Cervical cytology EQA – the Northern experience

The experience of the Northern region using the 1995 draft National Gynaecological Cervical Cytology External Quality Assurance (EQA) Scheme is given. Over three rounds, 390 staff reporting Cervical Cytology took part, and using a cumulative non-numeric marking scheme, five participants were deemed "unacceptable" at the end of three rounds. A total of 3450 responses were given to the 40 test slides used, with an overall false-positive rate of 5.9% and a false-negative rate of 1.4%. Grading was assessed for pathologists, and many appeared to perform badly by accumulating discrepancies for non-clinically-significant grading differences. The problems of slide selection/staining and grading consistency/accuracy are highlighted. This EQA scheme serves its dual function of education and identifying poor performance. It must be seen as a viable EQA scheme, although other options must be considered, given advances in technology.     

Utility of immunohistochemical markers in differentiating benign from malignant follicular-derived thyroid nodules

In virtual microscopy, a sequential process of captures of microscopical fields, allows to construct a virtual slide which is visualized using a specialized software, called the virtual microscopy viewer. This tool allows useful exploration of images, composed of thousands of microscopical fields of view at different levels of magnification, emulating an actual microscopical examination. The aim of this study was to establish the main pathologist's navigation patterns when exploring virtual microscopy slides, using a graphical user interface, adapted to the pathologist's workflow. Four pathologists with a similar level of experience, graduated from the same pathology program, navigated six virtual slides. Different issues were evaluated, namely, the percentage of common visited image regions, the time spent at each and its coincidence level, that is to say, the region of interest location. In addition, navigation patterns were also assessed, i.e., mouse movement velocities and linearity of the diagnostic paths. Results suggest that regions of interest are determined by a complex combination of the visited area, the time spent at each visit and the coincidence level among pathologists. Additionally, linear trajectories and particular velocity patterns were found for the registered diagnostic paths.     

iPathology cockpit diagnostic station: validation according to College of American Pathologists Pathology and Laboratory Quality Center recommendation at the Hospital Trust and University of Verona

Background

Validation of digital whole slide images is crucial to ensure that diagnostic performance is at least equivalent to that of glass slides and light microscopy. The College of American Pathologists Pathology and Laboratory Quality Center recently developed recommendations for internal digital pathology system validation. Following these guidelines we sought to validate the performance of a digital approach for routine diagnosis by using an iPad and digital control widescreen-assisted workstation through a pilot study.

Methods

From January 2014, 61 histopathological slides were scanned by ScanScope Digital Slides Scanner (Aperio, Vista, CA). Two independent pathologists performed diagnosis on virtual slides in front of a widescreen by using two computer devices (ImageScope viewing software) located to different Health Institutions (AOUI Verona) connected by local network and a remote image server using an iPad tablet (Aperio, Vista, CA), after uploading the Citrix receiver for iPad. Quality indicators related to image characters and work-flow of the e-health cockpit enterprise system were scored based on subjective (high vs poor) perception. The images were re-evaluated two weeks apart.

Results

The whole glass slides encountered 10 liver: hepatocarcinoma, 10 renal carcinoma, 10 gastric carcinoma and 10 prostate biopsies: adenocarcinoma, 5 excisional skin biopsies: melanoma, 5 lymph-nodes: lymphoma. 6 immuno- and 5 special stains were available for intra- and internet remote viewing. Scan times averaged two minutes and 54 seconds per slide (standard deviation 2 minutes 34 seconds). Megabytes ranged from 256 to 680 (mean 390) per slide storage. Reliance on glass slide, image quality (resolution and color fidelity), slide navigation time, simultaneous viewers in geographically remote locations were considered of high performance score. Side by side comparisons between diagnosis performed on tissue glass slides versus widescreen were excellent showing an almost perfect concordance (0.81, kappa index).

Conclusions

We validated our institutional digital pathology system for routine diagnostic facing with whole slide images in a cockpit enterprise digital system or iPad tablet. Computer widescreens are better for diagnosing scanned glass slide that iPad. For urgent requests, iPad may be used. Legal aspects have to be soon faced with to permit the clinical use of this technology in a manner that does not compromise patient care.

     

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.     

Immunohistochemical evaluation of HER-2/neu expression in infiltrating breast carcinoma: a study of reproducibility

OBJECTIVE: To determine interobserver and intraobserver reproducibility in the assessment of the HercepTest- and TAB250-immunostained slides. STUDY DESIGN: Three independent expert pathologists (two with and one without training in HercepTest assessment) evaluated the HercepTest and TAB250-immunostained slides of 108 infiltrating breast carcinomas with a triple-blind method. The evaluation was repeated, with the same method and sequence of view, after 60 days. RESULTS: Expert pathologists, after adequate training in HercepTest evaluation, could reach excellent interobserver (K=.911, P<.001) and intraobserver reproducibility (K of .863-.926; P <.001 for all). The percentage of disagreement in intraobserver reproducibility ranged from 0.9% to 3.7%. Interobserver and intraobserver reproducibility in the evaluation of TAB250-immunostained slides was good (K = .658, P < .001) and from good to excellent (K of .600-.895, P < .001 for all), respectively. CONCLUSION: Optimization of the level of accuracy in HercepTest evaluation is mandatory because the decision to initiate therapy with Herceptin depends on the result. Moreover, considering that the percentage of disagreement in intraobserver reproducibility ranges from 0.9% to 3.7%, it is advisable that two expert pathologists evaluate all HercepTest slides with a double-blind method. If there are discordant results, they must be discussed by the same pathologists.     

Automated Instrument for the Fluorescent Treponemal Antibody-Absorption Test and Other Immunofluorescence Tests

An automated diagnostic test instrument and its development program are described. The instrument automates the fluorescent treponemal antibody-absorption test for syphilis to the extent that only 4 hr of technician time is required to conduct approximately 200 tests daily. Evaluation to date suggests its efficacy. In addition, preliminary studies indicate the feasibility of detecting antibodies to Toxoplasma gondii, Plasmodium malariae, and nucleoprotein (antinuclear factor). The instrument would seem to have broad application for routine and research immunofluorescence testing. Two elements comprise the instrument: a slide processor and a microscope attachment. The slide processor is an electro-pneumatically actuated device which automatically feeds special laboratory slides, on which antigen or other reagents are prefixed, through a series of operations which provide reagent application, incubation, washing, drying, and stacking of the finished slides for readout. The instrument provides flexibility in that incubation time and temperature as well as point, sequence, and duration of reagent application can be varied to accommodate a variety of immunofluorescence techniques. The microscope attachment can be fitted to all conventional dark-field fluorescence microscopes and makes possible the reading of three to six slides per minute. The reacted slides from the processor are injected sequentially onto the stage of the microscope by movement of a lever. As injected, slides are automatically in visual focus; fine focus is occasionally required. Scanning of the reacted field is accomplished by means of the normal microscope controls. A buffered glycerol coupling is maintained between the darkfield condenser substage lens and the slide cover glass by means of a pushbutton-actuated feed system.     

Interpreting microbiopsies in cervical smears. A cytohistologic approach

OBJECTIVE: To assess interobserver variation in the diagnosis of thick tissue specimens (microbiopsies) in cytology smears and histologic sections taken from them, to evaluate the applicability of MIB-1 in histologic sections from microbiopsies and to evaluate whether processing microbiopsies in inconclusive smears has additional diagnostic value. STUDY DESIGN: Cytologic smears were selected in which there were diagnostic disagreements between pathologists and cytologists and microbiopsies were present. Interobserver variation among three pathologists and three cytologists in the diagnosis of these microbiopsies was investigated. The smears were processed for histologic sections, and interobserver variation between pathologist diagnoses were analyzed. An additional histologic slide stained for MIB-1 was used for consensus diagnosis. The consensus diagnosis was compared with available follow-up and its sensitivity and specificity determined. The value of applying the microbiopsy technique in slides diagnosed as inadequate or atypical squamous cells of undetermined significance (ASCUS) was analysed. RESULTS: From a series of 62,334 cervical smears, 49 with microbiopsies were selected. It was possible to derive histologic slides from 38 cases. Interobserver variability in the diagnosis of microbiopsies and histologic sections from them was moderate--kappa = .44 (SE = .06) and kappa = .44 (SE = .09), respectively. In the consensus meeting for all cases, a conclusive diagnosis was reached. The Pearson correlation coefficient between the consensus diagnosis and MIB-1 staining was r = .62. The sensitivity of the consensus diagnosis for the follow-up diagnosis was 71% and the specificity 60%. Diagnosis on approximately 50% of slides diagnosed as inadequate or ASCUS could be made. CONCLUSION: The histotechnical workup of microbiopsies is not difficult; however, their diagnosis can be a problem. Adequate diagnostic criteria are not available. Aided by MIB-1 staining, histologic sections from microbiopsies can be diagnosed, and the diagnoses correlated with follow-up in most cases. Processing of microbiopsies in smears with an inconclusive cytologic diagnosis or a diagnosis of ASCUS allowed correct diagnosis in 50% of cases in this study.     

Diagnostic parameters in liquid-based cervical cytology using a coagulant suspension fixative

OBJECTIVE: To evaluate in detail the morphology of cervical cell samples suspended in the coagulant fixative BoonFix (Finetec, Tokyo, Japan) in liquid-based Papspin slides (Thermo Shandon, Pittsburgh, Pennsylvania, U.S.A) to detect shifts in diagnostic parameters for infections and neoplasia. STUDY DESIGN: Split samples of 1,010 cases were collected. All Papspin slides were scanned with neural network technology. In 849 cases the diagnosis was "within normal limits"; in 22 cases it was preneoplasia. In 151 special cases conventional smears were compared with thin-layer slides. RESULTS: In 85% of the 151 special cases, a shift of the diagnostic parameter was observed in the Papspin slide. The parameter adhesion of inflammatory cells to epithelial cells was easier to discern in 94% of the cases, and adhesion of microorganisms varied 43-100%. Koilocytosis was more visible in 79%. Prominent nucleoli in atypical and malignant cells were enhanced in 50-100% of cases with preneoplasia. The fact that the cells on the Papspin slide were no longer present in diagnostic streaks posed a problem only in the case of follicular cervicitis. CONCLUSION: The shifts in parameters facilitated the diagnostic process. BoonFix permits the screening of liquid-based Papspin slides, which have proven to be well suited to automated neural network scanning.     

Architectural, morphometric and photometric features and their relationship to the main subjective diagnostic clues in the grading of prostatic cancer

Novel software was developed to perform quantitative measurements of architectural and nuclear features in tissue sections. A pilot study was then undertaken to determine the diagnostic relevance of these quantitative features in prostatic tissue and the relationship of these objective features to the subjective clues used by practicing pathologists in the grading of prostatic adenocarcinoma. From a group of 82 cases of adenocarcinoma of the prostate with long-term follow-up, a subset of 15 cases that included 5 each in Mostofi grades I, II and III was carefully selected for analysis. Consecutive sections from each case were stained with hematoxylin and eosin or the Feulgen stain for visual and cytometric evaluations, respectively. The most important differences in the objective architectural features observed between the Mostofi grade I and II cases were the number of nuclei per gland and their distance from the glandular center. Significant differences were also noted in gland size and the variation in gland size. The Mostofi grades were also significantly different in terms of quantitative high-resolution features measuring nuclear size and its variation, total nuclear DNA content and the proportion of very aneuploid nuclei. There was a fairly good agreement between many of the subjective diagnostic clues and their corresponding quantitative architectural and nuclear features. This work (1) significantly extended the capabilities of our PC-based microphotometer system to analyze glandular tissue specimens, (2) provided insight into the objective bases for the expert diagnosis of adenocarcinomas of the prostate and (3) gave preliminary evidence of the ability of quantitative architectural features and high-resolution cytometric features to discriminate between the major diagnostic categories of these lesions.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.     

Interlaboratory quality control in gynecologic cytopathology using the novel CONQUISTADOR software. Interobserver reproducibility in the Latin American screening study

OBJECTIVE: The recently developed software (CONQUISTADOR), capable of computing all intralaboratory and interlaboratory quality control (QC) indicators, was used to evaluate the diagnostic agreement among 4 cytology laboratories participating in the LAMS Study. STUDY DESIGN: The study was an interlaboratory exchange of specially designed 5 slide sets, each comprising 20 (conventional cytology) slides. At the first step, 80 slides (with "clear-cut" cases) were divided into four sets (A, B, C, D) of 20 specimens, each including inadequate and negative cases as well as in different proportions of all diagnostic TBS 2001 categories. In the second round, a fifth set (E) of 20 slides ("difficult cases") was designed, with all diagnostic categories, ASC and AGC included. Common measures of reproducibility (kappa and weighted kappa), accuracy (SE, SP, PPV, NPV) and 3 indices of diagnostic variability were calculated for sets A-D and set E, separately. RESULTS: For the 5 slide sets together, the weighted kappa was 0.8 (95% CI 0.76-0.85), which is the lower limit of the "almost perfect" ranking of kappa statistics, indicating an excellent interlaboratory agreement. The interlaboratory reproducibility was lower only for the difficult set (E). Similarly, the sensitivity for set E (70.0%) was lower than that (92.1%) for sets A-D. The diagnostic variability indices were not substantially different between the difficult (set E) and clearcut (sets A-D) cases. CONCLUSION: High interlaboratory reproducibility was obtained for sets A-D ("clear-cut" cases), while more interlaboratory variation was evident in the difficult samples. The new CONQUISTADOR software is a valuable tool in calculating the indicators needed in this intralaboratory and interlaboratory.     

Utility of 2-D and 3-D virtual microscopy in cervical cytology education and testing

OBJECTIVE: To evaluate the effectiveness of 3-D vs. 2-D virtual microscopy as adjuncts to education and assessment in cervical cytology. STUDY DESIGN: Five cervical cytology slides were acquired in 2-D; then the identical area of the slide was acquired in 3-D, resulting in 2 sets of virtual slides for comparison with the original glass slide. Seventy-nine paid volunteer cytologists and cytotechnology students participated. Approximately half were sent the 2-D set of slides via the Web, and the others a 3-D set of slides on a DVD. Evaluators examined the virtual slides and committed to an interpretation. After receipt of the original glass slides, a second interpretation was made, if different from the virtual slide interpretation. RESULTS: Diagnostic accuracy using virtual cytology slides was similar to that for glass slides (94% vs. 96%). There was no difference in diagnostic accuracy between 2-D and 3-D slides (p = 0.28); however, the ability to focus 3-D slides in the z-axis was strongly endorsed by the participants because of the uncertainty and frustration of having some cells out of focus on 2-D virtual slides. CONCLUSION: There was consensus that virtual cervical cytology slides would be a useful augmentation to education and testing.     

Implementation of LED Fluorescence Microscopy for Diagnosis of Pulmonary and HIV-Associated Tuberculosis in a Hospital Setting in Indonesia

Background

Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients.

Method

We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard.

Results

Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of ≥2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23±0.78 vs 5.82±1.60 minutes, P<0.01), while costs per slide were similar.

Conclusion

High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories.     

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.     

Computer-assisted sperm analysis of canine spermatozoa motility measurements

Computer-assisted sperm analysis (CASA) allows for the determination of specific motion characteristics of sperm cells in vitro. This study was designed to develop a system for the use of CASA to objectively evaluate canine sperm motility, and specifically to determine whether motility characteristics vary between individual dogs. Ejaculates from 10 dogs were collected weekly. Sperm cells were extended in a glucose-free TALP medium, placed on slides and videotaped at 200x. Videotaped samples were then analyzed by the Hamilton-Thorn Motility Analyzer, with 100 cells evaluated per slide. Two slides were made from each ejaculate. Motility characteristics that were evaluated included lateral head displacement, beat cross frequency, path velocity, path linearity, path straightness, percentage of motile cells, and percentage of progressively motile cells. Sperm cell morphology was also evaluated. Canine spermatozoa maintained good overall motility (mean +/- SD, 73 +/- 9%) during the procedure. Mean sperm motility and morphology measurements differed significantly between dogs (P<0.01). There was no difference (P>0.05) between the mean measurements of different ejaculates for an individual dog, or for different slides made from the same ejaculate. Mean motility values for the 10 dogs are reported. There was a significant but not strong correlation (r=0.44) between the percentage of progressively motile sperm cells and the percentage of sperm cells with normal morphology.     

Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology

A. Evered and N. Dudding Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology Objective: To evaluate virtual microscopy in terms of diagnostic performance and acceptability among practising cytologists. Methods: Twenty‐four experienced cytologists were recruited to examine 20 SurePath® cervical cytology slides by virtual microscopy. Diagnostic accuracy was compared with glass slide microscopy using an unbiased crossover experimental design. Responses were allocated a score of one for a correct identification of normal or abnormal (borderline/atypical changes in squamous or glandular cells or worse) and a score of zero for an incorrect response (a normal slide reported as abnormal or vice versa). Perceptions of virtual microscopy were assessed by questionnaire analysis. Results: Participants yielded a total of 285 responses for the virtual slide set and 300 for the glass slide set. The approximate time to screen a virtual slide was 18 minutes, compared with 8 minutes or less for a glass slide. Overall there was no significant difference between virtual microscopy and glass slide microscopy in terms of diagnostic accuracy (P = 0.22). Virtual microscopy under‐performed when images were captured over a narrow focal range (P = 0.01). Diagnostic accuracy of virtual microscopy equalled that of glass slide microscopy when participants were able to focus through the full thickness of the slide images (P = 0.07). The most common difficulties experienced by participants with virtual microscopy were freezing of the computer screen during image download, slow response of the computer during slide movement and, in some instances, ‘fuzzy’ images. Cytologists have a strong preference for glass slides over virtual microscopy despite the overall equal diagnostic performance of the two viewing modalities. Conclusions: Diagnostic accuracy of virtual microscopy can equal that of glass slide microscopy. However, without good computer network connections, wide focal range and software that permits effortless navigation across virtual slides, cytologists are unlikely to be convinced of the utility of this technology for cytology screening and diagnosis.     

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

BACKGROUND: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells. METHODS: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition. The ability of erythroid lineage cells to adhere to several surface modifications was examined. In situ hybridization methods were tested to find the best approach that is compatible with these cell preparations. RESULTS: The best glass slide coating for erythroid cells was found to be an antibody to glycophorin A, a red cell surface antigen. We were able to get excellent in situ hybridization signals in cells on flaskettes by modifying fixation and pretreatment parameters. CONCLUSIONS: The methods described here appear to be the best way of attaching a large number of erythroid lineage cells to slides and of detecting them by in situ hybridization.     

An update on the validation of whole slide imaging systems following FDA approval of a system for a routine pathology diagnostic service in the United States

Pathologists have used light microscopes and glass slides to interpret the histologic appearance of normal and diseased tissues for more than 150 years. The quality of both microtomes used to cut tissue sections and microscopes has improved significantly during the past few decades, but the process of rendering diagnoses has changed little. By contrast, major advances in digital technology have occurred since the introduction of hand held electronic devices, including the development of whole slide imaging (WSI) systems with software packages that can convert microscope images into virtual (digital) slides that can be viewed on computer monitors and via the internet. To date, however, these technological developments have had minimal impact on the way pathologists perform their daily work, with the exception of using computers to access electronic medical records and scholarly web sites for pertinent information to assist interpretation of cases. Traditional practice is likely to change significantly during the next decade, especially since the Federal Drug Administration in the USA has approved the first WSI system for routine diagnostic practice. I review here the development and slow acceptance of WSI by pathology departments. I focus on recent advances in validation of WSI systems that is required for routine diagnostic reporting of pathology cases using this technology.     

Correlation of modified Gleason grading of prostate carcinoma with age, serum prostate specific antigen and tumor extent in needle biopsy specimens

OBJECTIVE: To investigate the correlation of biopsy grade with age, serum prostate specific antigen (PSA) and biopsy tumor extent using the conventional and modified Gleason grading systems. STUDY DESIGN: A total of 828 consecutive needle biopsy specimens of prostate carcinoma were collected from the years 1995 and 2000 (graded with conventional Gleason grading) and 2006 and 2007 (graded with modified Gleason grading). RESULTS: Both conventional and modified Gleason grading correlated with age, serum PSA, percent positive biopsies and percent cancer length. In 2006-2007, the patients were on average younger and more biopsy cores were taken per patient. Serum PSA and percent positive cores were lower than in the 1995 and 2000 group, indicating a stage shift downward, but the Gleason scores were nevertheless higher. CONCLUSION: Conventional and modified Gleason grading both correlated with age, serum PSA and cancer involvement in needle biopsies. With modified Gleason grading there is a grade shift upward despite the downstaging that has been observed in recent years.     

Issues in using whole slide imaging for diagnostic pathology: “routine” stains,immunohistochemistry and predictive markers

The traditional microscope, together with the “routine” hematoxylin and eosin (H & E) stain, remains the “gold standard” for diagnosis of cancer and other diseases; remarkably, it and the majority of associated biological stains are more than 150 years old. Immunohistochemistry has added to the repertoire of “stains” available. Because of the need for specific identification and even measurement of “biomarkers,” immunohistochemistry has increased the demand for consistency of performance and interpretation of staining results. Rapid advances in the capabilities of digital imaging hardware and software now offer a realistic route to improved reproducibility, accuracy and quantification by utilizing whole slide digital images for diagnosis, education and research. There also are potential efficiencies in work flow and the promise of powerful new analytical methods; however, there also are challenges with respect to validation of the quality and fidelity of digital images, including the standard H & E stain, so that diagnostic performance by pathologists is not compromised when they rely on whole slide images instead of traditional stained tissues on glass slides.