Comparison of normal sperm morphology outcomes from two different computer-assisted semen analysis systems

By eliminating the human evaluation variable, it was possible to carry out an investigation into the 'true' association between normal sperm morphology outcomes assessed according to the World Health Organization guidelines and strict criteria. Two computer-assisted semen analysis systems were used, IVOS and Mika, to evaluate Diff-Quik and Papanicolaou stained slides. As expected, the mean normal sperm morphology outcomes for the World Health Organization classification evaluations were markedly higher for both the Diff-Quik (mean difference = 40.13%) and the Papanicolaou (mean difference = 32.55%) stained slides. The association between the outcomes were low for the Diff-Quik stained slides (r = 0.379) and poor for the Papanicolaou stained slides (r = 0.110). While the association achieved with the computer-assisted semen analysis systems using Diff-Quik stained slides was comparable to the association between the manual evaluations (r = 0.386), the manual evaluation of Papanicolaou stained slides produced a relatively good association (r = 0.690). Although the numbers were small, the results show the probability of poor class correlations. Approximately 40% of outcomes were incorrectly classed at a 14% (strict criteria) and 50% (World Health Organization guidelines) cut-off point for both staining methods. This study confirms the fundamental differences between the two classification systems. The results also indicate that, of the two stains used, Diff-Quik should be the preferred staining method for computer-assisted sperm morphology evaluations.     

Comparison of the ImmunoCyt test and urinary cytology with other urine tests in the detection and surveillance of bladder cancer

Despite several new urine markers urinary cytology remains the gold standard for the non-invasive detection of bladder carcinoma. The use of monoclonal antibodies against tumor associated antigens offers a promising approach to improve urinary cytology. The aim of this study was to compare fluorescence immunocytology (ImmunoCyt/Ucyt+ test), alone and in combination with the conventional cytology, with other urine markers. Urine samples from 126 patients undergoing cystoscopy were included in the study. Among them, 42 patients had urothelial carcinoma, two dysplasia, two other malignancies, and 78 had no evidence of bladder cancer. Urine samples were taken before any manipulation. We used the ImmunoCyt test and Papanicolaou staining for conventional cytology. The ImmunoCyt slides were examined under a fluorescence microscope. Evaluations of the tests were blinded to clinical and pathological data and were carried out by three independent observers. The results of cytology and ImmunoCyt were compared with the BTAstat, NMP22, Lewis X, 486p3/12, and Urovision tests. The sensitivity for the ImmunoCyt test was 78.3% and for conventional cytology 84.6%. The combination of ImmunoCyt and cytology showed a sensitivity of 89.1%. The specificity was 73.8% for the ImmunoCyt alone, 80.0% for the cytology, and 72.5% for the combination of ImmunoCyt and cytology. Sensitivities for the other tests were 68.8% for (FISH), 66.6% (BTA-Stat), 68.8% (486p3/12), 95.5% (Lewis X), and 71.1% for (NMP22). Specificity was 89.1% for (FISH), 78.2% (BTA-Stat), 76.4% (486p3/12), 32.8% (Lewis X), and 65.5% for (NMP22). Urinary cytology can be improved by immunostaining with monoclonal antibodies against tumor-associated antibodies. The combination of ImmunoCyt with conventional cytology offers a superior sensitivity to other commercial tests. The ImmunoCyt test provides a useful supplement to urinary cytology in the diagnosis of bladder cancer.M.I. Toma, M.G. Friedrich contributed equally to this study     

Monoclonal antibodies against transitional cell carcinoma for detection of malignant urothelial cells in bladder washing

The diagnosis of transitional cell carcinoma by cytological examination of exfoliated urinary cells is important in the early detection and followup of patients with this disease. Proper interpretation requires a skilled pathologist. Accuracy also is influenced by collection methods and nonmalignant pathological conditions of the bladder. An immunocytochemical technique using monoclonal antibodies G4 and E7 successfully identified tumor-associated antigens on the surface of transitional carcinoma cells obtained by bladder washings. The method, which uses immunoperoxidase staining, was compared to conventional Papanicolaou staining of bladder washings from 75 patients with and without transitional cell carcinoma. Patients were divided into 4 groups: group 1 (nontumor control)--15 patients with no pathological condition of the bladder or nonmalignant urological diseases, group 2 (nontransitional cell carcinoma)--19 patients with other urological malignancies, group 3-18 patients with active transitional cell carcinoma and group 4-23 patients with a history of transitional cell carcinoma but no evidence of tumor at the time of the washing. The incidence of positive staining in these groups was 0, 5, 78 and 0 per cent, respectively. The diagnostic value of immunoperoxidase staining was similar to that of Papanicolaou staining in the control group and in patients with high grade transitional cell carcinoma, and provided specific morphological criteria not possible by conventional cytology studies. Interpretation of immunoperoxidase staining was difficult in washings with a large number of inflammatory cells if endogenous peroxidase activity was not blocked properly. The application of the immunoperoxidase staining method for diagnosis of low grade tumor is under further investigation.     

Stellenwert der Urinzytologie in der Diagnostik von High-grade-Urotheltumoren*

Urinary cytology is a non-invasive adjunct to cystoscopy in the diagnosis of bladder cancer. In order to assess the diagnostic accuracy of urinary cytology as an office-based method for clinically relevant high grade (G3) bladder cancer, three nationwide cytology survey tests were performed. Urine specimens from seven patients, three patients with high grade (G3) bladder cancer and four patients with urinary tract infections, were collected. A total of 1,000 cytology slides were produced from each urine specimen. Each set contained five slides (two malignant, three benign) which were sent to all participating German urologists. Three nationwide tests were performed from 1998-2000. The specimen sets were kept the same for the first and second test and in the third test two new slides were introduced. In addition to validity, the reliability was calculated for the first and second test as interobserver and intraobserver reliability according to Cohen’s kappa statistics. Due to the change of two specimens in the third test in 2000 only sensitivity and specificity were calculated. A total of 335 urologists took part in the first survey test, 329 in the second and 292 in the third The sensitivity for G3 cytologies was 81.34% in the first, 87.08% in the second and 85.1% in the third survey test and the specificity was 85.87%, 83.58% and 89.15%, respectively. Interobserver reliability showed a weighted kappa value of 0.58 for the first and 0.59 for the second survey test. Calculation of intraobserver reliability was possible for 169 urologists taking part in the first and second survey test and showed a mean kappa value of 0.62. The results of the three nationwide urinary cytology tests indicate that urinary cytology has a high sensitivity in the detection of clinically relevant high grade bladder cancer. The kappa values achieved demonstrate a clear agreement of cytological diagnoses.     

Prognostic value of urinary cytology in bladder tumor]

Retrospective analysis was carried out to evaluate the prognostic value of urinary cytology in bladder tumor patients. All of the 170 patients (107 superficial (less than T2), 60 advanced (greater than T1) and 3 unknown (TX)), who were followed after their first occurrence, had histologically diagnosed transitional cell tumors. Recurrence rate for superficial cases was higher (p less than 0.05) when urinary cytology, either before or after the initial treatment, was positive than when negative. Patients with positive urinary cytology before the initial treatment proved to show poor prognosis compared to those with negative cytology (p less than 0.01), and many of the positive cases were with advanced cancer. Urinary cytology seemed to be indicative of the neoplastic potential of bladder tumor.     

Monoclonal antibody 486 P 3/12: a valuable bladder carcinoma marker for immunocytology

Monoclonal antibodies directed against tumor-associated antigens of bladder carcinoma were used to identify tumor cells in bladder washout specimens of 40 patients with bladder carcinoma (group 1), 41 with no bladder disease or with urinary tract infections (group 2), 41 who received long-term mitomycin C instillation therapy after excision of the tumors (group 3) and 39 who received no prophylaxis after excision of the tumors (group 4). In all groups the same bladder washout specimen was used for standard urinary cytological and immunocytological tests. True positive results were obtained in 90 per cent of the patients in group 1 according to our immunocytological criteria and in 43 per cent according to standard cytology studies. No urine specimens in group 2 (controls) were immunocytologically positive, while 16 of 41 in group 3 and 17 of 39 in group 4 were positive immunocytologically but only 4 and 5, respectively, were positive according to standard cytology studies. Further followup of these patients will show whether cells positive for monoclonal antibody 486 P 3/12 will permit early detection of recurrent bladder cancer and whether one can identify patients who require prophylaxis after removal of the superficial bladder tumors.     

Value of exfoliative cytology of the urinary bladder in occult bladder cancer and in the control of treated cancers

The exfoliative cytology of the urinary bladder is helpful in the detection of occult bladder carcinoma, which in according to definition is not recognizable without magnification, as well as in the follow-up of treated bladder carcinoma. Within a period of 5 years 23 occult bladder carcinomas were detected by cytological studies alone. The degree of malignancy of the established transitional cell carcinoma varied between G2 and G3. The latent period between the found cytological result and the histological confirmation amounted to 14 months. In our experience the diagnosis of occult bladder carcinoma can be established by exfoliative cytology of the urinary bladder, if invisible parts of tumor with moderate and high degree of malignancy communicate with the mucosa of the bladder. The cytological recognition of malignant cells of urothelium offers valuable support for further therapeutic procedures, if there is no cystoscopic evidence of tumor in the bladder. In addition to clinical diagnosis of bladder cancer exfoliative cytology of the urinary bladder should also be part of the follow-up of treated bladder cancer.     

DD23 Biomarker: a prospective clinical assessment in routine urinary cytology specimens from patients being monitored for TCC

BACKGROUND: A prospective clinical study was conducted to assess the ability of the DD23 murine monoclonal antibody to enhance detection of bladder cancer in routine alcohol fixed urine cytology samples. METHODS: Prospectively, 308 bladder cytology specimens were obtained from patients with a history of bladder cancer with a mean age of 71.4+/-11.9 (27% female, 73% male). Data included 121 biopsy-confirmed results and 187 cystoscopy results to assess presence or absence of cancer. Thirty-five normal cytology specimens were obtained from asymptomatic men and women between 55-85 years of age. Separate slides from the alcohol fixed cytology specimens were stained using the Papanicolaou (Pap) and Feulgen staining procedures. The DD23 assay was performed using an avidin-biotin alkaline phosphatase immunocytochemical procedure, with a single urothelial cell exhibiting intense immunostaining sufficient to make a positive call. RESULTS: Pap-Feulgen cytopathology for the 308 cases yielded an overall sensitivity of 65.5% and a specificity of 85.1%, and the DD23 biomarker alone yielded a sensitivity of 80.5% and a specificity of 59.7%. Analysis of the voided urines only (n=164) yielded sensitivities of 61.0% and 73.2% and specificities of 86.2% and 67.5% for cytopathology and DD23 alone, respectively. Results in 49 bladder wash urine cytology cases produced a sensitivity of 70.2% and 100% and specificities of 92.3% and 61.5% for cytopathology and DD23 alone, respectively. In 133 patients that underwent biopsy or had positive cystoscopy results, cytopathology yielded a sensitivity of 65.5% and a specificity of 69.6% while DD23 yielded a sensitivity of 80.5% and a specificity of 58.7%. In 25 biopsy-confirmed low-grade cancers, DD23 improved cancer detection from 32% to 72% when compared to cytopathology. The DD23 biomarker had a specificity of 85.7% in 35 age-matched normal asymptomatic control specimens. CONCLUSIONS: The DD23 biomarker is an adjuvant test that provides improved detection of bladder cancer in cytology specimens and enhances the sensitivity of the cytopathology diagnosis, especially in low-grade cancers.     

Comparison of two staining and evaluation methods used for computerized human sperm morphology evaluations

Summary. The purpose of the study was to analyse the agreement between computer analysed (Hamilton Thorne, IVOS Dimensions Version 3) normal sperm morphology and values obtained from 97 slides stained according to the Papanicolaou and Diff-Quik method. Liquefied semen samples were washed once by centrifugation and air dried smears on slides were made, which were stained according to the Papanicolaou and Diff-Quik method and analysed by computer. The paired t -test was used to assess whether any bias existed between the two methods. The limits of agreement were calculated using the Bland and Altman approach and a modification of this approach (mean-dependent limits). A significant bias of 1.6% was obtained in favour of higher normal sperm morphology percentages when using the Diff-Quik method. The standard limits of agreement were —13.4% to 16.6%, whereas the mean-dependent limits of agreement were 1.6% [5.8 + 0.6 (mean percentage normal morphology)]. Statistically, the Diff-Quik and Papanicolaou staining methods produce different normal sperm morphology profiles. These inherent differences may, therefore, require the establishment of new normal sperm morphology thresholds for male fertility, based on clinical data, when using the Diff-Quik staining method in conjunction with computerized analysis.     

Flow cytometry versus urinary cytology in the diagnosis and follow-up of bladder tumors: critical review of a 5-year experience

Summary The authors report the results of a 5-year experience with flow cytometry (FCM) in the diagnosis and evaluation of bladder tumors. FCM was applied to 400 patients (225 without a urinary tumor and 175 with a past or recent bladder tumor). For the patients without a tumor, bladder-irrigation-fluid FCM was positive in 22% of samples vs 1% of those tested with conventional cytology. The high rate of false-positive results may have been due to an imperfect quality of the samples, to the staining procedure and to the high amount of squamous cells. For 72 selected tumors in which the bladder washing was performed under specific requirements, the detection rate of FCM was lower than that of conventional cytology, including G1-grade tumors. A comparison between bladder-irrigation-fluid FCM and disaggregated-biopsy FCM revealed a 22% rate of discordant results. From these data it does not seem desirable tu use bladder-irrigation FCM instead of conventional cytology in routine urologic examinations. The use of this technique must be more selective in bladder tumor evaluation.     

The value of urinary cytology, serum and urinary carcinoembryonic antigen, rheumatoid factors, and urinary immunoglobulin concentration as tumor markers or prognostic factors in predicting progression of superficial bladder cancer

We followed 67 patients with superficial bladder cancer (TA, T1) for 12-48 months (mean, 26.8 months) after surgical removal of the tumors. Every 3-4 months, when the patients came for cystoscopic control, we evaluated serum and urinary carcinoembryonic antigen (CEA), urinary cytology, serum rheumatoid factors, and urinary IgG, IgA, and IgM concentrations. The purpose was to look for markers or prognostic factors in patients with superficial bladder carcinoma other than characteristics of the excised tumors themselves. We emphasized data that can be obtained on patient follow-up. Our results were correlated with tumor recurrence and tumor progression rates. In contrast with other reports, only urinary immunoglobulins (especially IgG) proved to have prognostic value. However, urinary CEA and (even more so) urinary cytology are fairly good tumor markers: they were positive when the bladder tumors were present and, in the case of recurrence, before recurrent tumors were visible.     

¿Cuál es el valor de la biopsia aleatoria en el seguimiento del carcinoma uroterial superficial de vejiga?

IntroductionTransitional cell carcinoma of the bladder represents a disease of entire urothelial tract. The follow up is very important to detect any lesion that might represent a progression or a local recurrence. Some authors recommend randomized biopsies as a routine workup, others recommend cystoscopies and urinary cytology as the main part of superficial bladder cancer follow up.Patients and MethodsForty nine patients with superficial bladder cancer were followed up during a ten-year period. Randomized biopsies and urinary cytology were harvested according to the international cancer protocol on bladder cancer.Results15 (1%) out of 1.489 randomized biopsies found to be positive to transitional cell carcinoma. Four out (10.5%) of 35 biopsies targeted to suspicious areas were positive to transitional cell carcinoma. 50 (17.4%) out of 288 cystoscopies with urinary cytology found to be positive to transitional cell carcinoma. Sensitivity and Specificity of biopsies (including randomized and targeted) were 31% and 85.2% respectively. Sensitivity and specificity of cystoscopies with urinary cytology were 48% and 86.5% respectively.ConclusionRandomized biopsies did not show to detect more local recurrence or progression when compared to the urinary cytology. Cystoscopies with urinary cytology have good sensitivity and specificity for detection of tumor recurrence during follow up of transitional cell carcinoma.     

The diagnostic value of flow cytometric DNA analysis in bladder cancer--comparison with conventional cytology]

The aim of this research was to assess the clinical value of flow cytometry (FCM) compared with conventional cytology in the detection and diagnosis of bladder cancer. The practical application of FCM combined with cytology was also investigated. Seventy patients with bladder cancer and 50 without neoplastic disease of the urinary tract were studied. The exfoliated cells used for FCM-DNA analysis and cytology by the Papanicolaou technique were obtained from voided urine and subsequent bladder irrigation specimens. The false negative rates for urine cytology, irrigation cytology, urine FCM, and irrigation FCM were 36%, 38%, 14% and 13%, respectively. Corresponding false positive rates were 2%, 0%, 14%, and 18%. When the above-mentioned examinations were paired, the combination of urine cytology and urine FCM appeared to be the most acceptable way to secure a correct diagnosis. Using this combination, the false negative and false positive rates were 4%, and 16%, respectively. When three or four examinations were combined, the false negative fate could not be improved despite the fact that the false positive rate worsened. It is noteworthy that FCM-DNA analysis seems to be less reliable in patients who have inflammatory diseases of the urinary tract or a history of intravesical chemotherapy.     

Detection of tumor cells in bladder washings by a monoclonal antibody to human bladder tumor-associated antigen

We have conducted two studies to evaluate the efficacy of using a specific monoclonal antibody (McAb) to detect exfoliated tumor cells in bladder washings. This is a preliminary step toward the development of immunological methods to improve the cytologic detection of bladder carcinoma. In this study, McAb 3G2-C6 was used. The McAb reacts to a bladder tumor-associated cell-surface antigen expressed in bladder tumors of various grades. Bladder washings from patients with and without carcinoma were stained with the McAb using two different indirect immunofluorescence methods (Methods A and B). The results of the immunological studies were compared with those obtained from the cytology laboratory and these in turn, were evaluated against the histopathological diagnosis of respective patients at the time the samples were taken. Immunofluorescence method A detected positive cells in 87% (56/64) of specimens from bladder cancer patients, including 18 of 19 from patients with grade 1 tumor. This method also had a low false-positive rate; only one of 17 specimens from patients with other urinary disorders had positively reacting cells. Immunofluorescence method B, evaluating a second group of specimens, detected positive cells in 68% (15/22) of specimens from patients with carcinoma, and in only one of 17 controls. However, it also identified positive cells in specimens from patients with chronic cystitis and urinary calculi. Overall the results of these studies indicate that the McAb method is superior to the routine cytology in detecting tumor cells in bladder washing specimens. More work must be done, however, to improve the specificity of the method before it can be used as an aid for routine tumor detection.     

Vergleich der perioperativen Methylenblau-gefärbten Schnellzytologie mit der nach Papanicolaou gefärbten Permanentzytologie in der Detektion von Patienten mit High-grade-Urothelkarzinomen der Harnblase

INTRODUCTION: The aim of the study was to investigate whether intraoperative methylene blue-stained and permanent Papanicolaou-stained urine cytologies show comparable accuracy in detection of high-grade urothelial carcinoma. PATIENTS AND METHODS: The study included 130 patients; 50 patients were without malignancy (25 follow-up, 25 with hematuria). In 80 patients transurethral resection due to urothelial carcinoma was performed. Per patient two cytology specimens were prepared: one immediate methylene blue-stained specimen, which was evaluated by the surgeon, and one Papanicolaou-stained permanent cytology slide, which was blinded and evaluated by one urologist. RESULTS: Cytology results of all patients without malignancy were unsuspicious irrespective of the staining method. Of 80 patients with urothelial carcinoma, 50 showed a low-grade tumor. Sensitivity of tumor detection was 20 and 30% for methylene blue/Papanicolaou-stained slides, respectively. Among 30 patients with high-grade carcinoma, 10 were detected by methylene blue cytology and 30 by Papanicolaou-stained slides, corresponding to a sensitivity of 40 and 100%, respectively. CONCLUSIONS: The results of standard Papanicolaou-stained urine cytology in the detection of clinically relevant high-grade urothelial carcinoma are excellent. The quality of cytological tumor detection by methylene blue-stained cytology made by different evaluators is insufficient in our opinion.     

Clinical evaluation of urinary nuclear matrix protein 22 as a marker for bladder cancer]

The purpose of this study is to evaluate the clinical usefulness of urinary nuclear matrix protein 22 (NMP22) as a marker for bladder cancer. We examined the positive rates of NMP22 test, urinary cytology and bladder tumor antigen (BTA) test, and compared the positive rate of NMP22 test with that in urinary cytology and BTA test. Urine samples were obtained from 50 patients with histologically confirmed bladder cancer before the treatment. The samples were examined by NMP22 test, urinary cytology and BTA test. In 50 patients with bladder cancer, the overall positive rate was 40% for NMP22 test, 40% for urinary cytology, and 16% for BTA test. A combination of NMP22 test and urinary cytology showed a significantly higher positive rate (54%) as compared to NMP22 test or urinary cytology alone. When NMP22 test and urinary cytology were compared for tumor size, number, shape, stage and grade, NMP22 test showed a significant higher positive rate than urinary cytology in grade 1 bladder cancer. In conclusion, although NMP22 test and urinary cytology gave a similar positive rate, a combination of NMP22 test and urinary cytology is more useful than the NMP22 test or urinary cytology alone for monitoring of bladder cancer.     

Clinical study of urinary cytology in bladder tumors--analysis of factors related with positivity of urinary cytology]

To clarify the clinical and pathological determinants affecting the sensitivity of urinary cytology, we reviewed cytological findings from 119 patients with bladder cancer who were initially treated between January 1982 and March 1988. Cytological specimens obtained from voided urine were stained by the Giemsa and Papanicolaou techniques, and were classified into three categories of malignant cells: positive, suspicious, and negative. Of 311 specimens examined, 114 (37%) were positive, 81 (26%) were suspicious, and the remaining 116 (37%) were negative. The overall positive rate, or sensitivity, was 50% (60 out of 119 patients). The sensitivities were 7% for patients with grade 1 tumors, 42% for grade 2 tumors, and 97% for grade 3 tumors. Univariate analysis by logistic regression analysis revealed that grade, stage, histological pattern of growth, size and number of tumors, and patient age were significantly related with the positivity of voided urinary cytology. The logistic regression model, as a multivariate analysis, demonstrated that grade was the most important determinant affecting the positive cytologic finding, followed by number of tumors with statistical significance. We conclude that the lower sensitivity in conventional urinary cytology for low-grade tumors necessitates new adjuncts, including immunocytochemistry and flow cytometry, to lower the false-negative rate.     

Sensitivity of exfoliative cytology in bladder cancer using the divided one-urine specimen examination

The urine of 104 patients with bladder cancer was examined by exfoliative cytology. Three staining methods (Giemsa, Papanicolaou and Oil Red O) were used and the results compared with the histological diagnosis. Giemsa was found to be superior to the other two methods but the highest sensitivity (which was statistically significant) was obtained by a combination of the three methods.     

How long is urinary cytology abnormal after flexible cystoscopy?

OBJECTIVE: To examine the urinary cytological changes caused by flexible cystoscopy and provide clinical guidelines for a reliable time interval for urinary cytological examination after flexible cystoscopy. PATIENTS AND METHODS: Forty-eight patients attending for flexible cystoscopy were recruited into the study. Each patient was asked to provide eight urine samples before, immediately after and at 1, 2, 7, 14 and 28 days after cystoscopy. Cytospin preparations of the urine samples were made and slides stained using the Papanicolaou stain. Cytology was analysed while unaware of sample origin, by three different cytopathologists. RESULTS: The cytological changes were characterized by a striking increase in cellularity immediately after flexible cystoscopy, mostly accounted for by urothelial cells. Consistent morphological changes included the formation of 'columnar' cells, papillary clusters, increased nucleo-cytoplasmic ratio and nuclear atypia. These changes were transient, with most disappearing within a day of flexible cystoscopy. CONCLUSIONS: There are cytological changes, on voided urine cytology, after flexible cystoscopy but they were transient, and urine sent more than a day after flexible cystoscopy should be free from artefactual change caused by instrumentation. These results suggest that clinicians sending urine for cytological analysis should provide information about the nature and timing of any endoscopy so as to avoid false-positive interpretations of urine cytology by the cytopathologist.     

Urinmarkersysteme zur Diagnostik des Urothelkarzinoms

Cystoscopy and urinary cytology are standard tools in the diagnostics of urothelial cancer of the urinary bladder; however, cystoscopy is invasive and urinary cytology lacks accuracy for the diagnosis of low grade tumors. More recently several alternative urinary test systems were developed with the aim to make the diagnostics of urothelial tumors more reliable; however, in general all protein-based point of care test systems have a high rate of false positive test results, especially in patients with benign disorders. Fluorescence in situ hybridization, which is highly sensitive and specific, may be a reasonable supplement to the diagnostic spectrum in patients after instillation therapy or bladder replacement. Additionally, there are several new test systems which still need to be tested in large clinical studies with respect to diagnostic accuracy.