Evaluation of sample stability and automated DNA extraction for fetal sex determination using cell-free fetal DNA in maternal plasma.

OBJECTIVE: The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. METHODS: A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. RESULTS: Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. CONCLUSION: We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Development and validation of multiplex real-time PCR assay for noninvasive prenatal assessment of fetal RhD status and fetal sex in maternal plasma.

OBJECTIVE: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. METHODS: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. RESULTS: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. CONCLUSION: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.

[Assessment of HPV detection assays for use in cervical cancer screening programs]. / Evaluación de las técnicas de detección del VPH en los programas de cribado para cáncer de cuello uterino.

OBJECTIVE: Detection of high-risk human papillomavirus types (HPV) infection is an important tool in the screening of cervical cancer and triage of cytological abnormalities. The different techniques for detection of this cancer need to be contrasted and validated for use in population screening. MATERIAL AND METHODS: Cervical cell samples were collected from 166 women attending a dermatology clinic in Oviedo (Spain). We evaluated the performance of three different assays for VPH detection. The methods utilized were 1) In-house PCR-EIA using LI consensus primers MY09/ MY11, 2) A PCR-reverse line blot hybridization (PCR-LBH) that uses LI consensus PGMY primers. 3) Hybrid Capture 2. All assays were performed blinded. The kappa statistic was used to test for global agreement between assay pairs. RESULTS: HPV DNA was detected in 24,7%, 25,3% and 29,5% of the women, respective to the assay. The overall agreement between the in-house PCR, PCR-LBH and HC2 was (73.5%) with all kappa values between assay pairs exceeding 0.56 (p<0.001). CONCLUSION: The three HPV assays were equally accurate in estimating high-risk HPV prevalence and HPV-related lesions. The method for HPV detection must be decided depending on the goals of the search (screening, follow-up or molecular studies).

Evaluación de las técnicas de detección del VPH en los programas de cribado para cáncer de cuello uterino / Assessment of hpv detection assays for use in cervical cancer screening programs

OBJETIVO: La identificación de la infección por tipos de alto riesgo del virus del papiloma humano (VPH) es una herramienta útil para el cribado de cáncer del cuello uterino. Las distintas técnicas aplicadas para su detección deben contrastarse y validarse para su empleo en la tamización poblacional. MATERIAL Y MÉTODOS: Se evalúan tres técnicas para la detección del VPH en 166 muestras cervicales procedentes de mujeres atendidas en una clínica de dermatología en Oviedo (España): a) PCR-EIA mediante consensos MY09/MY011; b) PCR con line blot hybridization (PCR-LBH) con consensos PGMY; y c) hybrid capture 2. RESULTADOS: El ADN-VPH se reconoció en 29.5 por ciento, 25.3 por ciento y 24.7 por ciento, de acuerdo con el ensayo. La concordancia global entre PCR-EIA, PCR-LBH y HC2 fue de 73.5 por ciento con los valores de kappa superiores a 0.56 entre los ensayos (p<0.001). CONCLUSIONES: La prevalencia de tipos de alto riesgo oncogénico así como de las lesiones fue similar en los tres ensayos. En virtud de que las técnicas son comparables, su elección debe basarse en las condiciones individuales de cada laboratorio y el volumen de muestras por procesar.

OBJECTIVE: Detection of high-risk human papillomavirus types (HPV) infection is an important tool in the screening of cervical cancer and triage of cytological abnormalities. The different techniques for detection of this cancer need to be contrasted and validated for use in population screening. MATERIAL AND METHODS: Cervical cell samples were collected from 166 women attending a dermatology clinic in Oviedo (Spain). We evaluated the performance of three different assays for VPH detection. The methods utilized were 1) In-house PCR-EIA using L1 consensus primers MY09/MY11, 2) A PCR-reverse line blot hybridization (PCR-LBH) that uses L1 consensus PGMY primers. 3) Hybrid Capture 2. All assays were performed blinded. The kappa statistic was used to test for global agreement between assay pairs. RESULTS: HPV DNA was detected in 24,7 percent, 25,3 percent and 29,5 percent of the women, respective to the assay. The overall agreement between the in-house PCR, PCR-LBH and HC2 was (73.5 percent) with all kappa values between assay pairs exceeding 0.56 (p<0.001). CONCLUSION: The three HPV assays were equally accurate in estimating high-risk HPV prevalence and HPV-related lesions. The method for HPV detection must be decided depending on the goals of the search (screening, follow-up or molecular studies).

Concordance of prevalence of human papillomavirus DNA in anogenital and oral infections in a high-risk population.

The concordance of the prevalence of human papillomavirus (HPV) DNA in 188 sex workers in five different locations was investigated. HPV was found in 43.6% of the women, and its prevalence at genital sites was similar. Prevalence was highest among women aged 20 years or younger but declined thereafter in specimens from all anogenital sites.