JPH01223329A - Particle counting method - Google Patents

Abstract

PURPOSE:To obtain a counting result corresponding to an automatic discrimination system in accuracy, by sending a particle signal to comparators to which four discrimination levels are set and counting a particle by the counter connected to the comparator. CONSTITUTION:When particle signals having the magnitudes corresponding to the sizes of particles are sent to comparators C1-C4, said particles signals are respectively compared with discrimination levels D1-D4 to emit signals which are, in turn, counted by the counters C11-C14 connected to the comparators. As mentioned above, by counting the particles while the particle signals are indivisually discriminated, at the finish time of measurement, a particle signal N1 whose magnitude is within a range exceeding a discrimination level D1 and equal to or less than a discrimination level D2, a particle signal N2 whose magnitude is within a range exceeding the level D2 and equal to or less than a discrimination level D3 and a particle signal N3 whose magnitude is within a range exceeding the discrimination level D3 and equal to or less than a discrimination level D4 are respectively obtained as data. When the data of the levels D1-D4 and the number of the signals N1-N3 are used, a histogram showing the number of the signals N1-N3 as an area can be drawn. The optimum discrimination level can be calculated by utilizing said histogram.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention estimates the optimal discrimination level when multiple types of particles, such as red blood cells and platelets, coexist in a measurement sample. This invention relates to a particle counting method that allows highly accurate counting results to be obtained.

[Conventional technology]

As a device that counts particles using a suspended liquid containing particles such as blood cells, when the particles are passed through the pores, a signal is emitted based on the electrical or optical difference between the particles and the liquid. Particle counters that count particles are known. In either case, a signal whose size corresponds to the size of the particle can be obtained.

For example, if you measure a blood cell suspension made by diluting human blood and amplify the signal, you will get a particle signal like the one shown in Figure 7. Normally, red blood cells are larger than platelets, so the red blood cell signal 31.33 large, platelet signal 30.32 is small,
Therefore, there are fixed discrimination methods and automatic discrimination methods as methods for discriminating signals of multiple types of particles obtained from one detection unit and obtaining the number of each particle.

The fixed discrimination method is a method in which a plurality of appropriate discrimination levels are set in advance and the magnitude of the signal is compared with the discrimination level and counted. In FIG. 7, the signal 31. 33 are counted as red blood cells, and signals 30, 31, 32, and 33 exceeding the discrimination level L are counted as red blood cells and platelets. Therefore, the count value of the signal that exceeded the discrimination level L1 was the number of red blood cells counted, and the value obtained by subtracting the count value of the signal that exceeded the discrimination level L from the count value of the signal that exceeded the discrimination level L was counted. This will be the platelet count.

The automatic discrimination method is a method that determines the optimum discrimination level after measurement and calculates the number of particles for each particle according to the discrimination level. Remember how many signals were obtained. After the measurement is completed, use those data to draw a distribution diagram of the frequency of particles against the signal size (particle size distribution diagram) as shown in Figure 9, and determine the optimal discrimination level Lx.
, L3 are determined, and the number of particles between the discrimination levels Lt and L3 or the number of particles at the discrimination level 8 or higher is obtained by calculation. To convert the signal magnitude into a digital signal, a comparator or an A/D converter provided with a large number of discrimination levels is used.

[Problem that the invention seeks to solve]

The fixed discrimination method has the advantage of being simple and inexpensive, and when the signal sizes of different types of particles have sufficient discrimination, that is, in FIG. 7, the red blood cell signal is a signal larger than the discrimination level L1. Therefore, if the platelet signal has a discrimination level and becomes a thicker signal smaller than 1+, even different particles can be correctly discriminated and correct counting results can be obtained.

However, as shown in FIG. 8, in the case of an abnormal specimen containing microcytic red blood cells or macrocytic platelets, the discrimination level is high, and a smaller red blood cell signal 35, a discrimination level L, and a larger platelet signal 36 are detected. There was a problem that accurate counting results could not be obtained.

On the other hand, the automatic discrimination method has the advantage that accurate counting results can be obtained even in the case of the above-mentioned abnormal specimens. 1st
The θ diagram shows the case where microcytic red blood cells are mixed into the platelet area.
Figure 11 shows a particle size distribution diagram in the platelet region for macrocytic platelets. In either case, the optimal discrimination level #, LS%L6, and Lv are automatically determined, and the respective discrimination levels If the number of particles between 4, L%, and Lh is calculated as the platelet count, and the number of particles with discrimination levels LS or higher and Lq or higher is calculated as red blood cells, accurate counting results can be obtained.・However, from a hardware perspective, parts for ^/D conversion, sufficient memory capacity, high-speed arithmetic processing equipment, etc. are required, and from a software perspective, data collection, determination of the optimal discrimination level, calculation of count values, etc. are required. Since a complicated program is required for data processing and calculation, there is a problem in that the particle counting device becomes large and expensive.

Therefore, an object of the present invention is to provide a particle counting method that can obtain counting results with an accuracy suitable for an automatic discrimination method by estimating and determining the optimum discrimination level using simple means and methods. do.

[Means for solving problems]

In order to achieve the above object, the particle counting method of the present invention, which will be described with reference to FIG. In a particle counting method in which an electrical signal corresponding to the particle size is emitted based on the difference in particle size, and this particle signal is counted, the particle signal is divided into four discrimination levels DI, D゜3, D, (
However, it should be sent to comparators C1, C, , C, , C4 in which D+<Dt<03<D3) is set, and counted by counters CIl, C1, Cts, CI4 connected to these comparators. Therefore, the size exceeds the discrimination level D, and the discrimination level 0! Number of particle signals in the following range N Number of particle signals whose size exceeds discrimination level D2 and is below discrimination level D2 Particles whose size exceeds discrimination level Ds and falls below discrimination level D4 Obtain the number of signals N3, and their discrimination levels D,,D! , N8, N4 and the number of particle signals N to N, , N3 When a histogram is drawn on the x-y coordinates, sample points A, B, and C that can show the characteristics of the histogram. By finding the interval number y=f (X) that passes through these sample points A, B, and C and is convex downward in the interval D1≦X≦04, the function in the interval; y=f(x
) is found to be the minimum true value, and this discrimination level is calculated by dividing the histogram into two. It is characterized in that the number of target particles is obtained using the number of particle signals belonging to the range between the two.

[Effect]

A particle signal having a size corresponding to the size of the particle is sent to the comparators CI-, C! , Cs, C4, the particle signal is compared with the discrimination levels D1, N2, N5, N4, respectively, and a signal is emitted, which is sent to the counter ClIC+ connected to the comparator.
It is counted by z, Cts, CI4. By counting the particle signals while individually discriminating them in this way, at the end of the measurement, the size exceeds the discrimination level D1 and reaches the discrimination level D! The number of particle signals N+ is in the following range, the number N8 of particle signals is in the range exceeding the discrimination level D3, the size is greater than the discrimination level D3, and the number N8 is in the range below the discrimination level D3, The numbers N and 1 of certain particle signals are obtained as data, respectively.

Discrimination level D+, Dt, 0, 04, number of particle signals N1
, N and N3, it is possible to draw a histogram expressing the number of particle signals N+, Nz, N as an area as shown in FIG. In order to quantify the features of this histogram, sample points A, B, and C that can represent the features of this histogram are selected in the 0 interval 0. The shape of the function is determined by passing through points A, B, and C, and the function y=f(x) is in the interval DI≦X≦D4. The minimum value of X can also be determined. In other words, the optimum discrimination level Dv is determined.

By dividing the histogram into two by a function of The number of particle signals belonging between levels D1 or N8 or OS or N4 is calculated.

Using the calculated number of particle signals, a measurement result of the target particle is outputted through processing such as correction.

〔Example〕

Examples of the present invention will be described below.

FIG. 1 shows a schematic diagram of an example of a particle counting device for carrying out the method of the present invention. 10 is a detection part, and the blood cell suspension obtained by diluting blood approximately 50,000 times, for example, is By introducing a blood cell into a pore of about 80 μm through which a constant current is passed, an electrical signal 12 is obtained based on the change in electrical resistance when blood cells pass through the pore and when they do not. This obtained particle signal 12 becomes larger as the size of the particle becomes larger.

The particle signal 12 is amplified by the amplifying section 14 to become an amplified particle signal 16, which is further converted into a square wave particle signal 20 having a peak value proportional to the size of the particle by the waveform processing section 18.
is converted to As shown in FIG. 2, when the particle passes through the center of the pore, a unimodal signal 16a is obtained, and the peak value ht of the signal is proportional to the particle size, and the particle is fine. If passing through the edge of the hole, a bimodal signal +6b
is obtained, and the bottom value ht of the signal is proportional to the size of the particle. Therefore, in any case, in order to obtain a signal whose size is proportional to the size of the particle, the waveform processing unit 18 has a signal If a means is provided to judge the shape and detect a peak or bottom, and a unimodal particle signal 16a is input, as shown in FIG. Wave 20a is output, bimodal particle signal +6
When b is input, a square wave 20b having the same peak value as the bottom value is output.

Particle signal 20 is input to comparators C0, C1, C8, C2, C4. The respective discrimination levels D@, DI, Dg, Ds, 04 of the comparators C+, Ct, Cx, C1, C4 connect the voltage supplied from the reference voltage source 22 to the resistors P0, R1.
, R, R3, and R4, the particle number 20 obtained is the discrimination level D0.0DI, 03, mouth,
When the peak value of the particle signal 20 exceeds the discrimination level, a pulse signal is output from each comparator. The signals output from the comparators C, C1, C2, C3, and C4 are outputted from the counters C1, C0, and CI, respectively.
! , C13, and CI4.

In this way, when the measurement is completed, the wave height value is the discrimination level. The number H0 of particle signals whose peak value exceeds the discrimination level Dt is stored in the counter C8°, the number M1 of particle signals whose peak value exceeds the discrimination level D1 is stored in the counter C0, and the number M2 of particle signals whose peak value exceeds the discrimination level Dt is stored in the counter C8. Counter C1! , the wave height value is at the discrimination level 0. The number M of particle signals whose peak value exceeds the discrimination level is stored in the counter CPFF, and the number M4 of particle signals whose peak value exceeds the discrimination level is stored in the counter C14.

The number Me, M of these particle signals is calculated by the arithmetic processing unit 24.
+, N2, N3, N4 are read out, the number N0 of particle signals in which the peak value of the particle signal 20 exceeds the discrimination level D and is below the discrimination level O1, and the number N0 of the particle signals exceeds the discrimination level D1 and is at the discrimination level 0. Number N1 of particle signals in the following range, discrimination level D! The number N of particle signals in the range exceeding the discrimination level Da and below the discrimination level D, and the number N of particle signals in the range exceeding the discrimination level D4, respectively, are No=A. - Gate 9, N+-M+ Mi, N2 Tomi M* Ms, N5
=FIsHa is calculated.

Using the discrimination level D0.0 and D5.04 and the data of the number N0, N2, and N of particle No. 13, the fourth
As shown in the figure, a histogram can be drawn in which the nodes are 0, D8, and D8.04. The horizontal axis X is the peak value of the particle signal 20, and the vertical axis y is the frequency.The number N0, N+, Hz, and N3 of 0 particle body numbers are expressed as areas. Furthermore, since the peak value of the particle signal 20 is proportional to the size of the particle, the horizontal axis X can be considered to be the size of the particle.

On this histogram, there are coordinates (IL, n+), (dt, chapter n), (d,) that suitably indicate the characteristics of the histogram.

3) Set up points A, B, and C and use them as sample points (dt<dx<ds) - The slope of the straight line connecting points A and B is (n
snow n+)/(dg - dt), and the slope of the straight line connecting points B and C is (ng-ni)/(ds dt), so in the case of dg-dt dg-dg "'■, sample point A , B, C, and there is a downwardly convex function in D1≦X≦D4 0 For example, quadratic interval number y = a
(x-e)" + r (however, aSeSf is an unknown number and a>0), it is possible to perform interpolation using points A, B, and C as sample points. Then, when DI≦X≦D, the number y between them The value e of X that minimizes =a(x-e)''+f is determined. That is, the function y#a(x-e)"
+f passes through points A, B, and C, so n, -a (d, -
e)”+ 1 ,, ■n*= a (dt e)
"+ f -■nz-a (ds-2)"+ f ・
The three equations =■ hold true. By eliminating a and f from these three equations and reorganizing them, the following equation for e is obtained.

By substituting the known data dd3, d8, nI3, and n snow into this equation, the value e of ,B,
In this example, the coordinates of C are dt = (D+
+ Di)/2, dt-(D*+Ds)/2,
ds−(Ds+D*)/2,n+”N+/(D
Wealth - D+), nt = Nt/ (Ds - D wealth),
N5-N! /(0,-C1,), and each discrimination level is .

-D+=Ds Di"Dn-Ds-d, so dl-D1+d/2...■, dx-D+
+3d/2...■, dl" D t + 5d/2-
■, nl-N1/+l=■, nt-Nx/d...'@,
n2=N3/d...■. By substituting these 0 to 0 expressions into the 0 expression, we obtain, and the ■ expression is N+-2Nt+N3> Q...
・It becomes Φ′.

In addition, the value of the [phase] formula is a certain 2 that is convex downward in the interval D1≦x:50a under the conditions of formula ■′ and formula ■2 to ■.
Given the quadratic function 7-g(X), this quadratic function y-g(x)
Assuming that has area equality with respect to the histogram,
That is, when the following equations ◎ to [phase] hold true, the quadratic function 7-g(X) becomes the same as the value of X that is minimum and minimum. Therefore, the [phase] equation can be regarded as the optimal discrimination level.

The value of e obtained in this way is expressed in the interval DI≦X≦D4
Considering that the interval number y = a (x-e)''+ 1 is convex downward in the interval 0.≦X≦D4, by comparing it with Dl and DI,
The optimal discrimination level Dv, which is the minimum value of
If +N3<Q, D, -D, and DI≦e≦
In the case of D4, that is, ■', using the 0 formula, 2N1 3
Nz +N3≧O and Nl −3Ng + 2Nz≧0
In the case of Dv! e-D1+d(ch■', if you use the [phase] formula, Nt-3Ns +2Ns < O, then D, L
04-DI+3d, which is 0 or more, is established when the condition of the Φ' expression is satisfied.

On the other hand, in the case of 1'-'-a = ", @ dg-dt ds dt, that is, if you use the formula ~■, Nt-2Nz ten N
, ≦0... [phase]', there is no downwardly convex function in %DI≦X≦D4, and the discrimination levels D1 and D
If tsOl, D, is set to an appropriate value, such a case will never occur as long as human blood is measured, so the device will issue an alarm as a measurement error. To calculate the total number of particles in D,
-D.

In this way, the optimal discrimination level Ov is the discrimination level 01
to D4.

Next, using the fact that the discrimination level Dv divides the histogram into two, the number of particle signals that belong between the discrimination level Ov and other discrimination levels is determined as follows. Discrimination level D!
Let the number of particle signals belonging between and DJ (where Dt < DJ) be expressed by Nmuj = OLL12・N
, becomes D! If ≦Dv<Di,” and knee〇,,,
, becomes.

Now, if [ ), < D, < Q, as shown in FIG.
Ntv-No+N+ + (Dv Dx)Nx/d
If this value is corrected by taking into account the dilution factor of the blood, the amount of diluted sample that has flowed through the pores, the simultaneous passage rate, etc., the number of platelets per unit volume of blood can be obtained, and the output device 2
6 is output.

Next, the results of implementing the method of the present invention will be explained. Fifth
Figure 6 shows blood cell signals in the platelet area, respectively, at 128
The graph shows the results of analysis using an A/D converter with a resolution of The discrimination level Dv was also found with high accuracy even when

In addition, the function for interpolating points A, B, and C as sample points is not limited to a quadratic function, but may be any function that is convex downward in Rho≦X≦04, and in addition, has symmetry at the minimum point. A good one is preferable.

In addition, sample points A, B, and C only need to show the characteristics of the histogram, and for example, n+:nz:n5=(
h):(-!L-h):(''-h)Nt DI Dt Os Dt Da-Ds(h
(is an arbitrary number). This means that the value of
This is because when the function is Y ``' a (x-e)'' + f, the value of e is determined regardless of the values of a and f.

Furthermore, the discrimination levels D1 and D4 can be arbitrarily set depending on the target particle size distribution state. Preferably, in the interval DI≦X≦D, the function y=
Set so that f(x) becomes minimum.

Furthermore, the number of particle signals that fall between two predetermined discrimination levels is used to detect whether the measured particles are macrocytic or microcytic compared to standard particles. It is also possible.

〔Effect of the invention〕

As explained above, in the particle counting method of the present invention, particle signals are compared with a small number of discrimination levels D1.0□, D3.04, and the number N of particle signals, which is a small number of multiple data obtained, is
Since counting results can be obtained by performing simple calculations using N and N, an inexpensive particle counting device can be constructed.

Furthermore, since the counting results can be calculated by estimating and finding the optimal discrimination level Dv between the discrimination level D and empty, highly accurate counting results can be obtained without any problems even in the case of abnormal samples. It will be done.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing an example of a particle counting device for carrying out the particle counting method of the present invention, and FIGS.
FIG. 4 is a histogram for explaining an embodiment of the method of the present invention, and FIGS. 5 and 6 are diagrams for explaining the function of waveform processing. A particle size distribution diagram showing the results of one example, FIGS. 7 and 8 are explanatory diagrams showing examples of particle signals, and FIGS. 9 to 11 show examples of particle size distribution diagrams. 10...detection section, 12.16.16a, 16b. 20.20a, 20b130.31.32.33.34
．． 35.36.37...Particle signal, 14...Amplification section, 18...Waveform processing section, 22...Reference voltage source, 24
... Arithmetic processing unit, 26... Output device, Do, D,
, Ot. D5, D4...discrimination level, Dv...optimum discrimination level, R6, R1, 6%R1, R4...resistance, C0,
C, C, C1, C4...Comparator, C+ss C1
1% C+z, cps, Cl c・'counter, gate N
6, M,, M,, N9, N., N1, N3, N,...
・Number of particle signals, A%BSC...Sampling point, Lo, L-2, L8, L4, L3, L-L? ...Discrimination level Figure 2 Figure C (U Child 35 Figure 7 Figure 9 Figure 11 Arrival H is also 7n large 3J Procedural amendment March 18, 1988 Me3-O!'' 2 Title of the invention Particle counting method 3 Relationship with the case of the person making the amendment Patent applicant 4, attorney 6 Number of inventions increased by amendment (1) Page 15 of the specification, line 6 from the bottom The eyes ``p,'' are ``
Corrected to "D4".

Claims (1)

[Claims] 1. A liquid in which particles are suspended is supplied to a detection unit, and an electrical signal corresponding to the size of the particles is emitted based on the electrical or optical difference between the particles and the liquid. In a particle counting method that counts particle signals, particle signals are divided into four discrimination levels (D_
1, D_2, D_3, D_4) (however, D_1<D_
2<D_3<D_4) and is counted by a counter connected to these comparators, the magnitude exceeds the discrimination level (D_1) and the discrimination level (
D_2) The number (N_1) of particle signals in the following range, the size of which exceeds the discrimination level (D_2) and the discrimination level (D_3)
), the number of particle signals (N_2) whose size is in the range below the discrimination level (D_3) and the number of particle signals (N_3) whose size is in the range below the discrimination level (D_4), and these discrimination levels (D_1 , D_2, D_3, D_4) and the number of particle signals (N_1, N_2, N_3) when a histogram is drawn on the x-y coordinates, a point (A, B, C) as sample points, and find a function y=f(x) that passes through these sample points (A, B, C) and is convex downward in the interval D_1≦x≦D_4, so that the interval D_1 ≦x≦
In D_4, the value of x that minimizes the function y=f(x) is determined as the optimal discrimination level (D_V), and this discrimination level (D_V) is calculated by dividing the histogram into two. A particle counting method characterized in that the number of target particles is obtained using the number of particle signals belonging to a level (D_V) and another discrimination level.