Direct quantification of fetal cells in maternal blood by real‐time PCR

Zhong, Xiao Yan; Holzgreve, Wolfgang; Hahn, Sinuhe

doi:10.1002/pd.1521

Cited by 28 publications

(22 citation statements)

References 21 publications

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“…The reaction was performed at the following conditions: a first denaturation step at 958C for 10 min, and 40 cycles of 1 min at 608C, followed by 15 s at 958C (12).…”

Section: Real-time Pcrmentioning

confidence: 99%

“…However, low numbers of fetal cells in predominant maternal blood cells (12) and low concentrations of cell free fetal DNA in earlier gestational ages have limited their use in clinical practice (13).…”

Section: Introductionmentioning

confidence: 99%

“…However, on the one hand, false allele generation and imbalance resulting from varying specificity, sensitivity, and preferential amplification of alleles can lead to misdiagnosis (18,19). On the other hand, WGA may not be suitable for the detection of rare targets in a predominant background, such as rare fetal cells in maternal blood (12), as pref-erential amplification of background molecules will make the identification of rare targets more difficult.…”

Section: Introductionmentioning

confidence: 99%

“…We used the model of cell free male fetal DNA in maternal plasma DNA to evaluate the strategy. The sex determining region (SRY) locus on the Y chromosome was used to identify fetal DNA, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used to identify total circulating cell free (ccf) plasma DNA, almost representing the maternal part (12). Using the Expand Long Template (ELT) PCR kit (Roche Diagnostics GmbH, Mannheim, Germany), we selectively pre-amplified only the SRY locus and assessed the efficiency of the pre-amplification by real-time PCR, for both SRY and GAPDH, in order to compare the quantities of pre-amplified fetal DNA with those of maternal total DNA without pre-amplification.…”

Section: Introductionmentioning

confidence: 99%

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A selected pre-amplification strategy for genetic analysis using limited DNA targets

Xia¹,

Radpour²,

Kohler³

et al. 2009

Clinical Chemistry and Laboratory Medicine

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Background: Limited DNA resources or limited DNA targets in predominant backgrounds for genetic tests can lead to misdiagnosis. We developed a strategy to selectively increase the amount of minor targets through a specific pre-amplification procedure. Methods: We used the model of circulating cell free (ccf) male fetal DNA as a minor target in the predominant maternal plasma DNA to evaluate the strategy. The sex determining region (SRY) locus on the Y chromosome was used to identify ccf fetal DNA, and the human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was used to identify ccf total DNA in maternal plasma. We selectively pre-amplified the minor target SRY locus using the Expand Long Template PCR system and assessed the efficiency of the pre-amplification by real-time PCR, for both SRY and GAPDH, to compare the quantities of pre-amplified fetal DNA with those of maternal total DNA without pre-amplification. Results: The selected pre-amplification increased the amount of ccf fetal DNA dramatically (Wilcoxon test: ps0.000, the fold changes11,596). After selected preamplification, a proportion of 2.19% of the ccf fetal minor part in the predominant maternal component was changed up to 25,334%. The increased amounts of ccf fetal DNA found with the pre-amplification are not correlated to the amounts found without the procedure (rs-0.017, ps0.949). Conclusions: This strategy may be useful in genetic analysis with limited DNA resources and limited DNA targets in predominant background molecules. However, this approach is not suitable for quantitative assessments, due to the fact that quantitative imbal-

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“…The reaction was performed at the following conditions: a first denaturation step at 958C for 10 min, and 40 cycles of 1 min at 608C, followed by 15 s at 958C (12).…”

Section: Real-time Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A selected pre-amplification strategy for genetic analysis using limited DNA targets

Xia¹,

Radpour²,

Kohler³

et al. 2009

Clinical Chemistry and Laboratory Medicine

Self Cite

View full text Add to dashboard Cite

show abstract

“…[8][9][10][11][12] So far, RHD qPCR has been used primarily to detect RHD sequences in cell-free fetal DNA, present in maternal plasma during pregnancy to predict the D blood group status of the fetus, without invasive procedures to obtain fetal genetic material [13][14][15] and for determination of (paternal) RHD zygosity. 16 concentrations from 900 to 50 nM and 250 to 50 nM, respectively.…”

Section: Introductionmentioning

confidence: 99%

Comparison of different blood sample processing methods for sensitive detection of low level chimerism byRHDreal-time PCR assay

et al. 2013

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Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

Devaney

Palomaki²,

Scott³

et al. 2011

JAMA

220

142

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Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997–April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%–96.1%) and specificity, 98.6% (95% CI, 98.1%–99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989–0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979–0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993–0.998) for real-time quantitative PCR (RTQ-PCR) (P=.02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965–0.998) (<7 weeks); AUC, 0.994 (95% CI, 0.987–0.997) (7–12 weeks); AUC, 0.992 (95% CI, 0.983–0.996) (13–20 weeks); and AUC, 0.998 (95% CI, 0.990–0.999) (>20 weeks) (P=.02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96.0%; specificity, 99.0%) outperformed conventional PCR (sensitivity, 94.0%; specificity, 97.3%). Testing after 20 weeks (sensitivity, 99.0%; specificity, 99.6%) outperformed testing prior to 7 weeks (sensitivity, 74.5%; specificity, 99.1%), testing at 7 through 12 weeks (sensitivity, 94.8%; specificity, 98.9%), and 13 through 20 weeks (sensitivity, 95.5%; specificity, 99.1%). Conclusions Despite interstudy variability, performance was high using maternal blood. Sensitivity and specificity for detection of Y chromosome sequences was greatest using RTQ-PCR after 20 weeks’ gestation. Tests using urine and tests performed before 7 weeks’ gestation were unreliable.

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Direct quantification of fetal cells in maternal blood by real‐time PCR

Cited by 28 publications

References 21 publications

A selected pre-amplification strategy for genetic analysis using limited DNA targets

A selected pre-amplification strategy for genetic analysis using limited DNA targets

Comparison of different blood sample processing methods for sensitive detection of low level chimerism byRHDreal-time PCR assay

Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

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