and the data processing unit 13 may be installed separately from the analysis module 100.
Although the reaction table 1 illustrated in
In order to simplify the operation control of the reaction table 1, it is preferable that the plurality of measurement ports 2 are installed at equal intervals on the reaction table 1. The reaction table 1 is rotated in one direction by the drive mechanism (not illustrated). In consideration of accessing to the measurement port 2 from the cell transfer unit 3, the sample dispensing unit 4, and the reagent dispensing units 5 and 6 and the operations of these units, it is preferable that the reaction table 1 intermittently performs rotation operations. The number of the measurement ports 2 installed in the reaction table 1 may be appropriately adjusted depending on the size of the reaction table 1, the cycle of the intermittent rotation operation, the time required for one rotation, and the like.
As illustrated in
The measurement port 2 includes a detection unit for measuring the coagulation reaction of the sample solution in the cell. The detection unit is preferably a mechanism for optically measuring the coagulation reaction of the sample solution. In the embodiment illustrated in
The detection unit sequentially obtains, as measurement data, the photometric quantity from the sample solution at preset time intervals. The time interval for obtaining data is preferably K/10 seconds (K=an integer of from 1 to 5), and is more preferably 0.1 seconds.
The cell transfer unit 3, the sample dispensing unit 4, the first reagent dispensing unit 5, and the second reagent dispensing unit 6 are arranged around the reaction table 1. The cell transfer unit 3 serves to supply a cell to the cell attachment portion 2a of the measurement port 2 from the cell housing unit 11, or to remove the cell from the cell attachment portion 2a. The sample dispensing unit 4 serves to dispense a subject specimen from the sample rack 7a and a diluent from the diluent rack 8 to the cell in the cell attachment portion 2a. The first reagent dispensing unit 5 and the second reagent dispensing unit 6 serve to dispense a first reagent of the first reagent installation unit 9 and a second reagent of the second reagent installation unit 10 to the cell in the cell attachment portion 2a, respectively.
In the analysis module 100 illustrated in
In
A cell supplied to the measurement port 2 is preferably a disposable cell. Preferably, the cells in the cell housing unit 11 are aligned by an alignment supplying unit (not illustrated), and thereafter supplied to the cell transfer unit 3. The cell transfer unit 3 supplies a cell to the cell attachment portion 2a. The cell after measurement is removed from the cell attachment portion 2a by the cell transfer unit 3, and is discarded to a cell discarding unit (not illustrated). Preferably, the cell transfer unit 3 includes a cell detection sensor (not illustrated) for detecting whether or not a cell is in the cell attachment portion 2a.
The sample rack 7 houses sample containers (blood collection tubes, sample cups, and the like) containing subject specimens. The sample rack 7 is arranged in the sample installation unit 20. In
The diluent rack 8 houses containers containing sample diluent used for dilution of the subject specimens. For example, the diluent rack 8 houses the containers containing normal plasma used for a cross mixing test. The sample rack 8 can house a maximum of five containers at the same time, and positions from 1 to 5 are sequentially assigned to the housing locations of the containers from the top toward the end in the Y axial direction (from the upper side to the lower side in the figure). In
The reagents used for preparation of the sample solution is housed in the first reagent installation unit 9 and the second reagent installation unit 10. In
The first reagent and the second reagent are used in accordance with test items. For example, in the case of measurement of prothrombin time (PT), only the first reagent is used, and in the case of measurement of activated partial thromboplastin time (APTT), the first reagent and the second reagent are used. Preferably, the first reagent and the second reagent which are used for analysis of the same sample are arranged in the respective reagent installation units so as to be arranged side by side. In this case, when only the first reagent is used for analysis, the adjacent position for the second reagent installation unit 10 is empty. The first reagent and the second reagent which are used for analysis are conveyed to a predetermined position (Pr) by movement of the first reagent installation unit 9 and the second reagent installation unit 10, and are sucked by the first reagent dispensing unit 5 and the second reagent dispensing unit 6, respectively.
Next, referring to
In
That is, supplying and removing of a cell to and from the measurement port 2 on the reaction table 1 by the cell transfer unit 3 are performed when the measurement port 2 reaches the position A. When the reaction table 1 is rotated from there, and the measurement port 2 to which the cell has been supplied reaches the position B, a subject specimen is dispensed to the cell by the sample dispensing unit 4. When the reaction table 1 is further rotated, and the measurement port 2 reaches the position C, the first reagent is dispensed to the cell by the first reagent dispensing unit 5. When the reaction table 1 is further rotated, and the measurement port 2 reaches the position D, when required, the second reagent is dispensed to the cell by the second reagent dispensing unit 6. When the required reagent has been dispensed to the cell, coagulation reaction measurement is started. When setting a longer heating time for the subject specimen, after the subject specimen is dispensed to the cell at the position B, the first reagent may be dispensed to the cell at the position D, and then, coagulation reaction measurement may be started. Alternatively, when setting a further longer heating time for the subject specimen, the cell to which the subject specimen has been dispensed at the position B can be passed through the positions C and D without dispensing the reagents. In this case, after dispensing the subject specimen to the cell, before dispensing the reagent to the cell, the reaction table 1 is rotated once, twice, or three or more times, during which the heating of the subject specimen is continued. When the cell reaches the position C or the position D for the second time, the third time, or the fourth or more time, the first reagent and, if necessary, the second reagent are dispensed to the cell, and then, coagulation reaction measurement is started. The reaction table 1 proceeds toward a measurement termination determination point (the position E, which will be described later) while being rotated. In a case where measurement is terminated at the position E, when the cell in the measurement port 2 is returned to the position A, the cell in the measurement port 2 is removed from the measurement port 2 by the cell transfer unit 3. While the position E is located downstream of the position D in the rotation direction of the reaction table 1, the position E is located upstream of the rotation direction of the reaction table 1 with respect to the position A. Preferably, the position obtained by shifting the position E by one cycle of the reaction table 1 in the rotation direction is the position A. That is, when the position A is the most upstream in the rotation direction of the reaction table 1, the position E is arranged at the most downstream (see
In an embodiment, supplying and removing of the cell to and from the measurement port 2 are both performed at the position A. In another embodiment, supplying and removing of the cell to and from the measurement port 2 are performed at another position on the reaction table 1. For example, supplying of a cell may be performed at the position A, and removing of the cell may be performed at the position E after completion of measurement.
The rotation speed (or the time required for one rotation) of the reaction table 1 is adjusted so as to be able to secure the time for, during one rotation, the attachment and detachment of the cell to and from the measurement port 2, dispensing of the subject specimen and the reagent to the cell, heating of the subject specimen and the sample solution, and coagulation reaction measurement of the sample solution. That is, while the reaction table 1 is rotated once, a new cell is supplied to the measurement port 2 after removing the measured cell, if necessary, a subject specimen and the reagent are dispensed to the cell and heated for a predetermined period of time, and the coagulation reaction is measured for a predetermined period of time.
In a preferred embodiment, the heating time of sample is from 40 to 60 seconds, the heating time of sample solution after addition of the reagent is from 160 to 180 seconds, and the measurement time of coagulation reaction is from 120 to 140 seconds. Accordingly, from 320 to 380 seconds, which is obtained by adding these, is the time required for analysis of one sample. When the interval of the intermittent rotation operation of the reaction table 1 is α (seconds), and the number of the measurement ports 2 on the reaction table 1 is β, the time for the reaction table 1 to be rotated once is [α×β] (seconds). Additionally, when the reaction table 1 has a disk-like shape, the rotation angle of the reaction table 1 in one intermittent rotation operation (one cycle) is [360°/β]. Appropriate ranges of α and β can be determined in consideration of the time required for one rotation, and the time required for one cycle of the reaction table 1 for performing predetermined analysis.
In a preferred embodiment, α=from 8 to 20, and β=from 16 to 47. However, the time [α×β] for the reaction table 1 to be rotated once is from 320 to 380 seconds.
In a more preferred embodiment, α=8 and β=from 40 to 47. In another more preferable embodiment, α=9 and B=from 36 to 42. In another more preferable embodiment, α=10 and B=from 32 to 38. In another more preferable embodiment, α=12 and B=from 27 to 31. In another more preferable embodiment, α=15 and β=from 22 to 25. In another more preferable embodiment, α=18 and B=from 18 to 21. In another more preferable embodiment, α=20 and B=from 16 to 19.
For example, in the case of the disk-like reaction table 1 including the 40 measurement ports 2 illustrated in
As illustrated in
As an example, a case will be described where the APTT of a subject specimen in P01 is measured by the analysis module 100 in
By repeating the supplying and removing of the cell to and from the cell attachment portion 2a, wear materials originating from the cell and the cell attachment portion 2a may be accumulated on the reaction table 1 (particularly, around the attachment portion 2a). In such a case, it is assumed that there is a risk that wear materials are mixed into the cell or enter between the cell attachment portion 2a and a cell outer surface, thereby negatively influencing photometric data. It is considered that such a risk becomes higher when the number of times of using the cell attachment portion 2a becomes larger. As an example of the countermeasure for reducing this risk, a method is considered that controls the operation of the reaction table 1 so that the number of times of using each measurement port 2 is made equal. For example, the measurement port 2 which is different from the measurement port 2 used in the last analysis may be used for the next analysis. Specifically, the measurement port 2 next to the measurement port 2 used in the last analysis can be used for the next analysis. Taking
A series of steps from the supplying of a cell to the measurement port 2 on the reaction table 1 to the removing of the cell after termination of measurement are illustrated in
In a preferred embodiment, in the data processing unit 13, a coagulation reaction curve is created from the time series data of the accumulated coagulation reaction. If necessary, the coagulation reaction curve may be differentiated to create the speed data or acceleration data of the coagulation reaction. These coagulation reaction curve, speed data and acceleration data thereof can be used for determination of termination of a coagulation reaction.
Arbitrary methods can be used for determination of termination of a coagulation reaction. For example, a coagulation reaction termination point Re may be determined in accordance with any criteria, such as a point of time at which a coagulation reaction curve R reaches a plateau, a point of time at which a first-order differential curve of R (the speed data of a coagulation reaction) is decreased to 0 or a constant value after reaching a peak (the maximum speed) (refer to JP-2020-068877), and the earliest point at which the ratio of the integrated value of R in a minute time period becomes less than a threshold value (WO 2021/132552). The detection of Re in the data processing unit 13 is performed in parallel with the measurement of coagulation reaction data (in real time, so to speak).
As an embodiment of the method of the present invention, an example of the detection procedure of Re based on the method described in WO 2021/132552 will be described in detail. In a coagulation reaction curve R(i) (i is a measuring point number), the ratio of the integrated value of R(i) in a minute time period is specified as an integration ratio Z(i), and is calculated by the following equation:
In the above equation, i represents the measuring point number, and m can be appropriately set in accordance with the measurement conditions, analysis items, and the like of a coagulation reaction, and is m=from 10 to 30, for example. R(i) at the earliest measuring point or point of time at which Z(i) becomes less than a threshold value Zs is detected as the coagulation reaction termination point Re. Although Zs may be appropriately set in accordance with an analysis item, Zs is greater than 1 and is 1.100 or less, and for example, in the case of APTT measurement, Zs is preferably 1.050 or less, and more preferably, Zs is in the range from 1.010 to 1.001. In order to prevent erroneous detection of Re due to an initial reaction abnormality and the like, it is preferable that the calculation of Z(i) is performed after i reaches a predetermined calculation starting point, and after R(i) becomes a predetermined value or more. In the present procedure, while measuring a coagulation reaction, R(i) is obtained and Z(i) is calculated for in parallel, and Re is detected.
When Re is detected, and it is determined that the coagulation reaction of the sample solution is completed, a signal of termination of reaction is sent from the data processing unit 13 to the control unit 12. When the reaction table 1 is rotated once and P01 reaches the position E, if where the control unit 12 has received the signal of termination of reaction, the control unit 12 terminates the measurement of the coagulation reaction of the sample solution in P01. In that case, the cell in P01 is removed from the measurement port 2 at the position A in the next cycle.
When the reaction table 1 is rotated once, and P01 reaches the position E for the first time, if Re has not been detected, the measurement of the coagulation reaction of the sample solution in P01 is continued. The cell in P01 is not removed from the measurement port 2, and the cell in P01 enters a second rotation on the reaction table 1 while the measurement is continued.
When the reaction table 1 is rotated twice, and P01 reaches the position E for the second time, if the control unit 12 has received the signal of termination of reaction, the measurement of the coagulation reaction of the sample solution in P01 is terminated, and the cell in P01 is removed from the measurement port 2 at the position A in the next cycle. In a case where Re is not detected even when the reaction table 1 is rotated twice, the cell in P01 enters a third rotation on the reaction table 1 to continue being measured. The cell can be left and rotated on the reaction table 1 in a similar procedure until the termination of reaction is detected, so as to continue the measurement of the coagulation reaction.
In this manner, the measurement of the cell attached to the measurement port 2 and containing the sample solution can be continued until the coagulation reaction is completed.
In some abnormal samples, Re cannot be detected since the amount of change in the measurement data due to a coagulation reaction is small. Based on the existence of such abnormal samples, an upper limit threshold value for the measurement time of a cell containing a sample solution may be set in advance. When the cell in P01 is rotated several times on the reaction table 1, and the measurement time reaches the upper limit threshold value, if the control unit 12 has not received the signal of termination of reaction, the control unit 12 can cause the cell in P01 to be removed from the measurement port 2, and can send a result that there is no signal of termination of reaction to the data processing unit 13. The upper limit threshold value for the measurement time can be appropriately set in accordance with the measuring method (for example, the PT measurement, the APTT measurement, the cross mixing test) of coagulation time.
The plurality of measurement ports 2 arranged on the circumference of the reaction table 1 sequentially arrive the positions from A to E with the rotations of the reaction table 1, and if cells are supplied there, the analysis step of the method of the present invention will proceed. Accordingly, the analysis module 100 can sequentially and continuously perform analysis of the coagulation reactions of a plurality of sample solutions. Although the analysis by the analysis module 100 can be performed in one or more measurement ports 2, it is not necessary to perform analysis in all of the plurality of measurement ports 2.
As an embodiment, a flow of the coagulation reaction analysis by the method of the present invention using the analysis module 100 illustrated in
S101: supply a cell to the measurement port 2 at the position A (a first cycle).
S102: dispense a sample to the cell at the position B (a second cycle).
Start reaction measurement (light measurement) after dispensing the first reagent to the cell in S104.
Start reaction measurement (light measurement) after dispensing the first reagent to the cell in S107.
S109: continuously monitor whether or not the coagulation reaction is completed based on the obtained measurement data, and when the reaction is completed, proceed to S110.
When the cell arrives at the position E with the reaction not being completed, proceed to S201.
S110: when the reaction is completed, calculate the coagulation time, and output the calculation result. When the reaction is not completed, the content (*) in accordance with the analysis result of measurement data is output.
*: example) a flag indicating “no reaction” or “in the middle of a reaction”, “the coagulation time>X seconds”, and the like
S111: terminate the measurement at the position E (a 40×Nth cycle, N≥1).
S112: remove the cell from the measurement port 2 at the position A (a 40×N+1th cycle, N≥1), and end the measurement.
S201: continue the measurement, and the cell enters the next rotation.
S202: continuously monitor whether or not the coagulation reaction is completed based on the obtained measurement data, and when the reaction is terminated, proceed to S110.
When the cell reaches the position E with the reaction not being terminated, proceed to S203.
S203: Determine whether or not the cell enters the next rotation based on a setting condition, and when the measurement is terminated, proceed to S110.
When the measurement is continued, proceed to S201.
Operation examples of the analysis module 100 illustrated in
Sample: a sample at the position 1 of the sample rack 7a
Analysis item: Two items, i.e., PT and APTT (hereinafter, AP) (it is assumed that the measured value of PT is the coagulation time.)
Reagent: PT reagent for P01 (one-reagent system), APTT reagent for P02 (two-reagent system).
Measurement time: until the reaction table 1 is rotated once, for both PT and AP Analysis Step)
PT: the cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P01 of the measurement port 2 at the position A.
PT: 50 μL of a sample sucked from the sample container housed in the sample rack 7 is dispensed to the cell in P01 at the position B. The dispensed sample is heated on the measurement port 2.
AP: the cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P02 of the measurement port 2 at the position A.
AP: 50 μL of the sample sucked from the sample container housed in the sample rack 7 is dispensed to the cell in P02 at the position B. The dispensed sample is heated on the measurement port 2.
PT: at the position C, the first reagent dispensing unit 5 dispenses, to the cell in P01, 100 μL of a predetermined amount of the PT reagent sucked from a first reagent container installed in the first reagent installation unit 9 to prepare a sample solution. The obtaining of measurement data (reaction measurement) is started immediately after the reagent is dispensed.
AP: at the position C, the first reagent dispensing unit 5 dispenses, to the cell in P02, 50 μL of a predetermined amount of the APTT reagent sucked from a first reagent container installed in the first reagent installation unit 9. The dispensed reagent is heated on the measurement port 2 together with the dispensed sample.
AP: at the position D, the second reagent dispensing unit 6 dispenses, to the cell in P02, 50 μL of a predetermined amount of calcium chloride liquid sucked from a second reagent container installed in the second reagent installation unit 10 to prepare a sample solution. The obtaining of measurement data (reaction measurement) is started immediately after the reagent is dispensed.
PT: The coagulation reaction progress status of PT is analyzed in real time based on the obtained time series measurement data, and when Re (the coagulation reaction termination point) is detected, the coagulation time is calculated, and the calculated coagulation time is output as the PT value.
AP: The coagulation reaction progress status of APTT is analyzed in real time based on the obtained time series measurement data, and when Re is detected, the coagulation time is calculated, and the calculated coagulation time is output as the APTT value.
PT: Since it is determined that the coagulation reaction is completed (Re is detected) when the position E is reached, the reaction measurement of P01 is terminated, and the obtained measurement data is saved.
PT: the cell in P01 which has reached the position A is removed and discard.
AP: Since it is determined that the coagulation reaction is completed (Re is detected) when the position E is reached, the reaction measurement of P02 is terminated, and the obtained measurement data is saved.
AP: the cell in P02 which has reached the position A is removed and discard.
The cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P01 of the measurement port 2 at the position A.
50 μL of a sample sucked from the sample container housed in the sample rack 7 is dispensed to the cell in P01 at the position B. The dispensed sample is heated in a reaction unit.
At the position C, the first reagent dispensing unit 5 dispenses, to the cell in P01, 50 μL of a predetermined amount of the APTT reagent sucked from a first reagent container installed in the first reagent installation unit 9. The dispensed reagent is heated on the measurement port 2 together with the dispensed sample.
At the position D, the second reagent dispensing unit 6 dispenses, to the cell in P01 of the reaction unit, 50 μL of a predetermined amount of calcium chloride liquid sucked from a second reagent container installed in the second reagent installation unit 10 to prepare a sample solution. The obtaining of measurement data (reaction measurement) is started immediately after the reagent is dispensed.
The reaction measurement is continued. The reaction table 1 performs intermittent rotation operations while obtaining measurement data from the cell in P01.
The coagulation reaction progress status of APTT is analyzed in real time based on the obtained time series measurement data, and when Re is detected, the coagulation time is calculated, and the calculated coagulation time is output as the APTT value.
Since it is determined that the coagulation reaction is not completed (Re is not detected) when the position E is reached, the reaction measurement is continued.
Since it is determined that the coagulation reaction is completed (Re is detected) when the position E is reached, the reaction measurement of P01 is terminated, and the obtained measurement data is saved.
The cell in P01 which has reached the position A is removed and discard.
In the cross mixing test, there are an immediate reaction and a delayed reaction depending on the difference in the heating time of a sample for measurement. Typically, the heating time of the sample for measurement in the delayed reaction in the cross mixing test is 2 hours. In the present operation example, a description will be given of the rapid method of the delayed reaction in the cross mixing test in which the heating time is reduced to approximately 13 minutes.
Heating time of sample: 45 seconds for the immediate reaction, which is the same as that in the normal APTT measurement (refer to
S: the cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P01 of the measurement port 2 at the position A.
S: 50 μL of a sample sucked from a container at the position 3 of the sample rack 7 is dispensed to the cell in P01 at the position B. The dispensed sample is heated on the measurement port 2.
M: the cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P02 of the measurement port 2 at the position A.
M: using the sample dispensing unit 4, first, normal plasma is sucked from a container housed at the position 2 of the diluent rack 8, and then, the sample is sucked from the container at the position 3 of the sample rack 7. At the position B, 50 μL, which is obtained by combining 25 μL of the normal plasma in a probe of the dispensing unit 4 and 25 μL of the sample, is dispensed to the cell in P02. The dispensed sample and normal plasma (mixed sample) are heated on the measurement port 2.
L: the cell transfer unit 3 supplies a new cell to the cell attachment portion 2a of P03 of the measurement port 2 at the position A.
L: 50 μL of normal plasma sucked from a container at the position 2 of the diluent rack 8 is dispensed to the cell in P03 at the position B. The dispensed normal plasma is heated on the measurement port 2.
Common to SML: Heating of the samples in each of the cells in P01, P02, and P03 is continued.
S: at the position C of the third rotation of the reaction table 1, the first reagent dispensing unit 5 dispenses, to the cell in P01, 50 μL of a predetermined amount of the APTT reagent sucked from the first reagent container installed in the first reagent installation unit 9. After the reagent is dispensed, heating of the sample (the dispensed reagent and the heated sample) in the cell in P01 is continued.
M: similar to the 87th cycle, 50 μL of the APTT reagent is dispensed to the cell in P02 at the position C. After the reagent is dispensed, heating of the sample (the dispensed reagent and the heated mixed sample) in the cell of P02 is continued.
L: similar to the 87th cycle, 50 μL of the APTT reagent is dispensed to the cell of P03 at the position C. After the reagent is dispensed, heating of the sample (the dispensed reagent and the heated normal plasma) in the cell of P03 is continued.
S: at the position D of the third rotation of the reaction table 1, the second reagent dispensing unit 6 dispenses, to the cell in P01, 50 μL of a predetermined amount of calcium chloride liquid sucked from the second reagent container installed in the second reagent installation unit 10 to prepare the sample solution. The obtaining of measurement data (reaction measurement) is started immediately after the reagent is dispensed.
M: Similar to the 106th cycle, 50 μL of calcium chloride liquid is dispensed to the cell in P02 at the position D to prepare a sample solution. The reaction measurement is started immediately after the reagent is dispensed.
L: Similar to the 106th cycle, 50 μL of calcium chloride liquid is dispensed to the cell in P03 at the position D to prepare a sample solution. The reaction measurement is started immediately after the reagent is dispensed.
(The Nth Cycle: Number of Cycles in which Second Reagent (Calcium Chloride Liquid) is Dispensed <N<Number of Cycles in which Cell is Removed)
Common to SML: for each of P01, P02, and P03, the coagulation reaction progress status is analyzed in real time based on the obtained time series measurement data, and when Re is detected, the coagulation time (APTT) is calculated, and the calculated result is output.
S: since it is determined that the coagulation reaction is already completed (Re is detected) when P01 reaches the position E of the third rotation of the reaction table 1, the reaction measurement is terminated, and the obtained measurement data is saved.
S: the cell in P01 which has reached the position A of the fourth rotation of the reaction table 1 is removed and discarded. M: since it is determined that the coagulation reaction is already completed (Re is detected) when P02 reaches the position E of the third rotation of the reaction table 1, the reaction measurement is terminated, and the obtained measurement data is saved.
M: the cell in P02 which has reached the position A of the fourth rotation of the reaction table 1 is removed and discarded. L: since it is determined that the coagulation reaction is already completed (Re is detected) when P03 reaches the position E of the third rotation of the reaction table, the reaction measurement is terminated, and the obtained measurement data is saved.
L: the cell in P03 which has reached the position A of the fourth rotation of the reaction table 1 is removed and discarded.
The same as that in Operation Example 3
(The Nth Cycle: Number of Cycles in which Second Reagent (Calcium Chloride Liquid) is Dispensed <N<Number of Cycles in which Cell is Removed)
Common to SML: for each of P01, P02, and P03, the coagulation reaction progress status is analyzed in real time based on the obtained time series measurement data, and when Re is detected, the coagulation time (APTT) is calculated, and the calculated result is output.
S: since it is not determined that the coagulation reaction is completed (Re is not detected) when the position E of the third rotation of the reaction table 1 is reached, the reaction measurement is continued.
M: since it is determined that the coagulation reaction is already completed (Re is detected) when P02 reaches the position E of the third rotation of the reaction table 1, the reaction measurement is terminated, and the obtained measurement data is saved.
M: the cell in P02 which has reached the position A of the fourth rotation of the reaction table 1 is removed and discarded. L: since it is determined that the coagulation reaction is already completed (Re is detected) when P03 reaches the position E of the third rotation of the reaction table 1, the reaction measurement is terminated, and the obtained measurement data is saved.
L: the cell in P03 which has reached the position A of the fourth rotation of the reaction table 1 is removed and discarded.
S: since it is determined that the coagulation reaction is already completed (Re is detected) when P01 reaches the position E of the fourth rotation of the reaction table 1, the reaction measurement is terminated, and the obtained measurement data is saved.
S: the cell in P01 which has reached the position A of the fifth rotation of the reaction table 1 is removed and discarded.
As illustrated in
Therefore, according to the method of the present invention, while measurement can be terminated in a relatively short time for a specimen with a normal coagulation reaction and a specimen with a slight extension of coagulation time, a long measurement time can be secured for a specimen with a serious abnormality in a coagulation reaction, and with significantly extended coagulation time. Accordingly, in the method of the present invention, even when continuously analyzing a specimen group which may include abnormal specimens with extended coagulation time as well as normal specimens, it is unnecessary to set a long measurement time tailored for abnormal specimens which may exist, the throughput for analysis is not reduced.
Further, when the method of the present invention is implemented, it becomes possible to perform a cross mixing test (the rapid method for immediate reaction and delayed reaction) in parallel with usual coagulation time measurement (PT, APTT, and the like). Therefore, various kinds of blood coagulation reaction analysis can be collectively performed, without causing a significant reduction in throughput. For example, when performing continuous APTT measurement on a large number of specimens in accordance with the method of the present invention by the analysis module 100 illustrated in
Additionally, in the analysis with the method of the present invention, the reaction table 1 for measuring the cell containing the sample solution is rotated only in one direction. Therefore, swinging of the cell and shaking of the sample solution in the cell, which may occur when the table is rotated in a reciprocated manner (for example, Patent Literatures 1 and 2), do not occur. Accordingly, blurring and noise in measurement values due to such swinging and liquid permeation can be prevented.
When using the analysis module 100 illustrated in
The coagulation reaction curves of a normal specimen and two abnormal specimens (abnormal specimens 1 and 2) with extended coagulation time were obtained by using the method of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-169648 | Oct 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/036831 | 9/30/2022 | WO |
