JPH0458574B2 - - Google Patents

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention uses fluorescence polarization measurement to measure the degree of stimulation of cells such as lymphocytes based on two parameters: the degree of fluorescence polarization and the amount of change per unit time. The present invention relates to a lymphocyte stimulation measurement device that performs two-dimensional judgment to determine the degree of cell stimulation.

[Background of the invention]

Lymphocytes are separated from the blood by centrifugation, this fractionated lymphocyte suspension is used as a sample, and a stimulating substance, for example, a phytohemagglutinin such as phytohemagglutinin, is added and reacted.
Juvenility occurs in healthy people, but not in patients with malignant tumors, so conventional methods have used this principle to distinguish between healthy people and patients with malignant tumors. It is being done.

Attempts to detect malignant tumors by measuring the intralymphocyte dynamics due to stimulus responses using fluorescence polarization were carried out by Drs. L. Cercek, B. Cercek, and Dr.
It is reported in the following literature by JAV Pritchard et al.

(1) Europ.J.Cancer Vol.13 P.903-915 (1977) (2) Br.J.Cancer Vol.38 P.339-343 (1978) All of the methods reported here are The degree of fluorescence polarization of stimulated lymphocytes P _CONT (abbreviation for Pcontrol)
The degree of fluorescence polarization of PHA (Phytohaemagglutimin) stimulated lymphocytes is determined by the ratio of P PHA to P _PHA , that is, P _PHA / P _CONT × 100 (%).
It is designed to represent the degree of stimulation of lymphocytes.
According to this method, the results shown in Figure 1 have been obtained, and the tendency is for patients with malignant tumors to
P _PHA /P _CONT ×100 (%) is distributed around 100 to 90%, and P _PHA /P _CONT × of healthy people and benign tumor patients
Since 100 (%) is distributed around 90-80%, it is possible to roughly classify healthy people and malignant tumor patients based on this.

However, as shown in Figure 1, data using only one parameter, P _PHA / P _CONT , shows that the difference between healthy people and patients with malignant tumors is
There is a problem that the values of P _PHA /P _CONT are distributed in an overlapping manner, and it is very difficult to clearly distinguish between the two.

Therefore, Cancer Basic Protein (CaBP), which specifically stimulates lymphocytes of patients with malignant tumors, has been developed.
Attempts have also been made to clarify the discrimination using CaBP (abbreviation for CaBP) based on a phenomenon opposite to the above-mentioned judgment, but there is a problem that it is not suitable for practical use because CaBP itself is difficult to obtain.

[Purpose of the invention]

An object of the present invention is to provide a practical lymphocyte stimulation measurement device that can clearly distinguish between healthy people or patients with benign tumors and patients with malignant tumors.

[Summary of the invention]

The present invention employs multiple parameters such as the degree of fluorescence polarization and the amount of change per unit time, and eliminates overlap in distribution data, making it possible to clearly distinguish between malignant tumor patients, healthy people, and benign tumor patients. be.

[Embodiments of the invention]

First, the principle underlying the present invention will be explained.

The dynamics of cells such as lymphocytes (cell fluidity) is
As observed by a fluorescence polarization measurement device, when cells are stimulated and intracellular fluidity increases, parallel components of light are depolarized and orthogonal components of light increase. On the other hand, if the cells are less stimulated, less light will be depolarized than in the former case.

This phenomenon is expressed by the degree of fluorescence polarization (P value) in equation (1) below.
It can be equivalently expressed by .

P=I−I _L ×G/I+I _L ×G ……(1) However, I: Polarized light intensity of parallel component I _L : Polarized light intensity of orthogonal component G: Instrument constant In the above equation (1), lymphocytes inspired,
When depolarization occurs, I _L increases and the P value decreases. Therefore, the value of P _CONT −P _PHA increases.

From this, the lymphocytes of healthy people and patients with benign tumors are stimulated by PHA, and thus exhibit a higher value of P _CONT -P _PHA than those of patients with malignant tumors.

On the other hand, when considering the amount of change per unit time, when cells are stimulated and the intracellular fluidity increases, and the fluorescence reaction described below is actively performed, the amount of change per unit time of the orthogonal component of light increases. , when the stimulation of the cell is small, the amount of change of the orthogonal component light per unit time is the same as in the case of no stimulation.

This fluorescence reaction is shown in equation (2).

FDA hydrolase――――――――――――――――――→ ――――――――――――――――――――→ (FIuoresceindiacetate) Fluorescein＋2CH ₃ COOH ...(
2) Here, FDA is a fluorescent precursor and does not inherently emit fluorescence. However, when it is taken into cells, it is broken down by hydrolytic enzymes in the cells and becomes fluorescein, which emits fluorescence. This hydrolysis reaction is
It is thought that the more stimulated the lymphocytes are, the more actively they are carried out.

This phenomenon can be equivalently expressed by the following equation (3).

ΔI _L ×G/ΔI ...(3) However, ΔI: Change amount of polarized light intensity per unit time of parallel component, ΔI _L : Change amount of polarized light intensity per unit time of orthogonal component, G: Equipment constant, Above In equation (3), in the case of lymphocytes from healthy people and benign tumor patients, ΔI _L increases because they are stimulated by PHA.

Therefore, ΔI _L ×G/ΔI (CONT−PHA) indicates a negative value.

Therefore, by using both the above-mentioned (1) parameter of the difference in the degree of fluorescence polarization and (2) parameter of the difference in the amount of change per unit time and representing them in two dimensions, it is possible to distinguish between healthy people and benign tumors. It becomes possible to clearly distinguish patients with malignant tumors from patients with malignant tumors.

Next, the present invention will be explained in detail based on specific examples.

FIG. 2 is a block diagram showing one embodiment of the present invention, showing stimulated lymphocytes (in a suspension state) or target unstimulated lymphocytes (in a suspension state).
is added to the FDA test solution, which is a fluorescent precursor,
It is housed in a cuvette 1 for measuring suspended samples. After measuring for a certain period of time, the suspension is filtered to remove only lymphocytes. This is done to compensate for the fluorescein (fluorescent substance) produced by the decomposition of FDA, which is not only localized within the cell but also leaks into the external solution. It is. The liquid after filtration is stored in a filtrate measurement cuvette 2 and undergoes fluorescence polarization.

In the fluorescence polarization measurement system, first, white light emitted from a light source 3 is separated into monochromatic light by an excitation side spectrometer 4. The excitation light passes through the excitation side polarizer 5 and is irradiated onto the measurement cuvette 1. In this case, the plane of polarization is normally set in the vertical direction in measurements. Furthermore, when measuring the device constant (G value), the plane of polarization is set in the horizontal direction.

Fluorescence emitted from the measurement sample by irradiation with excitation light passes through the fluorescence polarizer 6 and is guided to the fluorescence spectrometer 9. The fluorescence side polarizer 6 is configured to be able to rotate 0° parallel to the excitation side polarizer 5 and 90° perpendicular to the excitation side polarizer 5. The excitation side polarizer 5 and the fluorescence side polarizer 6 are rotated by an excitation side polarizer drive motor 7.
This is realized by applying a drive signal to the fluorescence side polarizer drive motor 8.

In this way, the optical signal obtained by the fluorescence side spectrometer 9 is detected by the photodetector 10, and the detected signal is amplified by the preamplifier 11, and then converted from an analog signal by the A/D converter 12. converted into a digital signal. This digital signal is then stored in the memory 13 and used in the calculation section 14 for calculation processing to be described later. The calculation result is displayed on the display section 1.
5 is displayed. The operation sequence of these signal processing systems and drive systems is controlled by a controller 16.

With such a system, measurement results as shown in the schematic diagram of FIG. 3 can be obtained.

Now, the degree of fluorescence polarization and the amount of change per unit time are calculated in the calculation unit 14 as follows.

In other words, I _L (T) at the midpoint of filtration during polarization measurement of a suspended sample solution: A is the value of the orthogonal component of the entire reaction solution, I is J is the value of the parallel component of the entire reaction solution, and is the polarization measurement of the filtrate. I _L (F): the value of the orthogonal component of the filtrate is B, I: the value of the parallel component of the filtrate is K, then the fluorescence polarization degree P value is P=I-I _L ×G/I+I _L × G, and is expressed by the following equation (4).

P=(A-B)-(J-K)×G/(A-B)+(
J-K)×G = C-L×G/C+L×G...(4) However, the device constant G is expressed as G=i _L /i=b/a...(5). Note that C=AB, L=JK.

The calculation unit 14 performs the calculation shown by this equation (4) on the non-pierced lymphocyte suspension and PHA for each sample.
The measurements of the stimulated lymphocyte suspension were carried out,
Find P _CONT and P _PHA . Furthermore, this P _CONT and P _PHA
Find the difference (P _CONT − P _PHA ).

Next, the polarized light intensity at the middle point of filtration is expressed as I _L =C=A-B I=L=JK.

Further, the polarized light intensity at the time of starting the measurement is expressed as I _LO =D−D×B/A _IO =M−M×K/J. Therefore, the amount of change per unit time is: ΔI _L =I _L -I _LO =C-(D-D×B/A) ΔL=I-I _O =L-(M-M×K/J)... ...(6), the calculation unit 14 calculates the amount of change per unit time for each of the unstimulated lymphocytes (CONT) and stimulated lymphocytes (PHA) based on this sixth equation, and then calculates the Find the ratio of the amount of change ΔI _L ×G/ΔI.

The calculation unit 14 blots the ratio of the degree of fluorescence polarization P and the amount of change per time calculated in this manner for each sample, with the former on the vertical axis and the latter on the horizontal axis, as shown in FIG. 4, for example, It is displayed on the display screen of the display unit 15.

As a result, the data for healthy people and benign tumor patients form a group in the upper left of the second quadrant, and the data for malignant tumor patients form a group on the right from the second quadrant to the first quadrant. The overlap in the distribution of patient fluorescence polarization measurements is significantly reduced, making it extremely easy to distinguish between the two.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, multiple parameters are used to eliminate overlap in the distribution of fluorescence polarization values, making it possible to clearly distinguish between healthy people, patients with benign tumors, and patients with malignant tumors. Furthermore, it is highly likely that it will be possible to make a distinction using only PHA, whereas conventional determinations were made using two stimulating reagents: PHA and CaBP, and this will have extremely excellent practical effects, such as simplification of measurement.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of measurement results by a conventional method, FIG. 2 is a block diagram showing an embodiment of the present invention, and FIG. 3 is a schematic diagram of signal processing in the present invention.
FIG. 4 is a diagram showing an example of a measurement result display according to the present invention. DESCRIPTION OF SYMBOLS 1... Cuvette for measuring suspended sample, 2... Cuvette for measuring filtrate, 3... Light source, 4... Excitation side spectrometer, 5... Excitation side polarizer, 6... Fluorescence side polarizer, 7... ... Excitation side polarizer drive motor, 8 ... Fluorescence side polarizer drive motor, 9 ... Fluorescence side spectrometer, 10
...Photodetector, 11...Preamplifier, 12...
A/D converter, 13... memory section, 14... calculation section, 15... display section, 16... controller.

Claims (1)

[Claims] 1. In a lymphocyte stimulation measurement device that measures the degree of stimulation of cells such as lymphocytes using fluorescence polarization, a first means for determining the difference between the degree of fluorescence polarization of stimulated lymphocytes and that of unstimulated lymphocytes. and a second means for determining the difference in the ratio of the change amount per unit time of the parallel component and the orthogonal component during reaction measurement of stimulated lymphocytes and unstimulated lymphocytes, and plots each calculation result. A lymphocyte stimulation measurement device characterized by determining the degree of stimulation of lymphocytes.