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=== Non-invasive extraction ===
Non-invasive [[prenatal testing]] can be used if the mother is RhD-<ref>{{Cite journal|last=Saramago|first=Pedro|last2=Yang|first2=Huiqin|last3=Llewellyn|first3=Alexis|last4=Walker|first4=Ruth|last5=Harden|first5=Melissa|last6=Palmer|first6=Stephen|last7=Griffin|first7=Susan|last8=Simmonds|first8=Mark|date=03Mar 2018|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|url=https://www.ncbi.nlm.nih.gov/pubmed/29580376|journal=Health Technology Assessment (Winchester, England)|volume=22|issue=13|pages=1–172|doi=10.3310/hta22130|issn=2046-4924|pmc=PMC5890172|pmid=29580376|via=}}</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|last=Carlson|first=Laura M.|last2=Vora|first2=Neeta L.|date=June 2017-6|title=Prenatal Diagnosis: Screening and Diagnostic Tools|url=https://www.ncbi.nlm.nih.gov/pubmed/28499534|journal=Obstetrics and Gynecology Clinics of North America|volume=44|issue=2|pages=245–256|doi=10.1016/j.ogc.2017.02.004|issn=1558-0474|pmc=PMC5548328|pmid=28499534|via=}}</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|last=Crowther|first=C. A.|last2=Keirse|first2=M. J.|date=2000|title=Anti-D administration in pregnancy for preventing rhesus alloimmunisation|url=https://www.ncbi.nlm.nih.gov/pubmed/10796088|journal=The Cochrane Database of Systematic Reviews|issue=2|pages=CD000020|doi=10.1002/14651858.CD000020|issn=1469-493X|pmid=10796088}}</ref>. 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>{{Cite journal|last=Brinc|first=Davor|last2=Lazarus|first2=Alan H.|date=2009|title=Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn|url=https://www.ncbi.nlm.nih.gov/pubmed/20008198|journal=Hematology. American Society of Hematology. Education Program|pages=185–191|doi=10.1182/asheducation-2009.1.185|issn=1520-4383|pmid=20008198}}</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|last=Alfirevic|first=Z.|last2=Sundberg|first2=K.|last3=Brigham|first3=S.|date=2003|title=Amniocentesis and chorionic villus sampling for prenatal diagnosis|url=https://www.ncbi.nlm.nih.gov/pubmed/12917956|journal=The Cochrane Database of Systematic Reviews|issue=3|pages=CD003252|doi=10.1002/14651858.CD003252|issn=1469-493X|pmc=PMC4171981|pmid=12917956}}</ref>. As the placental tissues are derived from [[Embryonic cell|embryonic cells]], hence, it contains foetal genetic information that can be used to determine the child’s RhD status<ref>{{Cite journal|last=Kickler|first=T. S.|last2=Blakemore|first2=K.|last3=Shirey|first3=R. S.|last4=Nicol|first4=S.|last5=Callan|first5=N.|last6=Ness|first6=P. M.|last7=Escallon|first7=C.|last8=Dover|first8=G.|date=May 1992-5|title=Chorionic villus sampling for fetal Rh typing: clinical implications|url=https://www.ncbi.nlm.nih.gov/pubmed/1375812|journal=American Journal of Obstetrics and Gynecology|volume=166|issue=5|pages=1407–1411|issn=0002-9378|pmid=1375812|via=}}</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 [[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 ====
== 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 reaction|polymerase chain reactions]] (PCR) mixture. The PCR mixture usually contains [[Taq polymerase|Taq DNA polymerase]], [[DNA primer|DNA primers]], [[Deoxyribonucleotide|deoxyribonucleotides]] (dNTP) and [[buffer solution]]<ref name=":5">{{Cite journal|last=Lorenz|first=Todd C.|date=2012-05-22|title=Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies|url=https://www.ncbi.nlm.nih.gov/pubmed/22664923|journal=Journal of Visualized Experiments: JoVE|issue=63|pages=e3998|doi=10.3791/3998|issn=1940-087X|pmc=PMC4846334|pmid=22664923}}</ref>. The DNA primers are specific for [[exon]] 7 and exon 10<ref>{{Cite journal|last=Hromadnikova|first=Ilona|last2=Vechetova|first2=Lenka|last3=Vesela|first3=Klara|last4=Benesova|first4=Blanka|last5=Doucha|first5=Jindrich|last6=Kulovany|first6=Eduard|last7=Vlk|first7=Radovan|date=Jul 2005-7|title=Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma|url=https://www.ncbi.nlm.nih.gov/pubmed/15980640|journal=Fetal Diagnosis and Therapy|volume=20|issue=4|pages=275–280|doi=10.1159/000085085|issn=1015-3837|pmid=15980640|via=}}</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|last=Dovč-Drnovšek|first=Tadeja|last2=Klemenc|first2=Polona|last3=Toplak|first3=Nataša|last4=Blejec|first4=Tanja|last5=Bricl|first5=Irena|last6=Rožman|first6=Primož|date=Feb 2013-2|title=Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasma|url=https://www.ncbi.nlm.nih.gov/pubmed/23637648|journal=Transfusion Medicine and Hemotherapy: Offizielles Organ Der Deutschen Gesellschaft Fur Transfusionsmedizin Und Immunhamatologie|volume=40|issue=1|pages=37–43|doi=10.1159/000345682|issn=1660-3796|pmc=PMC3636019|pmid=23637648|via=}}</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]] 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|last=Lee|first=Pei Yun|last2=Costumbrado|first2=John|last3=Hsu|first3=Chih-Yuan|last4=Kim|first4=Yong Hoon|date=2012-04-20|title=Agarose gel electrophoresis for the separation of DNA fragments|url=https://www.ncbi.nlm.nih.gov/pubmed/22546956|journal=Journal of Visualized Experiments: JoVE|issue=62|doi=10.3791/3923|issn=1940-087X|pmc=PMC4846332|pmid=22546956}}</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|last=Nadgeriev|first=M. K.|last2=Amelina|first2=O. P.|date=Jan 1966-1|title=[Complications in the transfusion of RH-incompatible blood]|url=https://www.ncbi.nlm.nih.gov/pubmed/4964445|journal=Sovetskaia Meditsina|volume=29|issue=1|pages=95–97|issn=0038-5077|pmid=4964445|via=}}</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 ===
=== 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|last=Kumpel|first=B. M.|date=Jul 1997-7|title=Monoclonal anti-D for prophylaxis of RhD haemolytic disease of the newborn|url=https://www.ncbi.nlm.nih.gov/pubmed/9269715|journal=Transfusion Clinique Et Biologique: Journal De La Societe Francaise De Transfusion Sanguine|volume=4|issue=4|pages=351–356|issn=1246-7820|pmid=9269715|via=}}</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|last=Brinc|first=Davor|last2=Lazarus|first2=Alan H.|date=2009|title=Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn|url=https://www.ncbi.nlm.nih.gov/pubmed/20008198|journal=Hematology. American Society of Hematology. Education Program|pages=185–191|doi=10.1182/asheducation-2009.1.185|issn=1520-4383|pmid=20008198}}</ref>. Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood<ref>{{Cite journal|last=Asfour|first=M.|last2=Narvios|first2=Aida|last3=Lichtiger|first3=Benjamin|date=2004-07-13|title=Transfusion of RhD-Incompatible Blood Components in RhD-Negative Blood Marrow Transplant Recipients|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1435608/|journal=Medscape General Medicine|volume=6|issue=3|pages=|issn=1531-0132|pmc=PMCPMC1435608PMC1435608|pmid=15520646|via=}}</ref>.
== References ==
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