Rh factor testing: Difference between revisions

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[[/Rh Factor Testing]]

'''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 Mar 7-03-07|website=BabyCenter|archive-url=|archive-date=|dead-url=|access-date=2019-03-07}}</ref><ref Marname=":0">{{Cite 7web|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>.

== Introduction ==

Rhesus factor testing utilizesutilises the genotyping technique to detect the presence of the 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 RhD gene in the individual’s genome, the presence of rhesus D (RhD) antigens can be inferred. Individuals with a positive RhD status has RhD antigens expressed on the cell membrane of their 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 fetusfoetus. By confirming the RhD status of both mother and fetus, precautions can be made if necessary to prevent any medical conditions caused by rhesus 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 rhesus factor which refers to the RhD antigen specifically is assayed. The RhD gene that codes for the RhD antigen is located on chromosome 1. RhD is a dominant gene, meaning that as long as at least one RhD gene is inherited from either parent, the RhD antigen is expressed<ref name=":2" />. Vice versa, if no RhD gene is inherited, no RhD antigen is produced.

== Extraction of test samples ==

Blood plasma are 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 fetalfoetal 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=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>. Blood samples can be obtained through venipuncture of the mother. Since plasma and other components of blood has different densities, centrifugation of blood samples with added anticoagulant (such as 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=PMCPMC4524540|pmid=26132177}}</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.

=== 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=PMCPMC5890172|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). These invasive procedures can be conducted on both RhD+ and RhD- mothers. After the invasive procedure, medications that prevent the Rh immunizationimmunisation are usually prescribed to RhD- mothers. This is done to avoid the production of maternal anti-D antibodies which may attack the foetal blood cells should the foetus be Rh incompatible with the mother.

=== Invasive extraction ===

==== Chorionic villus sampling ====

Chorionic villus sampling is usually done between the 10th and 12th13th week of pregnancy, it samples chorionic villi, which are tiny projections of 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=PMCPMC4171981|pmid=12917956}}</ref>. As the placental tissues are derived from embryonic cells, hence, it contains fetalfoetal 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. TranscervicalTrans-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. TransabdominalTrans-abdominal sampling requires the insertion of a needle through the abdomen and uterus to obtain placental tissue. Local anesthesiaanaesthesia can be applied to reduce pain from invasive procedures<ref>{{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 ====

Amniocentesis is another invasive procedure which can be used to collect foetal DNA samples. 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. The foetal genome can be found in these cells. Therefore, extracting amniotic fluid can the required foetal genetic material for the genotyping of RhD gene. Before amniocentesis commences, doctor will inject local anesthetics to the mother's abdomen. The doctor will then apply ultrasound to locate the foetus in the uterus. 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. The needle is used to withdraw trace amount of amniotic fluid. It will then be removed from the maternal body and the amniotic fluid extracted will be sent to laboratory for further testing<ref name=":3" />.

== Genotyping of RhD gene ==

The presence of 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 reactions (PCR) mixture. The PCR mixture usually contains Taq DNA polymerase, DNA primers, deoxyribonucleotides (dNTP) and buffer solution. 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. 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, DNA reference ladder, positive control containing DNA with RhD gene and the PCR product will be loaded onto the wells of the gel. An 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. Utilizing this property, DNA fragments with different molecular masses can be segregated. With the help of gel staining and visualizing devices such as UV transilluminators, RhD gene DNA fragments, if present, will be visible as a band with its corresponding molecular mass. Further DNA sequencing can be conducted to confirm that the sequence of product DNA fragments matches that of the RhD gene sequence.

== Clinical Applications ==