A prospective study of human papillomavirus (HPV) testing to resolve uncertainty in colposcopy.

OBJECTIVE: UK colposcopy services are seeing increased workloads, a large proportion of which are follow-up appointments. The English Cervical Screening Programme HPV Special Interest Group identified five subcategories of colposcopy clinic patients who often require prolonged follow-up regimes for low-grade abnormalities. Human papillomavirus (HPV) testing has a high negative predictive value, meaning that HPV-negative women are at very low risk of underlying disease. Our objectives were to quantify the number of HPV-negative women in each study subcategory and to evaluate the number who could potentially be discharged from colposcopy on the basis of their results. METHODS: Four colposcopy clinics prospectively identified women according to five categories over 12 months. All women underwent cytological testing and high-risk HPV (hrHPV) testing using the Hybrid Capture 2 test. Management outcomes and decisions based on a knowledge of the HPV status were recorded. RESULTS: Data available on 755 women showed that 422/755 (55.9%) and 260/755 (34.4%) had persistent cervical intraepithelial neoplasia grade 1 (CIN1) (Category 1) or a minor abnormality following treatment (Category 2), respectively. In Categories 1 and 2, 51.7% and 60.2%, respectively, were hrHPV negative. The rates with biopsies of CIN2 or worse (CIN2+) across the two categories were 3/355 (0.8%) and 21/291 (7.0%) for hrHPV-negative and hrHPV-positive women, respectively. CONCLUSION: The incorporation of hrHPV testing within organized cervical screening programmes has been widely accepted. hrHPV testing for the clinical scenarios outlined in this study detects women who are hrHPV negative and therefore at low risk of underlying disease, potentially reducing anxiety and inconvenience for women and costs to colposcopy services.

The volume perspective: a comparison of two excisional treatments for cervical intraepithelial neoplasia (laser versus LLETZ).

OBJECTIVE: To compare two excisional treatments, laser cone biopsy and large loop excision of the transformation zone (LLETZ), in terms of the volume of tissue removed, and to determine the relation between the height and the total volume of the cone specimen. DESIGN: Retrospective cross-sectional study. SETTING: Large teaching hospital in London. POPULATION: A total of 1136 eligible excisional treatments (laser cone or large loop excision of the transformation zone, LLETZ). METHODS: Eligible excisional treatments (laser cone or LLETZ) performed between 1 January 2002 and 31 December 2007 in our colposcopy unit were identified using the Infoflex(R) database. The total volume of the cone biopsy was calculated mathematically using the data provided in the histopathology reports. MAIN OUTCOME MEASURES: The volume of the cone biopsy was compared with the technique of excision and the histology grades. RESULTS: Three hundred and thirty-nine laser cone biopsies were performed, whereas 797 LLETZ biopsies were recorded, during the study period. There was no difference in the mean age in the two groups. However, there is a proportional increase in the volume of the cone as the height of the cone increases, and a significant number of the values are skewed, suggesting that the diameter of the base of the cone contributes significantly to the total volume. Laser cone biopsies (median volume 1.84 cm(3), 95% CI 1.98-2.54 cm(3)) account for a larger volume of tissue excised compared with LLETZ (median volume 0.78 cm(3), 95% CI 0.91-1.02 cm(3)) (P < 0.0001). This relationship is not altered when the two procedures are stratified for grade of lesion, i.e. excision for low-grade cervical intraepithelial neoplasia (CIN) (laser median volume 1.55 cm(3), 95% CI 1.46-2.06; LLETZ median volume 0.62 cm(3), 95% CI 0.73-0.88 cm(3)) (P < 0.0001) or high-grade CIN (laser median volume 1.84 cm(3), 95% CI 2.11-2.53 cm(3); LLETZ median volume 0.82 cm(3), 95% CI 0.94-1.07 cm(3)) (P < 0.0001). CONCLUSIONS: The volume of cervical tissue removed during laser conisation is significantly more than that removed with LLETZ. The indication of the cone biopsy does influence the volume of tissue removed.

HPV testing as an adjunct to cytology in the follow up of women treated for cervical intraepithelial neoplasia.

OBJECTIVE: To evaluate human papillomavirus (HPV) testing in combination with cytology in the follow up of treated women. DESIGN: A prospective study. SETTING: Three UK centres: Manchester, Aberdeen and London. POPULATION OR SAMPLE: Women treated for cervical intraepithelial neoplasia (CIN). METHODS: Women were recruited at 6 months of follow up, and cytology and HPV testing was carried out at 6 and 12 months. If either or both results were positive, colposcopy and if appropriate, a biopsy and retreatment was performed. At 24 months, cytology alone was performed. MAIN OUTCOME MEASURES: Cytology and histology at 6, 12 and 24 months. RESULTS: Nine hundred and seventeen women were recruited at 6 months of follow up, with 778 (85%) and 707 (77.1%) being recruited at 12 and 24 months, respectively. At recruitment, 700 women had had high-grade CIN (grades 2 or 3) and 217 had CIN1. At 6 months, 14.6% were HPV positive and 10.7% had non-negative cytology. Of those with negative cytology, 9% were HPV positive. Of the 744 women who were cytology negative/HPV negative at baseline, 3 women with CIN2, 1 with CIN3, 1 with cancer and 1 with vaginal intraepithelial neoplasia (VAIN)1 were identified at 24 months. Nine of 10 cases of CIN3/cervical glandular intraepithelial neoplasia (CGIN) occurred in HPV-positive women. At 23 months, cancer was identified in a woman treated for CGIN with clear resection margins, who had been cytology negative/HPV negative at both 6 and 12 months. CONCLUSIONS: Women who are cytology negative and HPV negative at 6 months after treatment for CIN can safely be returned to 3-year recall.

Quality control assurance in national screening programmes for cervical cancer.

The United Kingdom established a centrally controlled formal screening programme for cervical cancer in 1988. The programme has been an unprecedented success, with the incidence of cervical cancer falling by an accelerated rate of 7% a year. Underlying the success of the programme is a rigorous system of quality assurance at all levels of activity. Quality assurance assessment in performed for coverage of the target population, cytology reporting laboratories and colposcopy services. Each component in the system is examined annually by mandatory returns, and by regular on-site review of the clinical services by independent authorities. Processes also exist to establish and maintain the competency of all clinical and non-clinical staff involved in the programme. Using nationally published figures from the financial year 2001/2002, the different quality assurance strategies are described. The future development of quality assurance in the programme is then discussed.

The in vitro hydrodynamic characteristics of the porcine pulmonary valve and root with regard to the ross procedure.

OBJECTIVE: The hydrodynamic parameters and leaflet motion of the porcine pulmonary root and valve and the performance of the pulmonary autograft implanted in subcoronary position or as a free-standing root were investigated at systemic and pulmonary pressures in vitro. METHODS: Ten fresh pulmonary and aortic roots (anulus diameter, 20-25 mm) were tested in a pulsatile flow simulator. Five free-sewn pulmonary valves were implanted in aortic roots in the subcoronary position, and 5 pulmonary roots were implanted as free-standing roots. The external diameter of the roots was measured at the sinotubular junction in a pressure range of 0 to 120 mm Hg. The transvalvular gradient and regurgitation were measured, and the effective orifice area was calculated. The leaflet motion was recorded on video tape. RESULTS: The fresh pulmonary roots were more compliant than their aortic counterparts (33% +/- 3. 0% vs 7% +/- 1.5% with dilatation at 0-30 mm Hg and 46% +/- 8.4% vs 35% +/- 7.8% with dilatation at 0-120 mm Hg). The pulmonary roots had a lower pressure drop at systemic than at pulmonary pressures. The pressure drops of the pulmonary roots were also lower than those of the aortic roots in the systemic pressure range. The leaflet opening of the pulmonary valve was triangular, with low bending deformation at all pressures. Implanting the free-sewn pulmonary valve in the subcoronary position or the pulmonary root as a free-standing root did not affect the hydrodynamic parameters and leaflet motion adversely. CONCLUSION: The pulmonary valve and root could easily withstand aortic pressures in vitro. A biphasic dilatation curve ensures that higher pressures did not overdilate the pulmonary root. Moreover, valve performance was better at systemic pressures.

The effect of sizing on the hydrodynamic parameters of the Medtronic freestyle valve in vitro.

BACKGROUND: An in vitro model has been established to investigate the effect of sizing on the hydrodynamic characteristics and leaflet motion of the Medtronic Freestyle valve. METHODS: The valves were tested in fresh porcine aortic roots. Two or three different sizes of valves were implanted in the same aortic root one after the other. The compliance of the fresh aortic and the composite roots was measured in the pressure range of 0 to 120 mm Hg, and the composite roots were then tested in a pulsatile flow simulator. The transvalvular gradient and degree of regurgitation were measured and the effective orifice area and performance index were calculated. Leaflet motion was recorded on video. RESULTS: The fresh aortic roots dilated by average 39.4% as the hydrostatic pressure rose from 0 to 120 mm Hg. Implantation of the Medtronic Freestyle valve did not change the distensibility of the aortic root significantly. The sizing protocol did not affect significantly the hydrodynamic performance. However, a significantly lower open leaflet bending deformation was found in the undersized valves. Regurgitation was found only at 2-mm undersized valves. CONCLUSIONS: Leaflet motion of the Medtronic Freestyle valve in vitro was best if 1 mm undersized, and this may be beneficial to long-term durability.

Distribution of hepatic venous blood in the total cavo-pulmonary connection: an in vitro study.

OBJECTIVES: The objective of this project was to quantify the effects of geometry on the distribution of hepatic blood to the lungs in patients with a total cavo-pulmonary connection. The basis for this work is the supposition that hepatic blood is necessary for proper lung function. METHODS: Plastic models of these connections were made with varying degrees of offset between the inferior and superior vena cava and attached to an in vitro flow loop. Dye was injected into the inferior vena cava and its concentration quantified in each pulmonary artery. These data were converted to percentage concentration and distribution of hepatic blood to each lung. RESULTS: With no offset between the vena cava, hepatic blood distribution and concentration to each lung was similar to normal. For an offset of one or more diameters, hepatic blood tended to flow preferentially towards the nearest pulmonary artery with the opposite pulmonary artery exhibiting a deficit (<10% of normal). CONCLUSIONS: Distribution of hepatic blood to each lung was found to be a function of vena cava offset and pulmonary artery flow split. Under normal conditions, 60% of blood towards the right pulmonary artery, the hepatic blood distribution to both lungs could be maintained above 50% of normal if the inferior vena cava was offset towards the left pulmonary artery. Offsetting the inferior vena cava towards the right pulmonary artery jeopardized the delivery of hepatic blood to one lung.

The influence of size mismatch on the hemodynamic performance of the pulmonary autograft in vitro.

OBJECTIVES: We established an in vitro model to investigate the effect of size mismatch between the aortic and pulmonary root on the hydrodynamic performance and leaflet motion of the pulmonary autograft. METHODS: Ten fresh porcine pulmonary roots (annulus diameter: 19-25 mm) were tested in a pulsatile flow simulator. The autografts then were implanted in fresh porcine aortic roots (annulus diameter: 19-30 mm) and retested in the flow simulator. Three roots were oversized by 21-39%, three were undersized by 32-45% and there were four size for size implantations. The external diameter of the roots and autografts was measured at the sinotubular junction at hydrostatic pressures of 0 - 120 mmHg. The transvalvular gradient and regurgitation were also measured and the effective orifice area was calculated. The leaflet motion was recorded on video. RESULTS: The fresh pulmonary roots were more compliant than the fresh aortic roots (46 +/- 8.4% vs. 35 +/- 7.8% dilatation from 0 to 120 mmHg). The group of matching size autografts dilated by 43 +/- 4.9% in the same pressure range. The external diameter of the undersized autografts was 10 +/- 2.1% bigger than before implantation at 0 pressure and then the dilatation was 40 +/- 5.3% at 120 mmHg. The oversized implantation made the autografts 11 +/- 9.4% smaller in their relaxed state, but then they dilated by 65 +/- 11% as the pressure increased to 120 mmHg, resulting in a net dilatation of 54% over the original undilated state. The under or oversizing had little effect on the pressure gradient measured across the valves (5.6 +/- 2.57 mmHg before, 6.3 +/- 3.27 mmHg after implantation). Only the oversized valves showed significantly higher gradients than the native pulmonary valves. The effective orifice area of the undersized autografts was slightly bigger and the oversized autografts was slightly smaller after implantation, although the differences were not significant. The size mismatch did not cause regurgitation on the valves. The video images showed very low-open leaflet-bending deformation, both on the fresh pulmonary and the autograft valves. CONCLUSION: Under or oversizing the pulmonary autograft up to 40% of the annulus diameter did not affect the hydrodynamic parameters significantly. The compliance of the autograft root was able to compensate for the size mismatch without adversely influencing the valve performance.

The effect of sizing on the in vitro hydrodynamic characteristics and leaflet motion of the Toronto SPV stentless valve.

OBJECTIVES: We established an in vitro model to investigate the effects of valve sizing on the hemodynamic characteristics and leaflet motion of the Toronto SPV valve (St Jude Medical, Inc, St Paul, Minn). METHODS: Nine valves were first implanted in fresh porcine aortic roots and then retested in glutaraldehyde-treated porcine aortic roots. Three valves were 1- to 2-mm oversized, 3 were 1- to 2-mm undersized, and there were 3 size-for-size implantations. The elasticities of the aortic roots and the composite roots were measured in the pressure range between 0 and 120 mm Hg, and the composite roots were then tested in a pulsatile flow simulator. The transvalvular gradient and regurgitation were measured and the effective orifice area and performance index were calculated for each root. Leaflet motion was recorded on videotape. RESULTS: The external diameter of the fresh root increased by 35% as the hydrostatic pressure rose from 0 to 120 mm Hg, as compared with 11% for the glutaraldehyde-treated root. Valve implantation in the fresh root reduced the distensibility to 22% but did not change distensibility in the glutaraldehyde-treated root. The effective orifice area was dependent on the valve size, with the transvalvular gradient decreasing as the valve size increased. For the same size of valve the hydrodynamic parameters were slightly better if the valve was undersized by 1 mm. A significant difference in favor of the undersized valves was found in open-leaflet bending deformation. CONCLUSION: Leaflet motion of the stentless porcine aortic valve in vitro is improved if the valve is slightly undersized, and this may be beneficial to the long-term durability of the prosthesis.

Quantification of mitral regurgitation with MR phase-velocity mapping using a control volume method.

Reliable diagnosis and quantification of mitral regurgitation are important for patient management and for optimizing the time for surgery. Previous methods have often provided suboptimal results. The aim of this in vitro study was to evaluate MR phase-velocity mapping in quantifying the mitral regurgitant volume (MRV) using a control volume (CV) method. A number of contiguous slices were acquired with all three velocity components measured. A CV was then selected, encompassing the regurgitant orifice. Mass conservation dictates that the net inflow into the CV should be equal to the regurgitant flow. Results showed that a CV, the boundary voxels of which excluded the region of flow acceleration and aliasing at the orifice, provided accurate measurements of the regurgitant flow. A smaller CV provided erroneous results because of flow acceleration and velocity aliasing close to the orifice. A large CV generally provided inaccurate results because of reduced velocity sensitivity far from the orifice. Aortic outflow, orifice shape, and valve geometry did not affect the accuracy of the CV measurements. The CV method is a promising approach to the problem of quantification of the MRV.

Relation between human papillomavirus type 16 and potential for progression of minor-grade cervical disease.

We have previously reported that among 200 women referred for colposcopy with smears suggesting mild dyskaryosis, medium or high copy numbers of human papillomavirus type 16 (HPV16) DNA identified patients with current high-grade cervical disease. We have followed up 95 women from that group who had histologically proven mild-grade cervical disease (cervical intraepithelial neoplasia grade 1, n = 37) or wart virus infection (n = 12) or who had no evidence of cervical abnormality at study entry (n = 43). Kaplan-Meier survival analysis of the 70 months' follow-up was used to identify baseline features that might affect the risk of progression. 3 women were lost to follow-up; data were available for the remaining 92. Among the whole group the probability of remaining free of high-grade cervical disease was 0.71. Women with a histological diagnosis of minor-grade disease were more likely to progress to high-grade disease than those with no evidence of abnormality (proportion disease-free 0.52 vs 0.90, p = 0.004). Stratification of the group according to median age (28 years) revealed a weak association between age and disease progression (p = 0.04). There was no difference in disease-free probability between HPV16-positive and HPV16-negative women (0.75 vs 0.65, p = 0.19). Nor was there a significant difference in disease-free probability when the group was stratified by HPV16 viral burden. These data show that a histological diagnosis of minor-grade cervical disease is a better long-term predictor of disease progression than is HPV16 positivity, irrespective of copy number. These findings do not support the simple view that HPV16 alone is the cause of high-grade cervical disease, including cancer.

Sequence microheterogeneity in the long control region of clinical isolates of human papillomavirus type 16.

The polymerase chain reaction (PCR) has been used to amplify the long control region (LCR) of episomal human papillomavirus type 16 from cervical scrape DNA obtained from a woman with no evidence of cervical disease and a woman with cervical intraepithelial neoplasia grade 3 (CIN 3). An 883 base pair fragment containing the entire LCR was cloned into pUC13 and the DNA sequence determined for both isolates and compared with the prototype HPV type 16 LCR DNA sequence. Nucleotide variation was apparent in the LCRs derived from both women. In the case of the sample derived from the woman with no cervical disease, there were three nucleotide deletions, one insertion, four transversions, and three transitions (overall conservation: 98.7%). In contrast, the LCR derived from the woman with CIN 3 showed significantly more nucleotide variation with two nucleotide deletions, one insertion, nine transversions, and ten nucleotide transitions (overall conservation 97.6%). Using computer analyses coupled with available data from DNA footprint studies, the effects of these sequence variations on established transcription factor binding sequences were investigated. Cloning of the LCR derived from the woman without cervical disease into a chloramphenicol acetyl transferase (CAT) promoter screening vector, followed by transfection of HeLa cells with the LCR-CAT construct, revealed that the LCR was a functional promoter but was 4.6-fold less active than an equivalent SV40 early promoter-CAT construct.

Use of semi-quantitative PCR for human papillomavirus DNA type 16 to identify women with high grade cervical disease in a population presenting with a mildly dyskaryotic smear report.

The aim of this study was to assess whether qualitative or semi-quantitative detection of human papillomavirus type 16 (HPV 16) can help to identify women with major grade cervical intraepithelial neoplasia (CIN 2 and CIN 3) among those referred with a smear suggesting mild dyskaryosis. The study population consisted of 200 women sequentially attending the Royal Free Hospital colposcopy clinic. All women were investigated by cytology, colposcopy and, where appropriate, histopathology, and HPV 16 DNA was detected in cervical scrape samples using the polymerase chain reaction (PCR). A final clinical diagnosis of normal, wart virus infected (WVI), CIN 1, CIN 2 or CIN 3 was made in 179 women. On the basis of the qualitative PCR data, the presence of HPV 16 DNA was of borderline use in identifying women with high grade cervical disease [63/113 (normal/WVI/CIN 1) vs 46/66 (CIN 2/CIN 3); P = 0.065]. However, semi-quantitative PCR analysis showed that a high/medium HPV 16 result was significantly associated with high-grade disease [29/113 (normal/WVI/CIN 1) vs 38/66 (CIN 2/CIN 3); P = 0.0001]. Furthermore, semi-quantitative PCR and cytology were performed on the repeat smear taken immediately prior to colposcopy. The combined laboratory results show that 53/60 women with biopsy proven high-grade disease were identified, as were 26/95 women who were either normal or who had low grade cervical disease. The possibility of using such an approach for selecting women for more rapid or for routine colposcopy appointments in the two groups respectively is discussed.

Comparison of cervical cytology and the polymerase chain reaction for HPV 16 to identify women with cervical disease in a general practice population.

A comparison of the ability of cervical cytology and the polymerase chain reaction (PCR) for human papilloma virus type 16 (HPV 16) to identify women with cervical disease has been performed in a general practice population of 249 women, none of whom were believed to have current cervical disease prior to examination. Within this population, 29 women were found by colposcopy and subsequent histopathology to have evidence of cervical disease [5 with cervical intraepithelial neoplasia (CIN) 3; 8 with CIN 2; and 16 with CIN 1]. The prevalence of HPV 16 in this population was 18.9% (CIN 3, 80%; CIN 2, 50%, CIN 1, 12.5%, normal, 16.8%). Women with severe disease (CIN 2 and CIN 3) had a significantly higher incidence of HPV 16 DNA than those with mild cervical disease (CIN 1) or no cervical abnormality (P = 0.001). There was no significant difference in the ability of either PCR for HPV 16 or cytology to identify women with cervical disease. The combination of screening by cytology and the presence of HPV 16 DNA resulted in the identification of a higher proportion of the women with disease, but this observation did not reach statistical significance. Although the failure to detect disease by the two screening methods was similar, HPV 16 DNA positivity was associated with a higher false-positive rate for disease detection than cytology (P less than 0.03). The PCR assay for detecting HPV 16 in this investigation was shown to have a false-positive rate of 2.4% and a false-negative rate of 10.4%. The prospect of screening women for cervical disease using PCR for HPV 16 is discussed.

In vitro pulsatile flow hemodynamics of five mechanical aortic heart valve prostheses.

In vitro measurements of velocity, turbulent shear stress, effective orifice area (EOA), and regurgitant fraction were performed on five new-generation low-profile mechanical aortic heart valve designs under pulsatile flow conditions. These were: Medtronic-Hall tilting disc, St. Jude Medical bileaflet, Björk-Shiley Monostrut tilting disc, Omni-Carbon tilting disc, and Duromedics bileaflet. In general, bileaflet valves have larger EOAs than the tilting disc design, especially in the larger sizes, due to the larger opening angles and lack of obstructive struts. The regurgitant fractions range from 8% for 21-mm valves to 13% for the 29-mm sizes. This increase was largely due to an increase in leakage volume as opposed to closing volume. Furthermore, the leakage volumes increased as the mean aortic pressures increased. The tilting disc valves generally have better regurgitant characteristics compared to the bileaflet valve designs, due to lower leakage volumes and to the smaller opening angle of the occluder providing a more rapid closure of the valve. The velocity and shear stress measurements showed that none of the current valve designs are ideal: all designs create areas of stasis and/or regions of low-velocity reverse flow and regions of elevated turbulent shear stresses capable of causing sublethal and/or lethal damage to the formed elements of blood. It is therefore unlikely that these valve designs will eliminate the problems of hemolysis, thrombosis, and thromboembolic complications.

Treatment of cervical intraepithelial neoplasia (CIN) by radical electrocoagulation diathermy: 5 years' experience.

During the first 5 years of a colposcopy clinic established in a London teaching hospital 653 new patients were seen, usually referred because they had had an abnormal cervical smear report suggesting cervical intraepithelial neoplasia (CIN). Of 490 women treated within the district for CIN, 361 (73.7%) underwent radical electrocoagulation diathermy, 91 (18.6%) were treated by cone biopsy, 34 (6.9%) underwent hysterectomy and the other four patients were treated by cryocautery. Of the 245 women treated by radical electrocoagulation diathermy who have been followed for at least 12 months, persistent or recurrent disease has been found in only 15, a first time treatment success rate of 93.9%.

Human papillomavirus types 6 and 16 in multifocal intraepithelial neoplasias of the female lower genital tract.

Five women with multifocal intraepithelial neoplasia of the lower genital tract were investigated for the presence of human papillomavirus (HPV) infection by the method of DNA-DNA hybridization which detects the viral DNA. The DNA sequences of HPV types 6 and 16 were detected in each of the five patients and in each of the areas biopsied: cervix, vagina and vulva. DNA sequences of both viral types were also found in vulval intraepithelial neoplasia grades I-III and in cervical intraepithelial neoplasia grades I and III. The detection of HPV DNAs in multifocal lesions suggests a possible common aetiology for the lower genital tract intraepithelial neoplasias.