Maternal Contamination And The Sensitivity Of Genotyping

Maternal contamination of umbilical cord blood, chorionic villus, and amniocentesis samples is a well-documented problem that presents itself to laboratories conducting prenatal genetic testing (79-82). Because analysis by molecular diagnostic methodologies does not distinguish maternal from fetal DNA, maternal contamination presents a risk for misidentifica-tion of the fetal genotype and possible misdiagnosis. The reality of this problem is further emphasized when one considers that the cell counts of amniotic fluid samples range from several hundred cells/per milliliter prior to 10 wk gestation to several thousand cells/per milliliter prior to 20 wk gestation. Therefore, the introduction of even small volumes of maternal blood could cause the number of nucleated maternal cells to outnumber the fetal amniocytes, because the average leukocyte count in adults is approx 7 x 106 cells/mL (82). To address this issue, a variable number of tandem repeats (VNTR) analysis of defined sensitivity was conducted on actual fetal samples to define the rate and degree to which maternal contamination can be expected during routine diagnostic testing (11). Experiments were also conducted to determine the extent to which maternal contamination could be tolerated in PCR-based genotyping assays for RHC/c, RHD, RHE/e, Kell, HPA-1 (Pla), HPA-5 (Br), and FcyRIIIb. In each case, the ability to sensitively detect the possible alloantigen was evaluated, because its presence conveys risk of the immune cytopenic disorder.

The VNTR loci can be used to detect and estimate the degree of maternal contamination within individual fetal samples. The VNTR loci are highly polymorphic and are inherited in a

Mendelian fashion, making their analysis useful to confirm (or exclude) the source of template DNA. A total of 56 fetal samples (31 uncultured amniocyte, 6 cultured amniocyte, 18 fetal blood, and 1 chorionic villus) were analyzed. The VNTR loci employed, D1S80 and apoB, were amplified using primers that flank the repeat unit to generate variably sized products. Products were analyzed through polyacrylamide gel electrophoresis and silver staining (83-85). In fetal samples free of maternal contamination, only one of the two maternal bands will be detected in addition to the paternal band. In order to establish the sensitivity at which maternal contamination could be detected in fetal DNA samples, "contaminated" samples were created in cases for which there was adequate material by mixing 1% maternal DNA and 99% fetal DNA. Using this approach, 1% maternal DNA mixed with 99% fetal DNA was always detected in these experiments (Fig. 4) (11). Of the 56 fetal samples analyzed, maternal contamination was only observed in 4 uncultured amniocentesis samples (11). Maternal contamination of less than 1%, based on comparison of band intensities was observed in three of these samples (data not shown). In these cases, the fetuses were found at risk for their respective immune cytopenic disorder. Therefore, the level of maternal contamination present in these samples did not interfere with the detection of the paternal alloallele in the fetus. The fourth sample, which was visibly blood stained, possessed maternal contamination exceeding 50%, based on comparison of band intensities. A portion of this amniotic fluid sample was cultured and later found free of any detectable maternal contamination by VNTR analysis. This is consistent with the recent report of Winsor et al., which describes much higher maternal contamination rates in uncultured (21.4%) vs cultured (0.2%) amniotic fluid samples (86). In this particular case, the cultured and uncultured amniocentesis samples were subjected to HPA-1 genotyping. Both genotypings indicated that the fetus was not at risk for NATP.

In immune cytopenic disorders, a paternal allele encoding an alloantigen is responsible for the incompatibility between the mother and the fetus. Therefore, when identifying fetuses at risk, it is critical that this paternal alloallele be detected. To

Microsatellite Instability

Fig. 5. The sensitivity of ASPCR for detection of the JKB allele of the Kidd blood group system. Products of each analysis are run in pairs; the JKA reaction is in the first lane of each pair, and the JKB reaction is in the second lane of each pair. A 423-bp HGH control product is detected in each reaction possessing, the DNA template. Allele-specific amplification of either JKA or JKB generates a 97-bp product. -C: ASPCR reactions with no DNA template; Jka: ASPCR reactions with DNA template isolated from a Jka/Jka serotyped individual; 0: ASPCR reactions with DNA template isolated from a Jka/Jkb serotyped individual; 2-1024: ASPCR reactions with DNA template isolated from a Jka/Jkb serotyped individual serially diluted into DNA template isolated from a Jka/Jka serotyped individual (1:2 to 1 : 1024). The last lane is 100-bp DNA ladder size standard (Gibco/BRL, Gaithersburg, MD).

Fig. 5. The sensitivity of ASPCR for detection of the JKB allele of the Kidd blood group system. Products of each analysis are run in pairs; the JKA reaction is in the first lane of each pair, and the JKB reaction is in the second lane of each pair. A 423-bp HGH control product is detected in each reaction possessing, the DNA template. Allele-specific amplification of either JKA or JKB generates a 97-bp product. -C: ASPCR reactions with no DNA template; Jka: ASPCR reactions with DNA template isolated from a Jka/Jka serotyped individual; 0: ASPCR reactions with DNA template isolated from a Jka/Jkb serotyped individual; 2-1024: ASPCR reactions with DNA template isolated from a Jka/Jkb serotyped individual serially diluted into DNA template isolated from a Jka/Jka serotyped individual (1:2 to 1 : 1024). The last lane is 100-bp DNA ladder size standard (Gibco/BRL, Gaithersburg, MD).

determine the degree of maternal contamination in fetal samples that could be tolerated in ASPCR genotyping assays without the loss of detection of this paternal allele, maternal contamination of fetal samples was mimicked by diluting DNA isolated from a heterozygous individual into DNA isolated from a homozygous individual (undiluted to 1 : 1024) (86). Figure 5 illustrates an example of the results obtained when performing sensitivity studies using the Kidd ASPCR genotyping assay. In this example, DNA isolated from a person heterozygous for the Kidd antigen system (to mimick the fetus) is diluted into DNA isolated from a homozygous individual (to mimick the mother). The K1 allele is detectable to a point where the heterozygote DNA comprises only 0.2% of the template DNA in the reaction. Similar experiments have been conducted on all other ASPCR assays used for prenatal testing and indicate that >90% maternal contamination can be tolerated in these assays without the loss of detection of the paternal allele (86). Such samples would likely be blood stained. Therefore, a large safety margin exists when one considers the frequency and degree to which fetal amniocyte samples are contaminated and the ability of ASPCR assays to detect the offending paternal alloallele. It is advisable that laboratories using other typing methodologies define the sensitivities of the assays/technologies that they are employing. It is also advisable to confirm the fetal origin of DNA samples by VNTR analysis when a fetal sample genotypes compatible with the mother prior to withdrawing any further monitoring of the pregnancy.

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