Rh factor testing: Difference between revisions

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{{short description|Blood test}}

{{orphan|date=April 2019}}

'''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|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>.

== Background ==

Rhesus factor testing utilises the [[genotyping]] technique to detect the presence of the [[RHD (gene)|RhD gene]].<ref name=":1">{{cite journal | vauthors = Flegel WA | title = The genetics of the Rhesus blood group system | journal = Blood Transfusion = Trasfusione del Sangue | volume = 5 | issue = 2 | pages = 50–7 | date = April 2007 | pmid = 19204754 | pmc = 2535884 | doi = 10.2450/2007.0011-07 }}</ref>. By checking the existence of the RhD gene in the individual’sindividual's [[genome]], the presence of rhesus D (RhD) [[Antigen|antigensantigen]]s can be inferred. Individuals with a positive RhD status has RhD antigens expressed on the [[cell membrane]] of their [[Redred blood cell|red blood cells]]s, whereas Rhesus D antigens are absent for individuals with negative RhD status.<ref name=":2">{{Cite web|url=https://transfusion.com.au/blood_basics/blood_groups/inheritance_patterns|title=transfusion.com.au|website=transfusion.com.au|language=en|access-date=2019-04-08}}</ref>.

Rhesus factor testing is usually conducted on pregnant women to determine the RhD blood group of the mother and the foetus. By confirming the RhD status of both mother and foetus, precautions can be made if necessary to prevent any medical conditions caused by rhesus incompatibility.<ref name=":0" />

=== Rhesus factor ===

The entire [[Rh blood group system]] involves multiple antigens and genes.<ref name=":1" />. For Rh factor testing, however, only the rhesus factor which refers to the RhD antigen specifically is assayed.<ref name=":1" />. The RhD gene that codes for the RhD antigen is located on [[chromosome 1]].<ref name=":2" />. RhD is a dominant gene, meaning that as long as at least one RhD gene is inherited from either parent, the RhD antigen is expressed.<ref name=":2" />. Vice versa, if no RhD gene is inherited, no RhD antigen is produced.

== Extraction of test samples ==

[[Blood plasma]] is 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 foetal DNA.<ref name="Reliable Determination of Fetal RhD">{{cite journal | vauthors = Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P | title = Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma | journal = Transfusion Medicine and Hemotherapy | volume = 40 | issue = 1 | pages = 37–43 | date = February 2013 | pmid = 23637648 | pmc = 3636019 | doi = 10.1159/000345682 }}</ref>. 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 [[Ethylenediaminetetraacetic acid|EDTA]]) can segregate blood contents into multiples layers.<ref>{{cite journal | vauthors = Dagur PK, McCoy JP | title = Collection, Storage, and Preparation of Human Blood Cells | journal = Current Protocols in Cytometry | volume = 73 | pages = 5.1.1–16 | date = July 2015 | pmid = 26132177 | pmc = 4524540 | doi = 10.1002/0471142956.cy0501s73 | isbn = 9780471142959 }}</ref>. 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]].<ref>{{Cite web|url=https://www.bbts.org.uk/blog/noninvasive_prenatal_testing_for_fetal_rhesus-d_status_-_putting_the_/|title=BBTS {{!}} Non-invasive prenatal testing for fetal rhesus-D status - putting the {{!}}|website=www.bbts.org.uk|access-date=2019-04-09}}</ref>.

=== Non-invasive extraction ===

Non-invasive [[prenatal testing]] can be used if the mother is RhD-.<ref>{{cite journal | vauthors = Saramago P, Yang H, Llewellyn A, Walker R, Harden M, Palmer S, Griffin S, Simmonds M | title = High-throughput non-invasive prenatal testing for fetal rhesus D status in RhD-negative women not known to be sensitised to the RhD antigen: a systematic review and economic evaluation | journal = Health Technology Assessment | volume = 22 | issue = 13 | pages = 1–172 | date = March 2018 | pmid = 29580376 | pmc = 5890172 | doi = 10.3310/hta22130 }}</ref>. 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).<ref>{{cite journal | vauthors = Carlson LM, Vora NL | title = Prenatal Diagnosis: Screening and Diagnostic Tools | journal = Obstetrics and Gynecology Clinics of North America | volume = 44 | issue = 2 | pages = 245–256 | date = June 2017 | pmid = 28499534 | pmc = 5548328 | doi = 10.1016/j.ogc.2017.02.004 }}</ref>. These invasive procedures can be conducted on both RhD+ and RhD- mothers. After the invasive procedure, medications that prevent the Rh [[Immunization|immunisation]] are usually prescribed to RhD- mothers<ref>{{cite journal | vauthors = Crowther CA, Keirse MJ | title = Anti-D administration in pregnancy for preventing rhesus alloimmunisation | journal = The Cochrane Database of Systematic Reviews | issue = 2 | pages = CD000020 | date = 2000 | pmid = 10796088 | doi = 10.1002/14651858.CD000020 }}</ref>{{Update inline|reason=Updated version https://www.ncbi.nlm.nih.gov/pubmed/23450526|date = May 2019}}. This is done to avoid the production of maternal anti-D [[Antibody|antibodies]] which may attack the foetal blood cells should the foetus be Rh incompatible with the mother.<ref name="Mechanisms of anti-D action in the">{{cite journal | vauthors = Brinc D, Lazarus AH | title = Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn | journal = Hematology. American Society of Hematology. Education Program | volume = 2009 | pages = 185–91 | date = 2009 | pmid = 20008198 | doi = 10.1182/asheducation-2009.1.185 }}</ref>.

=== Invasive extraction ===

==== Chorionic villus sampling ====

[[Chorionic villus sampling]] is usually done between the 10th and 13th week of pregnancy, it samples [[chorionic villi]], which are tiny projections of [[Placenta|placental tissue]]<ref>{{cite journal | vauthors = Alfirevic Z, Sundberg K, Brigham S | title = Amniocentesis and chorionic villus sampling for prenatal diagnosis | journal = The Cochrane Database of Systematic Reviews | issue = 3 | pages = CD003252 | date = 2003 | pmid = 12917956 | pmc = 4171981 | doi = 10.1002/14651858.CD003252 }}</ref>{{Update inline|reason=Updated version https://www.ncbi.nlm.nih.gov/pubmed/28869276|date = May 2019}}. As the placental tissues are derived from [[Embryonicembryonic cell|embryonic cells]]s, hence, it contains foetal genetic information that can be used to determine the child’schild's RhD status.<ref>{{cite journal | vauthors = Kickler TS, Blakemore K, Shirey RS, Nicol S, Callan N, Ness PM, Escallon C, Dover G | title = Chorionic villus sampling for fetal Rh typing: clinical implications | journal = American Journal of Obstetrics and Gynecology | volume = 166 | issue = 5 | pages = 1407–11 | date = May 1992 | pmid = 1375812 | doi = 10.1016/0002-9378(92)91612-E }}</ref>. There are two types of chorionic villus sampling. Trans-cervical sampling involves inserting a [[catheter]] through the [[cervix]] into the [[placenta]] to obtain villi, [[ultrasound]] is used to guide the catheter to the site of sampling.<ref name=":4" />. Trans-abdominal sampling requires the insertion of a needle through the [[abdomen]] and [[uterus]] to obtain placental tissue.<ref name=":4" />. [[Local anesthesia|Local anaesthesia]] can be applied to reduce pain from [[Minimally invasive procedure|invasive procedures]].<ref name=":4">{{Cite web|url=https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/chorionic-villus-sampling-cvs|title=Chorionic Villus Sampling (CVS)|website=Johns Hopkins Medicine Health Library|language=en|access-date=2019-04-08}}</ref>.

==== Amniocentesis ====

[[Amniocentesis]] is another invasive procedure which can be used to collect foetal DNA samples.<ref>{{Cite web|url=https://www.healthline.com/health/amniocentesis|title=Amniocentesis: Purpose, Procedure and Risks|date=2012-07-18|website=Healthline|language=en|access-date=2019-04-09}}</ref>. This procedure is usually done between the 15th week to 20th week of [[pregnancy]].<ref name=":3">{{Cite web|url=https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=92&contentid=p07762|title=Amniocentesis - Health Encyclopedia - University of Rochester Medical Center|website=www.urmc.rochester.edu|access-date=2019-04-08}}</ref>. The purpose of AMC is to extract a small amount of [[amniotic fluid]] as foetal cells may be shed from the foetus and are suspended in the amniotic fluid.<ref name=":3" />. The foetal genome can be found in these cells. Therefore, extracting amniotic fluid can the required foetal genetic material for the genotyping of the RhD gene.<ref>{{cite journal | vauthors = Cotorruelo C, Biondi C, García Borrás S, Di Mónaco R, Martino W, Racca A | title = [Rh system genotyping in amniotic fluid] | journal = Medicina | volume = 61 | issue = 1 | pages = 76–8 | date = 2001 | pmid = 11265629 }}</ref>. Before amniocentesis commences, the doctor will inject local anaesthetics to the mother's abdomen.<ref name=":3" />. The doctor will then apply ultrasound to locate the foetus in the uterus.<ref name=":3" />. Under the guidance of the [[ultrasound imaging]], a long, thin, hollow needle will insert through the skin of the abdomen to the uterus of the mother.<ref name=":3" />. The needle is used to withdraw a trace amount of amniotic fluid.<ref name=":3" />. It will then be removed from the maternal body and the amniotic fluid extracted will be 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 [[Polymerasepolymerase chain reaction|polymerase chain reactions]]s (PCR) mixture. The PCR mixture usually contains [[Taq polymerase|Taq DNA polymerase]], [[DNA primer|DNA primers]]s, [[Deoxyribonucleotide|deoxyribonucleotidesdeoxyribonucleotide]]s (dNTP) and [[buffer solution]].<ref name=":5">{{cite journal | vauthors = Lorenz TC | title = Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies | journal = Journal of Visualized Experiments | issue = 63 | pages = e3998 | date = May 2012 | pmid = 22664923 | pmc = 4846334 | doi = 10.3791/3998 }}</ref>. The DNA primers are specific for [[exon]] 7 and exon 10.<ref>{{cite journal | vauthors = Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Kulovany E, Vlk R | title = Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma | journal = Fetal Diagnosis and Therapy | volume = 20 | issue = 4 | pages = 275–80 | date = Jul 2005 | pmid = 15980640 | doi = 10.1159/000085085 }}</ref>. Under different circumstances, primers for other regions of the RhD gene, such as [[intron]] 4 and exon 5, may also be used.<ref>{{cite journal | vauthors name= Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P | title = "Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma | journal = Transfusion Medicine and Hemotherapy | volume = 40 | issue = 1 | pages = 37–43 | date = February 2013 | pmid = 23637648 | pmc = 3636019 | doi = 10.1159"/000345682 }}</ref>. The mixture will be subjected to a series of PCR which is performed by a [[thermal cycler]].<ref name=":5" />. By the end of the PCR, the amount of RhD gene will be amplified if it is present. The product of the PCR will be analysed by [[gel electrophoresis]]. Before gel electrophoresis, [[Molecular-weight size marker|DNA reference ladder]], [[Positive control group|positive control]] containing DNA with RhD gene and the PCR product will be loaded onto the wells of the gel.<ref name=":5" />. An [[Electric current|electrical 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.<ref name=":6">{{cite journal | vauthors = Lee PY, Costumbrado J, Hsu CY, Kim YH | title = Agarose gel electrophoresis for the separation of DNA fragments | journal = Journal of Visualized Experiments | issue = 62 | date = April 2012 | pmid = 22546956 | pmc = 4846332 | doi = 10.3791/3923 }}</ref>. Utilising this property, DNA fragments with different molecular masses can be segregated. With the help of gel staining and visualising devices such as [[Transillumination|UV trans-illuminators]], RhD gene DNA fragments, if present, will be visible as a band with its corresponding molecular mass.<ref name=":6" />. Further DNA sequencing can be conducted to confirm that the sequence of product DNA fragments matches that of the RhD gene sequence.

== Clinical Applications ==

{{Citation needed span|text=Rh factor testing is crucial to the prevention of haemolytic conditions caused by the Rh incompatibility.|date=April 2019|reason=Needs reference to sources.}} The consequence of having haemolytic conditions can be dangerous or even lethal as it may lead to multiple complications.<ref>{{cite journal | vauthors = Nadgeriev MK, Amelina OP | title = [Complications in the transfusion of RH-incompatible blood] | journal = Sovetskaia Meditsina | volume = 29 | issue = 1 | pages = 95–7 | date = January 1966 | pmid = 4964445 }}</ref>. 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 [[Immunoglobulin G|IgG]] [[Rho(D) immune globulin|anti-RhD]] [[Antibody|antibodies]] will be developed in the RhD- individual’sindividual's body.<ref name=":7">{{Cite book|url=https://www.ncbi.nlm.nih.gov/books/NBK2269/|title=The Rh blood group|last=Dean|first=Laura|date=2005|publisher=National Center for Biotechnology Information (US)|language=en}}</ref>. The antibodies then attack red blood cells with attached RhD [[Antigen|antigensantigen]]s and lead to the destruction of these cells. This condition is known as a [[Hemolytic reaction|haemolytic reaction]].<ref name=":8">{{cite journal | vauthors = Strobel E | title = Hemolytic Transfusion Reactions | journal = Transfusion Medicine and Hemotherapy | volume = 35 | issue = 5 | pages = 346–353 | date = 2008 | pmid = 21512623 | pmc = 3076326 | doi = 10.1159/000154811 }}</ref> The destruction of red blood cells releases [[Hemoglobin|haemoglobin]] to the bloodstream. Haemoglobin may be excreted through [[urine]], causing [[Hemoglobinuria|haemoglobinuria]].<ref name=":8" />. The sudden release of haemoglobin will also pass through the liver and be metabolised into [[bilirubin]], which in high concentrations, accumulates under the skin to cause [[jaundice]].<ref name=":8" />. Liberation of blood cell debris into the circulation will also cause [[disseminated intravascular coagulation]].<ref>{{cite book |last=Costello|first=Ryan A. |last2=Nehring|first2=Sara M. | name-list-format = vanc |chapter =Disseminated Intravascular Coagulation (DIC)|date=2019 |url= http://www.ncbi.nlm.nih.gov/books/NBK441834/ |title=StatPearls |publisher=StatPearls Publishing |pmid=28722864|access-date=2019-04-09}}</ref>.

==== 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 colour and the patients may experience dizziness and confusion.<ref name=":9">{{Cite web|url=https://www.hopkinsmedicine.org/health/conditions-and-diseases/hemolytic-anemia|title=Hemolytic Anemia|website=Johns Hopkins Medicine Health Library|language=en|access-date=2019-04-09}}</ref>. [[Tachycardia]], the increase in heart rate, is also a symptom of the haemolytic disease.<ref name=":9" />.

=== 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, sensitising the mother to produce anti-RhD antibodies.<ref name=":7" />. 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 foetus as delivery occurs before the synthesis of antibodies by the mother.<ref name=":7" />. 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 foetus, causing the aforementioned haemolytic reaction in the foetus known as [[Hemolytic disease of the newborn|haemolytic disease of the newborn]].<ref name=":7" />. This disease is usually fatal for the foetus 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.<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|lungslung]]s or [[scalp]]. These are typically signs of [[hydrops fetalis]].<ref name=":10" />.

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" />.

=== 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 sensitised RhD- mother (previously conceived an RhD+ child) and the foetus being Rh+, medication such as [[Rho(D) immune globulin|anti-D immunoglobulin]] will be given to the RhD- mother.<ref>{{cite journal | vauthors = Kumpel BM | title = Monoclonal anti-D for prophylaxis of RhD haemolytic disease of the newborn | journal = Transfusion Clinique et Biologique | volume = 4 | issue = 4 | pages = 351–6 | date = July 1997 | pmid = 9269715 | doi = 10.1016/S1246-7820(97)80040-7 }}</ref>. Injecting RhD- mother with anti-D immunoglobulin has been proven effective in avoiding the sensitisation of RhD+ antigen, even though the mechanism of how this medication works remains obscure.<ref>{{cite journal | vauthors name= Brinc D, Lazarus AH | title = "Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn | journal = Hematology. American Society of Hematology. Education Program | volume = 2009 | pages = 185–91 | date = 2009 | pmid = 20008198 | doi = 10.1182"/asheducation-2009.1.185 }}</ref>. Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.<ref>{{cite journal | vauthors = Asfour M, Narvios A, Lichtiger B | title = Transfusion of RhD-incompatible blood components in RhD-negative blood marrow transplant recipients | journal = MedGenMed | volume = 6 | issue = 3 | pages = 22 | date = July 2004 | pmid = 15520646 | pmc = 1435608 }}</ref>.

== References ==