JPH035551B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for measuring lymphocytes derived from cancer disease, which is carried out using lymphocytes taken out from a subject.

[Prior Art] One method that has recently come into the spotlight as a diagnostic method for cancer, which is rapidly increasing in number, is a method using a photosensitizer and a laser beam. This involves intravenously administering a photosensitizer that has an affinity for tumors, allowing it to accumulate in the tumor over a long period of time, and then irradiating the affected area with a laser beam. Tumors are identified by observing fluorescence.

[Problems to be solved by the invention] However, this method is an in vivo measurement and requires about 3 days for accumulation.
This not only caused pain for the patient but also required a lot of effort.

Therefore, the present inventor focused on the fact that hematoporphyrin is ingested by lymphocytes taken out of the body, and added hematoporphyrin to the lymphocytes taken out of the body, and added hematoporphyrin to the lymphocytes taken out of the body. A method for measuring the amount of radicals produced using an electron spin resonance apparatus was filed as Japanese Patent Application No. 1983-83651.
The present inventor conducted further research and found that even better assay results could be obtained using hematoporphyrin derivatives than hematoporphyrin.

[Configuration to solve the problem] This method provides a method for measuring lymphocytes derived from cancer disease, in which a hematoporphyrin derivative is added to lymphocytes from a subject, and after incubation, only lymphocytes are separated. The method is characterized by the steps of quantitatively measuring the amount of radicals contained in the collected lymphocytes using an electron spin resonance device. Hereinafter, the present invention will be explained in detail using the drawings.

[Example] Hematoporphyrin derivatives are described in the magazine "Clinical Surgery" (Vol. 37, No. 4, pp. 517-522, April 1982).
March), “Chest” (Vol.81, No.3, MARCH 1982)
It was developed by Lipson et al. in 1960, and is made by treating hematoporphyrin hydrochloride extracted from blood hemoglobin with acetic acid and sulfuric acid. Some ingredients are mixed. The present inventors have discovered that this hematoporphyrin derivative, just like hematoporphyrin, generates π-cation radicals when irradiated with light, and that these radicals can be measured using an electron spin resonance device (ESR device). confirmed. Figure 1 shows the ESR of the above radical.
This shows an example of spectrum measurement, and the g value is 2.0015.
It is. Figure 2 shows the relationship between the ESR spectrum intensity (peak-to-peak value h in Figure 1) measured by irradiating light (ultraviolet light) to about 100μ of a lymphocyte buffer solution of hematoporphyrin derivatives and dilution concentration. It can be seen that, although a saturation phenomenon is observed in the high concentration region, overall there is a good linear relationship between the hematoporphyrin derivative concentration and the ESR signal intensity, just as in the case of hematoporphyrin. Note that the measurements were carried out at a low temperature of -100°C because the sensitivity decreases at room temperature.

FIG. 3 is a flowchart showing the procedure for implementing the method according to the present invention using an ESR device, (1)
Only lymphocytes are removed from the blood taken from the patient, for example, by centrifugation, and (2) this is
(3) incubate at 37°C for about 10 minutes, (4)
Centrifuge at 1500 rpm for 5 minutes to remove lymphocytes and wash with physiological saline. (5) After washing, suspend the lymphocytes in 300μ of physiological saline and perform ESR.
It is introduced into a device and quantitative measurements are performed while irradiating it with light (ultraviolet light).

FIG. 4 shows the ESR spectra of lymphocytes of a normal healthy person (bottom) and a cancer patient (top) measured according to the procedure shown in FIG. 3, respectively. The intensity (amount of radicals) exhibited by cancer-affected lymphocytes is about four times that of normal lymphocytes. In the case of patent application No. 1983-83651, which uses hematoporphyrin,
Since the difference was twice as large, the difference is larger in the present invention using hematoporphyrin derivatives, and the discrimination is more accurate.

In addition, according to the well-known calculation method of calculating the g value based on the distance between the focused spectrum peak and the reference peak (R in Fig. 4) of the standard sample,
When calculating the g value of the ESR signal shown by the lymphocytes in the figure,
Both signals in FIG. 4 are 2.006. π derived from the hematoporphyrin derivative shown in Figure 1
The ESR spectrum of a cation radical has a g value of
2.0015, the ESR signal shown by lymphocytes is not derived from π cation radicals. However, it is unclear what kind of substance the measured radicals originate from.

FIG. 5 shows the measurement results of blood collected from 21 normal healthy subjects, 26 cancer patients, and 5 patients with collagen disease using the procedure shown in FIG. Each measurement result is
Peak-peak value hr of the reference peak (R in Figure 4) due to a certain amount of standard sample mixed in during measurement
It is expressed as a relative intensity value in the form of a ratio.
From Figure 5, the relative intensity of lymphocytes from cancer patients is 0.8.
In contrast, lymphocytes from normal healthy people without cancer and collagen disease patients show lower values. Therefore, it can be seen that according to the differential test of the present invention, it is possible to test whether the lymphocytes are cancer patient's lymphocytes or not, using a certain level as the boundary line. Moreover, in the method of Japanese Patent Application No. 1983-83651 using hematoporphyrin, there was a portion where the values of cancer patients and normal healthy people overlapped to some extent near the boundary line, but in the present invention, such overlap is eliminated. It has become possible to more clearly determine whether a person has cancer or not.

Note that since the intensity of the reference peak changes depending on the amount of the standard sample mixed in, the relative intensity value also changes. Therefore, it goes without saying that a comparison of relative intensity values between a normal healthy person and a cancer patient must be performed by mixing the same amount of standard sample.

[Effects] As detailed above, according to the present invention, by using a hematoporphyrin derivative, a more accurate measuring method is realized than when using hematoporphyrin.

[Brief explanation of the drawing]

Figure 1 is a diagram showing an example of measuring the ESR spectrum of a hematoporphyrin derivative, Figure 2 is a diagram showing the relationship between the hematoporphyrin derivative concentration and the relative intensity of the ESR spectrum, and Figure 3 is a diagram showing a measurement example of the ESR spectrum of a hematoporphyrin derivative. Flowchart showing the procedure for carrying out the method, FIG. 4 is a diagram showing ESR spectra of lymphocytes of a normal healthy person (bottom) and a cancer patient (top) measured according to the procedure of FIG. 3, FIG. 5 is a diagram showing the measurement results of blood collected from 21 normal healthy subjects, 26 cancer patients, and 5 patients with collagen disease using the procedure shown in FIG. 3.

Claims (1)

[Claims] 1. Adding a hematoporphyrin derivative to lymphocytes taken out from a subject, (b) separating only lymphocytes after incubation, (c)
A method for measuring lymphocytes derived from cancer disease, which consists of several steps, in which the amount of radicals contained in separated lymphocytes is quantitatively measured using an electron spin resonance device. 2. Claim 1, in which separated lymphocytes are illuminated with light and quantitatively measured using an electron spin resonance device.
The method for measuring lymphocytes derived from cancer disease described in Section 1.