JPH04151541A - Cell analyzing apparatus - Google Patents

Abstract

PURPOSE:To improve the measuring accuracy for few blood corpuscles by performing the counting of the blood corpuscles beforehand, measuring the number of erythrocytes, the number of leukocytes and the number of lymphocytes, and determining the measuring conditions in analysis based on the measured results. CONSTITUTION:In a blood-cell data analyzing part 3, the measuring conditions for the next measurement are analyzed based on the counted data of blood cells. In a measuring-condition setting part 5, the measuring conditions of a measuring system are set based on the result of the analysis. Before the analysis of the cells in the blood, the number of erythrocytes, the number of leukocytes and the number of lymphocytes in the leukocytes are measured beforehand in a blood-corpuscle counting part 2. In the setting part 5, at first, the counted data of the blood corpuscles are analyzed, and the number of the erythroxytes, the number of leukocytes and the number of the lymphocytes in a unit volume are detected and compared with the number of the erythrocytes, the number of leukocytes and the number of the lymphocytes for ensuring the sufficient accuracy. Thus, the amount of the blood sample to be treated is determined. The conditions such as the elongation of the measuring time, the increase in sucking amount of the diluted blood to be measured, the increase in amount of determination and sending of the blood and the increase in flowing speed are determined. Thus, the measuring accuracy for the few blood corpuscles can be improved.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention irradiates individual blood cells with laser light and detects differences in blood cell types and functions from the scattered light and fluorescence intensity from the cells. It relates to a so-called flow cytometer for measuring.

[Conventional technology]

Conventional flow cytometers use a sheath flow system, in which a cell specimen prepared by diluting blood with physiological saline is surrounded by an external flow through a measurement flow cell. This is to irradiate each cell with a strong laser beam to obtain sufficient fluorescence intensity for measurement. Flow cytometers have a wide range of measurement targets, and were initially used for basic research, but in recent years, they have been used to measure subcentres of lymphocytes (subspecies, T cells, B cells, etc.) in white blood cells.
It has begun to be widely used in clinical laboratories in hospitals for applications such as testing of cells (e.g., white blood cells), classification of white blood cells themselves, and counting of reticulocytes.

Depending on whether the fluorescently labeled monoclonal antibody binds to the surface of lymphocytes, positivity or negativity for the antibody is determined by fluorescence intensity.

The preprocessing required for this measurement conventionally required long hours of work and skill, but recently it has been proposed by Analytical Clinical Laboratory, P 32 (Oct. 1988) (ACL
P 32. (Oct, 1988)), with the improvement of reagents and the development of pretreatment technology,
It's getting very easy.

[Problem to be solved by the invention]

However, in this test, depending on the blood sample, the number of lymphocytes may be very low or the red blood cells may not be sufficiently hemolyzed, so it may not be possible to accurately measure the positive rate, or ghost signals caused by light scattering from red blood cell fragments may affect the measurement. There is. - Under standard measurement conditions, blood specimens such as those described above may not be sufficient.

This will be explained using FIGS. 2(a) and (b). In Figure 2, a fluorescently labeled monoclonal antibody is applied to lymphocytes, the degree of binding of the antibody to lymphocytes is measured, and the presence of subsets (subspecies) of lymphocytes is determined from the ratio of positive and negative responses. As shown in FIG. 2(a), in the measurement, white blood cells are first classified into three types: lymphocytes, monocytes, and two granulocytes using two parameters, side scattering intensity and forward scattering intensity, by laser light.

Select the necessary lymphocytes and extract only the lymphocytes as shown in Figure 2 (
Create a histogram distribution of the fluorescence intensity shown in b) and calculate the ratio of the number of positive cells to the total number of lymphocytes.

There is no problem if the original number of lymphocytes is sufficient, but if the number is low, the height of the histogram will be low as shown by the dotted line in (b), and it may be impossible to distinguish between positive and negative lymphocytes, or the statistics may be difficult due to insufficient numbers. The error is also large, which reduces measurement accuracy.

Further, when red blood cell fragments due to poor hemolysis shown by the dotted line in (a) appear, they overlap with lymphocytes in a region, which results in an inability to accurately measure the number of lymphocytes and an increase in the negative rate. On the other hand, hemolysis may progress too much and the white blood cells themselves may be destroyed.

Such problems also occur in similar test items performed using a flow cytometer, such as white blood cell classification and reticulocyte counting.

In white blood cell classification, whether the number of white blood cells is high or low is a parameter that directly affects the measurement accuracy of the classification. Particularly when the number of white blood cells is small, it may not be possible to obtain sufficiently comprehensive data for each type of white blood cell, and due to statistical errors, each blood cell classification algorithm may not function properly. Furthermore, only certain white blood cells may be susceptible to hemolysis.

Blood specimens that cannot be measured under the uniform measurement conditions described above must be remeasured, but in order to do so, one more step is required.
It requires multiple blood sampling and pretreatment processes, and measurement conditions for the measuring device must be set separately. In facilities that handle a large number of blood specimens, this is a complicated and time-consuming process.

SUMMARY OF THE INVENTION An object of the present invention is to provide a blood cell analyzer that can automatically change and set measurement conditions to handle blood samples that cannot be handled under standard measurement conditions.

[Means to solve the problem]

In order to achieve the above purpose, before measuring with a blood cell analyzer, we perform a blood cell count to measure the number of red blood cells, white blood cells, and lymphocytes among white blood cells, and based on the measurement results, we measure blood cells. Be sure to determine the measurement conditions for analysis. In addition, during red blood cell hemolysis carried out in the pretreatment process, the hemolyzed state of blood is observed by light transmittance, and can be known from the measurement of the value or state of change in light transmittance. Once the blood has been sufficiently hemolyzed, the hemolysis step is completed and the next cell analysis begins.

The following conditions may be considered necessary for cell analysis.

The goal is to ensure a white blood cell or lymphocyte count that guarantees sufficient accuracy. Monitor the hemolyzed state of red blood cells to prevent insufficient or excessive hemolysis. For reticulocyte counting, it is necessary to ensure a sufficient number of red blood cells. Furthermore, if there is platelet aggregation, it will appear at the position of red blood cell debris as shown in FIG. 2(a), but this must also be removed by data processing.

In order to ensure the above measurement conditions, information measured by a blood cell count and a light transmittance measurement unit that monitors the state of hemolysis are used.

In normal blood cell counting, blood cell count, blood cell volume, and blood cell volume histogram information are obtained by measuring electrical resistance based on the Coulter principle. Deficiencies in the number of red blood cells and white blood cells can be determined from the number of blood cells per unit volume.

The number of lymphocytes can be determined from the white blood cell volume histogram. An example of a volume histogram is shown in FIG. The presence or absence of platelet aggregation indicates its presence between red blood cells and platelets in the red blood cell volume histogram.

The state of red blood cell hemolysis can be determined by measuring light transmittance. When a hemolytic agent is added to diluted blood, the light transmittance changes over time as shown in FIG. Initially, the transmittance is low, but
After a certain period of time has elapsed, the transmittance increases rapidly. After that, the light transmittance shows a tendency to saturate. The hemolytic operation is terminated at an appropriate point in time when the saturation tendency is reached, and the blood sample is sent for cell analysis and measurement is started.

Conditions for cell analysis are set as follows.

First, analyze the blood cell count data, determine the number of red blood cells, white blood cells, and lymphocytes per unit volume, and compare the numbers of red blood cells, white blood cells, and lymphocytes required for measurement accuracy to determine the amount of blood sample to be processed. decide.

Rather than setting measurement conditions for all blood specimens, it is basic to change the normal measurement conditions only for extremely small number of specimens and process blood cells to the same number as in normal subjects. . Possible measurement conditions include extending the measurement time, increasing the measured diluted blood suction volume, increasing the fixed amount of blood sent to the measurement system, increasing the flow rate, and controlling the sheath fluid volume.

If the measurement time is not changed, the fixed amount of blood sent to the measurement system will be increased, but if the sheath fluid flow rate is not changed, this will increase the diameter of the flow of the blood sample in the flow cell. In this case, there is an upper limit to the sample diameter due to the size of the optical system, especially the focused laser beam. On the other hand, when the sheath liquid flow rate is also increased, the sample diameter does not change, but the flow system control parameters increase.

For blood samples with platelet aggregation, the cell data processing is informed that the sample has platelet aggregation, and the platelet data is deleted during the processing.

[Effect]

It has a means for counting red blood cells, performs blood cell counting, and preliminarily measures the number of red blood cells, white blood cells, and lymphocytes among white blood cells before measurement with a blood cell analyzer, and includes a measurement condition setting section for blood cell analysis. The accuracy of blood cell analysis is improved by means of determining the amount of blood sample to be processed by comparing the measurement results with the number of red blood cells, white blood cells, and lymphocytes required for measurement accuracy, and by means of controlling the measurement system. characterized by things.

In addition, in the red blood cell hemolysis operation performed in the pretreatment process of a cell analyzer, by observing the hemolyzed state of blood using light transmittance and determining the value or change state of the light transmittance,
It is characterized by reducing measurement errors caused by red blood cell debris in cell analysis.

In this way, the measurement conditions for the next blood cell analysis measurement are judged and determined based on the previous blood cell measurement data, the measurement hardware is controlled, and the red blood cell hemolysis conditions in blood cell pretreatment are determined from the light transmittance measurement. It is possible to reduce the effects of blood specimens with a low number of white blood cells or lymphocytes, which have been a problem in the past, and the effects of blood hemolytic agents, which vary between individuals, and as a result, the measurement accuracy of the blood analyzer can be improved.

〔Example〕

The present invention will be explained below with reference to Examples.

(Example 1) An embodiment of the present invention will be explained with reference to FIG.

In the figure, 1 is a blood sample, 2 is a blood cell counter (counting)
3 is a blood count data analysis section, and 4 is a blood preprocessing section.

Reference numeral 5 denotes a measurement condition setting section for setting measurement conditions for the blood pretreatment section 4 and the cell analysis section 6. The measurement data from the cell analysis section 6 is processed by the cell data analysis section 7, and the measurement results are output from the control section 8 to the outside. The control unit 8 controls the entire measuring device.

The blood cell counting section 2 measures the number of red blood cells, white blood cells, lymphocytes, etc. In the blood count data analysis section 3, the blood cell counting section 2
From the data, the number of lymphocytes is determined from the percentage of lymphocytes in the white blood cell volume histogram shown in FIG. Platelet aggregation is determined by whether aggregated platelets are present in the intermediate region between the platelet distribution and the red blood cell distribution in the red blood cell volume histogram. The results of the blood count data analysis section 3 are transmitted to the measurement condition setting section 5, which determines the processing conditions for the cell analysis section 6 and blood pretreatment section 4.

The processing conditions include the following.

For treatment of minority blood cells, the measured blood cell count is increased.

That is, increase the measured aspiration amount of the measured hemodilution sample.

When increasing the measurement time, the flow rates per unit time of the blood sample and sheath fluid flowing through the measurement flow cell do not need to be changed. However, if the measurement time cannot be changed, there are two options: increasing the flow rate only for the blood sample, or increasing the sheath fluid flow rate as well. In the former case, the diameter of the blood sample flow in the measurement flow cell increases, and there are limitations on the settings of the optical system, including the laser beam. In the latter case, it is also necessary to control the flow of sheath fluid.

Since it is not possible to increase the amount of hemodilution sample to be measured without limit, it is necessary to set an appropriate upper limit. This judgment is also the job of the measurement condition setting section 5.

Since it is not practical to set continuous measurement conditions, it is sufficient to set uniform conditions for blood samples that fall within the normal range, and to set discrete conditions for other ranges.

When the number of white blood cells is small, it is also possible to set conditions by reducing the dilution ratio in the blood preprocessing section 4. Since the number of blood cells is originally small, it is rare for blood cells to pass through each other at the same time. However, it is necessary to be aware of changes in pretreatment conditions due to differences in dilution ratio.

To change the dilution conditions, the amount of blood aspirated and the amount of diluted liquid aspirated are controlled.

A time chart showing the entire process is shown in FIG.

(Example 2) The hemolyzing effect of red blood cell hemolysing agents varies depending on the type, and varies depending on the blood specimen, storage conditions, handling, and pretreatment conditions. As a result, the unlysed red blood cells and red blood cell fragments shown in FIG. 2 are mistakenly determined to be lymphocytes.

In the present invention, the state of these unhemolyzed red blood cells is monitored by measuring light transmittance. When the light transmittance exceeds a certain value,
Or, after entering a flat region after a large change in transmittance, the hemolytic operation is stopped after a predetermined period of time has elapsed.
Start cell analysis measurement. In this way, the conditions for hemolysis of red blood cells can be aligned, and cell analysis can be performed with less error.

The light transmittance measurement of a red blood cell sample uses a light lamp, semiconductor laser, LED, etc. as a light source.

A light source and a photodetector are set before and after the glass reaction container where blood pretreatment is performed, and the amount of transmitted light is measured to determine the light transmittance. The time change in the amount of transmitted light is measured from the start of hemolysis.

〔Effect of the invention〕

According to the present invention, there are effects as described below.

(1) The measurement accuracy of cell analysis using a small number of blood cells, which has been a problem in the past, has been improved by checking the number of target blood cells in advance using a different measurement method, and increasing the number of blood cells processed for cell analysis based on the results. To set measurement conditions,
The measurement accuracy of cell analysis can be improved.

(2) It has a means to measure the hemolyzed state of red blood cells while sequentially measuring changes in light transmittance, and since cells can be analyzed in a sufficiently hemolyzed state, there are fewer erroneous measurements in cell analysis due to insufficient red blood cell hemolysis during blood pretreatment. As a result, the accuracy of cell analysis can be improved.

(3) (1) and (2) automatically change and set the measurement conditions for each blood sample, eliminating the need for re-measurement or pre-processing, improving operability and shortening measurement time. can be measured.

[Brief explanation of drawings]

Figure 1 shows a block diagram showing the configuration of the present invention, and Figures 2 (a) and (b) show a method for measuring lymphocyte subset processing, where (a) shows lymphocytes being extracted using light scattering information. Parameter distribution diagram, (b) is a diagram showing a histogram of fluorescence intensity when a fluorescently labeled monoclonal antibody binds to lymphocytes, Figure 3 is a diagram showing an example of a histogram of white blood cell volume, and Figure 4 is a diagram showing blood FIG. 5 is a diagram showing the change in light transmittance over time when a hemolytic agent is applied to a diluted sample, and is a time chart of each process in FIG. 1. 1. Blood specimen, 2. Blood cell counter section, 3. Blood count data analysis section, 4. Blood preprocessing section, 5. Measurement condition analysis section, 6. Cells. Analysis Department, 7...Cell Data Analysis Department, Figure 2

Claims (1)

[Claims] 1. A blood count data analysis unit that analyzes measurement conditions for the next measurement based on blood cell count data, and measurement conditions that sets measurement conditions for the measurement system based on the analysis results. 1. A cell analysis device comprising a setting section and a control section for controlling a cell analysis section with measurement conditions thereof, and setting measurement conditions for each blood sample. 2. It has a blood preprocessing section consisting of a means for measuring the hemolyzed state of red blood cells by light transmittance and a means for identifying the degree of hemolysis from the data series of the measurement results, and starts cell analysis from the time hemolysis is completed. , a cell analysis device characterized by reducing analysis errors due to unhemolyzed red blood cells.