Rh factor testing

Last updated May 08, 2024

Rh factor testing, also known as Rhesus factor testing, is the procedure of determining the Rhesus D status of an individual (see Rh blood group system).^[1]^[2]

Background

Rhesus factor testing utilizes genotyping to detect the presence of the RhD gene. By checking the existence of the RhD gene in the individual's genome, the presence of Rhesus D (RhD) antigens can be inferred. Individuals with a positive RhD status have RhD antigens expressed on the cell membrane of their red blood cells, whereas Rhesus D antigens are absent for individuals with a negative RhD status.

Rhesus factor testing is usually performed on pregnant women to determine the RhD blood group of the mother and the fetus. By confirming the RhD status of both mother and fetus, precautions can be made, if necessary, to prevent any medical complications caused by Rhesus incompatibility.

Rhesus factor

The entire Rh blood group system involves multiple antigens and genes. For Rh factor testing, however, only the Rhesus factor correlated to the RhD antigen is assayed. The RhD gene that codes for the RhD antigen is located on chromosome 1. This chromosome contains gene instructions for making proteins in the body.^[3] RhD is a dominant gene, meaning that as long as at least one RhD gene is inherited from a single parent, the RhD antigen is expressed. Vice versa, if no RhD gene is inherited from either parent, no RhD antigen is produced.

Extraction of test samples

Non-invasive extraction

Blood plasma is commonly used as test samples for verifying the maternal RhD status. Blood plasma can also be used for determining the fetal RhD status if the mother is RhD- as maternal blood plasma contains maternal DNA and trace amounts of fetal DNA. In early pregnancy, around 3% of the mother’s free-cell DNA is from the fetus, and raises to 6-7% by late pregnancy.^[4] Blood samples can be obtained through venipuncture of the mother. Since plasma and other components of blood have different densities, centrifugation of blood samples with added anticoagulant (such as EDTA) can segregate blood contents into multiple layers. Blood plasma can then be isolated from the other components. It can be genotyped using real time PCR to determine the RhD status of the fetus.^[4] The method of extracting fetal DNA from maternal blood plasma is considered to be a type of non-invasive prenatal testing.

Invasive extraction

Non-invasive prenatal testing can be used if the mother is RhD-. However, in the case of maternal RhD status being positive, invasive prenatal testing may be used to determine the fetal RhD status instead. The two most common invasive methods of extracting fetal DNA are chorionic villus sampling (CVS) and amniocentesis (AMC). These invasive procedures can be conducted on both RhD+ and RhD- mothers. After the invasive procedure, medications that prevent the Rh immunization are usually prescribed to RhD- mothers. This is done to avoid the production of maternal anti-D antibodies which may attack the fetal blood cells should the fetus be Rh incompatible with the mother.

Chorionic villus sampling

Chorionic villus sampling is usually performed between the 10th and 13th week of pregnancy. It samples chorionic villi, which are tiny projections of placental tissue. The placental tissues are derived from embryonic cells, hence, they contain fetal genetic information that can be used to determine the child's RhD status. There are two types of chorionic villus sampling. Trans-cervical sampling involves inserting a catheter through the cervix into the placenta to obtain villi; an ultrasound is used to guide the catheter to the site of sampling. Trans-abdominal sampling requires the insertion of a needle through the abdomen and uterus to obtain placental tissue. Local anesthesia can be applied to reduce the pain from invasive procedures.

Amniocentesis

Amniocentesis is another invasive procedure which can be used to collect fetal DNA samples.^{[ medical citation needed]} This procedure is usually done between the 15th and 20th week of pregnancy. The purpose of AMC is to extract a small amount of amniotic fluid as fetal cells may be shed from the fetus and are suspended in the amniotic fluid. Since the fetal genome can be found in these cells, extracting amniotic fluid provides the required fetal genetic material for the genotyping of the RhD gene. Before amniocentesis commences, the doctor will inject local anesthetics to the mother's abdomen. The doctor will then use an ultrasound to locate the fetus in the uterus. Under the guidance of the ultrasound imaging, a long, thin, hollow needle will be inserted through the skin of the abdomen to the uterus of the mother. The needle is used to withdraw a trace amount of amniotic fluid. It is then removed from the maternal body and the extracted amniotic fluid is sent to the laboratory for further testing.

Genotyping of RhD gene

The presence of the RhD gene in an individual's genome is determined by genotyping. Firstly, the body fluid containing an individual's DNA will be extracted. DNA will then be isolated from unwanted impurities. The isolated DNA will then be mixed with various reagents to prepare the polymerase chain reactions (PCR) mixture. The PCR mixture usually contains Taq DNA polymerase, DNA primers, deoxyribonucleotides (dNTP) and buffer solution. The DNA primers are specific for exon 7 and exon 10. Under different circumstances, primers for other regions of the RhD gene, such as intron 4 and exon 5, may also be used. The mixture will be subjected to a series of PCR which is performed by a thermal cycler. By the end of the PCR process, the amount of RhD gene will be amplified if it is present. The product of the PCR will be analyzed by gel electrophoresis. Before gel electrophoresis, DNA reference ladder, a positive control containing DNA with RhD gene, and the PCR product will be loaded onto the wells of the gel. An electric current will be applied and the DNA fragments will migrate to the positive terminal as they are negative in charge. Since DNA fragments have different molecular sizes, the larger they are, the slower they migrate. Utilizing this property, DNA fragments with different molecular masses can be segregated. With the help of gel staining and visualizing devices such as UV trans-illuminators, RhD gene DNA fragments, if present, will be visible as a band with its corresponding molecular mass. Further DNA sequencing can be conducted to confirm that the sequence of product DNA fragments matches that of the RhD gene sequence.

Clinical Applications

Rh factor testing is crucial to prevent haemolytic conditions caused by the Rh incompatibility.^{[ citation needed ]} The consequence of having haemolytic conditions can be dangerous or even lethal as it may lead to multiple complications. Not only does Rh factor testing determine the rhesus status of the individuals, but also indicate the necessity for further medical intervention.

Prevention of Rh group incompatibility in blood transfusion

When RhD antigens on red blood cells are exposed to an individual with RhD- status, high-frequency of IgG anti-RhD antibodies will be developed in the RhD- individual's body. The antibodies then attack red blood cells with attached RhD antigens and lead to the destruction of these cells. This condition is known as a haemolytic reaction. The destruction of red blood cells releases hemoglobin to the bloodstream. Hemoglobin may be excreted through urine, causing haemoglobinuria. The sudden release of hemoglobin will also pass through the liver and be metabolized into bilirubin, which in high concentrations, accumulates under the skin to cause jaundice. Liberation of blood cell debris into the circulation will also cause disseminated intravascular coagulation.

Symptoms of Rh group incompatibility in blood donation

Patients receiving incompatible blood transfusion may have pale skin, splenomegaly, hepatomegaly and the yellowing of mouth and eyes. In addition, their urine may appear in dark color and the patients may experience dizziness and confusion. Tachycardia, the increase in heart rate, is also a symptom of the haemolytic disease.

Prevention of haemolytic disease of the newborn

In the case of pregnancy, when an RhD- mother carries an RhD+ fetus, some of the fetal red blood cells may cross the placenta into the maternal circulation, sensitizing the mother to produce anti-RhD antibodies. Since the mixing of fetal and maternal blood occurs mainly during separation of the placenta during delivery, the first RhD+ pregnancy rarely causes any danger to the fetus as delivery occurs before the synthesis of antibodies by the mother. However, if the mother were to conceive another RhD+ child in the future, the anti-RhD antibodies will cross the placenta to attack and lyse the red cells of the fetus, causing the aforementioned haemolytic reaction in the fetus known as haemolytic disease of the newborn. This disease is usually fatal for the fetus and hence preventive measures are conducted.

Symptoms of haemolytic disease of the newborn

Symptoms of the disease may vary in each pregnancy. They are usually not noticeable during pregnancy. However, prenatal tests may reveal yellow colouring of amniotic fluid, which is caused by the buildup of bilirubin.^[5] Splenomegaly, cardiomegaly and hepatomegaly may occur in the baby.^[5] Excessive tissue fluid may accumulate in the stomach, lungs or scalp. These are typically signs of hydrops fetalis.^[5]

After birth, the symptoms of the child are similar to that of incompatible blood transfusion in adults. The baby may have pale skin due to anaemia. The yellowing of the umbilical cord, skin and eyes, also known as jaundice, may arise within 24 to 36 hours of birth.^[5] Signs of hydrops fetalis such as the enlargement of spleen, heart and liver, along with severe edema, will continue after birth.^[5]

Medical Intervention

Normally, no extra medical intervention is required when maternal Rh status is RhD+, nor RhD- mothers going through first pregnancy. However, in the case of a sensitized RhD- mother (previously conceived an RhD+ child) and the fetus being Rh+, medication such as an anti-D immunoglobulin, called RhoGAM, will be given to the RhD- mother. Injecting RhD- mother with RhoGAM has been proven effective in avoiding the sensitisation of RhD+ antigen, even though the mechanism of how this medication works remains obscure.

This injection is given to the RhD- mother during the second trimester when there is incompatibility between her and the father. Another injection is given a couple days after delivery if the baby is found to be RhD+. These injections may also be given to RhD- mothers after a miscarriage/abortion,after injury to the abdomen, or after the prenatal tests mentioned before of amniocentesis and chorionic villus sampling (cite1). Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.

Related Research Articles

<span class="mw-page-title-main">Amniocentesis</span> Sampling of amniotic fluid done mainly to detect fetal chromosomal abnormalities

Amniocentesis is a medical procedure used primarily in the prenatal diagnosis of genetic conditions. It has other uses such as in the assessment of infection and fetal lung maturity. Prenatal diagnostic testing, which includes amniocentesis, is necessary to conclusively diagnose the majority of genetic disorders, with amniocentesis being the gold-standard procedure after 15 weeks' gestation.

Rh disease is a type of hemolytic disease of the fetus and newborn (HDFN). HDFN due to anti-D antibodies is the proper and currently used name for this disease as the Rh blood group system actually has more than 50 antigens and not only the D-antigen. The term "Rh Disease" is commonly used to refer to HDFN due to anti-D antibodies, and prior to the discovery of anti-Rh_o(D) immune globulin, it was the most common type of HDFN. The disease ranges from mild to severe, and occurs in the second or subsequent pregnancies of Rh-D negative women when the biologic father is Rh-D positive.

Prenatal testing is a tool that can be used to detect some birth defects at various stages prior to birth. Prenatal testing consists of prenatal screening and prenatal diagnosis, which are aspects of prenatal care that focus on detecting problems with the pregnancy as early as possible. These may be anatomic and physiologic problems with the health of the zygote, embryo, or fetus, either before gestation even starts or as early in gestation as practicable. Screening can detect problems such as neural tube defects, chromosome abnormalities, and gene mutations that would lead to genetic disorders and birth defects, such as spina bifida, cleft palate, Down syndrome, trisomy 18, Tay–Sachs disease, sickle cell anemia, thalassemia, cystic fibrosis, muscular dystrophy, and fragile X syndrome. Some tests are designed to discover problems which primarily affect the health of the mother, such as PAPP-A to detect pre-eclampsia or glucose tolerance tests to diagnose gestational diabetes. Screening can also detect anatomical defects such as hydrocephalus, anencephaly, heart defects, and amniotic band syndrome.

Chorionic villus sampling (CVS), sometimes called "chorionic villous sampling", is a form of prenatal diagnosis done to determine chromosomal or genetic disorders in the fetus. It entails sampling of the chorionic villus and testing it for chromosomal abnormalities, usually with FISH or PCR. CVS usually takes place at 10–12 weeks' gestation, earlier than amniocentesis or percutaneous umbilical cord blood sampling. It is the preferred technique before 15 weeks.

The amniotic fluid is the protective liquid contained by the amniotic sac of a gravid amniote. This fluid serves as a cushion for the growing fetus, but also serves to facilitate the exchange of nutrients, water, and biochemical products between mother and fetus.

Hemolytic disease of the newborn, also known as hemolytic disease of the fetus and newborn, HDN, HDFN, or erythroblastosis fetalis, is an alloimmune condition that develops in a fetus at or around birth, when the IgG molecules produced by the mother pass through the placenta. Among these antibodies are some which attack antigens on the red blood cells in the fetal circulation, breaking down and destroying the cells. The fetus can develop reticulocytosis and anemia. The intensity of this fetal disease ranges from mild to very severe, and fetal death from heart failure can occur. When the disease is moderate or severe, many erythroblasts are present in the fetal blood, earning these forms of the disease the name erythroblastosis fetalis.

Rh_o(D) immune globulin (RhIG) is a medication used to prevent RhD isoimmunization in mothers who are RhD negative and to treat idiopathic thrombocytopenic purpura (ITP) in people who are Rh positive. It is often given both during and following pregnancy. It may also be used when RhD-negative people are given RhD-positive blood. It is given by injection into muscle or a vein. A single dose lasts 12 weeks. It is made from human blood plasma.

In ABO hemolytic disease of the newborn maternal IgG antibodies with specificity for the ABO blood group system pass through the placenta to the fetal circulation where they can cause hemolysis of fetal red blood cells which can lead to fetal anemia and HDN. In contrast to Rh disease, about half of the cases of ABO HDN occur in a firstborn baby and ABO HDN does not become more severe after further pregnancies.

Hemolytic disease of the newborn (anti-Kell₁) is the second most common cause of severe hemolytic disease of the newborn (HDN) after Rh disease. Anti-Kell₁ is becoming relatively more important as prevention of Rh disease is also becoming more effective.

Hemolytic disease of the newborn (anti-Rhc) can range from a mild to a severe disease. It is the third most common cause of severe HDN. Rh disease is the most common and hemolytic disease of the newborn (anti-Kell) is the second most common cause of severe HDN. It occurs more commonly in women who are Rh D negative.

The Rh blood group system is a human blood group system. It contains proteins on the surface of red blood cells. After the ABO blood group system, it is the most likely to be involved in transfusion reactions. The Rh blood group system consisted of 49 defined blood group antigens in 2005. As of 2023, there are over 50 antigens among which the five antigens D, C, c, E, and e are the most important. There is no d antigen. Rh(D) status of an individual is normally described with a positive (+) or negative (−) suffix after the ABO type. The terms Rh factor, Rh positive, and Rh negative refer to the Rh(D) antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions and antibodies to the Rh(D) and Rh antigens confer significant risk of hemolytic disease of the newborn.

A nuchal scan or nuchal translucency (NT) scan/procedure is a sonographic prenatal screening scan (ultrasound) to detect chromosomal abnormalities in a fetus, though altered extracellular matrix composition and limited lymphatic drainage can also be detected.

Neonatal alloimmune thrombocytopenia is a disease that affects babies in which the platelet count is decreased because the mother's immune system attacks her fetus' or newborn's platelets. A low platelet count increases the risk of bleeding in the fetus and newborn. If the bleeding occurs in the brain, there may be long-term effects.

Hemolytic disease of the newborn (anti-RhE) is caused by the anti-RhE antibody of the Rh blood group system. The anti-RhE antibody can be naturally occurring, or arise following immune sensitization after a blood transfusion or pregnancy.

<span class="mw-page-title-main">Percutaneous umbilical cord blood sampling</span>

Percutaneous umbilical cord blood sampling (PUBS), also called cordocentesis, fetal blood sampling, or umbilical vein sampling is a diagnostic genetic test that examines blood from the fetal umbilical cord to detect fetal abnormalities. Fetal and maternal blood supply are typically connected in utero with one vein and two arteries to the fetus. The umbilical vein is responsible for delivering oxygen rich blood to the fetus from the mother; the umbilical arteries are responsible for removing oxygen poor blood from the fetus. This allows for the fetus’ tissues to properly perfuse. PUBS provides a means of rapid chromosome analysis and is useful when information cannot be obtained through amniocentesis, chorionic villus sampling, or ultrasound ; this test carries a significant risk of complication and is typically reserved for pregnancies determined to be at high risk for genetic defect. It has been used with mothers with immune thrombocytopenic purpura.

Immune tolerance in pregnancy or maternal immune tolerance is the immune tolerance shown towards the fetus and placenta during pregnancy. This tolerance counters the immune response that would normally result in the rejection of something foreign in the body, as can happen in cases of spontaneous abortion. It is studied within the field of reproductive immunology.

Cell-free fetal DNA (cffDNA) is fetal DNA that circulates freely in the maternal blood. Maternal blood is sampled by venipuncture. Analysis of cffDNA is a method of non-invasive prenatal diagnosis frequently ordered for pregnant women of advanced maternal age. Two hours after delivery, cffDNA is no longer detectable in maternal blood.

An Intrauterine transfusion (IUT) is a procedure that provides blood to a fetus, most commonly through the umbilical cord. It is used in cases of severe fetal anemia, such as when fetal red blood cells are being destroyed by maternal antibodies. IUTs are performed by perinatologists at hospitals or specialized centers.

<span class="mw-page-title-main">Ruth Darrow</span> American pathologist

Ruth Renter Darrow (1895–1956) was an American pathologist who was the first to identify the cause of hemolytic disease of the newborn (HDN). In 1938, three years prior to the discovery of antibodies against the Rh antigen, Darrow correctly hypothesized that the disease was caused by destruction of red blood cells due to antibodies in the mother's blood. Darrow's research was inspired by her personal experiences with the disease.

Noninvasive prenatal testing (NIPT) is a method used to determine the risk for the fetus being born with certain chromosomal abnormalities, such as trisomy 21, trisomy 18 and trisomy 13. This testing analyzes small DNA fragments that circulate in the blood of a pregnant woman. Unlike most DNA found in the nucleus of a cell, these fragments are not found within the cells, instead they are free-floating, and so are called cell free fetal DNA (cffDNA). These fragments usually contain less than 200 DNA building blocks and arise when cells die, and their contents, including DNA, are released into the bloodstream. cffDNA derives from placental cells and is usually identical to fetal DNA. Analysis of cffDNA from placenta provides the opportunity for early detection of certain chromosomal abnormalities without harming the fetus.

References

↑ "Blood test for Rh status and antibody screen". BabyCenter. 2019-03-07. Retrieved 2019-03-07.
↑ "Rh factor blood test - Mayo Clinic". www.mayoclinic.org. Retrieved 2019-04-08.
↑ "Chromosome 1: MedlinePlus Genetics". medlineplus.gov. Retrieved 2024-04-07.
1 2 nonacus-developer (2021-06-11). "Non-Invasive Fetal RhesusD Blood Genotyping". nonacus. Retrieved 2024-04-07.
1 2 3 4 5 "Hemolytic Disease of the Newborn (HDN) - Health Encyclopedia - University of Rochester Medical Center". www.urmc.rochester.edu. Retrieved 2019-04-09.

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[1] "Blood test for Rh status and antibody screen". BabyCenter. 2019-03-07. Retrieved 2019-03-07.

[:0-2] "Rh factor blood test - Mayo Clinic". www.mayoclinic.org. Retrieved 2019-04-08.

[3] "Chromosome 1: MedlinePlus Genetics". medlineplus.gov. Retrieved 2024-04-07.

[:1-4] 1 2 nonacus-developer (2021-06-11). "Non-Invasive Fetal RhesusD Blood Genotyping". nonacus. Retrieved 2024-04-07.

[:10-5] 1 2 3 4 5 "Hemolytic Disease of the Newborn (HDN) - Health Encyclopedia - University of Rochester Medical Center". www.urmc.rochester.edu. Retrieved 2019-04-09.

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