JPS62153758A - Method for measuring reticulocyte by flow sight meter - Google Patents

Abstract

PURPOSE:To improve the discrimination stability of a reticulocyte, by removing exciting beam from the beam emitted from the optical stimulus region of a flow cell arranged along the optical axis of exciting beam before measuring the forward scattering beam and forward fluorescence on the optical axis. CONSTITUTION:Exciting beam E' is incident to a flow cell 23 from a laser beam source 20 through a filter 21 and a lens 22. Exciting beam is incident and converged to the central part of a sheath liquid flowing so as to surround a liquid specimen and beam based on exciting beam E' in emitted beam is blocked by a beam stopper 24. Forward fluorescence F' and scattering beam S' are returned to parallel beam by a lens 25 and said parallel beam is passed through a slit plate 26 to be separated into two components of forward scattering beam S and forward fluorescence (f) by a dichroic lens 27. The forward scattering beam S is detected by a detection element 28 and displayed on a display part 37 through an amplifier 29, an A/D converter 30 and an analytical part 36. The forward fluorescence (f) is passed through a filter 31 and subsequently displayed on the display part 37 through an amplifier 34, an A/D converter 35 and the analytical part 36.

Description

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a technique for measuring reticulocytes in blood using a flow cytometer during blood analysis.

The uninvented method for measuring reticulocytes using a flow cytometer is extremely useful in the field of clinical testing when optically classifying and measuring formed components such as immature reticulocytes and other blood cells in blood samples.

BACKGROUND OF THE INVENTION Conventional techniques Blood analysis and measurement techniques using optical and flow cytometry methods, in which specific cells and particles are stained in advance using staining reagents and blood analysis of a specimen is performed, have been described in detail up to now.

For blood samples, the anisotropy ratio in the horizontal and vertical directions of the fluorescence emitted from fluorescently stained blood cells is measured (Japanese Patent Laid-Open No. 5
9-102159), a method for classifying and counting cells based on the fluorescence intensity distribution of cells (Japanese Patent Publication No. 54-14957)
Thiofurapine T, which has strong selectivity for reticulocytes, is used as a fluorescent staining reagent for cells.
(Refer to Japanese Patent Application Laid-Open No. 142465/1982), below, a device by the applicant "a" which attempts to receive narrow angle scattered light with high efficiency using a triangular flow cell (Machichiku Sho 60-66169)
(No. 60-123128), also unfiled by the applicant, using auramine Q, which is extremely selective for differentiated red blood cells, as a fluorescent staining reagent (Patent Application No. 123128/1983), and a method using a laser light source for fluorescence and scattered light. Examples include one that simultaneously measures light to identify and count reticulocytes (US Pat. No. 4,525,706).

For example, a measurement system having the basic configuration shown in FIG. 4 is used for the above-mentioned typical blood analysis using a flow cytometer. Excitation light E is incident from a laser light source 1 through a condenser lens 2 into the narrow stomach of a flow cell in which a blood sample is made to flow, and side scattered light and side fluorescence from this optical stimulation area are removed by a scattered light removal filter and a condenser. Lens 10.
Field aperture 11. After passing through the light receiving filter 12, only the side fluorescent light is measured by the photodetecting element 16, and at the same time, the flow cell 6 also goes straight, and part of the excitation light B among the 10 lights is removed by the χ beam stopper 4. After that, the condenser lens 5 and the field aperture 6 are removed. Then, the forward scattering source S is photometered by the photodetecting element 7, and each light intensity signal is assumed to be emitted from the same cell particle and is inputted to the analysis unit 15 via the transducers 14 and 8 to determine the blood cell type. Each particle is identified, counted, and displayed on the display or recorder 16.

In blood cell analysis using the above-mentioned flow cytometer, scattered light and fluorescent light as a response from the optical stimulation area travels to a photometric system arranged along a plurality of optical axes.

Light is received on each optical axis.

Therefore, as shown in FIG. 2(b) and FIG. ?
As for the single condenser lenses 2 and 5, which are arranged horizontally, the arrows indicate their m and focal positions. ＋L
Even if it is completed for each of 1 + L2, due to the above adjustment, 1 accumulated on the optical axis B, which is the same as the light @A.
Condenser lens 10 is also used for the photometry system of this lateral fluorescence F.
Adjustment must be made to eliminate the misalignment between the condenser lenses 2 and 5 in the direction of arrow L3, and correcting this misalignment on the base @B will again cause misalignment of the optical system. It becomes necessary to modify LL+L2 again. Thus, even after the optical axes A and B have been corrected for focus and minute deviations.

Furthermore, not only are these re-corrections required at least several times, but the optical axes of scattered light and fluorescent light are different, so changes over time [
As shown in the conventional method 1 on the left side of Figure 6, the waveform is biased toward a 5 degree deviation waveform (as shown by the dotted line) or a fluorescent signal (in some cases, the scattering source signal) with respect to the solid line signal during adjustment. appears. In this case, multiple chemical characteristics corresponding to specific blood cells or particles are required, and at the same time, it is necessary to periodically readjust the sensitivity deviation of the flow cytometer in the analysis section. Considerable errors were unavoidable in the identification and counting of these. In particular, this bias is a problem in conventional techniques that perform two-dimensional distribution analysis.

Next, a complex signal processing method is employed to perform accurate discrimination of reticulocytes from mature red blood cells and platelets by multiple detection information. For more information.

Please refer to the above-mentioned US Pat. No. 1,132,5706. This involves 1) creating a two-dimensional distribution map of the scattered light intensity and fluorescent light intensity, and 11) using the square separation criteria to separate red blood cells and platelets. 111) Correct the fluorescence intensity of one blood cell group from which platelets are removed using the scattered light intensity signal, and IV) find a frequency distribution curve for the corrected fluorescence intensity and use half of this distribution curve. The distribution curve of mature red blood cells is estimated as a normal distribution, and a group of signals that fall within the normal distribution curve is calculated as a reticulocyte group. This is the procedure. Not only is the above iV) particularly complicated, but it also assumes a normal distribution of the red blood cell fluorescence distribution, so there is a correlation between the fluorescence intensity of mature red blood cells and (immature) reticulocytes. This may lead to a non-negligible error in the blood cell analysis results based on this assumption. In particular, this tendency is thought to be related to the amount of staining at the time of staining, such as how large the specimens with a high proportion of reticulocytes are.

problem? One Means to Solve the Problem The present invention provides a method for measuring reticulocytes using a flow cytometer, regardless of the characteristics of the specimen, such as the size of the reticulocyte ratio. This makes it possible to minimize the occurrence of counting errors and to simplify the photometry optical system.

The second invention was to use a new fluorescent dye.

(1) In addition to improving the signal and making the discrimination process simple and stable, the excitation light A method for measuring reticulocytes using a flow cytometer that removes the top and measures reticulocytes by measuring forward scattered light and forward fluorescence on the same axis along the optical axis of excitation light. It is.

In the method for measuring reticulocytes using a flow cytometer according to the invention, first, 500 parts of a blood cell measurement reagent containing flavophosphine R4 as a main ingredient are mixed in a ratio of 1 part of an anticoagulant whole blood sample, and fluorescent staining 7 is performed. For details about reagents, please refer to the patent application in 1973.
9-201273, the sample is then measured. Flow cell 2 of flow cytometer as shown in Figure 1
6 from the v-sa light source 20 to the filter 21 . Excitation light E' is incident through the condenser lens 22χ. As the sample liquid in the flow channel flows, the central part of the sheath group serves as an optical stimulation region where the excitation source heat is focused, and the excitation light of the γ light emitted from the optical stimulation region is mainly focused. The light component is blocked by a beam stopper 24. The forward fluorescent light F' and the forward scattered light S' are transmitted through the condenser lens 25.
The beam is returned to a parallel beam, and after one step, it is narrowed down to a small diameter beam through a slit plate 26, and this is separated into two components, a beam scattering source S and a beam r, by a dichroic mirror 27. The forward scattered light is received by the photodetecting element 2B behind the dichroic mirror 27, amplified by the amplifier 29, and the forward scattered light intensity signal passes through 60 A/D converters, enters the analysis section 66, and is displayed on m3.
7 is displayed. - 10,000, the forward fluorescent light f reflected by the dichroic mirror 27 is filtered to remove stray light etc. by the filter '51, and then received by the light output element 32 and amplified by the amplifier 64, and the previous fluorescent light intensity signal is A/L) The signal enters the analyzer 6 through the converter 35 and is displayed on the display 67. Incidentally, the amplification degree of the forward scattered light intensity No. 13 and the amplification degree of the forward fluorescent light intensity can be controlled by the gain control 3B independently or in conjunction with each other, if necessary.

For production In the above measurement method using a flow cytometer.

As shown in Fig. 2(a), <,Lo setting-1,,,L,2
After adjustment, the forward fluorescent light and the forward scattered light are collected on the same axis as the optical axis of the excitation light incident on the flow cell 2, separated into wavelength ranges, and photometered. When the excitation light E' passes through the excitation light wavelength selection filter 21, it is narrowed down to a specific wavelength band from the emission wavelength of the light source 20, but after passing through the flow cell 260, it is cut off by the beam stopper 24. Slit plate 26? The dichroic mirror 2 is used as a point light source through
At step 7, the forward fluorescent light intensity is emitted, the main component of which is light with a wavelength that does not reach the absorption wavelength, and the forward scattered light, which is mainly composed of light with a wavelength that exceeds the absorption wavelength, is transmitted and divided into two parts. Photodetector 28.32
After detecting the scattered light and fluorescence, the original response of each blood cell is photoelectrically converted as an intensity signal, and these signals are A/D converted and then data recorded and analyzed in the analysis section 66, using a known method. Depending on the findings, they can be classified and counted as normal (mature) red blood cells, platelets, reticulated (immature) red blood cells, white blood cells, etc., or they can be fluorescently stained and counted.
In this case, counting reticulocytes among these blood cells is also important in clinical examination. As shown in Figure 2, the photoresponse of a single reticulocyte is determined by the conventional method, in which the photometric optical system is adjusted on different optical axes for six lenses centered on the flow cell. As shown by the corresponding peaks of scattered light and fluorescence on the right side of Figures 6(a) and (b), the bias was different, and whether it was ideally adjusted or not after 1 hour, for example, Figure 6 left $111 (
As shown in (a), the signal of the scattered light does not change the ratio, but as shown in (b) of the same figure, only the signal of the fluorescent light deviates considerably from the ideal and becomes 2Etjl.
As shown by the dotted line in ll(b), it becomes easier to beg for a lower price. Therefore, the fluorescence is attenuated and the intensity signal of the reticulocytes is confused with platelets and some normal red blood cells, making the discrimination unstable and resulting in a counting error. According to non-invention.

As shown in Figure 2 (a), the focusing system is focused on the same axis to the only element @.
In order to adjust the light and fl, the scattered light and fluorescence originating from the same blood cells have corresponding maximum peaks and attenuation slopes.
The corresponding peaks are the two in the center of Figure 6 (a) and (b).

They are detected as two points that fit together.

Effect of the invention The present invention has the following 9-bit unique effects 2.

(1) Blood cell discrimination stability was significantly improved.

Since fluorescent light and scattered light originating from the same blood cell can be measured simultaneously on one axis, it is not only possible to adjust the focusing optical system minutely and quickly to obtain the best sensitivity.

Even if there is some deficiency in this Lu adjustment, or if there is a slight adjustment over time, the intensity of scattered light and fluorescent light on one axis will fluctuate at the same time. Coupled with the improvement in the ratio of lf1 due to the effect of hypersensitivity, extremely stable discrimination of cloth red blood cells is possible even if the discrimination level is fixed.

(2) High sample processing speed.

When mature red blood cells and platelets simultaneously pass through the source stimulation area of the flow cell, the light response at this time is similar to that of reticulocytes, that is, light scattered by reticulocytes.

A signal χ that is confused with the fluorescence intensity signal is generated, and this confusion increases especially when the reticulocyte ratio is small and the flow diameter of the reagent in the flow channel is large. In order to avoid this problem in the conventional method, one attempt was made to narrow down the 70-cell tube to allow each cell to pass through one at a time to prevent simultaneous passage. Ta. According to the uninvented method, the stability of the discrimination level is improved, and the monitoring of mature red blood cell and platelet counts can be implemented to compensate for simultaneous passage without sacrificing sample processing speed. Sample processing can be performed with high efficiency.

(3) The number of man-hours required for adjusting the optical system has been reduced by one.

When scattered light and fluorescent light originating from specific blood cells are detected on two different optical axes, a modification of the photometric system on the -10,000 optical axis induces a modification of the σ-linked optical system on the other optical axis.

For this reason, it is necessary to repeat many corrections in order to reach the fine adjustment of the focusing position and optical axis that enables accurate measurement of the strength of both parties. Ta. In the present invention, since the photometric thread 7a is adjusted in one axis, the number of adjustment steps is minimized and the adjustment can be easily performed.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the basic configuration when carrying out the method for measuring reticulocytes using flow cytomake 2 according to the present invention;
Is the optical axis in Figure 2? 1 is a schematic diagram for comparison of photometric optical systems in the conventional method and the uninvented method, FIG. 6 is a peak waveform diagram for a similar comparison of detected light intensity signals, and FIG. 4 is a diagram of the conventional method. The basic configuration of a typical flow cytometer is shown below. Patent applicant Toa Medical Electronics Co., Ltd. (previously) Figure 3 Time

Claims (1)

[Claims] After substantially removing the excitation light emitted from the optical stimulation area of the flow cell disposed along the optical axis of the excitation light, the output light is emitted from the optical stimulation area along the optical axis. A method for measuring reticulocytes using a flow cytometer, characterized in that the number of reticulocytes is measured by photometrically measuring forward scattered light and forward fluorescent light on the same axis, distinguishing mature red blood cells and platelets from reticulocytes based on the photometric data.