Blood Group Genotyping Feb 2006

BLOOD GROUP & FETAL SEX GENOTYPING SERVICE

(Jump to Technical Aspects / Referring Samples)
Service Overview

Advances in molecular genetics enable the IBGRL to provide a service to predict the blood group status of fetuses at risk from haemolytic disease of the fetus and newborn (HDFN). This service builds on IBGRL's expertise in the molecular biology of human blood groups which dates back to the early 1980s, and we now aim to use this knowledge to provide a rapid, convenient and reliable service. The main purpose of the service is to identify women with antigen-positive fetuses who can then be informed and prepared for further careful monitoring during their pregnancy. It will also identify the pregnant women who have antigen-negative fetuses and who therefore are not at danger from HDFN. If these women are identified, further invasive procedures can be avoided. Two methods for determining the blood group of a fetus can be used. The non-invasive method for RhD analyses the small amount of fetal DNA that is present in the plasma of pregnant women. The second method, for RhD and also other antigens, analyses fetal DNA from a sample of amniotic fluid or chorionic villi obtained by amniocentesis or biopsy. Amniocentesis and placental biopsy are invasive procedures which carry a small but significant risk of miscarriage or trans-placental haemorrhage; for this reason the new non-invasive method is generally preferred.

We are also able to provide genotyping for most clinically important blood groups of patients who have been multi-transfused; these are patients who have not been serologically typed prior to transfusion. Genotyping provides a means of correctly typing these patients before improving the opportunities for providing matched red cells.

We provide a fetal sex genotyping service for those pregnancies affected by X-linked genetic conditions or when early treatment of the fetus differs according to fetal gender. This test can be performed earlier in pregnancy than blood group genotyping and samples can be accepted from 7 weeks gestation.

Charges

Fetal genotyping from maternal blood: £212.

Fetal sex typing from maternal blood: £212

Patient testing: £82

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Technical Aspects

Fetal Genotyping from Amniotic Fluid or Chorionic Villi

This method of fetal genotyping has been in use at IBGRL for a decade. Amniotic fluid and chorionic villus biopsy samples contain fetal cells which can be used as a source of fetal DNA for genotyping. This technology can be applied to most blood group polymorphisms.

Fetal Genotyping from Maternal Blood

This service was introduced in May 2001. Cell-free fetal DNA may be found in maternal plasma throughout pregnancy (Lo et al, Am. J. Hum. Genet. 62:768-775, 1998). This DNA can be obtained by maternal venepuncture without risk to the fetus. Fetal DNA in maternal plasma may be used to identify the blood group of the fetus, even in the presence of maternal DNA. At present the technique is validated for RhD, Rhc, RhE and K (Kell) typing. The amount of fetal DNA in maternal plasma is known to increase during pregnancy, therefore at early stages it may be too low to detect and we advise that the test be used only for pregnancies which have progressed beyond the 16th week for RhD, Rhc and RhE and the 20^th week for K.

RhD Typing

There are now known to be several genetic causes for the RhD-negative phenotype of an individual. These causes vary according to ethnic origin. In Caucasian RhD-negative individuals the RHD gene is nearly always absent, however the majority of RhD-negative black Africans have an intact but non-functional RHD gene, termed the RHD-pseudogene. This pseudogene contains a 37 base pair insert in exon 4 and a nonsense mutation in exon 6, which introduce stop codons preventing translation. Singleton et al. Blood, 95:12-18 (2000)

The non-invasive test from maternal plasma uses quantitative polymerase chain reaction (PCR) to amplify exon 10 of the RHD gene. This is reliable in all persons unless they have the RHD pseudogene (black Africans) or other rare Rh variants, so additional quantitative PCR tests are used to amplify exons 4 and 5 of only the active RHD gene and not the RHD-pseudogene. Quantitative PCR is also carried out to detect the CCR5 gene to confirm the presence of DNA in the sample, although this does not distinguish between maternal and fetal DNA. Another quantitative PCR detects the presence of the SRY gene on the Y chromosome, providing an internal control for fetal DNA, but only when the fetus is male. When the fetus appears to be RhD-negative and female, there remains a small possibility that no fetal DNA was isolated from the maternal plasma. In such a circumstance the test could give a false negative result, i.e. no RHD gene would be detected (because the mother is RhD-negative) even if the fetus was RhD-positive.

RhD typing from amniotic fluid, chorionic villi or DNA from multi-transfused patients uses a multiplex PCR to amplify intron 4 and exon 7 of the RHD gene because these regions show differences from the related RHCE gene. Thus, upon electrophoresis, the presence of PCR products representing intron 4 and exon 7 indicates the presence of the RHD gene and hence, a RhD-positive fetus. The PCR also includes primers to detect the RHD-pseudogene.

Rhc, RhC, RhE and Kell Typing (Maternal Blood)

These non-invasive tests from maternal plasma use quantitative PCR to amplify the appropriate exons in the RHCE gene for typing Rhc and RhE. RhC is genotyped by targeting a sequence in intron 2 of the RHCE gene that is always associated with RhC.

K genotyping is performed by detection of a single nucleotide polymorphism in exon 6 of the KEL gene.

Fetal Sex Typing (Maternal Blood)

This non-invasive test from maternal plasma uses quantitative PCR to amplify male specific DNA sequences found on the Y chromosome.

Rhc and RhC Typing (amniotic fluid, chorionic villi or DNA from multi-transfused patients)

Rhc typing is based on the use of an allele-specific primer to amplify the c-specific sequence of the RHCE gene. RhC typing detects a sequence present only in the C allele of RHCE.

Other Red Cell Antigens (amniotic fluid, chorionic villi or DNA from multi-transfused patients).

The molecular genetic basis of almost all blood group antigens has now been established. Armed with this information, IBGRL is able to type fetuses, donors, or patients for almost all antigens by allelic discrimination PCR assays or by direct sequencing. The following blood groups are routinely typed in the laboratory:

RhEe, RhCc, Fy, Jk, K/k, MN and Ss

Referring Samples Click the link below for further details on how to refer samples for investigation (pdf files)

User Guide and Sample Referral Forms

IMPORTANT: Hospitals and reference centres wishing to refer for the first time must contact the Blood Group Genotyping laboratory in the first instance to discuss the appropriateness of the proposed investigation. Samples referred without the consent of the Blood Group Genotyping laboratory may not be investigated.