Performance, Validity, Reliability, and Quality Assurance for Blot Typing of Human Papillomavirus DNA in Cervical Swab Samples
Date Issued
2007
Date
2007
Author(s)
Lin, Ching-Yu
DOI
zh-TW
Abstract
The aim of this study was to evaluate the performance, validity and reliability of the EasyChip® HPV Blot for Human papillomavirus (HPV) DNA genotyping in cervical swab samples, and assured the quality of HPV typing in large-scale community-based cervical neoplasia screening by conducting a quality assurance system. The performance of HPV Blot was assessed by analytic sensitivity、analytic specificity and sensitivity. The analytic sensitivity and specificity of HPV Blot was assessed by using 39 HPV genotype plasmids, and assessed sensitivity by using cell lines and cervical specimens, respectively. The 39 types of HPV (HPV 6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 72, 74, 82, CP8061, CP8304, L1AE5, MM4, MM7, and MM8) were assessed using plasmids DNA containing L1 region of HPV genome. Each plasmid was considered as a genome equivalent (geq). A clinical trial was conducted to assess the validity and reliability of HPV Blot. The gold standard of trial was 20 L1 type-specific parallel PCR. The assessment of validity was sensitivity, specificity and accuracy. The assessment of reliability was McNemar’s Test and Kappa statistic. The way of assessment was by subject and by type, respectively. The quality assurance system includes the maintenance of operating environment, assessment of reliability, assessment of reproducibility, and blot interpretation. The quality assurance standards include the assessment of seven extrinsic controls in operation and three intrinsic controls of HPV Blot in genotypes identification. Each batch experiment contained samples from 89 cervical specimens and seven extrinsic controls. The seven extrinsic controls were Caski cells, HeLa cells, Jurkat cells, male human blood cell DNA, sterile water, and paired sibling controls. The replicated aliquots of the same sample were a pair of sibling controls. Caski cells, HeLa cells, Jurkat cells and sibling controls were used simultaneously to assess entire procedures inclusive of DNA extraction, PCR, and HPV typing. Male human blood cell DNA and sterile water were used only in the assessment of PCR and HPV typing. One hundred batch experiments were conducted. Jurkat cells and male human blood DNA were used as an index of potential contamination with exogenous HPV sequences. Sterile water was used as an index of potential contamination with any exogenous DNA sequences.
The detection limits for analytical sensitivities of HPV Blot are 1 geq per PCR for HPV 16, 10 geq per PCR for HPV 6, 26, 31, 33, 37, 39, 43, 44, 45, 54, 55, 61, 67, 68, 69, 82, CP8061, CP8304, L1AE5, MM7 and MM8, 20 geq per PCR for HPV 11, 18, 32, 35, 42, 51, 52, 53, 56, 58, 59, 66, 70, 74 and MM4, and 50 geq per PCR for HPV 62 and 72. The overall analytical sensitivity of HPV detection is 1 to 50 copies of HPV geq for each sample. The analytic specificity of the HPV Blot is each of 39 HPV genotype probes that had no cross-reactivity with amplicons of other HPV genotypes. In cell lines, the detection limits for Caski and HeLa cells were 10–4 and 10–3 ng DNA, respectively, by the HPV Blot and 10–3 and 10–2 ng DNA, respectively, by the gel. The calculated copies are 1~10 for HPV 16 and 2~8 for HPV 18. Both copy numbers were expected on the basis of known HPV 16 and HPV 18 copy numbers in Caski cells (60–600 copies per cell) and HeLa cells (10–50 copies per cell). About 10 copies of HPV 16 and 18 viruses within cervical specimens were detected.
The sensitivity, specificity and accuracy of HPV Blot by subject are 97, 96 and 96.5%, respectively. The McNemar’s Test、Kappa and P value of HPV Blot by subject are 0.25, 0.93 and 0.62, respectively. The sensitivity, specificity and accuracy of HPV Blot by type are 90, 92 and 90.6%, respectively. The McNemar’s Test、Kappa and P value of HPV Blot by type are 2.81, 0.81 and 0.10, respectively. The comparison of HPV Blot and gold standard suggests that is excellent agreement. The sensitivity of HPV Blot by type is 100% for HPV 6, 11, 31, 35, 45, 59 and 66, 99~95% for HPV 16, 18, 33 and 58, 94~90% for HPV 51, 52, 53 and 70, 89~80% for HPV 39, 56 and 68, and less than 79% for HPV 62 and CP8061. The specificity of HPV Blot by type is 100% for HPV 6, 11, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 62, 66, 68, 70 and CP8061, and 99~95% for HPV 16. The accuracy of HPV Blot by type is 100% for HPV 6, 11, 35, 45, 59 and 66, and 99~95% for HPV 16, 18, 31, 33, 39, 51, 52, 53, 56, 58, 62, 68, 70 and CP8061. The type-specific agreement of HPV Blot is perfect (Kappa=1) for HPV 6, 11, 35, 45 and 59, excellent (0.99 < Kappa < 0.81) for HPV 16, 18, 31, 33, 39, 51, 52, 53, 58, 66, 68, 70, 62 and 56, good (0.8 < Kappa < 0.61) for HPV CP8061.
All experiments obeyed 4 criteria of quality assurance system as follows. Thus, all three HPV-negative extrinsic controls were HPV negative in 100 batch experiments. In other words, there was no HPV false-positive result. The HPV typing results for Caski, HeLa, and Jurkat cells indicated that the HPV Blot had 100% sensitivity and specificity for HPV 16, HPV 18, and GAPDH at a scale of 2000 cells. That five extrinsic controls met the requirements of quality assurance system in 100 batch experiments indicate that the HPV Blot had excellent reliability in operation. Intra-batch and inter-batch reproducibility of the HPV Blot were 98% and 97%, respectively. Reader identified three intrinsic controls of HPV Blot, so typing results of HPV Blot were confirmed. In the study, the qualities of identification were qualified.
In conclusion, the HPV Blot is a highly sensitive, accurate, reliable and reproducible tool for clinical and epidemiological studies on HPV infection. This study is conducted a quality assurance system comprising seven extrinsic controls and three intrinsic controls for HPV typing, which has been validated as an appropriate criterion for experiments and laboratory operation. These findings confirm that the HPV Blot is of great value for clinical HPV typing, follow-up of type-specific HPV persistence and development of type-specific HPV vaccines.
Subjects
人類乳突瘤病毒
效能
效度
信度
敏感度
特異性
正確性
品質保證
Human papillomavirus
Performance
Validity
Reliability
Sensitivity
Specificity
Accuracy
Quality Assurance
SDGs
Type
thesis
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