宫颈脱落细胞DNA倍体分析用于宫颈内瘤变筛查的价值探讨
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摘要
目的:
第一部分:观察手动与自动两种切片方法和组织种类对实测切片厚度的影响,为组织、细胞的形态结构和化学物质的定位与定性观察及定量分析结果的评价提供参考。
第二部分:
(1)应用细胞学检查、病理学诊断、细胞核DNA倍体分析和第二代杂交捕获试验方法检测HPV-DNA,并进行对比分析,为临床早期筛查宫颈癌及癌前病变提供一种快速而有效的辅助诊断的方法。
(2)有助于筛查出细胞学诊断不明确病例中有恶变潜能的风险人群,并为宫颈癌及癌前病变发展方向的预测、治疗效果及病变预后的评估提供更多有价值的肿瘤生物学信息。
方法:
第一部分:选取5只成年健康雄性SD大鼠的肝、胰腺、胃及肺4种组织,各组织单独包埋制成蜡块,预设电动切片机的切片厚度刻度值为3、5、7、10μm,分别作自动和手动两种方法切片,所获得的薄组织片经二次包埋、切片、HE染色,TIGER图像分析仪测量切片的实际厚度。
第二部分:对进行常规宫颈脱落细胞学普查的370例妇女,用宫颈刷取材,液基薄层制片备用。涂片分两组进行实验:其中一组采用巴氏法染色做常规细胞学检查;另一组经改良Feulgen法染色,进行细胞核DNA含量测量与倍体分析。对常规细胞学检查有异常的病例进行病理组织学检查。对检测到感染了高危型人乳头瘤状病毒(HR-HPV)的病例,将其检查结果与DNA倍体分析结果结合宫颈病变级别与年龄因素进行分析比较。
结果:
第一部分:
(1)手动和自动切片对组织切片的实测厚度有影响,组织种类对组织切片的实测厚度无影响。
(2)各组织切片的厚度测量均值均较切片机预设厚度值大,同一切片机预设厚度值条件下,自动切片的实测厚度均值均小于手动切片,且更接近切片机预设刻度值。
(3)无论手动或是自动切片的实测厚度均随着切片机预设切片厚度的增大而呈正比增加,其相关系数r均大于0.9830。
(4)自动和手动切片的实测厚度分别比切片机预设切片厚度要厚29-50%和48-91%,自动切片与手动切片的切片厚度均值相差25%,二者相对误差为61%。
(5)相同组织的组织切片实测厚度的极端离散趋势(极差)随切片机预设厚度值的增加而呈现上升趋势,且手动切片组的极差大于自动切片组。
第二部分:
(1) 370例受诊者经常规宫颈细胞学检查发现其中有362例(97.7%)异常:包括ASCUS 140例,占患病总数的37.8%;LSIL 190例,占51.4%;HSIL 31例,占8.4%;病理医生镜下观察诊断为:CINⅠ179例,占总患病例的48.4%;CINⅡ49例,占13.2%;经DNA倍体分析,370例样本其中258例(69.7%)为异常。以病理诊断结果为金标准,细胞核DNA倍体分析筛查CINⅡ以上宫颈病变的灵敏度为98.6%,而常规细胞学方法则为72.2%。
(2) 370例患者中有191例进行了HPV检测,其中有高危型HPV(HR-HPV)感染81例,将HPV检查结果与DNA倍体分析结果结合宫颈病变级别作分析比较,二者阳性检出率均与宫颈病变的程度呈正相关;大部分感染病例能检测到非整倍体。
(3) 191例有HPV检测结果的病例,以病理诊断结果为金标准计算,细胞核DNA倍体分析筛查CINⅡ以上宫颈病变的灵敏度为84.4%,而HR-HPV检测方法则为75.6%。
(4)不同年龄组间的高危型HPV检出率明显有差异,年轻组高于中老年组。而非整倍体的检出结果不受患者年龄因素的影响。
(5)细胞学诊断不明确的ASCUS病例,感染了HR-HPV且有异倍体的患者中,≥CINⅡ的检出率最高,未感染HR-HPV且未能检出异倍体患者中,无一例CINⅠ以上的病例。
结论:
第一部分:自动切片与手动切片的切片厚度相差25%,二者相对误差为61%,自动切片获得的组织切片的厚度精度较高,优于手动切片。
第二部分:
(1)宫颈脱落细胞DNA倍体分析较细胞学检查用于宫颈疾病筛查时灵敏度更高,细胞学检查结合DNA倍体分析是癌前病变和宫颈癌筛查的有效方法。
(2) HR-HPV感染率提高以及年轻患者HR-HPV感染阳性率较中老年高,是宫颈癌年轻化的两个重要因素。
(3) DNA异倍体检测结果在宫颈癌的不同发病年龄之间无差别,而HR-HPV阳性率在不同年龄之间有差别,所有HR-HPV感染病例中发现大部分有异倍体,而有异倍体病例中仅部分有HR-HPV感染。DNA倍体分析法较HR-HPV检测法灵敏度更高。
(4) HR-HPV感染随着细胞学或病理学检测宫颈病变级别的增高而增高。
(5)应用病理学检查、HR-HPV检查、细胞核DNA倍体分析三联法,为细胞学诊断不明确的ASCUS病例的鉴别及确诊提供参考,感染了HR-HPVH有异倍体的患者中,≥CINⅡ的检出率最高,未感染HR-HPV且未能检出异倍体患者中,无一例CINⅠ以上的病例。
Purposes:
Part one:To observe the effect of automatic and manual slicing methods and tissue types on the thickness variation of paraffin-embedded sections to provide a reference for observing the organization or the cell's morphology and the position or composition of chemical substances and also for the evaluation of the quantitative analysis results.
Part two:
(1) Application of cytology,pathology diagnosis,nuclear DNA ploidy analysis and second-generation hybrid capture test used to detect HPV-DNA,and make comparative analysis.To provide an effective and volant adjuvant diagnostic method for cervical cancer and cervical dysplasia screening.
(2) To provide an effective screening method for atypical squamous cells of undetermined significance cases which have the potential risk of malignant transformation,and to provide much more evaluating information for prognosis and evaluation of therapeutic efficacy.
Methods:
Part one:Liver,pancreas,stomach and lung samples were obtained from five healthy adult male rats.Each tissue was separately embedded in paraffin.Slices were cut automatically and manually at various thickness set points,3μm,5μm,7μm,10μm,by an automatic microtome.After the re-embedding vertical slicing and HE staining,the thickness of these slices was measured by a TIGER image analysis system.
Part two:Each of cervical samples from the women who accepted the conventional cast cell screening was made into liquid-based smear sample.The smear were separated into two groups for the experiment.One group were stained by papanicoloau stain to make conventional cytology screening.The other group were stained by Feulgen method and was analyzed by nuclear DNA ploidy analysis system.Chose the samples which were regarded as doubtful cases diagnosed by cytologic analysis, these samples were delivered to the pathohistology.The results of samples which were infected with human papillomavirus(HPV) were compared with those of DNA ploidy analysis in combination with the level of cervical lesions and age factor.
Results:
Part one:
(1) Only the automatic or manual slicing procedure exerted effect on the slice thickness.
(2) The mean measured thickness of each tissue section was bigger than the microtome set point.On condition of the same set point,the mean measured thickness of automatic section was smaller than manual section.
(3) No matter automatic or manual slice,the measured thickness has a direct ratio tendency to bigger along with the microtome set point become bigger.
(4) The thickness of auto-slice and manual slice were 29%～50%and 48%～91%, respectively,thicker than the nominal thickness set on the microtome.There is a 25%difference between the thicknesses of automatically cut slices and manually cut slices.The relative error rate of the two slicing methods is 61%.
(5) Dispersion tendency of the measured thickness of the same kind of tissue had an escalating trend along with the microtome set point become bigger,the rang was bigger when cut manual than cut automatic.
Part two:
(1) 362 abnormal cases from 370 doubtful cases were suspected ones,including 140 cases of atypical squamous intraepithelial undermined significance(ASCUS),190 cases of low-grade squamous intraepithelial lesion(L-SIL) and 31 eases of highgrade squamous intraepithelial lesion(H-SIL),were diagnosised by conventional cytology examination.23 eases of carcinoma in situ(CINⅢ) and 49 CINⅡwere ??detected among 370 women by pathohistology examination.258 abnormal cases were diagnosised by DNA image analysis.According to the criterion of pathohistology diagnosis,it can say that the sensitivity of DNA ploidy analysis was 98.6%,and sensitivity of conventional cytology was 72.2%.
(2) 191 cases in 370 patients made the HPV test,in which 81 cases were infected HR-HPV.Comparing the results of HPV detection with those of DNA ploidy analysis,there were a correlation between their positive detection rates and degree of dysplasia.Almost all cases with infection of high-risk human papillomavirus (HR-HPV) were aneuploidy.
(3) HPV test was made in 191 cases,calculated by the pathology diagnosis as the gold-standard,the sensitivity of nuclear DNA ploidy analysis in screening for cervical lesions higher than CINⅡwas 84.4%,while the HR-HPV detection was 75.6%.
(4) There was a directly correlation between the infection rate of HR-HPV and the age of patients.Youth group was higher than the older group.While the prevalence of aneuploidy was not influenced by the age of patients.
(5) Cytology diagnosed as uncertain cases such as ASCUS,the patients who had infected HR-HPV and aneuploid were tested positive,the detection rate was highest in equal or higher than CINⅡgroup.On the reverse side,no cases was higher than CINⅠ.
Conclusions:
Part one:There is a 25%difference between the thicknesses of automatically cut slices and manually cut slices.The relative error rate of the two slicing methods is 61%.The precision of automatic cut thickness is higher,therefore,is better than manual sectioning.
Part two:
(1) Nuclear DNA ploidy analysis is better for screening precancerous and carcinoma of the uterine cervix than conventional cytology.Provide an effective and volant adjuvant diagnostic method for cervical cancer screening.
(2) HR-HPV infection rate is increased as well as the HR-HPV positive detection rates of the youth group is higher than in middle-aged group,make important part in younger-cervical-cancer.
(3) Aneuploidy test results have no difference between the patient's vary age,while the positive of HR-HPV make the sense.Most of infected cases can find the aneuploidy,while there are only part of aneuploidy cases have infected HR-HPV. The sensitivity of DNA ploidy analysis is higher than the HR-HPV test.
(4) HR-HPV infection rate is increased as the cytology or pathology detection of cervical lesion become higher.
(5) Make reference for the differentiate and diagnosis of the cytologic uncertain cases such as ASCUS.
第一部分:观察手动与自动两种切片方法和组织种类对实测切片厚度的影响,为组织、细胞的形态结构和化学物质的定位与定性观察及定量分析结果的评价提供参考。
第二部分:
(1)应用细胞学检查、病理学诊断、细胞核DNA倍体分析和第二代杂交捕获试验方法检测HPV-DNA,并进行对比分析,为临床早期筛查宫颈癌及癌前病变提供一种快速而有效的辅助诊断的方法。
(2)有助于筛查出细胞学诊断不明确病例中有恶变潜能的风险人群,并为宫颈癌及癌前病变发展方向的预测、治疗效果及病变预后的评估提供更多有价值的肿瘤生物学信息。
方法:
第一部分:选取5只成年健康雄性SD大鼠的肝、胰腺、胃及肺4种组织,各组织单独包埋制成蜡块,预设电动切片机的切片厚度刻度值为3、5、7、10μm,分别作自动和手动两种方法切片,所获得的薄组织片经二次包埋、切片、HE染色,TIGER图像分析仪测量切片的实际厚度。
第二部分:对进行常规宫颈脱落细胞学普查的370例妇女,用宫颈刷取材,液基薄层制片备用。涂片分两组进行实验:其中一组采用巴氏法染色做常规细胞学检查;另一组经改良Feulgen法染色,进行细胞核DNA含量测量与倍体分析。对常规细胞学检查有异常的病例进行病理组织学检查。对检测到感染了高危型人乳头瘤状病毒(HR-HPV)的病例,将其检查结果与DNA倍体分析结果结合宫颈病变级别与年龄因素进行分析比较。
结果:
第一部分:
(1)手动和自动切片对组织切片的实测厚度有影响,组织种类对组织切片的实测厚度无影响。
(2)各组织切片的厚度测量均值均较切片机预设厚度值大,同一切片机预设厚度值条件下,自动切片的实测厚度均值均小于手动切片,且更接近切片机预设刻度值。
(3)无论手动或是自动切片的实测厚度均随着切片机预设切片厚度的增大而呈正比增加,其相关系数r均大于0.9830。
(4)自动和手动切片的实测厚度分别比切片机预设切片厚度要厚29-50%和48-91%,自动切片与手动切片的切片厚度均值相差25%,二者相对误差为61%。
(5)相同组织的组织切片实测厚度的极端离散趋势(极差)随切片机预设厚度值的增加而呈现上升趋势,且手动切片组的极差大于自动切片组。
第二部分:
(1) 370例受诊者经常规宫颈细胞学检查发现其中有362例(97.7%)异常:包括ASCUS 140例,占患病总数的37.8%;LSIL 190例,占51.4%;HSIL 31例,占8.4%;病理医生镜下观察诊断为:CINⅠ179例,占总患病例的48.4%;CINⅡ49例,占13.2%;经DNA倍体分析,370例样本其中258例(69.7%)为异常。以病理诊断结果为金标准,细胞核DNA倍体分析筛查CINⅡ以上宫颈病变的灵敏度为98.6%,而常规细胞学方法则为72.2%。
(2) 370例患者中有191例进行了HPV检测,其中有高危型HPV(HR-HPV)感染81例,将HPV检查结果与DNA倍体分析结果结合宫颈病变级别作分析比较,二者阳性检出率均与宫颈病变的程度呈正相关;大部分感染病例能检测到非整倍体。
(3) 191例有HPV检测结果的病例,以病理诊断结果为金标准计算,细胞核DNA倍体分析筛查CINⅡ以上宫颈病变的灵敏度为84.4%,而HR-HPV检测方法则为75.6%。
(4)不同年龄组间的高危型HPV检出率明显有差异,年轻组高于中老年组。而非整倍体的检出结果不受患者年龄因素的影响。
(5)细胞学诊断不明确的ASCUS病例,感染了HR-HPV且有异倍体的患者中,≥CINⅡ的检出率最高,未感染HR-HPV且未能检出异倍体患者中,无一例CINⅠ以上的病例。
结论:
第一部分:自动切片与手动切片的切片厚度相差25%,二者相对误差为61%,自动切片获得的组织切片的厚度精度较高,优于手动切片。
第二部分:
(1)宫颈脱落细胞DNA倍体分析较细胞学检查用于宫颈疾病筛查时灵敏度更高,细胞学检查结合DNA倍体分析是癌前病变和宫颈癌筛查的有效方法。
(2) HR-HPV感染率提高以及年轻患者HR-HPV感染阳性率较中老年高,是宫颈癌年轻化的两个重要因素。
(3) DNA异倍体检测结果在宫颈癌的不同发病年龄之间无差别,而HR-HPV阳性率在不同年龄之间有差别,所有HR-HPV感染病例中发现大部分有异倍体,而有异倍体病例中仅部分有HR-HPV感染。DNA倍体分析法较HR-HPV检测法灵敏度更高。
(4) HR-HPV感染随着细胞学或病理学检测宫颈病变级别的增高而增高。
(5)应用病理学检查、HR-HPV检查、细胞核DNA倍体分析三联法,为细胞学诊断不明确的ASCUS病例的鉴别及确诊提供参考,感染了HR-HPVH有异倍体的患者中,≥CINⅡ的检出率最高,未感染HR-HPV且未能检出异倍体患者中,无一例CINⅠ以上的病例。
Purposes:
Part one:To observe the effect of automatic and manual slicing methods and tissue types on the thickness variation of paraffin-embedded sections to provide a reference for observing the organization or the cell's morphology and the position or composition of chemical substances and also for the evaluation of the quantitative analysis results.
Part two:
(1) Application of cytology,pathology diagnosis,nuclear DNA ploidy analysis and second-generation hybrid capture test used to detect HPV-DNA,and make comparative analysis.To provide an effective and volant adjuvant diagnostic method for cervical cancer and cervical dysplasia screening.
(2) To provide an effective screening method for atypical squamous cells of undetermined significance cases which have the potential risk of malignant transformation,and to provide much more evaluating information for prognosis and evaluation of therapeutic efficacy.
Methods:
Part one:Liver,pancreas,stomach and lung samples were obtained from five healthy adult male rats.Each tissue was separately embedded in paraffin.Slices were cut automatically and manually at various thickness set points,3μm,5μm,7μm,10μm,by an automatic microtome.After the re-embedding vertical slicing and HE staining,the thickness of these slices was measured by a TIGER image analysis system.
Part two:Each of cervical samples from the women who accepted the conventional cast cell screening was made into liquid-based smear sample.The smear were separated into two groups for the experiment.One group were stained by papanicoloau stain to make conventional cytology screening.The other group were stained by Feulgen method and was analyzed by nuclear DNA ploidy analysis system.Chose the samples which were regarded as doubtful cases diagnosed by cytologic analysis, these samples were delivered to the pathohistology.The results of samples which were infected with human papillomavirus(HPV) were compared with those of DNA ploidy analysis in combination with the level of cervical lesions and age factor.
Results:
Part one:
(1) Only the automatic or manual slicing procedure exerted effect on the slice thickness.
(2) The mean measured thickness of each tissue section was bigger than the microtome set point.On condition of the same set point,the mean measured thickness of automatic section was smaller than manual section.
(3) No matter automatic or manual slice,the measured thickness has a direct ratio tendency to bigger along with the microtome set point become bigger.
(4) The thickness of auto-slice and manual slice were 29%～50%and 48%～91%, respectively,thicker than the nominal thickness set on the microtome.There is a 25%difference between the thicknesses of automatically cut slices and manually cut slices.The relative error rate of the two slicing methods is 61%.
(5) Dispersion tendency of the measured thickness of the same kind of tissue had an escalating trend along with the microtome set point become bigger,the rang was bigger when cut manual than cut automatic.
Part two:
(1) 362 abnormal cases from 370 doubtful cases were suspected ones,including 140 cases of atypical squamous intraepithelial undermined significance(ASCUS),190 cases of low-grade squamous intraepithelial lesion(L-SIL) and 31 eases of highgrade squamous intraepithelial lesion(H-SIL),were diagnosised by conventional cytology examination.23 eases of carcinoma in situ(CINⅢ) and 49 CINⅡwere ??detected among 370 women by pathohistology examination.258 abnormal cases were diagnosised by DNA image analysis.According to the criterion of pathohistology diagnosis,it can say that the sensitivity of DNA ploidy analysis was 98.6%,and sensitivity of conventional cytology was 72.2%.
(2) 191 cases in 370 patients made the HPV test,in which 81 cases were infected HR-HPV.Comparing the results of HPV detection with those of DNA ploidy analysis,there were a correlation between their positive detection rates and degree of dysplasia.Almost all cases with infection of high-risk human papillomavirus (HR-HPV) were aneuploidy.
(3) HPV test was made in 191 cases,calculated by the pathology diagnosis as the gold-standard,the sensitivity of nuclear DNA ploidy analysis in screening for cervical lesions higher than CINⅡwas 84.4%,while the HR-HPV detection was 75.6%.
(4) There was a directly correlation between the infection rate of HR-HPV and the age of patients.Youth group was higher than the older group.While the prevalence of aneuploidy was not influenced by the age of patients.
(5) Cytology diagnosed as uncertain cases such as ASCUS,the patients who had infected HR-HPV and aneuploid were tested positive,the detection rate was highest in equal or higher than CINⅡgroup.On the reverse side,no cases was higher than CINⅠ.
Conclusions:
Part one:There is a 25%difference between the thicknesses of automatically cut slices and manually cut slices.The relative error rate of the two slicing methods is 61%.The precision of automatic cut thickness is higher,therefore,is better than manual sectioning.
Part two:
(1) Nuclear DNA ploidy analysis is better for screening precancerous and carcinoma of the uterine cervix than conventional cytology.Provide an effective and volant adjuvant diagnostic method for cervical cancer screening.
(2) HR-HPV infection rate is increased as well as the HR-HPV positive detection rates of the youth group is higher than in middle-aged group,make important part in younger-cervical-cancer.
(3) Aneuploidy test results have no difference between the patient's vary age,while the positive of HR-HPV make the sense.Most of infected cases can find the aneuploidy,while there are only part of aneuploidy cases have infected HR-HPV. The sensitivity of DNA ploidy analysis is higher than the HR-HPV test.
(4) HR-HPV infection rate is increased as the cytology or pathology detection of cervical lesion become higher.
(5) Make reference for the differentiate and diagnosis of the cytologic uncertain cases such as ASCUS.
引文
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[1]Bray F,Loos AH,McCarron P,et al.Trends in cervical squamous cell carcinoma incidence in 13 European countries:changing risk and the effects of screening[J].Cancer Epidemiol Biomarkers Prev,2005,14(3):677-686.
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[5]郎景和.迎接子宫颈癌预防的全球挑战与机遇[J].中华妇产科杂志,2002,37(2):129-131.
[6]Liu S,Semenciw R,Probert A,Mao Y.Cervical cancer in Canada:changing patterns in incidence andmortality[J].Int J Gynecol Cancer,2001,11:24-31.
[7]Sasieni PD,Cuzick J,Lynch-Farmery E.Estimating the efficacy of screening by auditing smear histories of women with and without cervical cancer.The National Co-ordinating Network for Cervical Screening Working Group[J].British Journal Of Cancer,1996,73(8):1001-1005.
[8]Alfred B(o|¨)cking,Vu Quoc Huy Nguyen.Diagnostic and Prognostic Use of DNA Image Cytometry in Cervical Squamous Intraepithelial Lesions and Invasive Carcinoma[J].Cancer(Cancer Cytopathology),2004,102(1):41-54.
[9]Ito T,Ishizuka T,Suzuki K,et al.Cervical cancer in young Japanese women[J].Arch Gynecol Obstet,2000,264(2):68-70.
[10]Frega A,Stentella P,De Ioris A,et al.Young women,cervical intraepithelial neoplasia and human papillomavirus:risk factors for persistence and recurrence[J].Cancer Letters,2003,196(2):127-134.
[11]Duensing S,Munger K.Centro some abnormalities and genomic instability induced by human papilloma virus on proteins[J].Prog Cell Cycle Res,2003,5:383-391.
[12]Evans MF,Adamson CS,Papillo JL,et al.Distribution of human papillomavirus types in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology and correlations with patient age and biopsy outcomes[J].Cancer,2006,106(5):1054-1064.
[13]Walboomers JM,Jacobs MV,Manos MM,et al.Hunan papilloma virus is a necessary cause of invasive cervical cancer worldwide[J].Pathol,1999,189:12-19.
[14]Moodley J.Combined oral contraceptives and cervical cancer[J].Curr Opin Obstet Gynecol,2004,16(1):27-29.
[15]Ddak D,Samudovsky M.Cervical cancer in a female-to2-male trans-sexual[J].Eur J cancer,2004,40(11):1795-1796.
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