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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|title=Blood test for Rh status and antibody screen|last=|first=|date=2019-03-07|website=BabyCenter|archive-url=|archive-date=|dead-url=|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>.
== IntroductionBackground ==
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=April 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=PMCPMC2535884PMC2535884|pmid=19204754|via=}}</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" />
== 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>{{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=PMCPMC3636019PMC3636019|pmid=23637648|via=}}</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|last=Dagur|first=Pradeep K.|last2=McCoy|first2=J. Philip|date=2015-07-01|title=Collection, Storage, and Preparation of Human Blood Cells|url=https://www.ncbi.nlm.nih.gov/pubmed/26132177|journal=Current Protocols in Cytometry|volume=73|pages=5.1.1–16|doi=10.1002/0471142956.cy0501s73|issn=1934-9300|pmc=PMCPMC4524540PMC4524540|pmid=26132177|via=}}</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|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=03 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=PMCPMC5890172PMC5890172|pmid=29580376}}</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=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=PMCPMC5548328PMC5548328|pmid=28499534}}</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=PMCPMC4171981PMC4171981|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=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}}</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=PMCPMC4846334PMC4846334|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=PMCPMC3636019PMC3636019|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=PMCPMC4846332PMC4846332|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=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}}</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’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|antigens]] and lead to the destruction of these cells. This condition is known as a [[Hemolytic reaction|haemolytic reaction]].<ref name=":8">{{Cite journal|last=Strobel|first=Erwin|date=2008|title=Hemolytic Transfusion Reactions|url=https://www.ncbi.nlm.nih.gov/pubmed/21512623|journal=Transfusion Medicine and Hemotherapy: Offizielles Organ Der Deutschen Gesellschaft Fur Transfusionsmedizin Und Immunhamatologie|volume=35|issue=5|pages=346–353|doi=10.1159/000154811|issn=1660-3796|pmc=PMCPMC3076326PMC3076326|pmid=21512623}}</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>{{Citation|last=Costello|first=Ryan A.|title=Disseminated Intravascular Coagulation (DIC)|date=2019|url=http://www.ncbi.nlm.nih.gov/books/NBK441834/|work=StatPearls|publisher=StatPearls Publishing|pmid=28722864|access-date=2019-04-09|last2=Nehring|first2=Sara M.}}</ref>.
==== Symptoms of Rh group incompatibility in blood donation ====
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