JPH0249664B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of reacting in vitro with lymphocytes in a specimen using a compound that has an affinity for lymphocytes of cancer patients, and measuring the fluorescence intensity of the reacted compound. Therefore, this method is useful as a method for testing cancer patients.

Among the rapidly increasing methods for diagnosing cancer diseases, one of the methods that has recently attracted attention is a method using a photosensitizer and a laser beam. This method involves intravenously administering a photosensitizer that has an affinity for tumor cells, allowing it to accumulate in the tumor over a long period of time, then irradiating the affected area with a laser beam and observing the fluorescence emitted by the photosensitizer. This is a method of identifying tumors based on the However, since this method involves measurement in vivo, it causes a great deal of pain to the patient and requires a lot of labor.

The present inventors conducted research to improve these points, and discovered that hematoporphyrin or hematoporphyrin derivatives react with lymphocytes and cancer cells of cancer patients taken out of the body.
We have completed a technology to test cancer diseases by measuring the amount of hematoporphyrin that has reacted with these lymphocytes and cancer cells using an electron spin resonance (ESR) device, and have applied for a patent on this technology (patent application). Showa 58-
No. 83651 and Patent Application No. 58-243453). Thereafter, the present inventors conducted further research and found that similarly good results could be obtained by measuring fluorescence intensity, and based on this knowledge, they completed the present invention.

That is, the present invention involves bringing a compound that has an affinity for lymphocytes derived from a cancer patient and having the ability to emit fluorescence into contact with lymphocytes to be measured, and then measuring the fluorescence intensity. The present invention relates to a characteristic method for measuring lymphocytes derived from cancer patients.

Compounds that have an affinity for lymphocytes of cancer patients and have the ability to emit fluorescence (hereinafter referred to as fluorescent compounds) include hematoporphyrin, its derivatives, acridine orange, and pheophorbide a. can be used. Derivatives of hematoporphyrin are (R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₈ are −CH ₃ , −
CH ₂ CH ₃ , −CH(OH)CH ₃ ,
R ₆ and R ₇ are -(CH ₂ ) _o COOH (n=2 to 4).
Z is a divalent metal atom such as Fe ⁺⁺ , Co ⁺⁺ , Mn ⁺⁺ or a hydrogen atom. ) etc.

The lymphocytes to be measured are collected from the subject. This collection is performed, for example, by heparin blood sampling. In order to improve measurement accuracy, lymphocytes are preferably separated from a sample such as blood before being brought into contact with the above-mentioned fluorescent compound. In addition, macrophages in particular are the cause of false positives, so they should be removed in advance. It is preferable. A common method for removing macrophages is to adsorb them to a monocyte removal agent or Cephadex. For example, use a monocyte-depleting agent at 8-12% of whole blood.
(v/v) and left at 35-38°C for several minutes to 30 minutes with stirring as necessary to adsorb macrophages. By layering the separated solutions and centrifuging them, macrophages can be removed and lymphocytes can be separated. When using Cephadex, when white blood cells (including granulocytes, neutrophils, macrophages, lymphocytes, etc.) prepared by specific gravity centrifugation are introduced into the Cephadex column, cells other than lymphocytes will be adsorbed to Cephadex. Therefore, it is sufficient to collect the flow-through fraction that flows out of the column.

The lymphocytes thus separated are washed with a phosphate buffered saline solution (PBS solution), etc., as necessary, and adjusted to a predetermined concentration with a PBS solution, etc. Needless to say, the appropriate range of this concentration is determined by the fluorescence measuring device, the type of specimen, the fluorescent compound used, etc.

The method for bringing a fluorescent compound into contact with lymphocytes is to create a mixed solution state of the two. Taking the case of using hematoporphyrins as an example, for example, a hematoporphyrins solution is brought into contact with lymphocytes. Add to the liquid and heat at room temperature or around 37℃ for 5~
It is sufficient to incubate for about 20 minutes.

After the reaction, unreacted fluorescent compounds are usually removed by centrifugal washing, and then the fluorescence intensity is measured. However, when using a flow cytometer, which will be described later, lymphocytes can be measured individually, making it possible to count lymphocytes that have reacted with a fluorescent compound without removing unreacted fluorescent compounds. It is possible. However, even in this case, it is preferable to remove unreacted fluorescent compounds from the viewpoint of improving identification.

The fluorescence intensity can be measured using a known fluorescence measurement device, for example, it is possible to measure it with a fluorescence photometer, but in that case, there is a problem that the measurement accuracy is poor due to interference between lymphocytes. There is a point. The present inventors discovered the above fact and were able to solve this problem and improve measurement accuracy by using a flow cytometer (FCM).

This FCM uses a hydrodynamic focusing method to make cells flow almost in a line at a uniform speed.
This device measures the scattered light and fluorescent light of each cell by irradiating this sample stream with laser light, and is particularly effective in that it can simultaneously measure the number of cells that emit fluorescent light. For example, argon laser light, krypton laser light, etc. may be used as the laser light.

As described above, the method of the present invention allows cancer testing to be performed easily and quickly without causing physical pain to the subject.

Although the cancer testing method has been mainly described above, the method of the present invention is not limited thereto, and its principles can be applied to various tests, diagnoses, and the like.

Examples are shown below.

Example 1 In this example, measurements were performed on 25 cancer patient samples, 15 benign disease patient samples, and 20 normal person samples.

Lymphocytes were prepared from 5-10 ml of heparinized whole blood. That is, 500 to 1000 μl of monocyte removal reagent (manufactured by IBL) containing 5% silica was added to whole blood,
The mixture was mixed by inversion at 37°C for 30 minutes. Separate this L
(manufactured by Moto Roire Chemical Co.) and centrifuged to collect lymphocytes. This lymphocyte
After centrifugal washing with PBS solution (0.008 mol, PH7.4, 0.8% NaCl), the final concentration was approximately 10 ⁶ cells/ml.
It was suspended using PBS solution.

50 μl of 0.005M hematoporphyrin solution was added to this lymphocyte suspension, and the mixture was incubated at 37° C. for 10 minutes. Subsequently, the lymphocytes were washed three times with PBS solution, the concentration was adjusted to about 10 ⁶ cells/ml, and the fluorescence intensity was measured by FCM.

EPICSV ^TM manufactured by Coulter was used for FCM, and 590 nm red light was measured using argon laser light for excitation of hematoporphyrin.

Figure 1 shows the relationship between fluorescence intensity and cell number obtained for one normal patient (a) and two cancer patients (b) and (c). As shown in the figure, there are many lymphocytes positive for hematoporphyrin in cancer patients.

According to the measurement results of all samples, the ratio of hematoporphyrin-positive cells among lymphocytes of cancer patients was 5.71±
It was 2.59% (mean value ± 1SD), which was significantly higher than 1.89 ± 0.47% in patients with benign disease and 1.66 ± 0.56% in normal subjects. This distribution is
As shown in the figure.

Note that the same results as above were obtained for each hematoporphyrin exemplified in general formula (B).

Example 2 Using acridine orange or pheophorbide a instead of hematoporphyrin, Example 1
When measured under almost the same conditions as above, the following results were obtained.

Fluorescent compound Normal subjects (n=20) Cancer patients (n=
25) Acridine Orange 1.78±0.63% 4.25±
2.32% Hueophorbide a 1.74±0.61% 4.39±
2.17%

[Brief explanation of the drawing]

FIG. 1 shows an example of the results of measuring the fluorescence intensity of lymphocytes reacted with hematoporphyrin using a flow cytometer, where the vertical axis shows the number of cells and the horizontal axis shows the fluorescence intensity. FIG. 2 shows the distribution states obtained by measuring the percentage of positive lymphocytes in normal subjects, benign disease patients, and cancer patients.

Claims (1)

[Claims] 1. A method for measuring lymphocytes from a cancer patient, which comprises bringing hematoporphyrin, its derivatives, acridine orange, or pheophorbide a into contact with lymphocytes in a specimen, and then measuring the fluorescence intensity.