Method of analyzing reaction rate in chemical analysis

The present invention relates to a method of analyzing a reaction rate in chemical analysis, in which a plurality of time region bands wherein the reaction rate is measured are set to be different in time series, a sample high in active value is calculated based on the data contained in the region band positioned forward in time series, and a sample low or normal in active value is calculated based on the data contained in the region positioned backward in the time series.

BACKGROUND OF THE INVENTION 
The invention relates to improvement in a method of analyzing a reaction 
rate in chemical analysis used, for example, in the field of medical 
checks. 
In modern medical diagnosis, the checks of tumors for example typically 
urine and blood is one of indispensable factors. In these checks, the 
sample to be checked and reagents are distributed into a reaction cell 
that is moved in a reaction tank whose temperature is kept constant, then 
after the reaction, the resulting liquid to be measured is illuminated by 
a photometric light, the absorbance is detected, and thus, for example, 
the active amount of an enzyme in a serum is measured. In this case, in 
the analytical method using a conventional reaction rate measuring 
process, a sample and all reagents required for measurement are mixed, and 
after a prescribed period of the reaction has passed, the absorbance of 
the reaction liquid is detected. This "prescribed period" is generally 
called "lag time" and this lag time has the following three meanings: 
(1) The time period which goes from the time when reagents are added to the 
time when the change in temperature caused thereby stops. 
For example, when an enzyme reaction is measured, it is required to keep 
the temperature constant, and therefore the value of the temperature in 
the reaction cell during the reaction is to be kept constant at all times. 
However, in the case wherein the temperature of a reagent that is added in 
the final stage is different from that constant temperature, since the 
temperature in the cell changes naturally at the time of the addition, 
generally the measurement is carried out taking the period required for 
settling of that change in temperature into consideration. 
(2) The time period required for the stabilization of a reaction liquid 
after the stirring. 
For example, after the final reagent is added, the sample and the reagent 
ar stirred well. At that time, a state unfavorable for the measurement of 
absorbance, for example, a state wherein bubbles suspend in the reaction 
liquid continues for a while. Consequently, for accurate measurement the 
system must wait until the unstable state of the reaction liquid due to 
the stirring, for example, the presence of bubbles in the reaction liquid 
disappears. 
(3) Lag time of the reaction. 
For example the analysis of glutamic oxaloacetic transaminase (GOT) in 
serum can be expressed by the following two step separate reactions (i.e. 
equations): 
##STR1## 
In the measurement of enzyme reactions by using NADH, if the reactions 
include the dehydrogenation reaction of the coenzyme NADH indirectly or 
directly, the measurement is carried out at around 340 nm. Here, the first 
reaction cannot be detected optically, but the produced oxaloacetic acid 
can be related to the second reaction thereby enabling an optical 
measurement. 
In this case, the reduction type coenzyme (NADH) has absorption in the 
ultraviolet range, the change in absorbance that takes place when the NADH 
changes to NAD.sup.+ according to the equation (2) is utilized for the 
measurement of the enzyme active amount of the above GOT, and in order to 
measure that reaction, the second reaction must be waited until the first 
reaction proceeds. That is, the reaction according to the second reaction 
equation (2) is required to be waited until oxaloacetic acid is produced 
enough to reach the maximum rate in the conversion of NADH to NAD.sup.+ 
during the reaction of the equation (1). Generally, the lag time including 
this waiting time until said maximum rate is obtained is called lag time 
of the reaction. Accordingly, so long as the accuracy of measurement is to 
be as great as possible, it is required to secure a lag time that will be 
long enough to expect normal proceeding of reactions with respect to all 
factors. 
Therefore, in conventional methods of measuring reaction rates, the 
above-mentioned lag time after the addition of all the reagents required 
for measurement was preset at a longest period in which reactions will 
proceed as prescribed, and the measured value of the absorbance during 
that period was excluded in the essential calculation of the measurement 
of the absorbance. 
SUMMARY OF THE INVENTION 
However, for example, in measurement of samples high in active value, since 
the proceeding of the reaction is high, if the lag time is preset based on 
the above concept, NADH required for the proceeding of the reaction 
disappears in the lag time. Thus, if the calculation of the measurement is 
carried out based on the data obtained in the measurement region band, an 
incorrect data will be obtained. Therefore, in conventional methods of 
measuring reaction rates, the reaction limit level was set based on the 
unit of the absorbance, and when the NADH concentration lowered below a 
certain level in the measurement region, an alarm or information that the 
particular sample was too active to be measured was issued. Accordingly, 
in the conventional method, it had a defect that there inevitably happened 
a limit on the range of the measurement, and a highly active sample could 
not be measured. 
Taking the above situation into consideration, this invention has been 
completed, and the object of the present invention is to provide a novel 
method of analyzing a reaction rate in chemical analysis wherein a 
plurality of measurement region bands required for measurement of a 
reaction are set thereby allowing the range of measurement to be widened. 
To attain the above object, first the present invention provides a method 
of analyzing a reaction rate in chemical analysis wherein first a sample 
and a reagent are reacted, and by measuring the reaction rate thereof, 
characteristic values such as the active value and the concentration 
concerning the sample to be measured are measured and analyzed, 
characterized in that a plurality of measurement region bands different in 
time series in which the said reaction rate will be measured are provided, 
the measurement of a sample high in active value is calculated by using 
data included in the region band in a forward position in said time 
series, and the measurement of a sample normal or low in active value is 
calculated by using data included in the region band in a backward 
position in said time series. 
Secondly, the present invention provides a method of analyzing a reaction 
rate in chemical analysis as will be set forth below wherein first a 
sample and a reagent are reacted, and by measuring the reaction rate 
thereof, characteristic values such as the active value and the 
concentration concerning the sample to be measured are measured and 
analyzed, characterized in that a plurality of measurement region bands 
different in time series in which the said reaction rate will be measured 
are provided, the measurement of the characteristic values concerning said 
sample to be measured is carried out by the measurement mode that uses the 
region band positioned backward in said time series, and when the number 
of the effective measured data obtained in said measurement mode is less 
than a predetermined value, the analysis is automatically carried out 
based on the measurement mode that uses the region band positioned forward 
in said time series. 
In the present invention, a plurality of measurement region bands different 
in time series are provided where data for the calculation of the reaction 
rate are collected, and one of the measurement region bands is a region 
band positioned forward in the time series, and the other is a region band 
positioned backward in the time series, so that the measurement of a 
sample high in active value can be carried out in the region band 
positioned forward in said time series, while the measurement of a sample 
normal or low active value can be carried out in the region band 
positioned backward in said time series, thereby extending the limit of 
the measurement.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention will now be described in more detail with reference 
to illustrated embodiments, but before that an automatic analytical 
apparatus for carrying out the present method of analyzing a reaction rate 
is described with reference to FIG. 4. 
The automatic chemical analytical apparatus designated generally 1 consists 
of a thermostatic tank 10 having a structure known per se, for example, in 
the shape of a circle, a reaction line 20 made up of reaction cells Sn 
(wherein n is 1 to 51) that are for example 51 in number and can be 
rotated in said thermostatic tank 10 in the direction shown by the arrow 
according t a certain principle, a first reagent distributing apparatus 
21, a sample distributing apparatus 22, a second reagent distributing 
apparatus 23, a first stirring apparatus 24, a second stirring apparatus 
25, and a suitable reaction cell washing apparatus 26 that are arranged 
respectively at prescribed positions A to F around said thermostatic tank 
10, and a suitable absorbance measuring apparatus 30 made up of a light 
source lamp section 31 and a photometric section 32 with said reaction 
line 20 between them. In this case, said first reagent distributing 
apparatus 21, said sample distributing apparatus 22, said second reagent 
distributing apparatus 23, said first stirring apparatus 24, said second 
stirring apparatus 25, said reaction cell washing apparatus 26, and said 
absorbance measuring apparatus 30 are apparatuses of known types having 
functions and structures known per se respectively. 
When the reaction line 20 is in the state of stop, a first reagent is 
distributed from the first reagent distributing apparatus 21 into the 
reaction cell Sn situated opposite to position A, a sample to be measured 
is distributed into the reaction cell Sn situated opposite to a position B 
from the sample distributing apparatus 22, and a second reagent is 
distributed from the second reagent distributing apparatus 23 into the 
reaction cell Sn situated opposite to a position C. Stirring of the sample 
and the first reagent is effected in the reaction cell Sn situated 
opposite to a position D by the first stirring apparatus 24, and stirring 
in the reaction cell Sn situated opposite to a position E is effected by 
the second stirring apparatus 25 after the distribution of the second 
reagent. After these operations have been completed, the reaction line 20 
is rotated one and half rotation and 1/2 pitch to be moved to the next 
position. 
In this way, during the one rotation of each reaction cell Sn through the 
thermostatic tank 10, the measurement of the absorbance of the reagent or 
the mixture liquid of the sample with the reagent contained in the cell is 
carried out by the action of the absorbance measuring apparatus 30. 
Therefore, if each reaction cell Sn is assumed to rotate one turn in the 
thermostatic tank 10 in, for example, 18 sec, data of the absorbance can 
be obtained from the mixture liquid of the sample and the reagent in the 
reaction cell in every 18 sec. 
Now, the present method of analyzing a reaction rate that uses the 
automatic chemical analytical apparatus 1 having the above constitution 
will be described with reference to the time-absorbance change diagram 
shown in FIG. 1. 
After a first reagent is distributed into a reaction cell Sn at the first 
reagent distributing position (position A) (After the time period t.sub.1 
has expired), the absorbance Q.sub.1 is measured. Then the absorbance 
Q.sub.2 in the amount of time (t.sub.2) that the reaction line needs to 
rotate the reaction cell Sn to the position next to the first reagent 
distributing position is measured, and the absorbance Q.sub.3 at the next 
time (t.sub.3) is measured. Thus, after (time t.sub.4) a sample is 
distributed at the sample distributing position (position B), the 
absorbance Q.sub.4 is measured, after (time t.sub.5) the stirring at the 
first stirring position (position D) is effected, the absorbance Q.sub.5 
is measured, and every time (each of time t.sub.6 to time t.sub.19) when 
the reaction cells Sn are rotated, each of the absorbances Q.sub.6 to 
Q.sub.19 of the solutions in the reaction cells Sn are measured. 
After (time t.sub.20) a second reagent is distributed at the second reagent 
distributing position (position C), the absorbance Q.sub.20 is measured, 
after (time t.sub.21) stirring at the second stirring position (position 
E) is carried out, the absorbance Q.sub.21 is measured, and every time 
(each of time t.sub.22 to time t.sub.38) when the reaction cells Sn are 
rotated, each of the absorbances Q.sub.22 to Q.sub.38 in the reaction 
cells Sn is measured. All of the data of the measurements of the 
absorbances measured at these points are stored in suitable memory and 
regenerating means (not shown) such as a computer. 
Under these conditions for the measurement of absorbance, the period from 
the time t.sub.20 when the second reagent is distributed into the reaction 
cell Sn to the time t.sub.23 is set as lag time Tr, and a measurement 
region band X.sub.1 where the change in absorbance of a sample low in 
active value will be calculated is set in the region band from the time 
t.sub.24 to the time t.sub.38. A measurement region band X.sub.2 where the 
change in absorbance of a sample high in active value is set in the region 
band (including the lag time band) from the time t.sub.20 to the time 
t.sub.38. In other words, the measurement time region bands X.sub.1, 
X.sub.2 are previously set such that the reaction rates can be calculated 
on the basis of all the absorbances from the absorbance Q.sub.24 measured 
at the time t.sub.24 to the absorbance Q.sub.38 measured at the time 
t.sub.38 in the case of a sample low in low activity, and on the basis of 
all the absorbances from the absorbance Q.sub.20 measured at the time 
t.sub.20 to the absorbance Q.sub.38 measured at the time t.sub.38 in the 
case of a sample high in active value. W in FIG. 1 is the previously set 
measurement range of absorbance, and Q.sub.U is its upper limit value and 
Q.sub.L is its lower limit value. X.sub.2 is defined as the region band 
positioned forward in time series, and X.sub.1 is defined as the region 
band positioned backward in time series. 
Thus, in the case of a sample (e.g., M.sub.1, and M.sub.2) low in active 
value, since the states of changes in absorbance for time in the 
measurement region band X from the time t.sub.24 to the time t.sub.38 
become approximately constant (linear), the measured values of the 
absorbances therein fall in the above absorbance measurement range W 
thereby exhibiting the measurement effect, while, in the case of a sample 
(e.g., N.sub.1) high in active value, since major part of NADH is consumed 
already at the point beyond the above-mentioned lag time t.sub.23, the 
values of the absorbances concerning the measurement after the time 
t.sub.26 will be lower than the above-mentioned lower limit Q.sub.L, and 
therefore the accurate measured value cannot be calculated, thereby 
resulting in a data error. 
However, since effective data greater than the above-mentioned lower limit 
value Q.sub.L are in the data of the measurement of absorbances measured 
after the time t.sub.21, and remain in the above-mentioned memory and 
regenerating means, if the effective measured data are used in the 
measurement of a sample high in active value, calculation of measurement 
accurate enough can be carried out. 
The present invention has been completed with attention paid to the above 
point, and the present first invention is constituted such that when a 
sample high in active value is measured, the measurement data of 
absorbances measured in the measurement region band X.sub.2 set in time 
series before the measurement region band X.sub.1 for a sample low in 
active value are utilized. 
Next, the second constitution of the present invention and its action will 
be described with reference to FIGS. 2 and 3. 
In the case of FIG. 2, the operation of measurement of absorbance is 
started with the measurement mode that uses the measurement region band 
X.sub.1 having the lag time for the case of a sample low in active value, 
and in the case that absorbance measured values lower than the 
above-mentioned lower limit Q.sub.L began to increase during the 
measurement, and the measurement must be carried out with the number of 
effective absorbance measured values (absorbance data) higher than the 
lower limit value Q.sub.L being, for example, only 2 (absorbance data 
Q.sub.24, and Q.sub.25 in the time t.sub.24 and the t.sub.25) as shown in 
a curve N.sub.2, suitable means is used to convert that state to an 
electrical signal, then it is inputted into a suitable counter means or 
comparative means for discrimination to expand (or shift) automatically 
the measurement region band to a region band X.sub.3 positioned forward in 
time series, and the calculation of the measurement is carried out on the 
basis of the measurement mode that uses absorption data Q.sub.23 in the 
time t.sub.23 contained in the lag time. 
In FIG. 3, as shown by a curve N.sub.3, in the case where the absorption 
data Q.sub.25 is below the lower limit value Q.sub.L, a measurement region 
band X.sub.4 positioned further forward in time series is set, and the 
absorption data Q.sub.24, Q.sub.23, and Q.sub.22 at the time t.sub.24, the 
time t.sub.23, and the time t.sub.22 are incorporated from said memory and 
regenerating means to carry out the calculation of the measurement. In 
this way, the measurement region band is successively moved until the 
measurement region band is expanded or shifted to a region where three 
absorption bands Q.sub.20, Q.sub.21, and Q.sub.22 of the time T.sub.20, 
the time T.sub.21, and the time t.sub.22 can be used, and the calculation 
of measurement is carried out. The present second invention is constituted 
as mentioned above. That is, in this measurement mode, the absorbance data 
in the lag time initially set are successively incorporated on the basis 
of a certain standard, so that the measured values can be calculated by 
using the initial measurement data of the reaction. 
As the standard for the incorporation in this case, the incorporation is 
successively effected so that effective absorption data may be 3 or more 
in the illustrated embodiment. However, various incorporation methods are 
possible; for example, when the absorption changes of the absorption data 
Q.sub.24 and the absorption data Q.sub.25 at the time t.sub.24 and the 
time t.sub.25 exceed a certain value, the absorption change between the 
absorption data Q.sub.22 at the time t.sub.22 contained in the lag time 
and the absorption data at the time t.sub.23 and the absorption change 
between the absorption data Q.sub.25 and the absorption data Q.sub.26 
positioned afterward in time series are compared, and when the compared 
result is below a certain ratio, the absorption data in the lag time are 
utilized. 
Although, in the above embodiment, the absorption data Q.sub.20, the 
absorption data Q.sub.21, and the absorption data Q.sub.22 are used as a 
final combination, other combination, for example, a combination of the 
absorption data Q.sub.19, the absorption data Q.sub.20, and the absorption 
data Q.sub.21 including the absorption Q.sub.19 at the time when the final 
reagent is absent can be used. 
In the illustrated embodiment, the number of absorption Q.sub.n used in the 
calculation is 3, but the number may be 2 at the lowest. The present 
invention is not limited to the embodiments described above, but various 
modifications may be made without departing from the sprit and scope of 
the present invention. For example, in the illustrated embodiments, 
although the time width of the measurement region band X.sub.1 in the case 
of a sample low in active value, and the time width of the measurement 
region band X.sub.2 in the case of a sample high in active value are set 
to be different, they may be set to be the same or narrow, and the 
measurement region band X.sub.2 of the sample high in active value may be 
positioned more forward in time series than the measurement region band 
X.sub.1 of the sample low in active value. The type and the structure of 
the automatic chemical analytical apparatus to which the present method of 
analyzing a reaction rate is applied are not limited to those of the 
illustrated embodiments, and a suitable type and structure thereof can be 
used.