Comparison of manual and automated methods of liquid-based cytology. A morphologic study

OBJECTIVE: To evaluate the morphologic characteristics of gynecologic samples prepared by 3 different methods of liquid-based cytology. STUDY DESIGN: Cytologic samples from representative cases of each diagnostic category of squamous epithelial lesion, prepared by automated and manual liquid-based systems, were analyzed by 3 laboratories in the United States, Portugal and Brazil. The systems included: ThinPrep (automated, U.S. Food and Drug Administration approved; Cytyc Corp., Boxborough, Massachusetts, U.S.A.), Autocyte PREP (South American system, manual; TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) and DNACITOLIQ (manual; Digene Brazil, S?o Paulo, Brazil). A panel of 16 morphologic parameters was evaluated: cellularity, clean background, uniform distribution, artifacts, cellular overlapping, architectural and cytoplasmic distortion, cytoplasmic vacuolization, cellular elongation, imprecise cytoplasmic borders, folded cytoplasmic borders, nuclear hyperchromasia, coarse chromatin, prominent nucleolus, irregular nuclear borders, atypical mitosis and inflammatory infiltrate. Negative, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) cases were included. Cases without biopsies were confirmed by consensus. RESULTS: Cellularity was adequate in all samples. Clean background was observed in the vast majority of samples with all liquid-based systems. Uniform distribution was frequently found in ThinPrep and Autocyte PREP samples but not in DNACITOLIQ. Artifacts were not present in DNACITOLIQ samples, rare in ThinPrep and observed in 8 (34.7%) Autocyte PREP. Cellular overlapping was observed in all 3 system samples: 11 (31.42%) cases in ThinPrep, 16 (69.56%) in Autocyte PREP and 17 (68%) in DNACITOLIQ System. Architectural and cytoplasmic distortion were present in 3 cases of HSIL (13%) and cytoplasmic vacuolization in 2 cases of LSIL and 1 HSIL of Autocyte PREP. Cellular elongation was found in 13 (56.5%) Autocyte PREP and in 5 (20%) DNACITOLIQ samples. Inflammatory infiltrate was found in all 3 system samples but with more frequency in the Autocyte PREP (69.56%) and DNACITOLIQ System (72%). CONCLUSION: This study clearly indicated that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation. The choice of method will depend on price, availability and procedures involved.     

Evaluation of a centrifuge method and thin-layer preparation in urine cytology

OBJECTIVE: To evaluate and compare the quality and cost of urine cytology using the Cytospin method (Shandon, ThermoElectron Corporation, Waltham, Massachusetts, U.S.A.) and the AutoCyte PREP (TriPath Imaging, Burlington, North Carolina, U.S.A.) in a general laboratory. STUDY DESIGN: A study of differences between the Cytospin method and AutoCyte PREP in the areas of specimen preparation, staining, number and quality of diagnostic cells, background, screener preference, and cost was undertaken over a 3-month period in 2000. Sixty fresh voided urine samples from 25 patients with known transitional cell carcinoma were prepared by the Cytospin method and the AutoCyte PREP according to the manufacturers' instructions. RESULTS: The Cytospin method had longer preparation time but shorter screening time than the AutoCyte PREP. The number of diagnostic cells was higher in the Cytospin method. Fixation quality and staining clarity were better in the Cytospin method. Qualitative assessment of cell arrangements, cell and nuclear size and shape, nuclear/cytoplasmic ratio and nuclear membrane irregularity showed no significant differences between the 2 methods. Cellular details and nuclear chromatin patterns were clearer and better preserved in the Cytospin method, but the AutoCyte PREP showed less blood and inflammatory cells and debris. CONCLUSION: In the majority of cases the screeners preferred the Cytospin method due to its better overall cytologic quality. However, the amount of blood, inflammation and debris was much lower in the AutoCyte PREP. This reduced the need to make a second, diluted specimen and made turnaround time faster. The AutoCyte PREP was 7 times more expensive than the Cytospin method.     

Applicability of liquid-based cytology to the assessment of DNA content in cervical lesions using static cytometry

OBJECTIVE: To evaluate the nuclear DNA content of cervical lesions in liquid-based cytologic specimens prepared for static cytometry. STUDY DESIGN: The DNA content of cervical lesions was evaluated in cervical samples prepared with the Autocyte PREP liquid-based cytology system (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.). A series of 47 samples stained with the Papanicolaou method (chronic cervicitis, n = 15; cervical intraepithelial neoplasia [CIN] 1, n = 25; CIN 2, n = 5; CIN 3, n = 2) were collected from consecutive women enrolled in an ongoing screening study at Leonor Mendes de Barros Hospital, S?o Paulo, Brazil, in 2002. Each residual sample was processed according to the Feulgen-thionin method (TriPath Imaging). Ploidy evaluation was performed using the CAS 200 image analysis system and Quantitative DNA Measurement software 3.0 (version 8.1) (Becton Dickinson, San Jose, Califoria, U.S.A.). Cellular ploidy was analyzed from atypical nuclei, and the DNA index was obtained using histograms for interpretation. RESULTS: All chronic cervicitis cases were diploid. Of the CIN 1 cases, 44% were diploid, 12% tetraploid, 32% aneuploid and 12% polyploid (diploid plus tetraploid). CIN 2 lesions were diploid in 60% and aneuploid in 40% of cases, whereas all CIN 3 lesions (100%) were aneuploid. CONCLUSION: The liquid-based cytologic samples proved to be suitable and highly useful for DNA analysis by image cytometry, which was capable of discriminating CIN 3 lesions from CIN 1 and 2 but not CIN 1 from 2 lesions. Aneuploidy was closely associated with CIN 3 lesions.     

Fine needle aspiration cytology of lymph node: experience of 2 university hospitals with conventional smears and liquid-based cytology

OBJECTIVE: To compare 2 different series of fine needle aspirations (FNA) performed by conventional smears (CS) and liquid-based cytology (TriPath PREP). STUDY DESIGN: From January 1, 2001, to December 31, 2005, we selected 139 FNA samples of lymph node, if the diagnosis was histologically proven after the initial cytologic diagnosis. Samples came from 2 university hospitals from Brussels: UZ-Brussel (n=96) using liquid-based cytology (LBC) and Hospital Erasme ULB (n=43) using CS. RESULTS: The number of inadequate samples was greater for LBC than CS, but there was no statistical difference (17.7% vs. 4.7%, p = 0.059). No differences were found between LBC and CS in sensitivity (respectively, 85.0% vs. 85.2%), specificity (84.2% vs. 85.7%) and efficiency (84.8% vs. 85.4%). CONCLUSION: Despite the cost, the efficiency of lymph node FNA cytology is identical between the CS and LBC performed by the TriPath PREP system. The quality of the smears, both LBC and CS, depends mainly on practitioner dexterity. Nevertheless, one advantage of LBC is that it provides an ancillary technique to improve FNAC diagnosis. In the future, a combination of these technologies with FNAC alone could in some cases take the place of surgical lymph node excision.     

Evaluation of thin-layer methods in urine cytology

Conventional cytospin smears prepared from urinary tract specimens were compared with two new thin layer techniques, i.e. ThinPrep and AutoCyte PREP. Cellularity, cell preservation, background features, detection rate, screening time and ease of preparation were evaluated. Thin-layer techniques when applied to urine cytology were found to improve cell yield and cell preservation, and reduce background artefact. The reporting rate for abnormal urothelial cells was comparable to conventional cytospin smears, as was screening time. Laboratory staff found the methodologies to be practicable and easily incorporated into a large routine diagnostic service. We conclude that a one-slide thin-layer urine preparation is comparable to four cytospin slides in the detection of urothelial abnormalities, and that both ThinPrep and AutoCyte PREP have comparable features.     

Accuracy of thin-layer cytology in patients undergoing cervical cone biopsy

OBJECTIVE: To compare the accuracy of thin-layer cytology with Autocyte PREP (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with conventional smears in 500 women undergoing cervical cone biopsy. STUDY DESIGN: The study was performed among 500 consecutive women presenting for cone biopsy for high grade cervical intraepithelial neoplasia (CIN) on biopsy in 350 (70%) and discrepant cytology/colpohistology in 150 (30%). Before performing a cone biopsy, two cervical samples were collected for conventional smears and thin-layer cytologic slides, with randomization of the order. Conventional smears were stained and diagnosed at Pasteur Cerba, while thin-layer cytologic slides were processed at a local TriPath office (Meylan, France) and sent in a masked fashion for screening at Pasteur Cerba. Any slides initially read as normal were reviewed again and reported without knowledge of the other cytologic or cone biopsy data. The final cytologic diagnoses for the two methods were compared with histopathology of the cone biopsy. RESULTS: The conventional smear was unsatisfactory in 58 (11.6%) of cases, while there were 4 (0.8%) unsatisfactory thin-layer cytologic slides (P < .001). Endocervical cells were missing from 31 (6.2%) of conventional smears and 34 (6.8%) of thin-layer cytologic slides. For the pooled data, sensitivities of conventional smear and thin layer for detecting high grade CIN (0.82% and 0.86%, respectively) were similar as were specificities (0.40% and 0.43%, respectively). When first samples were compared, the sensitivities of the conventional smear and thin layer for high grade CIN were 0.79% and 0.89%, respectively (P = .02), with corresponding specificities of 0.41% and 0.36% (P < .01). CONCLUSION: When controlled for sample order, the sensitivity of thin-layer cytology for detecting high grade CIN was significantly higher than that of conventional smears in patients with previous abnormal cytology, but at the expense of specificity.     

Processing liquid-based gynecologic specimens: comparison of the available techniques.

OBJECTIVE: To develop a cytopreparation protocol suitable for satisfactory processing by the AutoCyte PREP System with the gynecologic specimens collected in PreservCyt fluid for the ThinPrep machine. STUDY DESIGN: The residue of a number of gynecologic specimens collected in PreservCyt and processed by ThinPrep were processed by AutoCyte PREP. Some modifications were made in the cytopreparatory protocol in order to obtain satisfactory specimens. RESULTS: The ThinPrep and AutoCyte PREP specimens were examined independently. The results were comparable, with a high degree of concordance between the two techniques. CONCLUSION: Gynecologic specimens collected in PreservCyt and following the ThinPrep specimen collection protocol can be processed using the AutoCyte PREP System. Minor protocol modifications provided comparable diagnostic material. Additional studies are needed to explore the feasibility of this approach and fulfill the various U.S. regulatory agency requirements for the liquid-based Pap test.     

Laboratory Test of an Automated Cell Analysis System For Cervical Screening

An automated cell analysis system (Autoplan-MIAC) for the early detection of precancerous lesions of the cervix was tested under semi-routine conditions in a clinical cytology laboratory. A set of 1500 specimens, highly enriched with abnormal cases, was analysed. Cervical scrapings were collected in suspension and processed by cytocentrifugation for microscopy. Two slides were prepared from each sample: one for staining according to Papanicolaou for the visual reference diagnosis and one for Feulgen staining for automated analysis. the specimens were evaluated in two ways: the first one, which is referred to as the automated machine classification system (AMC), classifies the specimens according to the number and ratio of selected objects (alarms) and is a fully automated system. the second system classifies the specimens after visual evaluation of the stored alarms as they are displayed on a TV monitor, and is designated the interactive machine classification system (IMC). the AMC results showed a false positive rate of 16.5% when the cut-off threshold was selected so that all 117 positively diagnosed specimens were classified ‘positive’ by the system. In that case 87.4% of the CINI and 96.9% of the CINII cases were AMC-positive. the IMC results showed a false positive rate of 2.5%, when 86.3% of the CIN I cases, 96.9% of the CIN II cases and all CIN III and invasive carcinoma cases were positively classified.     

Use of automated primary screening on liquid-based, thin-layer preparations

OBJECTIVE: To evaluate the accuracy of the AutoPap System (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) (TriPath) in screening AutoCyte PREP liquid-based, thin-layer preparations by comparing the final cytologic diagnoses with instrument slide classification results. STUDY DESIGN: A total of 9,665 AutoCyte PREP thin-layer slides were first independently screened to establish a final cytologic diagnosis (reference diagnosis). The slides were then processed on the AutoPap System. Each slide successfully processed was reported into result categories. The generated report gave a ranking score for each slide designated for "review." Slides designated "no further review" (NFR) were also listed in the report. The reported results were then compared to the reference cytologic diagnoses. RESULTS: Of 9,665 slides initially submitted to the AutoPap, 8,688 (90.8%) were qualified for scanning, and 884 (9.2%) were definitely classified as process review or rerun and excluded from the study. Of high grade squamous intraepithelial lesions and greater (HSIL+), 85.2% were ranked in the first rank, 12.7% in the second, one (2.1%) in the third, none in the fourth and fifth and none in the NFR category. Of low grade squamous intraepithelial lesions, 47.4% were ranked in the first rank, 20.8% in the second, 10.6% in the third, 10.1% in the fourth, 5.3% in the fifth and 5.8% in NFR. Of atypical squamous cells of undetermined significance and atypical glandular cells of undetermined significance, 53.6% were ranked in the first rank, 22.5% in the second, 12.4% in the third, 5.4% in the fourth, 3.8% in the fifth and 2.3% in NFR. Considering a cutoff value at < or = 3rd rank, 84% of cervical abnormalities (RR 6.52, 95% CI 4.96-8.66) and 100% of HSIL+ were identified. CONCLUSION: The AutoPap demonstrates a high capability for detecting cervical abnormalities on AutoCyte PREP thin-layer slides. HSIL+ was associated with the highest instrument scores.     

Split-sample analysis of discarded cells from liquid-based Pap smear sampling devices

OBJECTIVE: To examine cells that were retained on sampling devices used to collect ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A) Pap smears in order to evaluate both the number and significance of cells that are routinely discarded with these devices after liquid-based specimens are collected. STUDY DESIGN: One hundred Pap smears from 100 women were prospectively procured after gynecologic Pap smears were collected for the ThinPrep Pap test. The sampling end of the collection devices was cut off and placed in a vial that contained SUREPATH preservative fluid (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A). The residual cell samples were processed using the SurePath PREPSTAIN slide processor (TriPath). A single liquid-based slide was prepared from the sampling devices from each of the 100 specimens collected. The slides produced from the discarded devices were reviewed for the following: squamous cells, endocervical component, epithelial cell abnormalities and miscellaneous findings. The slides prepared from the "throw-away" (TA) material were subsequently compared with the primary ThinPrep Pap smear slide. RESULTS: Twenty-five percent of the TA samples had an equal or greater number of squamous cells per high-power microscopic field when compared to the primary ThinPrep slide, with 8% of the TA slides demonstrating greater overall cellularity. An endocervical component was present on 27 of 66 cervical samples (40.9%). Three of five cases (60%) interpreted as atypical squamous cells of undetermined significance had similar cells on the TA slides. Two cases of atypical glandular cells of undetermined significance had no abnormal cells on the TA slides. Twelve of 14 cases (85.71%) of low grade squamous intraepithelial lesion contained similar cells on the TA slides. Two of four cases (50%) of high grade squamous intraepithelial lesion also had similar abnormal cells on the TA slides. Miscellaneous findings included 1 case of benign endometrial cells and 4 Candida infections present on both preparations, along with 1 case of Trichomonas vaginalis organisms present on the ThinPrep slide only. In 1 specimen, several multinucleated histiocytic giant cells were present only on the TA slide. CONCLUSIONS: Specimens prepared from TA collecting devices used for the ThinPrep Pap test are less sensitive than the primary specimen for the detection of cervical lesions. This is in contrast to split-sample studies involving ThinPrep and conventional smears. Our study documented the presence of normal and abnormal cells discarded from ThinPrep sampling devices in a high percentage of cases. Discarded abnormal cells on the TA slides were, however, few when compared to the primary specimen, with only 1 exception involving a high grade lesion.     

Conventional Pap smear and liquid-based cytology as screening tools in low-resource settings in Latin America: experience of the Latin American screening study

OBJECTIVE: To evaluate the performance of the conventional Pap test and liquid-based cytology (LBC) in an ongoing multicenter trial testing optional screening tools (cytology, screening colposcopy, visual inspection with acetic acid, visual inspection with Lugol's Iodine, cervicography and Hybrid Capture II [HCII] (Digene Brazil, S?o Paulo, Brazil) conventional and self-sampling), for cervical cancer in Brazil and Argentina. STUDY DESIGN: A cohort of 12,107 women attending four clinics (Campinas, S?o Paulo, Porto Alegre, Buenos Aires) were randomized into the 8 diagnostic arms. Women testing positive with any of the tests were referred for colposcopy, and cervical biopsies were used as the gold standard to assess performance characteristics of the diagnostic tests. Conventional Pap smears were sampled by all clinics (n = 10,240), and LBC (Autocyte PREP, [TriPath Imaging, Burlington, North Carolina, U.S.A.], n=320, and DNA-Citoliq [Digene Brazil], n =1,346) was performed by 1 of the clinics. RESULTS: Conventional Pap smears showed no squamous intraepithelial lesions (normal) in 8,946 (87.4%) and LBC in 1,373 (82.4%). Using high grade squamous intraepithelial lesions (HSIL) as the cutoff, Pap smears predicted high grade (cervical intraepithelial neoplasia [CIN] 3) with OR 63.0 (95% CI, 36.90-107.70), standard error (SE) 59%, SP 97.8%, positive predictive value (PPV) 68.1% and negative predictive value (NPV) 96.7%. The same figures for Autocyte PREP were: OR 9.0 (95% CI, 2.43-33.24), sensitivity (SE) 33.3%, specificity (SP) 100%, PPV 100% and negative PV (NPV) 88.8%. DNA-Citoliq detected CIN 3 as follows: OR 11.8 (95% CI 2.60-53.26), SE 40.0%, SP 94.6%, PPV 40.0% and NPV 94.6%. Lowering the cutoff to low grade squamous intraepithelial lesions increased SE and NPV but compromised SP and PPV. The detection rates for high grade lesions after an atypical squamous cells of undetermined significance diagnosis were similar with the 3 techniques. In our settings, the 3 methods of cervical cytology were slightly different in performance. The conventional Pap smear had the highest SE, while Autocyte PREP had 100% SP and PPV in detecting CIN3 with the HSIL cutoff. All 3 tests had lower SE but higher SP as compared to HCII.     

Evaluation of the AutoCyte SCREEN system in a clinical cytopathology laboratory

OBJECTIVE: To compare the AutoCyte SCREEN (AutoCyte, Burlington, North Carolina, U.S.A.) system with manual screening by experienced cytotechnologists using thin-layer preparations that had been previously extensively studied and their cytologic abnormalities well defined. STUDY DESIGN: AutoCyte PREP (AutoCyte) samples prepared for a previous split-sample study comparing thin-layer preparations to conventional smears were used. These 1,992 AutoCyte PREP samples were in a cohort the abnormal findings of which had been confirmed via independent review by two sets of pathologists. For the current study, these samples were remasked and evaluated by the AutoCyte SCREEN system in a clinical laboratory. The instrument scanned each slide and selected six overview fields and 120 single objects for storage and display. The computer classified each slide in one of the following categories: abnormal, uncertain, normal or unsatisfactory. Independently for each case, a cytotechnologist evaluated the six fields and 120 objects selected by the instrument as abnormal, normal or unsatisfactory. For those cases classified as uncertain by AutoCyte, the technologist then reexamined the cellular displays and entered a consensus classification. These results were then compared to those of an independent review by cytotechnologists of the identical set of slides using routine manual screening. RESULTS: The AutoCyte SCREEN selected 35% of slides for manual review. Technologist and computer rendered equivalent classifications in 79%. Of the total slides screened by the AutoCyte SCREEN, 57% were classified as "uncertain," and 88% of these were subsequently classified as normal by consensus. Using the well-defined abnormal values of the cellular sample as a basis for calculation, the AutoCyte SCREEN-assisted practice had a diagnostic sensitivity of 85% and diagnostic specificity of 97.6%. Comparable values for manual screening of the identical cellular sample were a diagnostic sensitivity of 80% and specificity of 97.4%. CONCLUSION: The AutoCyte SCREEN achieves comparable or greater sensitivity in detecting cervical abnormalities in comparison with manual screening. When combined with the substantial advantage of thin-layer preparations over conventional smears, the AutoCyte SCREEN provides a screening system of superior sensitivity over conventionally prepared and examined cervical smears.     

An Evaluation of Some Commercial Test Papers and Tablets for the Determination of Glucose in Urine

The commercial test papers, Tes-Tape, Clinistix, Uristix and Combistix, and the tablet preparation, Clinitest, were evaluated as indicators of glucose in urine by means of a quantitative automated glucose oxidase procedure for glucose determination. The semiquantitative Tes-Tape yielded very low values on urine specimens when compared with the quantitative method. More reliable results could be obtained with this product if the urine specimens were first treated with a mixed bed resin to remove inhibitors of the glucose oxidase peroxidase system. The qualitative test papers, Clinistix, Uristix and Combistix, yielded responses in closer agreement with the automated data, the best performance being obtained with Clinistix. The semiquantitative Clinitest tablets generally yielded more accurate results on a direct urine test than did Tes-Tape, although the Clinitest tablet is designed to measure total reducing substances rather than glucose alone.     

The role of cytology in the diagnosis and follow-up of patients with cervical adenocarcinoma

A review was undertaken of the 121 patients with cervical adenocarcinoma, ranging in age from 10 to 91 years, who had cytology specimens examined in this laboratory. All but two had cervicovaginal smears. Fifty-eight patients had cervical smears taken prior to treatment; 91% of these contained malignant cells. The commonest histologic subtype was mucinous adenocarcinoma (61 patients; 52%), followed by endometrioid carcinoma (26 cases; 22%), adenosquamous carcinoma (16 cases; 14%) and clear-cell carcinoma (12 cases; 10%). The cytologic characteristics were evaluated and correlated with the histopathology. During the follow-up period after definitive treatment, 119 patients had gynecologic smears taken; 24% were positive, representing 64% of those cases with central recurrences. In two patients, the abnormal smear was the first indicator of recurrence. Nine patients had 11 nongynecologic specimens revealing metastatic disease. Notable in this study were the 18 asymptomatic women, of whom 15 also had either no visible cervical lesion or minimal changes associated with benign conditions; cytology was abnormal in all but two. Cytology was effective in detecting disease. Its usefulness continued during the follow-up period in identifying or confirming recurrent or metastatic disease.     

The preparation of cervical scrape material for automated cytology using gallocyanin chrome-alum stain.

A method is described for preparing cervical scrape specimens for automated analysis on the Cerviscan prescreening system. In order to reduce the cellular clumping found in cervical scrape material, cells are collected in suspension, syringed to disaggregate the cell clumps, and then pipetted onto a glass to give a monolayer of cells. The cells are then stained with gallocyanin chrome-alum to give the required quantitation of nucleic acid content, using a rapid staining procedure. Experimental results are given which show that specimens prepared by this method are more suitable for automated analysis than the conventional Papanicolaou stained preparation.     

New red blood cell lysing fixative for use in fine needle aspiration and fluid cytology

OBJECTIVE: To describe a new red blood cell (RBC) lysing fixative, Devine's lysing solution (DLS), that increases the diagnostic utility of fine needle aspiration (FNA) and fluid cytology. STUDY DESIGN: Twenty bloody FNA cases were fixed with either DLS or 95% ethanol, and the ability to render a diagnosis on these materials was analyzed. DLS was compared to the red cell lysing fixative CytoRich Red (CRR) (TriPath Care Technologies, Burlington, North Carolina, U.S.A.) in its ability to lyse RBCs by mixing human RBCs with the U266 multiple myeloma cell line. DLS was compared to CRR in its ability to suitably preserve materials for Papanicolaou and immunocytochemical analysis. RESULTS: Comparison of DLS with 95% ethanol fixation in 20 bloody FNA cases prepared in duplicate showed that DLS reduced from 17 to 3 the number of cases that had RBCs obscuring > or = 25% of the diagnostic material. In 3 cases, DLS enabled the rendering of a definitive diagnosis of malignancy, which could not be made on the ethanol-fixed material. DLS was compared to CRR, and both fixatives were similarly effective at lysing RBCs, preserving the cellular morphology of diagnostic cells in FNA and fluid cytology, and preserving cells for use in immunocytochemistry. CONCLUSIONS: DLS increases the visualization of diagnostic cellular material when compared to ethanol fixation. DLS is comparable to CRR in RBC lysing ability, diagnostic cell preservation and preservation of materials for immunocytochemistry.     

Collection fluid helps preservation in voided urine cytology

Objectives:  Degenerative change caused by delay in processing contributes to false-negative and false-positive diagnosis of urothelial carcinoma in cytology. The aim of the study was to see if the use of a collection fluid for urine samples made a significant difference to urine cytology diagnosis, and if one was better suited for routine use in the hospital laboratory. Three cell collection fluids were evaluated by analysing the preservation and degeneration of cells in urine samples, as was the routine preparation which did not use a collection fluid.
Methods:  In the design study 50 voided urine specimens were taken at random from the hospital haematuria clinic. Three commercially available collection fluids cytolyt^TM, cytospin^® and cytoRich^®Blue and the hospital's routine conventional preparation of urine were compared. The degree of degeneration, and so preservation, was assessed by a table of chosen criteria; then ranked and analysed by Friedman's nonparametric test, at P  = 0.05. A second table showing the cell content of each slide was also made.
Results:  These showed no significant diagnostic difference between the collection fluids, but there was a significant difference between the collection fluids and the routine preparation. Minor differences that do not affect diagnosis, such as crystals and ghost red blood cells, were noted in cytospin^® and cytoRich^®Blue.
Conclusion:  It is recommended that a collection fluid is used. This choice should be made after health and safety issues and cost are considered.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.     

Use of mailed urine specimens in diagnosing urothelial carcinoma by cytology and DNA image analysis

OBJECTIVE: To evaluate the usefulness of urine specimens collected via a mailer system and analyzed by cytology and DNA ploidy for the detection of urothelial carcinoma (UC). STUDY DESIGN: We retrospectively reviewed the diagnoses of 91 mailed urine specimens received from 72 patients, 67% of whom had a history of UC. The specimens were fixed in an equal volume of 50% ethanol solution before being mailed. The cytologic findings were interpreted in conjunction with DNA ploidy image analysis. We compared these initial diagnoses with those of follow-up examinations, including biopsies, cystoscopic findings and urinary cytology/DNA ploidy analyses. In addition, to examine the quality of the mailed samples, 3 cytopathologists performed a blinded assessment of cytologic slides of 20 mailed and 17 fresh urinary samples for bacterial overgrowth, urothelial degeneration, and presence of proteinaceous material and crystals. RESULTS: Follow-up was available for 68 of the 91 mailed specimens. The sensitivity for detecting UC using mailed urine specimens that were analyzed by both cytology and DNA ploidy was 61%, while specificity was 92%. The levels of bacterial overgrowth and urothelial degeneration in the mailed specimens were not significantly greater than in the fresh specimens (p>0.05). The levels of proteinaceous material and crystals were significantly higher in the mailed specimens (p<0.05). CONCLUSION: The results of combined cytology and DNA ploidy image analysis by using mailed urine samples were comparable to those of fresh urine specimens for the detection of UC reported in previous publications. The increase in crystals and proteinaceous material did not impede diagnostic interpretation. The mailing system is a reliable and convenient method of monitoring and triaging patients with UC or related symptoms.     

Comparison of ThinPrep and conventional preparations on fine needle aspiration cytology material

OBJECTIVE: To compare the various cytologic features on ThinPrep 2000 (TP) (Cytyc Corporation, Marlborough, Massachusetts, U.S.A.) and conventional preparation (CP) specimens from fine needle aspiration cytology (FNAC) material by a semiquantitative scoring system. STUDY DESIGN: In this prospective study a total of 71 consecutive cases were included. In each case, two passes were performed. The first pass was used for conventional preparations, with direct smears made and fixed immediately in 95% alcohol for Papanicolaou stain. For TP preparation a second pass produced material for processing in the ThinPrep 2000. The TP and CP slides were studied independently by two observers and representative slides of CP and TP compared for cellularity, background blood and necrotic cell debris, cell architecture, informative background, presence of monolayer cells, and nuclear and cytoplasmic details by a semiquantitative scoring system. Statistical analysis was performed by Wilcoxon's signed rank test on an SPSS program (Chicago, Illinois, U.S.A.). RESULTS: TP preparations contained adequate diagnostic cells in all cases and were tangibly superior to CP preparations concerning monolayer cells, absence of blood and necrosis, and preservation of nuclear and cytoplasmic detail (statistically significant, Wilcoxon's signed rank test, P < .000). CONCLUSION: TP preparations are superior to conventional preparations with regard to clear background, monolayer cell preparation and cell preservation. It is easier and less time consuming to screen and interpret TP preparations because the cells are limited to smaller areas on clear backgrounds, with excellent cellular preservation. However, TP preparations are more expensive than CP and require some experience for interpretation.