A study of cancer screening in human placenta extract intradermal reaction

A study of cancer screening in human placenta extract intradermal reaction

Zhang Jianlin, Institute of Epidemiology and Emblem Biology, Chinese Academy of Preventive Medicine

Wenlin Huang, Wuhan Institute of Virology, Chinese Academy of Sciences

The First People's Hospital of Shashi, Hubei Province, Huang Fengzhu

In 1962, Matsuhara found that human placenta and cancer tissue are similar in biology. He extracted antigen from human placenta instead of cancer tissue and conducted skin test on patients. The positive rate of early cancer that has not yet metastasized can be as high as 94,5%. Therefore, Japan's National Cancer Center has adopted this method for cancer screening since 1972, and some domestic units have also carried out some reports, but it has not been popularized so far. The reason is that there are few cases and there are still some problems that are difficult to popularize technically, such as its safety, especially the carrying of hepatitis B antigen, the titer determination of each product needs to be carried out on healthy people, and the shelf life, etc. We have studied and solved these problems and laid the foundation for the promotion of this method, which is reported as follows.

1. Materials and Methods

Preparation of 1.1 Human Placenta Extract: Take fresh and healthy human placenta from the first parturient born at full term. Each placenta serum is checked by radioimmunoassay to see if it has hepatitis B surface antigen. Only HBsAg negative patients can use it. Cut the placenta to remove the sheep's skin and blood vessels, cut it into small pieces, rinse while squeezing, straight into 3 times the amount of anhydrous ethanol, mix well and place it in a 40C refrigerator overnight. Filter with filter paper the next day, place the filtered sediment together with the filter paper in a 40C refrigerator for shade drying, peel off and weigh, add 0.4 phenol and 0.9 NaCl distilled water to make it 3%(W/V). After the solution is in the refrigerator overnight, grind and mix it evenly with a glass tissue grinder, filter out Han, and the clarified solution after suction filtration is the extract of fetal human disc.

The above extract was inoculated in broth medium and high layer solid puncture culture for 10 days, no seedling growth. And 10 mice weighing 18-20g were taken, one group was not injected with control, and the other group was injected with 1:8 diluted human placenta extract for safety test. After the injection of 30min, 24h, 48h, 72h until 10 days, two groups of mice had no abnormal reaction.

1.2 titer determination and storage: The reported titer determination method at home and abroad is to dilute the extract to different concentrations, then inoculate 7~8 healthy people at each concentration, and select the concentration with skin reaction size between 18-20mm as the appropriate concentration (the concentration is the titer). However, this method is more troublesome and not easy to popularize, so we switched to a 72-type spectrophotometer and used the double-shrinking urine method to have no blood in the water. After weighing, add a double amount of 50C distilled water, stir for 1min in a stirrer (12000r/min) for 2 times in total, form a homogenate, and freeze at-20C overnight. The next day, melt at 40C and room temperature, add 50C distilled water, make 3 times the weight of placenta, stir evenly, correct PH to 6.0, place 400C water bath, stir once every hour in the direction of the clock. After 10h 40C refrigerator overnight. After taking it out, it is filtered with 2 layers of gauze, the sediment is discarded, the PH of the filtrate is adjusted to 6.4 with 0,1N NaOH, the refrigerator is used overnight, the next day it is centrifuged at 3000r/min for 7-8min, the supernatant is stored at 40C, the precipitate is added with 2 times of distilled water, the PH of the supernatant is adjusted to 6.8 with NaOH, the sediment is discarded, the PH of the supernatant is adjusted to 7.2 is adjusted to in the refrigerator overnight, and centrifuged. Finally, the minimum sediment obtained by centrifugation is mixed with the supernatant with pH 6.4 stored in the refrigerator, the volume is measured, and the measurement O.D. is added instead of the human rest injection test method. Different concentrations (1:4,1:6,1:8) are injected into healthy people to observe the reaction, and the value of O.D.540 is measured at the same time. It is found that the skin reaction size is exactly 18~20mm at 1:6, o.D. for 0.06.

In this test, the potency of the stock solution after 4 months of preservation needs to be diluted 1:5, while the stock solution after 7-12 months of preservation needs to be diluted 1:3.

1.3 skin test method: after 75% alcohol elimination, 0.2ml of human placenta extract was injected intradermally with a tuberculous bacillus syringe on the 1/3 palmar side of the middle of the forearm of the patient, and the reaction was observed and recorded after 4 hours.

2 Results

2.1 reaction safety animal A total of 455 patients and healthy people were observed in 3 years. One case developed systemic allergy 72 hours after intradermal injection because he did not ask about the skin allergy history in detail in advance, and his body temperature slightly increased. He recovered after general desensitization treatment. The remaining 454 had no reaction.

2.2 diagnosis results

2.2.1 The control group (non-tumor patients and healthy people) consisted of 160 people, of which 25 people had a skin reaction with a maximum diameter of more than 25mm, with a positive rate of 15.6. Among the 25 patients, 18 patients, 7 of whom were chronic lymphadenitis, lymph node tuberculosis, others were chronic breast hyperplasia, liver abscess, etc. The average size of the longest diameter was 28.6mm.

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

2.2.3 91 cases were observed in group II (patients treated with anti-cancer), with an average positive rate of 49.5.

2.2.4 Observation group III (untreated patients with benign tumors) a total of 28 cases, positive 8 people, the average positive rate of 30.7.

2.2.5 The positive rate of the 4 groups was statistically tested (X2). The positive rate of the control group and the observation group I was significantly different from that of the bowel cancer. Although the clinical suspicion of malignant tumor, but the skin test negative, with other methods finally denied malignant tumor typical cases in 9 cases. Patients with cervical cancer in Yuguan Township, Wufeng County, Yichang Prefecture, Hubei Province. Among them, 7 people were positive for skin test, 1 person was suspicious and 1 person was negative. However, among the 24 non-cancer women, except for 3 suspicious, there was no positive reaction in 1 case, and the coincidence rate was higher.

Diagnosed with cancer, 3 patients with positive skin test were desensitized by 3mg of desensitization, and the second skin test remained positive. In the non-cancer patients, 7 cases of skin test positive by desensitization test, of which 5 people turned negative, 2 people remained positive, 2 people 1 for allergic constitution. The reaction of placental skin test was proved to be specific by desensitization test.

There is no report on the preservation of human placenta extract in China. In this study, 40C preservation and dilution were compared. The results showed that the effect of diluted human placenta extract decreased significantly after 4 months (1:4), O.D. decreased to 0.07 before preservation, 0.03 after 4 months, 0.06 before preservation and 0.015 after 4 months. The undiluted stock solution was stored at 40C for 4 months, and then diluted when used temporarily, the titer did not decrease significantly. The effect of longer preservation remains to be seen.

In short, this method is simple and easy to use, and has a high coincidence rate for early detection of malignant, especially digestive and reproductive tract cancers. Our study not only used 10 times more people than other reporters (P<0.001); There was also a difference in the positive rate between group I and group II (P<0.001). There was no significant difference between the control group and the observation group III.

3. Discussion

A simple and convenient method for cancer generalization is an important means for early detection of cancer. Although a few units in China have tested the intradermal reaction of human placenta extract, it has not been popularized and applied due to poor safety, difficulty in measuring titer and few observation cases (dozens of cases> and other reasons. A total of 455 cases were observed in this paper, the average positive rate of early cancer without metastasis was 83.6%, especially the early digestive tract cancer and urogenital system cancer, the positive rates were 91.3~92.8% respectively. Even if there is a statistically significant difference between the positive rate of advanced cancer and non-tumor patients (15.6), there are 18 typical cases of clinically suspected malignant tumor, positive skin test, and finally confirmed by pathology and other methods. Among them, 16 cases of Chen X X clinically suspected intestinal cancer, but X-ray did not support it, but skin test was positive. Finally, sigmoidoscope was used to confirm the reliability of this method and improve it, especially for its safety, human placental blood in advance with radioimmunoassay of hepatitis B antigen, at the same time to measure the light density method instead of the determination of potency on the human body, as well as the study of the preservation time and methods, these are conducive to the further intermediate test and large-scale promotion of this method.

A simple and convenient method for cancer generalization is an important means for early detection of cancer. Although a few units in China have tested the intradermal reaction of human placenta extract, it has not been popularized and applied due to poor safety, difficulty in measuring titer and few observation cases (dozens of cases> and other reasons. A total of 455 cases were observed in this paper, the average positive rate of early cancer without metastasis was 83.6%, especially the early digestive tract cancer and urogenital system cancer, the positive rates were 91.3~92.8% respectively. Even if there is a statistically significant difference between the positive rate of advanced cancer and non-tumor patients (15.6), there are 18 typical cases of clinically suspected malignant tumor, positive skin test, and finally confirmed by pathology and other methods. Among them, 16 cases of Chen X X clinically suspected intestinal cancer, but X-ray did not support it, but skin test was positive. Finally, sigmoidoscope was used to confirm the reliability of this method and improve it, especially for its safety, human placental blood in advance with radioimmunoassay of hepatitis B antigen, at the same time to measure the light density method instead of the determination of potency on the human body, as well as the study of the preservation time and methods, these are conducive to the further intermediate test and large-scale promotion of this method.