Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

Kelly A. Sillence; Llinos A. Roberts; Heidi J. Hollands; Hannah P. Thompson; Michele Kiernan; Tracey E. Madgett; Welch C Ross; Neil D. Avent

doi:10.1373/clinchem.2015.239137

Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

Kelly A. Sillence, Llinos A. Roberts, Heidi J. Hollands, Hannah P. Thompson, Michele Kiernan, Tracey E. Madgett, Welch C Ross, Neil D. Avent^*

^*Corresponding author for this work

School of Biomedical Sciences

University Hospitals Plymouth NHS Trust

Research output: Contribution to journal › Article › peer-review

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Abstract

Abstract BACKGROUND Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping. METHODS Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P &lt; 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (&lt;2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (&lt;2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.

Original language	English
Pages (from-to)	1399-1407
Number of pages	0
Journal	Clinical Chemistry
Volume	61
Issue number	11
DOIs	https://doi.org/10.1373/clinchem.2015.239137
Publication status	Published - 1 Nov 2015

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10.1373/clinchem.2015.239137

239137 clin chem 2015

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@article{34b1c0cadf5e4b1a8b4c49aef9ccd56f,

title = "Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR",

abstract = "Abstract BACKGROUND Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping. METHODS Blood samples were collected in EDTA tubes and Streck{\textregistered} Cell-Free DNA{\texttrademark} blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital{\texttrademark} PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA. ",

author = "Sillence, {Kelly A.} and Roberts, {Llinos A.} and Hollands, {Heidi J.} and Thompson, {Hannah P.} and Michele Kiernan and Madgett, {Tracey E.} and Ross, {Welch C} and Avent, {Neil D.}",

year = "2015",

month = nov,

day = "1",

doi = "10.1373/clinchem.2015.239137",

language = "English",

volume = "61",

pages = "1399--1407",

journal = "Clinical Chemistry",

issn = "0009-9147",

publisher = "Oxford University Press",

number = "11",

}

TY - JOUR

T1 - Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

AU - Sillence, Kelly A.

AU - Roberts, Llinos A.

AU - Hollands, Heidi J.

AU - Thompson, Hannah P.

AU - Kiernan, Michele

AU - Madgett, Tracey E.

AU - Ross, Welch C

AU - Avent, Neil D.

PY - 2015/11/1

Y1 - 2015/11/1

N2 - Abstract BACKGROUND Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping. METHODS Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.

AB - Abstract BACKGROUND Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping. METHODS Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR). RESULTS The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay. CONCLUSIONS qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.

U2 - 10.1373/clinchem.2015.239137

DO - 10.1373/clinchem.2015.239137

M3 - Article

SN - 0009-9147

VL - 61

SP - 1399

EP - 1407

JO - Clinical Chemistry

JF - Clinical Chemistry

IS - 11

ER -

Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR

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