IBGRL Molecular Diagnostics



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  Molecular Diagnostics

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, RhC 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, RhE and RhC and the 20th week for K (Kell).

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.
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). 
IBGRL is able to type donors and patients for almost all antigens by allelic discrimination PCR assays or by direct sequencing. Two levels of genotyping are available:

  • Standard genotype (RhD, C, c, E, e, K/k, Fya/b, Jka/b, M/N, S/s, U-, Uvar)

  • Extended genotype (haemoglobinopathy test array including common RhD, C and e variants, V, VS, hrB, hrS , or standard genotype plus FyX, Kpa/b, Jsa/b, Lua/b, Dia/b, Coa/b, Doa/b, LWa/b, Sc as required)





Referring Investigations

Click here for further details on how to refer samples for investigation

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

By email:        Molecular.Diagnostics@nhsbt.nhs.uk
By phone:      +44 (0)117 921 7572
By FAX:         +44 (0)117 912 5782
By Post:        

Molecular Diagnostics IBGRL,
NHS Blood and Transplant

North Bristol Park



BS34 7QH

Who We Are

The Molecular Diagnostics Department of IBGRL undertakes genetic investigation to identify women with antigen-positive fetuses who are at risk of haemolytic disease of the fetus and newborn (HDFN). Upon identification, expecting mothers can then be informed and prepared for further careful monitoring during their pregnancy. 
The Molecular Diagnostics Department also identifies pregnant women who have antigen-negative fetuses and who therefore are not at danger from HDFN.

Genotyping is also provided for the most clinically important blood groups of patients who have been multi-transfused; but not serologically typed prior to transfusion.

The laboratory conducts a fetal sex genotyping service for 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.