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== Introduction ==
Rhesus factor testing utilises the [[genotyping]] technique to detect the presence of the [[RHD (gene)|RhD gene]]<ref name=":1">{{Cite journal|last=Flegel|first=Willy A.|date=2007-4|title=The genetics of the Rhesus blood group system|url=https://www.ncbi.nlm.nih.gov/pubmed/19204754|journal=Blood Transfusion = Trasfusione Del Sangue|volume=5|issue=2|pages=50–57|doi=10.2450/2007.0011-07|issn=1723-2007|pmc=PMCPMC2535884|pmid=19204754}}</ref>. By checking the existence of the RhD gene in the individual’s [[genome]], the presence of rhesus D (RhD) [[Antigen|antigens]] can be inferred. Individuals with a positive RhD status has RhD antigens expressed on the [[cell membrane]] of their [[Red blood cell|red blood cells]], 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" />
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== Extraction of test samples ==
[[Blood plasma]]
=== Non-invasive extraction ===
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==== 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|last=Cotorruelo|first=C.|last2=Biondi|first2=C.|last3=García Borrás|first3=S.|last4=Di Mónaco|first4=R.|last5=Martino|first5=W.|last6=Racca|first6=A.|date=2001|title=[Rh system genotyping in amniotic fluid]|url=https://www.ncbi.nlm.nih.gov/pubmed/11265629|journal=Medicina|volume=61|issue=1|pages=76–78|issn=0025-7680|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’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=PMCPMC4846334|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=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}}</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=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=PMCPMC3636019|pmid=23637648}}</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=PMCPMC4846332|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 ==
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=== 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’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
==== Symptoms of Rh group incompatibility in blood donation ====
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=== 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
== References ==
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