Study on cancer screening by intradermal reaction of human placenta extract

 

Institute of Epidemiology and Embryology, Chinese Academy of Preventive Medicine, Zhang Jianlin

 

Wuhan Institute of Virology, Chinese Academy of Sciences

 

Shashi First People's Hospital, Hubei Province

 

In 1962, Songyuan Zhengxiang discovered that human placenta and cancer tissue have similar biological aspects. He extracted the antigen from human placenta instead of cancer tissue, and performed a skin test on the patient. The positive rate of early cancer that has not been transferred can be as high as 94. 5%. Therefore, the National Cancer Center of Japan has used this method for cancer screening since 1972. Some units in China have also carried out some reports, but they have not been promoted yet. The reason is that there are fewer cases and there are some technical problems. Problems that are difficult to promote, such as its safety, especially hepatitis B antigen carrying questions, each product titer needs to be measured in healthy people, as well as the shelf life and so on. We have studied and solved these problems and laid the foundation for the promotion of this law. The report is as follows.

 

1. Materials and methods

 

1.1 Preparation of human placenta extract: take the human placenta of the first healthy mother in the fresh healthy full-term, and check the placenta serum by radioimmunoassay to check whether it has hepatitis B surface antigen. Only HBsAg-negative can be used. Cut the placenta into the alpaca and blood vessels, cut into small pieces, squeeze while rinsing, straight into 3 times the amount of absolute ethanol, and mix and place in a 40C refrigerator overnight. The next day, it was filtered with filter paper. The filtered sediment was placed in a 40 C refrigerator with the filter paper, dried, peeled off, and added with 0.4% phenol, 0.9% NaCl distilled water to make 3% (W/V). After the solution refrigerator is overnight, it will be ground and mixed with a glass tissue grinder, the filter is removed, and the clear liquid is the fetal body plate extract after suction filtration.

 

The above extract was inoculated into a broth medium and a solid layer puncture culture for 10 days, and no seedling growth was observed. Ten mice weighing 18-20 g were taken, one group was not injected with the control, and the other group was injected with a 1:8 dilution of the human placenta extract for safety test. After 30 minutes, 24 hours, 48 ​​hours, and 72 hours after observation, the mice in the two groups had no abnormal reaction.

 

1.2 Determination and preservation of titer: The method for measuring the titer of domestic and international replenishment is to dilute the extract to different concentrations, and then inoculate 7~8 healthy people at a concentration, and select the concentration of skin reaction just between 18-20mm. It is a suitable concentration (this concentration is the potency). However, this method is more troublesome and difficult to promote. Therefore, we switched to the 72-type spectrophotometer, and the double-retraction method has no blood color in the water. After weighing, a 50 C distilled water was added, and the mixture was stirred for 1 min in a stirrer (12000 r/min) for 2 times to form a homogenate, and frozen at 20 C overnight. The next day, melt at 40C and room temperature, add 50C distilled water, make it 3 times the weight of the placenta, stir evenly, correct PH to 6.0, set 400C water bath, stir once every hour clockwise. After 10 hours, the 40C refrigerator was overnight. After taking out, it was filtered with 2 layers of gauze, the sediment was discarded, the pH of the filtrate was adjusted to 6.4 with 0,1N NaOH, the refrigerator was overnight, and the next day was centrifuged at 3000 r/min for 7-8 min, the supernatant was stored at 40 C, and the precipitate was added with 2 times of distilled water. Adjust the pH to 6.8 with NaOH, centrifuge as above, discard the sediment, adjust the pH to 7.2 in the supernatant, then set the refrigerator overnight, centrifuge as above, and adjust the pH of the supernatant to 8.5. Finally, the least precipitate obtained by centrifugation is mixed with the supernatant stored in the refrigerator at pH 6.4. The volume is measured and the OD is measured instead of the human injection test at different concentrations (1:4, 1:6, 1: 8) Inject a healthy person to observe the reaction, and simultaneously measure the OD540 value, and find that the skin reaction size is exactly 18 to 20 mm and the OD is 0.06 at 1:6.

 

In this test, the titer should be preserved by the method of liquid storage, which should reach 0.05O.D., the stock solution after 4 months of storage should be diluted 1:5, and after 7-12 months of storage, it should be diluted 1:3.

 

1.3 skin test method: in the patient's forearm in the middle of the 1/3 palm side, after 75% alcohol elimination, the human placenta extract 0.2ml was injected intradermally with a tuberculosis syringe. After 4 hours, the reaction was observed and recorded.

 

2 results

 

2.1 Responsive safety During the 3 years, 455 cases of various patients and healthy people were observed. One case did not have detailed history of skin allergies in advance. Systemic allergies occurred 72 hours after intradermal injection, and the body temperature increased slightly. After general desensitization treatment That is to heal. The remaining 454 people did not have any reaction.

 

2.2 diagnosis results

 

2.2.1 The control group (non-tumor patients and healthy people) a total of 160 people, of which 25 patients with a maximum skin reaction diameter of more than 25mm, the positive rate was 15.6%. Among the 25 patients, 18 were patients, 7 of whom were chronic lymphadenitis and lymphatic tuberculosis, and the other were chronic breast hyperplasia and hepatic abscess. The average length of the longest diameter was 28.6 mm.

 

2.2.2 Observed group I (not diagnosed as malignant tumor without anti-cancer treatment) a total of 191 people, including 128 cases of early cancer, 63 cases of advanced cancer. A total of 107 cases of early cancer 4 h skin reaction more than 25 mm, the positive rate of 83.6%. Among them, the positive rate of early digestive tract cancer was as high as 92.8% (52/56), followed by 91.3% (21/23) of early life and death urinary system cancer. The positive rate of advanced cancer was low, and the average positive rate was only 39.7%. The average positive rate in the early and late stages was 69.6%.

 

2.2.3 A total of 91 patients in group II (patients treated with anticancer) were observed, with an average positive rate of 49.5%.

 

2.2.4 Observed group III (untreated benign tumor patients) a total of 28 cases, 8 positive, the average positive rate was 30.7%.

 

2.2.5 Comparison of positive rate of 4 groups After statistical test (X2), the positive rate of the control group and the observation group I was significantly different as intestinal cancer. Although clinically suspected of malignant tumors, the skin test is negative, and 9 cases of typical cases of malignant tumors are finally rejected by other methods. Cervical cancer patients in Yuguan Township, Wufeng County, Yichang District, Hubei Province. Among them, 7 people were positive for the skin test, 1 was suspicious, and 1 was negative. Among the 24 non-cancer women, except 3 were suspicious, none of them had a positive reaction and the coincidence rate was high.

 

The diagnosis was cancer, 3 cases of skin test positive patients, desensitization of 3 mg of dexamethasone, and the second skin test remained positive. In 7 patients with non-cancerous patients, the skin test was positive, and 5 of them were negative, 2 remained positive, and 1 of 2 was allergic. The desensitization test proved that the placental skin test reaction was specific.

 

There is no report on the preservation of human placenta extract in China. In this study, 40C preserved and diluted solutions were compared. The results showed that the human placenta extract preserved after dilution showed a significant decrease after 4 months (1:4), and the OD before storage was 0.07, which decreased to 4 months later. 0.03, while the 1:6 dilution was 0.06 before storage and fell to 0.015 after April. The undiluted stock solution was stored at 40 °C for 4 months, and diluted at the time of use, the titer did not decrease significantly. The effect of preservation for a longer period of time remains to be seen.

 

In short, this method is simple and easy to perform, and has a high coincidence rate for early detection of malignancy, especially cancer of the digestive tract and genital tract. Our study was not only 10 times more human than other reporters (P<0.001); the positive rate between the observation group I and the current group II was also significantly different (P<0.001); the control group was no significant compared with the observation group III. difference.

 

  3 Discussion

 

Simple method for cancer screening is an important means of early detection of cancer. Although a few units have tested the intradermal reaction of human placenta extract, the safety is poor, the measurement price is difficult, and the number of observations is small (tens of cases). Such reasons have not been promoted and applied. A total of 455 cases were observed in this study. The average positive rate of non-metastatic early cancer was 83.6%, especially in early digestive tract cancer and genitourinary cancer, and the positive rate was 91.3~92.8%, respectively. Even advanced cancer and non- There was also a statistically significant difference in the positive rate of tumor patients (15.6%). There were 18 cases of clinically suspected malignant tumors, positive skin tests, and finally confirmed by pathology. Among them, 16 cases of Chen XX were clinically suspected. Intestinal cancer, but X-ray does not support, but the skin test is positive. Finally, the reliability of this method is confirmed by sigmoidal confirmation, and the improvement is made. Especially for its safety, human placental blood is previously used to detect hepatitis B antigen. At the same time, the photometric density method is used instead of measuring the titer on the human body, and the preservation time and method are studied, which are beneficial to the further intermediate test and large-scale promotion of this method.