Rh factor testing: Difference between revisions

Revision as of 03:34, 5 February 2024

Revision as of 00:14, 7 April 2024

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== Background ==

Rhesus factor testing utilises theutilizes [[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's [[genome]], the presence of rhesusRhesus D (RhD) [[antigenAntigen|antigens]]s can be inferred. Individuals with a positive RhD status hashave 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 a 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 conductedperformed on pregnant women to determine the RhD blood group of the mother and the foetusfetus. By confirming the RhD status of both mother and foetusfetus, precautions can be made, if necessary, to prevent any medical conditionscomplications caused by rhesusRhesus 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 rhesusRhesus factor which referscorrelated to the RhD antigen specifically is assayed.<ref name=":1" /> 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 nameweb |title="Chromosome 1:2" 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.<ref name=":2" /> 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]] is 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 foetalfetal DNA.<ref name="ReliableIn Determinationearly ofpregnancy, Fetalaround RhD">{{cite3% journalof |the vauthorsmother’s = Dovčfree-Drnovšekcell T,DNA Klemencis P,from Toplakthe Nfetus, Blejecand T,raises Briclto I,6-7% Rožmanby Plate | titlepregnancy.<ref name=":1">{{Cite Reliable Determination of Fetal RhD Status by RHD Genotyping from Maternal Plasmaweb | journal last= Transfusion Medicine and Hemotherapynonacus-developer | volume date= 402021-06-11 | issue title=Non-Invasive 1Fetal |RhesusD pagesBlood = 37–43Genotyping | date url= February 2013https://nonacus.com/blog-non-invasive-fetal-rhesusd-blood-genotyping/ | pmid access-date= 236376482024-04-07 | pmc website= 3636019nonacus | doi language= 10.1159/000345682 en-US}}</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 multiplesmultiple layers.<ref>{{cite journal | vauthors = Dagur PK, McCoy JP | title = Collection, Storage, and Preparation of Human Blood Cellsplasma |can journalthen =be Currentisolated Protocolsfrom inthe Cytometryother | volume = 73 | pages = 5components.1.1–16 |It datecan =be Julygenotyped 2015using |real pmidtime =PCR 26132177to |determine pmcthe =RhD 4524540status |of doi =the 10fetus.1002/0471142956.cy0501s73 | isbn<ref name=":1" 9780471142959 }}</ref> Blood plasma can then be isolated from the other components for rhesus factor testing. The method of extracting foetalfetal 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>

=== Invasive extraction ===

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== 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]]s (PCR) mixture. The PCR mixture usually contains [[Taq polymerase|Taq DNA polymerase]], [[DNA primer]]s, [[deoxyribonucleotide]]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 | s2cid = 25607502 }}</ref> Under different circumstances, primers for other regions of the RhD gene, such as [[intron]] 4 and exon 5, may also be used.<ref name="Reliable Determination of Fetal RhD">{{cite journal |vauthors=Dovč-Drnovšek T, Klemenc P, Toplak N, Blejec T, Bricl I, Rožman P |date=February 2013 |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 |doi=10.1159/000345682 |pmc=3636019 |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 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]] 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 ==

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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 a sensitisedsensitized RhD- mother (previously conceived an RhD+ child) and the foetusfetus being Rh+, medication such as an [[Rho(D) immune globulin|anti-D immunoglobulin]], called RhoGAM, 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 immunoglobulinRhoGAM has been proven effective in avoiding the sensitisation of RhD+ antigen, even though the mechanism of how this medication works remains obscure.<ref name="Mechanisms of anti-D action in the"/> 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>

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.<ref>{{Cite web |title=Rh incompatibility Information {{!}} Mount Sinai - New York |url=https://www.mountsinai.org/health-library/diseases-conditions/rh-incompatibility |access-date=2024-04-07 |website=Mount Sinai Health System |language=en-US}}</ref>   Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.

== References ==