Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears.

AIMS: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. METHODS: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of beta-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. RESULTS: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. CONCLUSION: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

Improved endocervical sampling and HPV viral load detection by Cervex-Brush Combi.

OBJECTIVE: Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. METHODS: Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. RESULTS: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). CONCLUSION: Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.

Sequential triage in the first trimester may enhance advanced ultrasound scanning in population screening for trisomy 21.

OBJECTIVE: To design a trisomy 21 screening protocol for sequential triage in the first trimester, and to evaluate whether it reduces the need for advanced ultrasound scanning to such an extent that this could be dealt with by a limited number of well-trained sonographers only. METHODS: Screening results of 31 trisomy 21 affected pregnancies and 16 096 unaffected pregnancies from the first trimester screening program of Algemeen Medisch Laboratorium in Antwerp, Belgium, were used to define high-risk, intermediate-risk and low-risk groups. A serum screening result (age, pregnancy-associated plasma protein-A (PAPP-A) and free beta-human chorionic gonadotropin (beta-hCG)) of >or=1 : 30 and/or a nuchal translucency thickness (NT) measurement of >or= 3.5 mm were classified as high risk. A serum screening result of < 1 : 1000 together with an NT of < 3.5 mm were classified as low risk. Other results were considered intermediate risk, for which further advanced ultrasound screening would be indicated. This protocol was then evaluated prospectively in another population of 13 493 first-trimester pregnancies. RESULTS: Of the total population, 1.9% was identified as being high risk (14 trisomy 21 pregnancies and 222 unaffected pregnancies; prevalence, 1 : 17), 59.6% was identified as being low risk (three trisomy 21 pregnancies and 9615 unaffected pregnancies; prevalence, 1 : 3206) and 38.4% was identified as being intermediate risk (10 trisomy 21 pregnancies and 6190 unaffected pregnancies; prevalence, 1 : 620). A similar distribution was found in the prospective arm of the study. There was no reduction of overall screening performance compared with our current first-trimester combined screening program. The number of intermediate-risk pregnancies was sufficiently low as to enable advanced ultrasound scanning by well-trained sonographers only. CONCLUSION: In population screening for fetal trisomy 21, sequential triage in the first trimester can be achieved using very simple methods. Pregnancies at high or at low risk can be identified easily and the number of pregnancies at intermediate risk can be reduced sufficiently to enable advanced ultrasound scanning by well-trained sonographers only. A prospective study is needed to evaluate the performance of this approach and to compare its results with current combined or integrated screening algorithms.

Audit on nuchal translucency thickness measurements in Flanders, Belgium: a plea for methodological standardization.

OBJECTIVES: To audit nuchal translucency thickness (NT) measurements for fetal aneuploidy screening in Flanders, and to estimate the impact of small variations in NT measurement on the screening result of two first-trimester screening algorithms: maternal age + NT (Algorithm A), and maternal age + NT + pregnancy associated plasma protein-A + free beta-human chorionic gonadotropin (Algorithm B). METHODS: We used the database of first-trimester combined screening, as collected by the General Medical Laboratory AML in Antwerp, Belgium, between 1 January 2001 and 1 April 2004. Audit was performed by establishing a delta-NT distribution curve for one trainee of The Fetal Medicine Foundation (FMF) and for a group of 263 other sonographers, in comparison with the FMF reference values. Risks for fetal aneuploidy were calculated at a cut-off value of 1 : 300 for Algorithm A and 1 : 150 for Algorithm B. These risks were recalculated in both algorithms after a modeled increase of all NT values by 0.1 or 0.2 mm. RESULTS: In a total of 592 measurements performed by the FMF trainee, the 5th, 50th and 95th percentiles of delta-NT measurements were at -0.41, +0.03 and +0.68 mm, respectively. These values were close to the FMF reference values. The screen-positive rate for this set of data was 4.4% (26/592) in both algorithms. For the 12 555 measurements of the 263 other sonographers, the 5th, 50th and 95th percentiles of delta-NT were at -0.81, -0.14 and +0.73 mm, respectively, which clearly indicates underestimation of NT in the lower range. In this set of data the screen-positive rate was 3.5% for both algorithms (439/12 555 for Algorithm A and 436/12 555 for Algorithm B). Also in this group, 5% (59/1186) of negative screening results at maternal age > or = 35 years in Algorithm A became positive after a modeled 0.1-mm increase in NT, whereas this was only in 1.2% (134/11 369) of tests at maternal age < 35 years (P < 0.0001). The overall increase of screen-positive rate in Algorithm A after an NT modification of +0.1 mm was 1.2% (152/12 555), significantly more than in Algorithm B (86/12 555; 0.7%) (P < 0.0001). CONCLUSION: In Flanders, there is a systematic underestimation of NT in comparison with the FMF reference range. Attempts to change these measurements according to the FMF criteria are crucial. This will mainly influence the screening results of women at advanced maternal age and of NT-based algorithms without the use of other parameters.

Cervical cytological screening and human papillomavirus DNA testing in Flanders.

The causal relationship between genital human papillomavirus (HPV) infection and cervical dysplasia/carcinoma has been recognised for some time. The aim of this study was to document the occurrence and distribution of HPV infection in the five provinces of the Flemish region in Belgium and to correlate the HPV DNA test results with the cytological results on simultaneously performed thin layer preparations of cervical cells. Out of a total screened group of 105107 samples, 1978 samples with cytological abnormalities were tested for HPV DNA using the MY09/MY11 consensus PCR. The mean age of the whole group was 36.9 years. The LSIL group, with a mean age of 33.6 years, was significantly younger than the other groups. There was no significant difference in HPV prevalence among the provinces. In four out of five provinces the HPV prevalence reached 100% in high-grade lesions. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology (17.9% WNL < 51.1% ASCUS < 83.8% LSIL < 97.2% HSIL). Three peaks of HPV DNA positivity were observed, a first at 22 yrs (82%), a second at 47 yrs (60%) and a third in women older than 65 yrs (52%). These results shed more light on HPV prevalence in Flanders and show that the MY09/MY11 consensus primer based detection system is very suitable for the detection of HPV infections in Flanders.

Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool?

AIMS: To test the ability of Ki-67 to detect cytological lesions in a screening setting and its use as a surrogate marker of human papillomavirus (HPV) infection. METHODS: A study of liquid based cytology, HPV DNA testing by MY09/MY11 consensus polymerase chain reaction (PCR), type specific PCRs, and Ki-67 immunocytochemistry on a randomly selected series of 147 patients. RESULTS: Comparison of the number of Ki-67 immunoreactive cells/1000 cells in the different cytological groups showed that the HSIL group yielded a significantly higher mean count than did the other groups. The number of Ki-67 immunoreactive cells/1000 cells was significantly higher in HPV-16 positive samples than in samples containing infections with other high risk types. Receiver operating characteristic curves indicated a test accuracy (area under curve) of 0.68, 0.72, and 0.86 for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL), and high grade squamous intraepithelial lesions (HSIL), respectively. Thresholds for 95% sensitivity were 0.07, 0.08, and 0.15 Ki-67 immunopositive cells/1000 cells for ASCUS, LSIL and HSIL, respectively. The threshold for 95% specificity was 1.9 Ki-67 immunopositive cells/1000 cells. CONCLUSIONS: Ki-67 immunocytochemistry can be applied to liquid based cytology. The accuracy and diagnostic indices of the test are good when compared with those of other techniques. As part of a panel of screening procedures, it could be used as an adjunct to liquid based cytology to identify HSIL, and as a surrogate marker of HPV-16 infection.

Thin-layer liquid-based cervical cytology and PCR for detecting and typing human papillomavirus DNA in Flemish women.

The objective of this study was to document the occurrence and to correlate the prevalence of different human papillomavirus (HPV) types with the cytological results on simultaneously performed thin-layer preparations in a large population of Flemish women. During 1 year, 69 290 thin-layer preparations were interpreted using the Bethesda classification system. Using an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Reflex HPV testing was performed on cytologically abnormal samples and on an age matched randomly selected control group with normal cervical cytology (n=1351). Correlation between cytology, age and prevalence for the 14 different high-risk HPV types is given. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology. Coinfection with multiple HPV types also increased with cytological abnormalities, and was highest in HSIL (16.7%). In Flanders, HSIL was most often associated with HPV types 16, 33, 35, 31, 18 and 51. Using thin-layer liquid-based cytology and PCR to detect HPV, it is feasible to screen large numbers of women.