Rhesus incompatibility is one of the most common causes of hemolytic disease in newborns. However, it is possible to overcome undesirable reactions during pregnancy and childbirth by using anti-Rh immunoglobulin. If the father is heterozygous for the RHD gene and the mother is Rh negative, there is a 50% chance that the baby will be Rh positive. Prenatal diagnosis of RHD status in these cases is of extreme importance, because if a negative Rh factor of the fetus is detected, further tests to determine the antibody titer and therapeutic procedures become unnecessary. If a Rh-positive fetus is detected, it will be necessary to conduct pregnancy with appropriate precautions and prescribe anti-Rh immunoglobulin for prevention.

Traditionally, the Rh factor of the fetus is established when testing amniotic fluid, chorionic villi, or umbilical cord blood. However, to obtain material for the study, invasive procedures (chorionic biopsy, amniocentesis, or corodocentesis) are necessary. These procedures are quite dangerous, and, in addition, can lead to an increased risk of maternal sensitization. To prevent these risks, several research groups studied the possibility of determining the RHD status of the fetus by fetal cells isolated from the blood of a pregnant woman. The main problem with this approach is the difficulty in obtaining a sufficient number of cells from the blood of a pregnant woman. Several studies have shown the feasibility of isolating dissolved fetal DNA from maternal plasma for genetic testing. In this study, we used fetal DNA from the blood plasma of a pregnant Rh-negative woman to determine the Rh factor of the fetus.  

Purpose of the study:

To evaluate the method of non-invasive diagnosis of the Rh factor of the fetus by the blood of a pregnant woman using new diagnostic kits for identification of the Rh factor gene (RHD) of the fetus in the mother’s blood “DNA-Rhesus of the child” produced by LLC Gentekhnologiya (Russia).  

Materials and methods:

To determine the specificity of the used diagnostic PCR systems, we used 10 samples of RHD positive and 10 samples of RHD-negative freshly harvested umbilical cord blood (the Rh factor was determined by a serological method using cyclones). To determine the diagnostic value of the test system, we used blood samples of one hundred pregnant Rh-negative women with a volume of 5 ml. Twenty women were in the first trimester of pregnancy (7-12 weeks), thirty-eight in the second trimester (13-24 weeks) and 42 in the third trimester (25-40 weeks). Women donated blood either during standard examinations, or purposefully for entry into the study. In each case, women signed informed consent.  

Sample preparation for research:

Blood samples were collected in tubes containing EDTA as an anticoagulant. Then the tubes were centrifuged at 1600 g for 10 minutes and carefully, without capturing the leukocyte layer, plasma was collected in 1.5 ml disposable microcentrifuge tubes. Further, the plasma was either immediately used for DNA isolation, or stored at a temperature of minus 40 C for subsequent isolation.

DNA isolation:

DNA was isolated from 1 ml of plasma by modifying the isolation protocol using the QIAamp Viral RNA Mini Kit (Qiagen) isolation kit. The volume of the DNA solution was 60 μl  

Rh factor gene detection:

In order to identify the Rh factor gene, real-time PCR was used using diagnostic kits for identification of the Rh factor gene (RHD) of the fetus in the mother’s blood “DNA Rhesus of the baby” produced by Gene-Technology LLC (Russia, RU No. ФСР2010 / 09565) . In order to control the quality of DNA extraction, the GAPDH gene (glyceraldehyde 3-phosphate dehydrogenase) was used. Two exons of the Rh factor gene were amplified. The conclusion about the positive Rh factor of the fetus was made on the basis of a reaction with GAPDH and two exons simultaneously. The conclusion about the negative Rh factor was made during PCR with GAPDH and the absence of reaction in test tubes with exons of the Rh factor gene. The study was performed on a BioRad IQ5 amplifier with real-time detection.

Results:

To determine the possibility of using the diagnostic system at the first stage, we used genomic DNA obtained from 20 Rh-positive and Rh-negative cord blood samples. We obtained a complete correspondence between the obtained genetic results and serological results.

To determine the sensitivity of the test system, we used control DNA diluted in deionized water and isolated from Rh negative samples of genomic DNA. The lower the DNA concentration, the more PCR cycles were required to obtain a detectable amount of fluorescent molecules. The limit of sensitivity of the method was a concentration of 10 copies of DNA per ml.

All 100 women who entered the study were Rh-negative by serological analysis. For 60 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. The results of the correlation of the genetic method for maternal and serological blood are presented in table 1.

DNA analysis of 37 samples indicated a positive Rh factor of the fetus, in 23 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 match (with a negative Rh factor, according to the test system, the actual Rh factor was 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. Thus, the sensitivity and specificity of the diagnostic method used was 100%.  

Discussion of the results:

A successful clinical study of a new diagnostic method proves the possibility of its use in practice. The relevance of the introduction of this innovative methodology for the early determination of the Rh factor of the fetus in Rh-negative pregnant women is obvious, because it allows you to determine the Rhesus affiliation of the fetus in early pregnancy and thus identify pregnant women in whom the Rhesus factor of the fetus and its own are incompatible. Such women need special medical supervision and prophylaxis of the Rh conflict for the entire period of pregnancy in order to exclude the hemolytic disease of the newborn.

Another group of Rh-negative pregnant women, who, as a result of applying this technique, establishes that the fetus is also Rh-negative (and there are about 50% of the total number), does not need special medical supervision, because Rhesus conflict is excluded from them.

When determining the Rh factor of the fetus in a Rh-negative pregnant woman in the early stages, several problems are solved at once.

1. Psychological - women pregnant with a Rh-negative fetus will not worry about a possible pregnancy pathology;
2. Economic:

- as a result of determining a Rh-negative fetus, half of the women will not need to regularly take tests to determine the antibody titer (now all Rh-negative pregnant women do it 10-12 times per pregnancy at the expense of the state);

- there is no need for preventive measures using an expensive drug - anti-Rhesus immunoglobulin for all Rh-negative pregnant women, thereby halving the amount of allocated budgetary funds or funds of pregnant women themselves.

New diagnostic kits for identification of the Rh factor gene (RHD) of the fetus in the mother’s blood “DNA Rhesus of the baby” produced by Gentekhnologiya LLC (Russia) can be recommended for implementation in medical practice by obstetrician-gynecologists for early non-invasive diagnosis of the Rh factor of the fetus by blood a pregnant woman.

Table 1.

The results of correlation of the genetic and serological method for evaluating the Rh factor in maternal blood.
Comparison of the Rh factor of a born child with the results of DNA analysis.