In Russia, the percentage of Rh sensitized women reaches 1.2%. In developed countries, the number of women with Rh sensitization is 0.1–0.2%. Such a low percentage was achieved due to the introduction of specific prophylaxis into clinical practice by introducing anti-Rhesus immunoglobulin to non-sensitized Rh negative women. The difference in the number of sensitized women arose for several reasons, one of which is the high cost of anti-Rhesus immunoglobulin, which you have to buy at your own expense. Although this drug should be administered only to those Rh-negative pregnant women whose fetus is Rh-positive.

Contrary to the belief that the placenta forms a barrier between the mother and the baby, this is not so. Numerous studies have shown that both whole fetal cells and extracellular fetal DNA pass through the placenta and circulate in the maternal bloodstream.

Intact fetal cells circulating in the maternal bloodstream are an attractive target for non-invasive prenatal diagnosis, namely for the diagnosis of the fetus Rh factor, fetal sex and chromosomal abnormalities by simple karyotyping. Although the existence of fetal cells in maternal blood has been known for more than a century, directly intact fetal nuclear red blood cells were not used for prenatal diagnosis until 1990. Since that time, the isolation and detection of fetal cells from the mother’s blood began to be intensively investigated, and various methods for the isolation of fetal cells began to appear, which were successful to varying degrees. However, the research results were disappointing. This was due to insufficiency of intact fetal cells circulating in the maternal bloodstream (about 1 cell per milliliter of maternal blood), and low research efficiency. Although some fetal cells (particularly fetal nuclear red blood cells) have a relatively short life span in maternal blood, other types of fetal cells can remain in maternal blood decades after pregnancy and are potentially capable of causing false positive results in subsequent pregnancies. Thus, despite the fact that modern studies are trying to improve the technique of sorting cells, most recent scientific studies focused on the cell-free DNA of the fetus in the mother’s blood, which is present in sufficient quantities.

A huge amount of human DNA is located inside the cell, but the presence of a small amount of cfDNA in the circulation of both healthy and sick women was discovered in 1947. Although their biological source and potential function remain undetermined, it is suggested that this is a product of apoptosis, resulting in fragmentation and exit of chromosomal DNA from the cell. The presence of fetal DNA in maternal circulation was demonstrated by Lo et al in 1998.

Fetal DNAs are formed by apoptosis of placental cells (trophoblast) obtained from and make up approximately 3–6% of total bcDNA in maternal blood flow at the beginning and end of pregnancy, respectively (the remaining 94–97% are maternal cfDNAs).

Fetal DNA can be detected from the 4th week of gestation, although its concentration increases reliably only from the 8th week with an increase in gestational age, from 16 fetal genomes per milliliter of mother’s blood in the first trimester of pregnancy to 80 in the third trimester), with an acute peak during the last 8 weeks of pregnancy. Unlike fetal cells, ccfDNA is rapidly excreted from the mother’s blood with a half-life of 16 minutes and is not determined 2 hours after delivery.

The purpose of the study is to evaluate the method of non-invasive diagnosis of the Rh factor of the fetus by the blood of a pregnant woman.

Materials and research methods

Fetal DNA from the blood plasma of a pregnant Rh-negative woman was used to determine the Rh factor of the fetus. The study involved 150 pregnant Rh-negative women. 7 ml venous blood samples were used. In each case, women signed informed consent.

To identify the Rh factor gene, real-time PCR was used using diagnostic kits for identification of the fetus Rh factor gene in the mother’s blood “DNA-Rhesus of the baby” produced by Gene-Technology LLC (Russia, RU No. ФСР2010 / 09565).

Determination of the Rh factor of the fetus by the blood of a pregnant Rh-negative woman is based on the fact that extracellular fetal DNA is present in the blood plasma of a pregnant woman.

For the correct determination of the Rh factor of the fetus by the blood of a pregnant woman, the following conditions must be observed:

1. Pregnancy is from 10 weeks.
2. Proper plasma handling, DNA isolation as quickly as possible.
3. DNA isolation using reliable technology.
4. Organization of work in a PCR laboratory in accordance with applicable national standards.
5. Follow the instructions carefully.

The study is carried out in several stages:

1. Blood sampling and plasma secretion. The test material for the analysis is the venous blood of a pregnant Rh-negative woman. Blood is donated on an empty stomach, collected in a tube with an anticoagulant EDTA in an amount of 7-10 ml. Within 24 hours from the time of blood collection, plasma should be taken and transferred to a new tube. Plasma should be delivered to the laboratory within 16-24 hours after taking the material.
2. DNA isolation. It is carried out by the column-adsorption method using the recommended sets of kits designed to isolate circulating nucleic acids from biological fluids according to the manufacturer's method.
3. Real-time amplification with detection. Sample preparation is standard for PCR. Reactions are prepared for the direct detection of the Rh factor gene in the test sample, as well as control reactions confirming the correctness of the study. The reaction takes from 1.5 to 2 hours (depending on the rate of change of temperature with an amplifier).
4. Registration and accounting of analysis results.

Research results and discussion

All 150 women who entered the study were Rh-negative by serological analysis. For 115 of them, the Rh factor of the born child became known.

The Rh factor of the children born was evaluated by the serological method in the hospital.

DNA analysis of 76 samples indicated a positive Rh factor of the fetus, in 39 cases a negative Rh factor was established. The results of the analysis were confirmed by a blood test of children after their birth. In one case, the result of the analysis did not coincide (with a negative Rh factor, according to the test system, the actual Rh factor turned out to be Rh positive). It should be noted that the gestational age for this patient was 9 weeks, while the minimum period guaranteeing the correctness of the analysis results is 10 weeks. Therefore, this result cannot be used to evaluate the analytical characteristics of the test system. In two children, the Rh factor was determined incorrectly in the hospital. After receiving the result of the analysis for determining the Rh factor of these children, which does not coincide with our results, they took a buccal scraping and carried out a genetic determination of the Rh factor. The result of this analysis confirmed the correctness of the Rh factor of the fetus determined by us for the blood of a pregnant woman. 1.8% (2 children) - incorrect determination of the Rh factor of children in the hospital. Thus, the sensitivity and specificity of the diagnostic method used was 100%.

Conclusions

• new diagnostic kits for identifying the Rh factor gene (RHD) of the Russian-made Rh baby DNA can be recommended for introduction into medical practice by obstetrician-gynecologists for early non-invasive diagnosis of the Rh factor in the blood of a pregnant woman;
• determination of the Rh factor in early pregnancy avoids expensive drug prophylaxis;
• administration of an anti-Rhesus immunoglobulin to all Rh-negative women who are pregnant with a Rh-positive fetus will subsequently lead to a decrease in the percentage of Rh-sensitized women in Russia.