Comparison of human papillomavirus testing strategies for triage of women referred with low-grade cytological abnormalities.

AIM: To compare triage strategies using different human papillomavirus (HPV) consensus and genotyping tests and a p16(INK4a) test. METHODS: 1228 women referred with a borderline or single mildly dyskaryotic smear. Samples were taken at colposcopy using PreservCyt. Tests included Hybrid Capture 2, Abbott RealTime PCR, BD HPV, Cobas 4800, PreTect HPV-Proofer, APTIMA and p16(INK4a). Results were based on the worst histology within 9 months. RESULTS: 97/1228 (7.9%) women had CIN3+ (203/1228 (17%) CIN2+). HPV testing alone using Hybrid Capture 2, Abbott RealTime PCR, BD HPV, Cobas 4800 or APTIMA had a sensitivity for CIN3+ ranging from 99.0% to 100.0% and specificity for

Comparison of seven tests for high-grade cervical intraepithelial neoplasia in women with abnormal smears: the Predictors 2 study.

High-risk human papillomavirus (HPV) DNA/RNA testing provides higher sensitivity but lower specificity than cytology for the identification of high-grade cervical intraepithelial neoplasia (CIN). Several new HPV tests are now available for this purpose, and a direct comparison of their properties is needed. Seven tests were evaluated with samples in liquid PreservCyt transport medium from 1,099 women referred for colposcopy: the Hybrid Capture 2 (Qiagen), Cobas (Roche), PreTect HPV-Proofer (NorChip), Aptima HPV (Gen-Probe), and Abbott RealTime assays, the BD HPV test, and CINtec p16(INK4a) cytology (mtm laboratories) immunocytochemistry. Sensitivity, specificity, and positive predictive value (PPV) were based on the worst histology found on either the biopsy or the treatment specimen after central review. Three hundred fifty-nine women (32.7%) had CIN grade 2+ (CIN2+), with 224 (20.4%) having CIN3+. For detection of CIN2+, Hybrid Capture 2 had 96.3% sensitivity, 19.5% specificity, and 37.4% PPV. Cobas had 95.2% sensitivity, 24.0% specificity, and 37.6% PPV. The BD HPV test had 95.0% sensitivity, 24.2% specificity, and 37.8% PPV. Abbott RealTime had 93.3% sensitivity, 27.3% specificity, and 38.2% PPV. Aptima had 95.3% sensitivity, 28.8% specificity, and 39.3% PPV. PreTect HPV-Proofer had 74.1% sensitivity, 70.8% specificity, and 55.4% PPV. CINtec p16(INK4a) cytology had 85.7% sensitivity, 54.7% specificity, and 49.1% PPV. Cytology of a specimen taken at colposcopy (mild dyskaryosis or worse) had 88.9% sensitivity, 58.1% specificity, and 50.7% PPV. Our study confirms that, in a referral setting, HPV testing by a number of different tests provides high sensitivity for high-grade disease. Further work is needed to confirm these findings in a routine screening setting.

Multiplex ligation-dependent probe amplification (MLPA): a reliable alternative for fetal chromosome analysis?

OBJECTIVE: To determine whether molecular karyotyping using multiple ligation probe amplification (MLPA) is a reliable alternative for quick and accurate diagnosis of fetal chromosomal abnormalities. METHODS: MLPA, using specialised probe sets designed to detect aneuploidy, major chromosomal rearrangements and recognised microdeletion syndromes, was used to analyse chorionic villi or amniocytes left after traditional karyotyping of 476 fetuses for clinical indications. RESULTS: An abnormal result was obtained in 190 cases, including 124 trisomies, 21 sex chromosome anomalies, 14 triploidies, and 31 rearrangements or mosaics. All trisomies were detected by all three techniques, but triploidies were only detected by karyotyping and QF-PCR. In 19 of the 31 cases of rearrangements or mosaicism there was an uncertain or high risk of adverse outcome. Traditional karyotyping detected 13 of the 19 pathogenic rearrangements, MLPA detected 18, and QF-PCR did not detect any. CONCLUSION: MLPA, using specialized probe sets, detects more chromosomal rearrangements, conferring significant risk of adverse outcome than karyotyping. A combination of qfPCR and MLPA could be a good, rapid alternative to current practice. In the future, used in conjunction with non-invasive prenatal diagnosis based on cell free fetal DNA it might provide a rapid and efficient approach to fetal karyotyping.

Incidence of placental mosaicism leading to discrepant results between QF-PCR and karyotyping in 22,825 chorionic villus samples.

OBJECTIVE: To review the frequency and analyse the origin of completely discrepant results observed between QF-PCR and karyotyping in chorionic villus samples (CVS) as a result of placental mosaicism. Also, to assess QF-PCR results for biallelic or triallelic patterns and determine their significance. METHODS: Between May 2002 and December 2009, 22 825 CVS were received at TDL Genetics for processing by QF-PCR and karyotype. The QF-PCR and karyotype data were compared to determine the incidence of discrepant results. RESULTS: Of the 22 825 samples received, 22 779 (99.8%) gave concordant results between the PCR and karyotype, and 46 samples (0.2%) gave discordant results. Of these discrepant cases, 5 displayed triallelic peaks and 41 displayed biallelic peaks. All discordant results are due to the presence of placental mosaicism, a known limitation of using this sample type for prenatal diagnosis. CONCLUSION: This retrospective study of placental mosaicism in CVS is the largest single centre study to date and provides a figure for the occurrence of completely discrepant results between QF-PCR and karyotype due to placental mosaicism. This study also demonstrates that the presence of triallelic peaks at QF-PCR is not sufficient to exclude the presence of placental mosaicism.

Performance of the Abbott RealTime high-risk HPV test in women with abnormal cervical cytology smears.

HPV DNA testing is known to be much more sensitive than cytology, but less specific. A range of HPV and related tests in 858 women referred for colposcopy because of an abnormal smear were evaluated to compare the performances of these tests. This article compared the Abbott test to other tests which had been previously evaluated. This test was a real true test for 14 high-risk HPV types. The Abbott test was found to be highly sensitive for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) (98.9%) with a specificity of 31.5%. These numbers were comparable with the Qiagen HC2 test, the Roche Linear Array and Amplicor tests, and the Gen-Probe APTIMA test. Differences between these tests appeared to be related mostly to the choice of cutoff level. An added feature of the Abbott test was the provision of type specific results for HPV 16 and 18.

A novel method for extracting DNA from chorionic villus samples for use in CVS-PCR, which ensures complete villus dissociation.

OBJECTIVE: To demonstrate that glass disruption beads dissociate chorionic villus samples releasing DNA from mesenchymal and cytotrophoblast cells that is suitable for processing by CVS-PCR (rapid molecular aneuploidy testing). This method is quicker than conventional methods and may limit discrepancies between PCR and karyotype in certain types of placental mosaicism. METHOD: DNA was extracted from villus samples by mechanical disruption of the cells using glass beads. This method was compared to collagenase incubation followed by chelex extraction of the digested villus. PCR data generated were compared using standard criteria. RESULTS: DNA extracted by glass bead disruption generated data of equivalent quality to that obtained from DNA extracted using conventional collagenase and chelex-based extraction method. The case study demonstrates probable cytotrophoblast enrichment of a sample when processed by collagenase digestion and chelex incubation. Re-extraction of the digested sample by glass bead disruption resulted in cytotrophoblast and mesenchyme cells contributing to the supernatant. CONCLUSION: Glass bead disruption of chorionic villus samples is an effective, inexpensive and rapid DNA extraction method that dissociates villus ensuring that DNA from both cytotrophoblast and mesenchyme cells is represented in the supernatant. Extracted DNA produced is suitable for CVS-PCR and can be stored stably at - 20 degrees C.

Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears.

BACKGROUND: The detection of high-risk human papillomavirus (HPV) DNA provides higher sensitivity but lower specificity than cytology for the identification of high-grade cervical intraepithelial neoplasia (CIN). This study compared the sensitivity and specificity of several adjunctive tests for the detection of high-grade CIN in a population referred to colposcopy because of abnormal cytology. METHODS: 953 women participated in the study. Up to seven tests were carried out on a liquid PreservCyt sample: Hybrid Capture II (Digene), Amplicor (Roche), PreTect HPV-Proofer (NorChip), APTIMA HPV assay (Gen-Probe), Linear Array (Roche), Clinical-Arrays (Genomica), and CINtec p16INK4a Cytology (mtm Laboratories) immunocytochemistry. Sensitivity, specificity, and positive predictive value (PPV) were based on the worst histology seen on either the biopsy or the treatment specimen after central review. RESULTS: 273 (28.6%) women had high-grade disease (CIN2+) on worst histology, with 193 (20.2%) having CIN3+. For the detection of CIN2+, Hybrid Capture II had a sensitivity of 99.6%, specificity of 28.4%, and PPV of 36.1%. Amplicor had a sensitivity of 98.9%, specificity of 21.7%, and PPV of 33.5%. PreTect HPV-Proofer had a sensitivity of 73.6%, specificity of 73.1%, and PPV of 52.0%. APTIMA had a sensitivity of 95.2%, specificity of 42.2%, and PPV of 39.9%. CINtec p16INK4a Cytology had a sensitivity of 83.0%, specificity of 68.7%, and PPV of 52.3%. Linear Array had a sensitivity of 98.2%, specificity of 32.8%, and PPV of 37.7%. Clinical-Arrays had a sensitivity of 80.9%, specificity of 37.1%, and PPV of 33.0%.

Complete discrepancy between abnormal fetal karyotypes predicted by QF-PCR rapid testing and karyotyped cultured cells in a first-trimester CVS.

A chorion villus sample (CVS) biopsied at 11 weeks' gestation for raised nuchal translucency, revealed monosomy X (presumptive 45,X karyotype) by QF-PCR for rapid aneuploidy testing for chromosomes 13, 18, 21, X and Y. Long-term culture gave the karyotype: 47,XY,+ 21[66]/49,XYY,+ 21,+ 21 [22]. This discrepancy prompted redigestion of the combined residual villus fragments from the original QF-PCR assay. The repeat QF-PCR assay identified the presence of trisomy 21 and a Y chromosome consistent with a 47,XY,+ 21 karyotype. A double non-disjunction event early in embryogenesis in a 47,XY,+ 21 conceptus with subsequent cell lineage compartmentalisation of the three observed cell lines (45,X; 47,XY,+ 21 and 49,XYY,+ 21,+ 21) would account for these results. This is the first reported case to describe complete discrepancy at diagnosis between abnormal karyotypes detected by QF-PCR rapid aneuploidy testing and a cultured karyotype in the same CVS.