Rh factor testing: Difference between revisions

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'''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]]).<ref>{{Cite web|url=https://www.babycenter.com/0_blood-test-for-rh-status-and-antibody-screen_1480.bc|archive-url=https://web.archive.org/web/20070911115522/http://www.babycenter.com/0_blood-test-for-rh-status-and-antibody-screen_1480.bc|url-status=usurped|archive-date=September 11, 2007|title=Blood test for Rh status and antibody screen|date=2019-03-07|website=BabyCenter|access-date=2019-03-07}}</ref><ref name=":0">{{Cite web|url=https://www.mayoclinic.org/tests-procedures/rh-factor/about/pac-20394960|title=Rh factor blood test - Mayo Clinic|website=www.mayoclinic.org|access-date=2019-04-08}}</ref>

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[[/Rh Factor Testing]]

== Background ==

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). Rhesus factor testing utilizes the [[genotyping technique]] to detect the presence of the [[RHD (gene)|RhD gene]]. By checking the existence of the RhD gene in the individual’sindividual's [[genome]], the presence of rhesusRhesus D (RhD) antigens[[antigen]]s can be inferred. Individuals with a positive RhD status hashave RhD antigens expressed on the [[cell membrane]] of their [[red blood cellscell]]s, whereas Rhesus D antigens are absent for individuals with a negative RhD status.

Rhesus factor testing is usually conductedperformed 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 conditionscomplications caused by rhesusRhesus incompatibility.

=== Rhesus factor ===

The entire [[Rh blood group system]] involves multiple antigens and genes. For Rh factor testing, however, only the rhesusRhesus factor which referscorrelated to the RhD antigen specifically 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.<ref>{{Cite web |title=Chromosome 1: MedlinePlus Genetics |url=https://medlineplus.gov/genetics/chromosome/1/ |access-date=2024-04-07 |website=medlineplus.gov |language=en}}</ref> RhD is a dominant gene, meaning that as long as at least one RhD gene is inherited from eithera 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 are commonly used as test samples for verifying the maternal RhD status. Blood plasma can also be used for determining the foetal RhD status if the mother is RhD- as maternal blood plasma contains maternal DNA and trace amounts of fetal DNA. Blood samples can be obtained through venipuncture of the mother. Since plasma and other components of blood has different densities, centrifugation of blood samples with added anticoagulant (such as EDTA) can segregate blood contents into multiples layers. Blood plasma can then be isolated from the other components for rhesus factor testing. The method of extracting foetal DNA from maternal blood plasma is considered to be a type of non-invasive prenatal testing.

[[Blood plasma]] areis commonly used as test samples for verifying the maternal RhD status. Blood plasma can also be used for determining the foetalfetal 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.<ref name=":1">{{Cite web |last=nonacus-developer |date=2021-06-11 |title=Non-Invasive Fetal RhesusD Blood Genotyping |url=https://nonacus.com/blog-non-invasive-fetal-rhesusd-blood-genotyping/ |access-date=2024-04-07 |website=nonacus |language=en-US}}</ref> Blood samples can be obtained through [[venipuncture]] of the mother. Since plasma and other components of blood hashave different densities, [[centrifugation]] of blood samples with added [[anticoagulant]] (such as [[Ethylenediaminetetraacetic acid|EDTA]]) can segregate blood contents into multiplesmultiple layers. Blood plasma can then be isolated from the other components. forIt rhesuscan factorbe testinggenotyped using real time PCR to determine the RhD status of the fetus.<ref name=":1" /> The method of extracting foetalfetal DNA from maternal blood plasma is considered to be a type of non-invasive [[prenatal testing]].

== Non-invasive extraction ==

Non-invasive prenatal testing can be used if the mother is RhD-. However, in the case of maternal RhD status being negative, invasive prenatal testing may be used to determine the foetal RhD status instead. The two most common invasive methods of extracting foetal 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 foetal blood cells should the foetus be Rh incompatible with the mother.

=== Invasive extraction ===

Non-invasive [[prenatal testing]] can be used if the mother is RhD-. However, in the case of maternal RhD status being negativepositive, invasive prenatal testing may be used to determine the foetalfetal RhD status instead. The two most common invasive methods of extracting foetalfetal 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 [[Antibody|antibodies]] which may attack the foetalfetal blood cells should the foetusfetus be Rh incompatible with the mother.

==== Chorionic villus sampling ====

[[Chorionic villus sampling]] is usually doneperformed between the 10th and 12th13th week of pregnancy,. itIt samples [[chorionic villi]], which are tiny projections of [[Placenta|placental tissue]]. As theThe placental tissues are derived from [[embryonic cellscell]]s, hence, itthey containscontain fetal genetic information that can be used to determine the child’schild's RhD status. There are two types of chorionic villus sampling. TranscervicalTrans-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. TransabdominalTrans-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 [[Minimally invasive procedure|invasive procedures]].

==== Amniocentesis ====

[[Amniocentesis]] is another invasive procedure which can be used to collect foetalfetal DNA samples.^{[<nowiki/>[[Wikipedia:Identifying reliable sources (medicine)|''medical citation needed'']]]} This procedure is usually done between the 15th week toand 20th week of [[pregnancy]]. The purpose of AMC is to extract a small amount of [[amniotic fluid]] as foetalfetal cells may be shed from the foetusfetus and are suspended in the amniotic fluid. TheSince the foetalfetal genome can be found in these cells. Therefore, extracting amniotic fluid canprovides the required foetalfetal 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 applyuse an ultrasound to locate the foetusfetus in the uterus. Under the guidance of the [[ultrasound imaging]], a long, thin, hollow needle will insertbe 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 willis then be removed from the maternal body and the extracted amniotic fluid extracted will beis sent to the laboratory for further testing.

== Genotyping of RhD gene ==

The presence of the RhD gene in an individual’sindividual's genome is determined by [[genotyping]]. Firstly, the body fluid containing an individual’sindividual'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 reactionsreaction]]s (PCR) mixture. The PCR mixture usually contains [[Taq polymerase|Taq DNA polymerase]], [[DNA primersprimer]]s, deoxyribonucleotides[[deoxyribonucleotide]]s (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 analysedanalyzed by [[gel electrophoresis]]. Before gel electrophoresis, [[Molecular-weight size marker|DNA reference ladder]], a [[Positive control group|positive control]] containing DNA with RhD gene, and the PCR product will be loaded onto the wells of the gel. An electrical[[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 [[Transillumination|UV transilluminatorstrans-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 the prevention ofprevent haemolytic conditions caused by the Rh incompatibility.{{Citation needed|date=May 2024}} 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 donationtransfusion ===

When RhD antigens on red blood cells are exposed to an individual with RhD- status, high-frequency of [[Immunoglobulin G|IgG]] [[Rho(D) immune globulin|anti-RhD]] [[Antibody|antibodies]] will be developed in the RhD- individual’sindividual's body. The antibodies then attacksattack red blood cells with attached RhD antigens[[antigen]]s and lead to the destruction of these cells. This condition is known as a [[Hemolytic reaction|haemolytic reaction]]. The destruction of red blood cells releases haemoglobin[[hemoglobin]] to the bloodstream. HaemoglobinHemoglobin may be excreted through [[urine]], causing [[Hemoglobinuria|haemoglobinuria]]. The sudden release of haemoglobinhemoglobin 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 and circulatory shock]].

==== Symptoms of Rh group incompatibility in blood donation ====

== Prevention of haemolytic disease of the newborn ==

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.

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 haemolyse the red cells of the fetus, causing the aforementioned haemolytic in the fetus known as haemolytic disease of the newborn. This disease is usually fatal for the fetus and hence preventive measures are conducted.

=== Prevention of haemolytic disease of the newborn ===

== Medical Intervention ==

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 haemolyselyse the red cells of the fetus, causing the aforementioned haemolytic reaction in the fetus known as [[Hemolytic disease of the newborn|haemolytic disease of the newborn]]. This disease is usually fatal for the fetus and hence preventive measures are conducted.

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 the a sensitized RhD- mother (previously conceived an RhD+ child) and the fetus being Rh+, medication such as anti-D immunoglobulin will be given to the RhD- mother. Injecting RhD- mother with anti-D immunoglobulin has been proven effective in avoiding the sensitization of RhD+ antigen, even though the mechanism of how this medication works remains obscure. Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.

==== Symptoms of haemolytic disease of the newborn ====

= References =

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.<ref name=":10">{{Cite web|url=https://www.urmc.rochester.edu/encyclopedia/content.aspx?ContentTypeID=90&ContentID=P02368|title=Hemolytic Disease of the Newborn (HDN) - Health Encyclopedia - University of Rochester Medical Center|website=www.urmc.rochester.edu|access-date=2019-04-09}}</ref> Splenomegaly, [[cardiomegaly]] and hepatomegaly may occur in the baby.<ref name=":10" /> Excessive [[tissue fluid]] may accumulate in the [[stomach]], [[lung]]s or [[scalp]]. These are typically signs of [[hydrops fetalis]].<ref name=":10" />

1. Blood test for Rh status and antibody screen. BabyCenter. [date unknown]. [accessed 2019 Mar 7]. https://www.babycenter.com/0_blood-test-for-rh-status-and-antibody-screen_1480.bc

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 [[Anemia|anaemia]]. The yellowing of the [[umbilical cord]], skin and eyes, also known as jaundice, may arise within 24 to 36 hours of birth.<ref name=":10" /> Signs of hydrops fetalis such as the enlargement of [[spleen]], [[heart]] and [[liver]], along with severe [[edema]], will continue after birth.<ref name=":10" />

2. Rh factor blood test. Mayo Clinic. 2018 Jun 14. [accessed 2019 Mar 7]. https://www.mayoclinic.org/tests-procedures/rh-factor/about/pac-20394960

=== Medical Intervention ===

3. Non-invasive prenatal testing for fetal rhesus-D status - putting the NICE guidance into practice. British Blood Transfusion Society. [date unknown]. [accessed 2019 Mar 7]. https://www.bbts.org.uk/blog/noninvasive_prenatal_testing_for_fetal_rhesus-d_status_-_putting_the_/

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 the a sensitized RhD- mother (previously conceived an RhD+ child) and the fetus being Rh+, medication such as an [[Rho(D) immune globulin|anti-D immunoglobulin]], called RhoGAM, will be given to the RhD- mother. Injecting RhD- mother with anti-D immunoglobulinRhoGAM has been proven effective in avoiding the sensitizationsensitisation of RhD+ antigen, even though the mechanism of how this medication works remains obscure. Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.

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.

4. Stickler T. Healthline. Prenatal Screening Tests: Types, Diagnosis, and Takeaways. 2016 Mar 23. [accessed 2019 Mar 7]. https://www.healthline.com/health/pregnancy/prenatal-testing#second-trimester

== References ==

5. Inheritance patterns of blood groups. Australian Red Cross Blood Service. [date unknown]. [accessed 2019 Mar 7]. https://transfusion.com.au/blood_basics/blood_groups/inheritance_patterns

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8. Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Kulovany E, Vlk R. Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma. Fetal Diagn Ther. 2005; 20 (4): 275-280.

9. Dovč-drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P. Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma. Transfus Med Hemother. 2013; 40 (1): 37-43.

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11. Saramago P, Yang H, Llewellyn A, Walker R, Harden M, Palmer S, Griffin S, Simmonds M. High-throughput, non-invasive prenatal testing for fetal rhesus D status in RhD-negative women: a systematic review and economic evaluation. Health Technology Assessment. 2018; 22 (13): 1-172.

12. Ross H. Amniocentesis: Purpose, Procedure and Risks. Healthline. 2016 Jan 11. [accessed 2019 Mar 7]. https://www.healthline.com/health/amniocentesis#complications

13.Chorionic Villus Sampling. Johns Hopkins Medicine. [date unknown]. [accessed 2019 Mar 7]. https://www.hopkinsmedicine.org/healthlibrary/test_procedures/gynecology/chorionic_villus_sampling_cvs_92,p07769

14. Amniocentesis. University of Rochester Medical Center. [date unknown]. [accessed 2019 Mar 7]. https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=92&contentid=p07762

15. Kickler TS, Blakemore K, Shirey RS, Nicol S, Callan N, Ness PM, Escallon C, Dover G. Chorionic villus sampling for fetal Rh typing: clinical implications. American Journal of Obstetrics and Gynecology. 1992; 166(5): 1407-1411.

16. Van den veyver IB, Subramanian SB, Hudson KM, Werch J, Moise KJ Jr, Hughes MR. Prenatal diagnosis of the RhD fetal blood type on amniotic fluid by polymerase chain reaction. Obstetrics and Gynecology. 1996; 87(3): 419-422.

17. Brinc D, Lazarus AH. Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn. Hematology. American Society of Hematology. Education Program. 2009; 2009 (1): 185-191.