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Maternal–Fetal Medicine
singletons, and day 38 in all women studied. Since the fetal circulation is time interval between obtaining the specimen and running the assay.
not established until 28–30 days post-conception, the hematopoietic This problem has also been reported elsewhere. Whereas ccff DNA is
system is unlikely to be the source. Since the concentration of ccff DNA in stable, over time maternal white cells break down and release DNA and
amniotic fluid is roughly 200 times higher than that found in maternal the excess maternal DNA can inhibit the detection of the fetal RHD. As a
plasma, direct transfer may be also be a minor source.
9,10
result of these false tests, most laboratories will not run the assay if the
specimen is more that five days old.
12,13
How Well Does the Testing Work?
Previous reluctance to accept pre-natal testing was due to the lack of An English study reported a series of 1997 women tested before 28
accuracy with the early testing schemes. The first generation of test weeks’ gestation. False-negative results were obtained in only three
results targeted only exons four, seven or 10/3UTR, and the results were samples (0.2%). All three specimens were delayed in transport before
inaccurate in the Black population because of the variants noted above. testing. They postulated that the risk for a future pregnancy being
Better and more accurate primers needed to be used. affected by hemolytic disease of the fetus/newborn because of a failure
to give RhD immune globulin based on their detection rate of 99.7%
would be one in 86,000 RhD-negative women. Given a 90% successful
treatment rate, the risk for a fetal/neonatal loss would then be one in
The majority of the reported studies have
860,000 RHD-negative women.
4
been performed by realtime polymerase
The majority of the reported studies have been performed by realtime
chain reaction, which requires multiple
PCR, which requires multiple amplification reactions to detect all targets
amplification reactions to detect all
for each patient sample. In the US, Sequenom
®
(Sequenom, Inc., San
Diego, CA) has taken advantage of its MassARRAY™ system’s
targets for each patient sample.
multiplexing capability, and has developed a prototype single-tube RHD
genotyping test to non-invasively detect specific sequences within exons
four, five, and seven of the RHD gene and RHDψ, as well as three
The results of routine testing in Belgium between 2002 and 2006 were Y-chromosome genes (SRY, DBY, and TTTY) from ccff DNA in maternal
reported in 2008. The study evaluated 563 pregnant mothers and their serum. Of the approximately 500 clinical samples tested for pre-clinical
581 babies using realtime polymerase chain reaction (PCR) and targeting performance evaluation, the fetal RHD status of all samples was correctly
exons four, five, and 10 of the RHD gene combined with targeting an identified by comparing the SEQureDx™ RHD test results with the results
SRY gene sequence. The concordance rate was 99.8%, with one unusual previously obtained using a realtime PCR assay.
14
false-positive. Three discordant results (0.5%) between predicted ccff
DNA results and cord blood were resolved by baby venipuncture samples Conclusion
that confirmed the results of the ccff DNA testing. Discordant cases due Pre-natal RHD fetal typing is used routinely in Europe but not in the US.
to an error in cord blood typing may be secondary to poor identification An American College of Obstetricians and Gynecologists (ACOG) Practice
or mixture of cord blood with maternal blood. Another discrepancy was Bulletin in 2006
15
states that “detection of fetal D by molecular analysis
found when the ccff DNA fetal testing indicated an RHD-positive of maternal plasma or serum can be assessed in the second trimester
fetus and the newborn was RHD-negative. Further evaluation showed with greater that 99% accuracy… However, it should be noted that this
that the mother had previously received a kidney transplant from a is not a widely used clinical tool.” RhD immune globulin is a blood
RHD-positive male. Patients who have solid-organ transplants may have product that is expensive and there is a worldwide shortage. Its use has
microchimerism in peripheral blood and plasma.
11
been associated with the transmission of viral diseases (hepatitis C)
and it is subject to possible prion (variant Creutzfeld-Jacob disease)
Another series reported in 2008 from Germany tested 1,113 women contamination. Since about 40% of RhD-negative women carry an
between six and 32 weeks of pregnancy and found a sensitivity of RHD-negative fetus, each year at least 190,000 women in the US are
99.7%. At least one of the false-negative results (the newborn was unnecessarily exposed to these complications. The time has come to start
found to be RhD-positive) was thought to be secondary to the excessive clinically using ccff DNA to pre-natally type RHD status in the US. ■
1. Bowman JM, Hemolytic Disease. In: Creasy RK, Resnik R (eds), 1228–33. 11. Minon JM, Gerard G, Senterre JM, et al., Routine fetal RHD
Maternal–Fetal Medicine, Philadelphia: WB Saunders Company, 6. Daniels G, Finning K, Martin P, Summers J, Fetal RhD genotyping: genotyping with maternal plasma: a four-year experience in
1994;711–43. A more efficient use of anti-D immunoglobulin, Transfusion Belgium, Transfusion, 2008;48:373–81.
2. Moise KJ, Management of Rhesus alloimmunization in pregnancy, Clinique et Biologique, 2007;14:568–71. 12. Muller SP, Bartels I, Stein W, et al., The determination of the fetal
Obstetrics Gynecology, 2008;112:164–76. 7. Cerrato PL, The battle against Rh disease is about to get a lot D status from maternal plasma for the decision making on Rh
3. National Center for Health Statistics, National Vital Statistics easier, Contemporary OB/GYN, 2006. prophylaxis is feasible, Transfusion, 2008:1–10.
Reports, 2007;56:6. Available at: www.cdc.gov/nchs 8. Lo YMD, Zhang J, Leung TN, et al., Rapid clearance of fetal DNA 13. Van der Schoot CE, Hahn S, Chitty LS, Non-invasive prenatal
4. Finning K, Martin P, Sumers J, et al., Effect of high throughput from maternal plasma, Am J Hum Genetics, 1999;64:218–24. diagnosis and determination of fetal Rh status, Semin Fetal
RHD typing of fetal DNA in maternal plasma of anti-RhD 9. Bianchi DW, Avent ND, Costa JM, van der Schoot CE, Noninvasive Neonatal Med, 2008;13:63–8.
immunoglobulin in RhD negative pregnant women: prospective prenatal diagnosis of fetal Rhesus D, Obstetrics Gynecology, 14. Poster presentation, Society for Maternal-Fetal Medicine, Dallas,
feasibility study, BMJ, 2008; ePub. 2005;106:841–4. 2008.
5. Kenny-Walsh E, Clinical outcomes after hepatitis C infection from 10. Bianchi DW, Circulating fetal DNA: Its origin and diagnostic 15. ACOG practice bulletin, Management of alloimmunization in
contaminated anti-D immune globulin, New Eng J Med, 1999;340: potential – A review, Placenta, 2004;25:S93–101. pregnancy, Obstetrics Gynecology, 2006;108:457–64.
36 US OBSTETRICS & GYNECOLOGY
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