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=== 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|
==== 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|
==== Amniocentesis ====
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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"/> 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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=== 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]]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 |
==== Symptoms of Rh group incompatibility in blood donation ====
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