Rh factor testing: Difference between revisions

Revision as of 16:00, 8 April 2019

Revision as of 14:42, 9 April 2019

Line 6:

== 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 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 fetusfoetus, 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]]<ref name=":2" />. 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 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=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 [[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=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 [[Immunization|immunisation]] are usually prescribed to RhD- mothers. 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.

=== 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=PMCPMC4171981|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. Trans-abdominal sampling requires the insertion of a needle through the [[abdomen]] and [[uterus]] to obtain placental tissue. [[Local anesthesia|Local anaesthesia]] can be applied to reduce pain from [[Invasive procedure|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<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. 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 anestheticsanaesthetics 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 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]]. 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, [[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. 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. UtilizingUtilising this property, DNA fragments with different molecular masses can be segregated. With the help of gel staining and visualizingvisualising devices such as [[Transillumination|UV transilluminatorstrans-illuminators]], 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 ==

Rh factor testing is crucial to the prevention of haemolytic conditions caused by the Rh incompatibility. The consequence of having haemolytic conditions can be dangerous or even lethal as it may lead to multiple complications. 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 donationtransfusion ===

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. The antibodies then attacks red blood cells with attached RhD [[Antigen|antigens]] and lead to the destruction of these cells. This condition is known as [[Hemolytic reaction|haemolytic reaction]]. The destruction of red blood cells releases [[Hemoglobin|haemoglobin]] to the bloodstream. Haemoglobin may be excreted through [[urine]], causing [[Hemoglobinuria|haemoglobinuria]]. The sudden release of haemoglobin will also pass through the liver and be metabolizedmetabolised into [[bilirubin]], which in high concentrations, accumulates under the skin to cause [[jaundice]]. Liberation of blood cell debris into the circulation will also cause [[disseminated intravascular coagulation]] and [[Circulatory Shock|circulatory shock]].

==== Symptoms of Rh group incompatibility in blood donation ====

Patients receiving incompatible [[blood transfusion]] may have pale skin, [[splenomegaly]], [[hepatomegaly]] and the yellowing of mouth and eyes. In addition, their urine may appear in dark colour and the patients may experience dizziness and confusion. [[Tachycardia]], the increase in heart rate, is also a symptom of the hemolytichaemolytic disease.

=== Prevention of haemolytic disease of the newborn ===

In the case of pregnancy, when an RhD- mother carries an RhD+ fetus, some of the fetal red blood cells may cross the placenta into the maternal circulation, sensitizingsensitising the mother to produce anti-RhD antibodies. Since the mixing of fetal and maternal blood occurs mainly during separation of the placenta during delivery, the first RhD+ pregnancy rarely causes any danger to the fetusfoetus as delivery occurs before the synthesis of antibodies by the mother. However, if the mother were to conceive another RhD+ child in the future, the anti-RhD antibodies will cross the placenta to attack and haemolyselyse the red cells of the fetusfoetus, causing the aforementioned haemolytic reaction in the fetusfoetus known as [[Hemolytic disease of the newborn|haemolytic disease of the newborn]]. This disease is usually fatal for the fetusfoetus and hence preventive measures are conducted.

==== Symptoms of haemolytic disease of the newborn ====

Symptoms of the disease may vary in each pregnancy. They are usually not noticeable during pregnancy. However, prenatal tests may reveal yellow colouring of amniotic fluid, which is caused by the buildup of bilirubin. Splenomegaly, [[cardiomegaly]] and hepatomegaly may occur in the baby. Excessive [[tissue fluid]] may accumulate in the [[stomach]], [[Lung|lungs]] or [[scalp]]. These are typically signs of [[hydrops fetalis]].

After birth, the symptoms of the child are similar to that of incompatible blood transfusion in adults.  The baby may have pale skin due to anemia[[Anemia|anaemia]]. The yellowing of the [[umbilical cord]], skin and eyes, also known as jaundice, may arise within 24 to 36 hours of birth. Signs of hydrops fetalis such as the enlargement of [[spleen]], [[heart]] and [[liver]], along with severe [[edema]], will continue after birth.

=== 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 the a sensitizedsensitised RhD- mother (previously conceived an RhD+ child) and the fetusfoetus being Rh+, medication such as [[Rho(D) immune globulin|anti-D immunoglobulin]] will be given to the RhD- mother. Injecting RhD- mother with anti-D immunoglobulin has been proven effective in avoiding the sensitizationsensitisation of RhD+ antigen, even though the mechanism of how this medication works remains obscure. Anti-D immunoglobulin injection is also offered to RhD- individuals who have been mistakenly transfused with RhD+ blood.

== References ==