Discussion

Fetal DNA has been known to be present in maternal blood since the work of Lo et al.[9] in 1997. They first showed that Y-chromosomal sequences could be amplified from DNA isolated from the plasma of pregnant women carrying a male fetus. Fetal DNA is present in maternal serum at 5 weeks' gestation[10] and the concentration of fetal DNA increases with gesta-tional age. Fetal DNA represents 3.4% (0.4-12%) and 6.2% (2.3-11.5%) of total plasma DNA in the first and third trimesters of pregnancy, respectively.[11] The origin of fetal DNA is still unclear. One or more mechanisms might be involved in this process: Fetal DNA might be released from apoptotic fetal cells circulating in maternal blood, or by trophoblasts (by lysis of the cells at the fetal-maternal interphase, or after entrapment in the maternal lung); it could also passively cross the placenta. Regardless of its origin, fetal DNA is rapidly cleared (mean half-life, 16 min)[12] and it has been shown not to persist in maternal blood after pregnancy.[13]

Although anecdotal diagnosis has been made using circulating fetal DNA (myotonic dystrophy;[14] achon-droplasia.[15]), most of the studies on the accuracy of PCRs based on fetal circulating DNA have focused on Y-chromosome and RhD. In the past few years, studies have reported a 100% accuracy in fetal sex detection using a PCR technique in the first trimester.[16,17] This technique has now modified the prenatal diagnosis of X-linked disorders,[18] as well as the management of pregnancies at risk for congenital adrenal hyperplasia.[19] The use of cell-free DNA in maternal plasma and serum for noninvasive fetal RhD genotyping has been achieved independently by five groups.[3,4,20-22] Faas et al.[4] reported a highly sensitive but conventional PCR specific for RHD exon 7. The RhD genotype was correctly predicted in 31 second-trimester pregnancies. Lo et al. used a real-time RHD PCR technique on exon 10 in 57 RhD-negative women at different gestational ages. Two false-negative results were obtained in the first trimester. Results of Bischoff et al.[20] were less accurate, with 30% false negatives. In their PCR, input DNA was isolated from only 10 pL of serum, and this could explain the low sensitivity observed in this study.[2] Zhong et al.[21] analyzed 22 pregnancies and obtained one false-positive result, but they used a multiplex nested PCR using Y-chromosome-specific and RhD-specific primers. Our team studied 106 sera from RhD-negative pregnant women in the first trimester, and maternal serum results were in complete concordance with those obtained on fetal cells isolated from amniotic fluid and with the RhD serotype of the newborn.[22]

The assay was based on real-time PCR targeted at the 3' end (exon 10) region of the RHD gene. Being a closed-tube system, real-time PCR offers, to date, the highest level of safety and represents the most secure amplification procedure. Total DNA was extracted

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Fig. 1 Detection of the RHD gene in maternal serum using real-time PCR. Two patients' samples are tested in quadruplicate—one giving a positive result, and the second giving a negative one. (View this art in color at www.dekker.com.)

from 400 pL of serum using a silica-based commercial procedure and was then eluted with 50 pL of an elution buffer, of which 10 pL was used for the assay. Amplification as carried out in a LightCycler® instrument for 50 cycles. Each sample was analyzed, in duplicate, by different DNA extraction methods. Figure 1 illustrates typical results.

Van Der Schoot et al.[2] report a 100% concordance over 100 plasma samples (details on gestational age are lacking). The discrepancy on first-trimester determination of fetal RhD status might be a result of poor amplification efficiency. The real-time PCR assay (our team uses FRET instead of TaqMan technology) achieves a high level of sensitivity associated with a high level of safety because it is a closed-tube system; no false-positive results related to PCR product carryover are observed.[22] Poor efficiency of DNA recovery, or an inhibitor effect of DNA extract may also explain low sensitivity. As a control for amplifiability, the use of an endogenous gene (such as p-globin gene) seems inappropriate1-3-1 because it evaluates mainly maternal DNA, which is present in large amounts in the plasma. For this reason, our group has proposed the addition of a low amount of a heterologous DNA as an internal control. Finally, the use of maternal serum instead of plasma used by Lo et al. may also explain these discrepancies.

One of the concerns for this technique is the internal control for the presence of fetal DNA. We postulate that fetal DNA was always present in maternal serum and that this control was not necessary. Van Der Schoot et al. consider that when no RhD-specific signal is obtained, the presence of fetal DNA in the plasma has to be confirmed by another fetus-specific DNA sequence from a highly polymorphic paternal antigen (STR markers) or from the Y-chromosome (e.g., SRY), which could be used only in women bearing a male fetus. However, most of those assays that have already been described are multiplex PCRs in which sensitivity is low because paternal alleles are difficult to detect within the high background of maternal alleles and because of poorer amplification.[21] Notwithstanding this, Pertl et al.[23] successfully applied multiplex fluorescent PCR of STR on fetal DNA in maternal plasma, but gestational age in this study was 34 weeks and over—a time when fetal DNA is particularly high in maternal serum, which is not the case in the first trimester.[2] To Van Der Schoot et al.'s point of view, only positive results are meaningful until allele-specific amplification strategies allow more sensitive systems. We do agree in theory with this statement, but our personal experience (over 250 cases with 100% accuracy) has shown that if internal control of the amplification is used, negative result is still of clinical value.

Another issue is genotyping errors caused by rare variants of the RHD gene. These variants could lead to false-positive or false-negative results. A fetus carrying an RhD-negative allele might be typed RhD-positive when the RhD-specific PCR assays are based on polymorphic sites still present in the silent allele. In already sensitized RhD-negative pregnant women, a false-positive result will not have much consequences as maternal IgG titer will be followed as usual and will not lead to any invasive intervention. In case of unsensitized women, unnecessary Ig injection will be performed if needed, which is acceptable. In the Caucasian population, RhD negativity is usually caused by deletion of the RHD gene, but negativity could also result from aberrant RHD alleles that do not lead to RhD expression. In the African black population, the variant RHD genes r's (=CdeS) and RHDC can underlie serologic RhD negativity.[2] In the Caucasian population, 14 different RhD-negative RHD alleles have been described,[24] but the frequency of these alleles is extremely low (1/1500); therefore they do not influence the accuracy of RHD-specific PCR assays. It has been suggested that different regions of the RHD gene should be examined to increase the accuracy of RhD genotyping. However, the assay described by our group was only targeted at the 3' untranslated region specific to the RHD gene (exon 10) and no false positive was detected. Lo et al.[3] and Zhang et al.[25] also used single RHD gene region assay on exons 10 and 7, respectively. The population of those studies is mainly Caucasian, which could explain the absence of false positives. Van Der Schoot et al.[2] have designed primers and probes located in exon 7. This exon is present in almost all RhD-positive RHD alleles (except some rare variants). False-positive results will be obtained, such as with the primers used by our team located in exon 10, in fetuses carrying only the silent RHDC gene.

RhD positivity might be missed when the fetus is carrying an aberrant allele, which still leads to RhD positivity, although not all RhD-specific polymorphic nucleotides are present.[2] This kind of variant might lead to alloimmunization in an RhD-negative mother, but HDN in a fetus whose red blood cells have a partial D antigen is rare. Van de Schoot et al. suggest that for immunized mothers, no aberrant RhD alleles should be missed; therefore a multiplex PCR approach on more than one region of the RHD gene has to be taken and the RHDC gene has to be recognized. From our point of view, the sensitivity of a multiplex PCR will be less than the actually reported protocols, and the frequency of these aberrant alleles is so small that sensitivity of the test should be preferred. However, in a non-Caucasian population, the existence of those variants should be kept in mind.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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