全自动DNA图像分析在乳腺细针穿刺细胞学诊断中的应用

目的探讨全自动DNA图像分析（DNA-ICM）系统在乳腺细针穿刺细胞学（FNAC）诊断中的应用价值。方法对95例乳腺肿块患者行细针穿刺,对所获细胞标本分别行常规细胞学诊断及DNA—ICM检测,比较两种诊断方法与组织病理学诊断结果的符合率。结果以组织病理诊断为标准,DNA—ICM与常规细胞学诊断符合率差异无统计学意义[88．4％（84／95）比94．7％（90／95）1（χ^2=2．457,P=0．117）;DNA—ICM检测排除了常规细胞学诊断的2例假阳性（可疑癌细胞）。DNA-ICM检测中,浸润性导管癌异常DNA含量的检出率高于浸润性小叶癌[90．3％（65／72）比50．0％（4／8）]（Fisher精确概率法,P=0．011）。结论DNA—ICM可以降低FNAC的假阳性率,但其对二倍体肿瘤诊断存在局限性。     

前列腺癌的细胞核DNA图像分析研究

目的探讨DNA图像分析技术在前列腺癌(PC)诊断中的应用价值。方法应用计算机图像分析技术测定对5例正常前列腺(NP)和30例PC的标本DNA倍体。结果5例NP中均为整倍体(2C和3~4C),30例PC中有23例出现非整倍体,并且随着PC分级的增高,其倍体率也增大。结论细胞核DNA含量和倍体测定是反映前列腺癌细胞增殖能力的重要生物学定量指标,能较客观反映PC的预后,为正确诊断恶性肿瘤提供依据。     

全自动DNA图像分析技术提高胸水细胞病理学的阳性检出率

目的:探讨全自动DNA图像分析技术（DNA-ICM）在胸水细胞病理学检查中的临床应用价值。方法:选择558例病例,分别采用薄层液基细胞学联合细胞蜡块技术（TCT+CB）和Feulgen染色的DNA-ICM技术进行检测,并将两种结果进行分析。结果:558例中,TCT+CB检出阳性173例,阴性385例,阳性检出率为31.00%（173/558）,阴性检出率为69.00%（385/558）;DNA-ICM检出阳性253例,阴性305例,阳性检出率为45.34%（253/558）,阴性检出率为54.66%（305/558）;DNA-ICM在恶性肿瘤诊断中的敏感性、特异性分别为95.95%（166/173）、77.40%（298/385）。DNA-ICM阳性且TCT+CB诊断为阴性的45例病例中,经临床综合诊断为阳性的32例。结论:细胞DNA-ICM的敏感性和特异性均较高,而且可有效提高胸水细胞学的阳性检出率,有效减少细胞病理学诊断的漏检。     

尿脱落细胞液基薄层细胞学检测联合DNA定量分析法筛查泌尿系统肿瘤

目的探讨尿脱落细胞薄层细胞学检测(TCT)联合细胞定量DNA分析技术筛查泌尿系统恶性肿瘤的作用。方法在284例正常健康体检人群、102例普通泌尿系统门诊患者及29例膀胱癌患者中,取尿液的脱落细胞,以TCT技术制片,同时行细胞病理检查和DNA定量分析,计算各组的阳性率,各组间的检出率以2比较统计学意义。结果体检组肿瘤细胞0例,可疑细胞1例,DNA定量分析阳性1例,可疑2例;门诊组未见肿瘤细胞,DNA定量分析可疑3例;膀胱癌组肿瘤细胞阳性率为79.3%(23/29),DNA定量分析阳性率为82.8%(24/29)。膀胱癌组尿肿瘤细胞和DNA定量分析阳性率高于体检组和门诊组,2分析表明差异有统计学意义(P<0.01)。结论在没有血尿等临床表现和影像学异常发现前,TCT联合DNA定量分析筛查泌尿系统恶性肿瘤效果明确,简便易行。     

DNA cytometry and cytology by quantitative fluorescence image analysis in symptomatic bladder cancer patients

A semi-automated quantitative fluorescence image analysis (QFIA) technique was developed with the Leitz TAS-Plus to detect bladder cancer using hyperploidy in urinary cells. Absolute nuclear fluorescence intensity (ANFI) (emission at 540 nm with excitation at 436 nm) of individual acridine-orange-stained cells was quantitated using (1) QFIA and (2) simple filter microspectrofluorophotometry (SFM). Both methods employed an internal phosphor particle standard which, when once calibrated against the DNA content of normal cells, obviates the necessity of routinely calibrating against normal cells in each sample. Results of SFM and QFIA were compared with routine Papanicolaou (Pap) cytopathology, using histopathology as the diagnostic standard in 272 samples from 67 symptomatic patients. The sensitivities for detecting low-grade transitional-cell carcinoma were 86% for SFM, 76% for QFIA, and 33% for Pap cytology. QFIA and SFM were significantly more sensitive at detecting bladder cancer than was Pap (0.01 greater than p greater than 0.001). Comparison of sensitivity obtained with bladder washings and urine samples showed that noninvasively obtained urines can be used. ANFI also detected recurrent and precancerous bladder lesions and kidney, ureter, and prostate lesions. This approach may prove generally useful in quantifying biochemical and immunological probes and should be broadly applicable as a research tool for studying the relationship of biochemical markers in the pathogenesis of disease and as a test for cancer control.     

液基细胞学结合DNA倍体定量分析在宫颈病变中的应用

宫颈癌是妇女最常见的恶性肿瘤,为我国妇女恶性肿瘤的第一位。如何早期诊断癌前病变和宫颈癌是防治的关键。近年来推广Bethesda报告系统（The Bethesda System,TBS）和液基薄层细胞学（Liquidbased cytology test,LCT）技术正逐步应用于临床,很大程度上克服了传统巴氏诊断系统和细胞学技术的不足,明显提高了标本的满意度及宫颈异常细胞检出率。同时结合应用DNA倍体定量分析对宫颈癌和癌前病变有更高的预测值,为宫颈癌早期诊断及降低患病率提供理论依据。     

Flow cytometry in exfoliative cytology of malignant tumor

Exfoliative cells from 170 cases of malignant tumor and 70 cases of normal tissue were studied by flow cytometry. The results showed that 155/170 (91.2%) cases were positive and 15/170 (8.8%) were false-negative; 4/70 (6.0%) cases were false-positive. The authors consider that flow cytometry has achieved a diagnostic accuracy comparable to the conventional cytology. Flow cytometry is a useful supplementary method in the diagnosis of tumors.     

Flow cytometry of prostate cancer: relationship of DNA content to survival

Between March 1970 and December 1978 there were 366 patients with prostatic cancer treated by 125I seed implants and pelvic lymph node dissection. All had a minimum of 5 years follow-up. One hundred thirty-three patients had metastatic prostatic cancer in lymph nodes (Stage D1) at the time of lymph node dissection and seed implantation. Ninety-one of the 133 patients were judged to have sufficient metastatic prostatic cancer in their nodal tissue (greater than 50% replacement with tumor) to justify flow cytometric cellular DNA measurements on the involved paraffin-embedded nodal tissue. Nine patients were excluded due to uninterpretable DNA histograms leaving 82 patients for analysis. Forty-nine patients had aneuploid and 33 had diploid tumors. There was no statistical bias between the aneuploid and diploid groups due to age (P = 0.970, chi 2 test), time between diagnosis and implantation (P = 0.217, chi 2 test), number of positive nodes (P = 0.669, two-sample t test of means), or tumor grade (P = 0.332, chi 2 test). Median survival time of the aneuploid and diploid groups was 5.0 and 8.8 years, respectively (P = 0.0109, log rank test). Cox regression analysis confirmed the effect of aneuploidy versus diploidy on survival by controlling for other potentially confounding variables (age, time from diagnosis to implantation, number of positive nodes, and grade). Grade as a predictor of survival did not approach statistical significance in this series of relatively small size (P = 0.116). Thirty-eight of the 82 patients had moderately differentiated neoplasms. Nineteen of these were aneuploid and 19 diploid. The median survival was 5.8 and 9.1 years, respectively, for these grade-matched aneuploid and diploid groups (P = 0.039, log rank test). We conclude that flow cytometric DNA measurements on archived paraffin-embedded tumor in nodal metastases appear to be a strong predictor of survival for Stage DI prostatic cancer.     

Measurement of cellular DNA content as an adjunct to diagnostic cytology in malignant effusions

We reviewed the final diagnosis and outcome of 119 patients who developed serous effusions. In addition to routine cytological examination, the cellular DNA content of fluid samples aspirated from the effusions was measured using flow cytometry in order to determine whether the detection of aneuploid cells could aid in diagnosis or serve as a guide to prognosis. The final diagnosis of 35 patients was non-malignant and a further 40 patients with biopsy-proven cancer had cytologically negative effusions. In all of these cases flow cytometry revealed the presence of diploid cells only. The effusions from 36 cancer patients were reported by cytology to contain a variable proportion of malignant cells, and aneuploid cells were detected in 23 of these samples, the remainder containing only diploid cells. Of 8 effusions where cytology was equivocal, one contained aneuploid cells and clinical outcome subsequently showed that all 8 were malignant. Median survival of patients with cancer was 3 months, and a positive cytology had no influence on survival. However, of the patients with positive cytology, those whose effusions contained aneuploid cells had a poorer short-term prognosis than those cases where only diploid cells could be detected (median survival 1.5 vs 4 months). Measurement of cellular DNA content using flow cytometry can occasionally confirm cancer in a cytologically equivocal effusion, but the negative results in 13 out of 36 (36.1%) effusions where cytology was reported as positive suggests that it has only a limited role in this clinical setting, using currently available techniques.     

DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study

Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease. In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974. Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch. Thirty-micron sections were cut from each plug for dewaxing and disintegration. The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry. In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern. No apparent correlation was found between the histopathological grade and the DNA ploidy. Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy. The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.     

The diagnostic value of flow cytometric DNA measurements in follicular tumors of the thyroid gland

Flow cytometric analysis of DNA content was performed on single or multiple samples from 34 thyroidectomy specimens. There were 29 thyroids with diploid DNA content, comprising 15 nonneoplastic lesions, 5 follicular adenomas, 1 medullary carcinoma, and 8 papillary carcinomas. Aneuploid DNA pattern was observed in five cases, including one metastatic mammary carcinoma. The initial histologic diagnoses in the remaining four aneuploid thyroid lesions were follicular carcinoma in one and follicular adenoma in three. The abnormal DNA pattern in the three follicular "adenomas" prompted a review of their aspiration cytologic and histologic features. The fine-needle aspiration biopsy was performed in two of the three cases and showed evidence of a follicular neoplasm with significant nuclear atypia in both. Histologic review of the three lesions led to a modified diagnosis of noninvasive low grade follicular carcinoma in all three. Flow cytometric analysis of DNA content may prove to be a highly useful adjunct in the evaluation of follicular thyroid tumors. Long-term clinical follow-up is warranted to document the clinical significance of these observations.     

Laser scanning cytometry for the detection of neoplasia in urologic cytology specimens

BACKGROUND: The objective of the current pilot project was to assess the efficacy of laser scanning cytometry (LSC) for DNA ploidy analysis of atypical urologic cytology specimens to enhance the distinction between benign and malignant changes. METHODS: Forty selected urologic cytology specimens that previously had been categorized as normal, atypical, or malignant were studied. Nuclear propidium iodide and fluorescence intensity measurements were converted to pixel values, which were used to create scattergrams that excluded debris and cell clusters from ploidy analysis, creating a gated (isolated) region of predominantly single cells for LSC ploidy analysis. Integral histograms then were created to show the number of cells present in diploid, tetraploid, and aneuploid peaks; these histograms also were used to assess DNA ploidy. RESULTS: Ten normal specimens, 10 malignant specimens, and 20 atypical specimens were examined to assess the efficacy of LSC ploidy analysis. Normal and malignant specimens generated reference histograms for comparison with the atypical specimens and exhibited 90% specificity and 100% sensitivity. Ten atypical aneuploid specimens had histogram and scattergram patterns similar to those produced by malignant specimens and, using the cytometer's relocation feature, the presence of atypical cells was confirmed in the aneuploid regions. CONCLUSIONS: The authors determined that DNA ploidy analysis of atypical urologic cytology specimens using LSC is a useful adjunct tool for identifying malignant specimens that lack sufficient cytologic criteria for diagnosis by light microscopy alone. However, LSC is time consuming and requires expensive equipment.     

Accuracy of fine needle aspiration cytology of salivary gland lesions: routine diagnostic experience in Bangkok, Thailand

Fine needle aspiration (FNA) cytology is well accepted as a safe, reliable, minimal invasive and cost-effective method for diagnosis of salivary gland lesions. This study evaluated the accuracy and diagnostic performance of FNA cytology in Thailand. A consecutive series of 290 samples from 246 patients during January 2001-December 2009 were evaluated from the archive of the Anatomical Pathology Department of our institution and 133 specimens were verified by histopathologic diagnoses, obtained with material from surgical excision or biopsy. Cytologic diagnoses classified as unsatisfactory, benign, suspicious for malignancy and malignant were compared with the histopathological findings. Among the 133 satisfactory specimens, the anatomic sites were 70 (52.6%) parotid glands and 63 (47.4 %) submandibular glands. FNA cytological diagnoses showed benign lesions in 119 cases (89.5 %), suspicious for malignancy in 3 cases (2.2 %) and malignant in 11 cases (8.3%). From the subsequent histopathologic diagnoses, 3/133 cases of benign cytology turned out to be malignant lesions, the false negative rate being 2.2 % and 1/133 case of malignant cytology turned out to be a benign lesion, giving a false positive rate was 0.8%. The overall accuracy, sensitivity, specificity, positive predictive value and negative predictive value were 97.0% (95% CI, 70.6%-99.4%), 81.3% (95% CI, 54.4%-96.0%), 99.1% (95% CI, 95.4%-100%), 92.9% (95% CI, 66.1%-99.8), 97.5% (95% CI, 92.8%-99.5%), respectively. This study indicated that FNA cytology of salivary gland is a reliable and highly accurate diagnostic method for diagnosis of salivary gland lesions. It not only provides preoperative diagnosis for therapeutic management but also can prevent unnecessary surgery.     

Magnetic resonance spectroscopy of the malignant prostate gland after radiotherapy: a histopathologic study of diagnostic validity

PURPOSE: Accurate spatial representation of tumor clearance after conformal radiotherapy is an endpoint of clinical importance. Magnetic resonance spectroscopy (MRS) can diagnose malignancy in the untreated prostate gland through measurements of cellular metabolites. In this study we sought to describe spectral metabolic changes in prostatic tissue after radiotherapy and validate a multivariate analytic strategy (based on MRS) that could identify viable tumor. METHODS AND MATERIALS: Transrectal ultrasound-guided prostate biopsies from 35 patients were obtained 18-36 months after external beam radiotherapy. One hundred sixteen tissue specimens were subjected to 1H MRS, submitted to histopathology, and analyzed for correlation with a multivariate strategy specifically developed for biomedical spectra. RESULTS: The sensitivity and specificity of MRS in identifying a malignant biopsy were 88.9% and 92% respectively, with an overall classification accuracy of 91.4%. The diagnostic spectral regions identified by our algorithm included those due to choline, creatine, glutamine, and lipid. Citrate, an important discriminating resonance in the untreated prostate gland, was invisible in all spectra, regardless of histology. CONCLUSIONS: Although the spectral features of prostate tissue markedly change after radiotherapy, MRS combined with multivariate methods of analysis can accurately identify histologically malignant biopsies. MRS shows promise as a modality that could integrate three-dimensional measures of tumor response.     

Clinical DNA flow cytometry

Modal DNA values of tumours from various sites may exhibit (1) a diploid-near diploid distribution, (2) an exponential distribution in the tetraploid to triploid range, or (3) a log-normal distribution in the triploid to tetraploid range. Examples of these various types of distribution are non-Hodgkin's lymphoma (1), aneuploid prostate carcinoma (2), aneuploid colon, breast, cervix and testicular carcinomas (3). These differences indicate dissimilarities in tumour development. In aneuploid tumours from the same site both tetraploid exponential and triploid-tetraploid log-normal distributions may occur. In bladder carcinoma these are related to grade. Modal DNA values in tumours are 10% higher than would be expected from modal chromosome numbers. This difference seems not to be due to a relative increase in large-sized chromosomes or due to technical shortcomings. Chromosome studies also show the possibility of the existence of near diploid malignant cells in grossly aneuploid tumours. Modal DNA values are connected with functional tumour properties by the proportion of S-phase cells. The significance of the latter is exemplified by follow-up of patients with bladder and cervix carcinoma.     

DNA image analysis combined with routine cytology improves diagnostic sensitivity of common bile duct brushing

BACKGROUND: Cytologic evaluation of common bile duct brushings has a low sensitivity for diagnosing malignancy because of scant cellularity, poor cellular preservation, or sampling errors occur. The aim of this study was to evaluate whether cytology combined with image analysis improves the diagnostic accuracy of bile duct brushing in comparison with cytology alone. METHODS: Forty-nine specimens of bile duct brushings obtained from 45 patients during endoscopic retrograde cholangiopancreatography were evaluated using cytology and image analysis. Specimens were classified as negative, atypical, suspicious, or malignant by using cytologic evaluation. DNA histograms were classified as diploid (D), broad diploid (BD), aneuploid (A), or tetraploid (T). Degree of hyperploidy (DH), representing cells with a DNA content > 5C was evaluated using a cutoff value of > or = 1%. Final diagnosis of cancer was based on tissue specimens that were obtained by fine-needle aspiration or surgical biopsy and clinical fol- low-up. RESULTS: Thirty-four patients ultimately proved to have a malignancy. Cytology revealed 19 negative cases, 15 atypical cases, 9 suspicious cases, and 6 malignant cases. Together, suspicious and malignant cytology cases yielded a sensitivity of 44% and a specificity of 100% for a cytologic diagnosis of cancer. The DNA histogram pattern was D in 24 cases, BD in 9 cases, and A in 16 cases. BD and A patterns were significantly associated with malignancy (P < 0.001). A DH > or = 1% was noted in 22 cases. DH alone had a sensitivity of 62% and a specificity of 91% and was significantly associated with malignancy (P < 0.004). Atypical cytology alone had a false-negative rate of 29%, but in combination with a DH > or = 1%, the false-negative rate decreased to 7%. Additionally, when the authors combined atypical, suspicious, and malignant cytology with a DH > or = 1%, the diagnostic sensitivity increased to 88%, but the specificity decreased to 73%. CONCLUSIONS: Combined cytology and image analysis of bile duct brushing increased diagnostic sensitivity compared with cytology alone. The findings suggest that image analysis may help select patients having atypical cytology who should undergo a more rigorous evaluation for malignancy. A larger prospective study of the usefulness of combined cytology and image analysis of bile duct brushing is warranted.