Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience.

BACKGROUND: Despite being deliberately targeted to common chromosome aneuploidies, the rapid quantitative fluorescent polymerase chain reaction (QF-PCR) tests can detect the majority of chromosome abnormalities in prenatal diagnosis. The main advantages of this assay are low cost, speed and automation allowing large-scale application. METHODS: We developed a QF-PCR test that was applied on 43 000 clinical samples reporting results in 24 h. Most common indications were biochemical risk (32%) and advanced maternal age (30%). Samples were also tested by cytogenetic analysis and the results compared. RESULTS: Aneuploidies involving chromosomes 21, 18, 13, X and Y were detected with 100% specificity. Several cases of partial trisomies and mosaicism were also identified. Overall 95% of clinically relevant abnormalities were readily detected and termination of affected pregnancies could be performed without waiting for the cytogenetic results. CONCLUSIONS: Our study supports the possibility of reducing the load of prenatal cytogenetic tests if the pregnancies are carefully monitored by non-invasive screening. In case of abnormal QF-PCR results, medical action can be taken within few hours from sampling. In cases of negative QF-PCR results, cytogenetic analyses might only be performed for fetuses with ultrasound abnormalities. In countries where large-scale cytogenetic tests are not available, QF-PCR may be used as the only prenatal diagnostic procedure.

[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.

X chromosome dosage by quantitative fluorescent PCR and rapid prenatal diagnosis of sex chromosome aneuploidies.

During the past few years, rapid prenatal diagnosis of chromosome aneuploidies has been successfully achieved by quantitative fluorescent PCR (QF-PCR) amplification of chromosome-specific small tandem repeats (STR). This approach has proven to be very useful in clinical settings, since it allows the detection of major numerical disorders in a few hours after sampling. For the detection of Turner's syndrome (45,X), several highly polymorphic STR on the X chromosome are needed in order to reduce the likelihood that a normal female might be homozygous for all sequences and, consequently, that the test could fail to discriminate between samples retrieved from a Turner's and a normal female fetus. Here we report a new method for rapid and accurate detection of X chromosome copy number in prenatal samples that does not depend on STR heterozygosity. The test is based on QF-PCR amplification of the X-linked HPRT together with the autosomal D21S1411 used as internal control for quantification. In the course of this study, this assay allowed the prenatal diagnosis of a rare case of a normal female homozygous for four selected highly polymorphic X chromosome STR, as well as the assessment of the normal chromosome complement of a fetus homozygous for five chromosome 21 markers.