Fetal Genotyping from Amniotic Fluid or Chorionic Villi
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
Fetal Genotyping from Maternal Blood
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).
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
This non-invasive test from maternal plasma
uses quantitative PCR to amplify male specific DNA sequences found on the Y
Rhc and RhC Typing (amniotic fluid, chorionic villi or DNA from multi-transfused
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
Standard genotype (RhD,
C, c, E, e, K/k, Fya/b, Jka/b,
M/N, S/s, U-, Uvar)
(haemoglobinopathy test array including common RhD, C and e variants, V,
VS, hrB, hrS
, or standard genotype plus FyX,
LWa/b, Sc as required)
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.
HOW TO CONTACT US
By email: Molecular.Diagnostics@nhsbt.nhs.uk
By phone: +44 (0)117
+44 (0)117 912 5782
NHS Blood and Transplant
Who We Are
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
also provided for the most clinically important blood groups of patients
who have been multi-transfused; but not serologically typed prior to
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