Comparison of the Cellient(™) automated cell block system and agar cell block method.

OBJECTIVE: To compare the Cellient(TM) automated cell block system with the agar cell block method in terms of quantity and quality of diagnostic material and morphological, histochemical and immunocytochemical features. MATERIALS AND METHODS: Cell blocks were prepared from 100 effusion samples using the agar method and Cellient system, and routinely sectioned and stained for haematoxylin and eosin and periodic acid-Schiff with diastase (PASD). A preliminary immunocytochemical study was performed on selected cases (27/100 cases). Sections were evaluated using a three-point grading system to compare a set of morphological parameters. Statistical analysis was performed using Fisher's exact test. RESULTS: Parameters assessing cellularity, presence of single cells and definition of nuclear membrane, nucleoli, chromatin and cytoplasm showed a statistically significant improvement on Cellient cell blocks compared with agar cell blocks (P < 0.05). No significant difference was seen for definition of cell groups, PASD staining or the intensity or clarity of immunocytochemical staining. A discrepant immunocytochemistry (ICC) result was seen in 21% (13/63) of immunostains. CONCLUSION: The Cellient technique is comparable with the agar method, with statistically significant results achieved for important morphological features. It demonstrates potential as an alternative cell block preparation method which is relevant for the rapid processing of fine needle aspiration samples, malignant effusions and low-cellularity specimens, where optimal cell morphology and architecture are essential. Further investigation is required to optimize immunocytochemical staining using the Cellient method.

Interrater agreement in the clinical evaluation of acute pediatric asthma.

Prior studies of observer agreement of the clinical exam of children with asthma have focused on small numbers of specially trained observers, often in the setting of clinical trials. Our objective was to evaluate interobserver reliability in the physical exam of acute pediatric wheezing and asthma among a large group of diverse examiners, in a setting of routine clinical practice, and without prior special training. The setting was a large urban children's hospital. Observers were attending pediatric emergency physicians and fellows; hospital respiratory therapists; and emergency department (ED) nurses. Patients were children receiving nebulized medications for wheezing in the ED or inpatient asthma unit. Pairs of observers simultaneously but independently rated work of breathing, wheeze, decreased air entry, prolonged expiration, breathlessness, respiratory rate, mental status, and global (or overall) severity using a structured exam template. A total score for each exam was also evaluated. A total of 230 pairs of observations were performed; mean patient age was 5.3 years. For all pairs, the weighted kappa statistics for the exam components ranged from 0.61 to 0.74 (moderate or substantial agreement). The global severity category and total score had weighted kappas of 0.80 and 0.82, respectively (excellent agreement). Agreement was generally somewhat lower for unlike (different profession) observer pairs than for like observer pairs, but remained acceptable. Agreement in two age groups (< or = 3 years old and > or = 4) was at least moderate for all exam components analyzed. Spearman rank correlations between individual exam components and the global assessments of patient severity were all greater than 0.5, indicating at least moderate to good correlations. We found substantial interobserver agreement among a broad range of examiners in the components of the clinical examination of acute wheezing in both younger and older children. This is contrary to the commonly held observation that the poor interobserver reliability of physical exam findings in asthma may limit their usefulness as asthma outcome measures. Support for use of a structured respiratory exam format or template in asthma guidelines was also shown.

Short-term outcomes after acute treatment of pediatric asthma.

CONTEXT: The short-term course of pediatric patients after emergency department (ED) treatment for acute asthma has not been comprehensively documented; most previous studies have limited outcomes to ED length-of-stay, hospital admission, and relapse. OBJECTIVE: To describe symptom persistence, medication use, functional disability, follow-up, and relapse in these children in the 2 weeks after acute treatment and ED discharge. DESIGN: Randomly selected, prospective cohort from September 1996 to August 1997; follow-up telephone interviews at 1 and 2 weeks. Setting. A large, inner-city children's hospital emergency department. Patients. Random sample of pediatric asthma visits requiring ED treatment but not admission; 457 were eligible, 388 with complete follow-up (85%); final sample included 367 patients after multiple visits deleted. MAIN OUTCOME MEASURES: Details of symptom persistence, functional disability, medication use, relapse, and routine follow-up. RESULTS: Results included significant morbidity: 23% (95% confidence interval [CI]: 19, 27) with cough and 12% (95% CI: 9, 15) with wheeze persistent at 2 weeks; 20% (95% CI: 16, 24) with decreased activity at 1 week; 45% (95% CI: 39, 51) missed >2 and 24% (95% CI: 19, 29) >/=5 days of school or day care; 17% (95% CI: 13, 21) spent >/=3 days in bed; 54% (95% CI: 47, 60) of caretakers missed at least 1 and 18% (95% CI: 13, 24) missed >2 days of school or work; and 32% (95% CI: 28, 38) of patients were still using greater than baseline medication at 2 weeks. Reported relapse rates were averaged at 13% (95% CI: 10, 17) with 3% (95% CI: 1, 5) admitted. Routine office follow-up was poor: 29% (95% CI: 25, 34) had had a visit; 48% (95% CI: 43, 54) reported no visit/none planned. CONCLUSIONS: A considerable proportion of inner-city pediatric patients discharged from the hospital from the ED after standard treatment for acute asthma had poor short-term outcomes. Conventional markers of successful ED treatment, such as avoiding hospital admission or relapse, do not adequately describe outcomes of acute care. The patient-oriented measures described here may provide more useful indicators of outcome in the evaluation of acute asthma care.

MSH6 and MSH3 are rarely involved in genetic predisposition to nonpolypotic colon cancer.

A set of 90 nonpolypotic colon cancer families in which germ-line mutations of MSH2 and MLH1 had been excluded were screened for mutations in two additional DNA mismatch repair genes, MSH6 and MSH3. Kindreds fulfilling and not fulfilling the Amsterdam I criteria, showing early and late onset colorectal (and other) cancers, and having microsatellite stable and unstable tumors were included. Two partly parallel approaches were used: genetic linkage analysis (19 large families) and the protein truncation test (85, mostly smaller, families). Whereas MSH3 was not involved in any family, a large Amsterdam-positive, late-onset family showed a novel germ-line mutation in MSH6 (deletion of CT at nucleotide 3052 in exon 4). The mutation was identified through genetic linkage (multipoint lod score 2.4) and subsequent sequencing of MSH6. Furthermore, the entire MSH6 gene was sequenced exon by exon in families with frameshift mutations in the (C)8 tract in tumors, previously suggested as a predictor of MSH6 germ-line mutations; no mutations were found. We conclude that germ-line involvement of MSH6 and MSH3 is rare and that other genes are likely to account for a majority of MSH2-, MLH1-mutation negative families with nonpolypotic colon cancer.

Continuing education for cytotechnologists: the Australian experience.

The development of a continuing education scheme for cytotechonologists in Australia is described. The process involved the establishment of a working party with Terms of Reference to review current practice in Australia and existing schemes in other parts of the world. The scheme developed takes the form of a continuing education diary that provides guidelines on the various forms of continuing education activity and corresponding credit points. The diary also provides for a record of activity to be kept. The scheme requires bi-annual submission of personal activity which is logged into a national database. A peer profile is provided and successful achievement is marked by the issue of a certificate of participation. The programme has achieved a 57% compliance in its first year of operation.

Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis.

The distal portion of chromosome 1p is one of the most commonly affected regions in human cancer. In this study of hereditary and sporadic colorectal cancer, a region of frequent deletion was identified at 32.2 centimorgans from 1ptel. Deletion breakpoints clustered in the vicinity of or inside the gene RIZ, which encodes a retinoblastoma protein-interacting zinc finger protein. Sequence analysis revealed frequent frameshift mutations of the RIZ gene. The mutations consisted of 1- or 2-bp deletions of a coding (A)(8) or (A)(9) tract and were confined to microsatellite-unstable colorectal tumors, being present in 9 of 24 (37.5%) primary tumors and in 6 of 11 (54.5%) cell lines; in 2 cell lines the mutation was homozygous/hemizygous. The mutations apparently were selected clonally in tumorigenesis, because similar poly(A) tracts in other genes were not affected. Two alternative products of the gene exist, RIZ1, which contains a PR (PRDI-BF1-RIZ1) domain implicated in tumor suppressor function, and RIZ2, which is lacking this motif. Furthermore, the C-terminal region, which contains the poly(A) tracts, includes a PR-binding motif, possibly mediating interactions with other proteins or with RIZ itself (oligomerization). Four of eleven microsatellite-unstable colorectal cancer cell lines, three of which had frameshifts, showed reduced or absent mRNA expression of RIZ1. In a cell line that is homozygous/hemizygous for the typical frameshift mutation, immunoblotting showed truncated RIZ protein, whereas adenovirus-mediated RIZ1 expression caused G(2)/M arrest and apoptosis. We propose that RIZ is a target of the observed 1p alterations, with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.

Evaluation of the CytoRich technique for cervical smears.

The aim of the study was to assess the ability of the CytoRich System to prepare optimal gynaecological smears for diagnosis. The diagnostic results obtained from evaluating 1,325 matched slide-pairs, prepared using conventional methods and thin-layer technology, were compared. Cytological material for study was obtained using the combined spatula-cytobrush sampling technique. An assessment of the pitfalls associated with the interpretation of these smears was also undertaken. Diagnostic agreement was achieved in 1,272 of the 1,325 matched slide-pairs (96.0%), and these included 1,172 negative, 50 atypical, 24 low-grade squamous intraepithelial lesion (LSIL), 24 high-grade SIL (HSIL), and two malignancies. A total of 1,309 cases showed the same diagnosis within one diagnostic grade for an agreement of 98.8%. Evaluation of the 53 discordant diagnoses revealed that the conventional smear identified a significantly greater number of abnormal smears than the CytoRich technique (P < .001). It is suspected that the use of the combined spatula-cytobrush sampling technique did not provide adequate residual specimen for CytoRich after conventional smear preparation. This limitation is evidenced by the fact that the CytoRich preparations showed a lower yield of endocervical cells (P < .001) and infectious organisms (P < .001) than was demonstrated on conventional smears. Despite a number of diagnostic pitfalls associated with the interpretation of thin-layer smears, these preparations were easier and faster to screen and showed well-preserved and evenly distributed cells. Thin-layer smears were also characterised by a marked reduction in thick cell groups, air drying artifact, and obscuring inflammation and blood. The results confirm the limitation of the combined spatula-cytobrush technique in these types of comparative studies.

Effectiveness of automated cervical cytology rescreening using the AutoPap 300 QC System.

This study assesses the performance of the AutoPap 300 QC System in identifying false-negative (FN) smears in a slide population previously screened as normal and compares the detection rate to that achieved with a random rescreen of the same slide population. A total of 1,840 "normal" smears were rescreened both manually and by the AutoPap 300 QC System. Overall, a total of 7 FN slides were detected. At QC selection rates of 30% and 20% the device achieved sensitivities for detection of FN smears of 57.19% (4/7) and 42.8% (3/7), respectively. This represents a three- to fourfold enrichment in the number of FN smears over that obtained by a random rescreen of a similar proportion of cases. None of the FN slides were identified by either method at a 10% rescreening rate. The ability of the device to detect slides previously classified as abnormal (n = 139) and FN (n = 40) was also studied. The overall sensitivity to abnormal smears at QC selection rates of 10%, 20%, and 50% was 61.9%, 77.0%, and 94.2%, respectively. Improved sensitivity to smears classified as LSIL or worse (n = 112) was obtained for corresponding selection rates (61.6%, 75.9%, and 93.8%). Sensitivity to FN slides classified as LSIL or worse (n = 17) for QC selection rates of 10%, 20%, and 50% was 29.4%, 70.6%, and 88.2%, respectively. The sensitivity and specificity of the device to an adequate squamous and endocervical cell component was also determined. At predetermined thresholds, the overall sensitivity to slides with an inadequate squamous cell component (n = 55) and to those smears with an endocervical cell component (n = 1.587) was 81.8%, and 82.7% respectively. The study demonstrated that the AutoPap 300 QC System is superior to human random rescreen for the identification of FN smears although only a marginal improvement was noted due to the small sample. Further studies are required using a larger number of smears to fully assess the value of the device in quality control mode. The device also has the potential to improve the accuracy of specimen adequacy determinations and to serve as a useful adjunct to existing quality control measures designed to monitor individual performance and reporting accuracy.

Mixture risk assessment: a case study of Monsanto experiences.

Monsanto employs several pragmatic approaches for evaluating the toxicity of mixtures. These approaches are similar to those recommended by many national and international agencies. When conducting hazard and risk assessments, priority is always given to using data collected directly on the mixture of concern. To provide an example of the first tier of evaluation, actual data on acute respiratory irritation studies on mixtures were evaluated to determine whether the principle of additivity was applicable to the mixture evaluated. If actual data on the mixture are unavailable, extrapolation across similar mixtures is considered. Because many formulations are quite similar in composition, the toxicity data from one mixture can be extended to a closely related mixture in a scientifically justifiable manner. An example of a family of products where such extrapolations have been made is presented to exemplify this second approach. Lastly, if data on similar mixtures are unavailable, data on component fractions are used to predict the toxicity of the mixture. In this third approach, process knowledge and scientific judgement are used to determine how the known toxicological properties of the individual fractions affect toxicity of the mixture. Three examples of plant effluents where toxicological data on fractions were used to predict the toxicity of the mixture are discussed. The results of the analysis are used to discuss the predictive value of each of the above mentioned toxicological approaches for evaluating chemical mixtures.

Malignant mesothelioma: cytologic diagnosis with histologic, immunohistochemical, and ultrastructural correlation.

The differential diagnoses of malignant mesothelioma in serous effusions include adenocarcinoma and reactive mesothelial cells. While several cytologic features are of predictive value in separating these entities, immunostaining and ultrastructural examination are important adjuncts that increase the diagnostic yield. Many of the cytomorphologic features can be correlated with immunohistochemical and ultrastructural findings. Most important among these is the ultrastructural demonstration of long, often branching microvillous processes in malignant mesothelial cells. Corresponding microvilli can be visualized by immunostaining for epithelial membrane antigen in both cell block preparations from effusions and biopsy specimens, allowing the identification of malignant mesothelioma. In addition, the circumferential distribution of these immunostained microvilli in cells dispersed in stromal connective tissue identifies them as malignant mesothelial cells, corresponding to the ultrastructural appearance of aberrant microvilli, which project through deficiencies in the basal lamina. These microvilli show interdigitation with stromal collagen fibers, a phenomena not observed in adenocarcinoma.

Cytopathology of malignant mesothelioma: a stepwise logistic regression analysis.

Twenty-four cytologic features, previously reported to be useful in the distinction of malignant mesothelioma, adenocarcinoma, and benign mesothelial proliferation in serous effusions were assessed. Forty-four cases of malignant mesotheliomas, 46 cases of metastatic adenocarcinomas, and 30 cases of benign mesothelial proliferations were examined for these parameters. When these cytologic features were subjected to a stepwise logistic regression analysis, five features were selected to distinguish malignant mesothelioma from adenocarcinoma. These were true papillary aggregates, multinucleation with atypia, cell-to-cell apposition, acinus-like structures, and balloon-like vacuolation, the latter two features being characteristic of adenocarcinoma. The four variables selected to distinguish malignant mesothelioma from benign mesothelial proliferations were nuclear pleomorphism, macronucleoli, cell-in-cell engulfment, and monolayer cell groups, the latter being a feature of benign proliferations. Using these selected variables, the logistic model correctly predicted 95.4% of cases of malignant mesothelioma versus 100% of adenocarcinoma and 100% of malignant mesotheliomas versus 90% of benign mesothelial proliferations. The results of regression analysis suggest that many of the previously described cytologic features are not important diagnostic discriminators.

Use of morphometry in cytological preparations for diagnosing follicular non-Hodgkin's lymphomas.

Morphometric techniques were applied to cytocentrifuge smears from 27 patients with histologically confirmed follicular non-Hodgkin's lymphoma to determine the usefulness of this method in diagnosis. Analysis of quantitative data confirmed that most subtypes were classified correctly on the basis of the proportion of large cells, small cleaved, and large non-cleaved cells, and nuclear, nucleolar, and cytoplasmic features. Morphometry is a more objective and reproducible technique than manual cell counting methods, and the use of cytocentrifuge smears has several advantages compared with conventional histological sectioning. Data derived with the aid of morphometry may be of value in follow up clinicopathological studies.

Quantitation of nuclear shape in non-Hodgkin's lymphoma. Alternate shape descriptors.

The nuclear contour index (NCI) has been used extensively to quantitate cellular shape in non-Hodgkin's lymphoma (NHL). This disease entity has a mixed cellular component, with nuclei exhibiting a spectrum of shapes; the main disadvantage of conventional shape descriptors, such as NCI, is that similar values are assigned to these widely divergent shapes. In this study, two alternative shape indices, measures of convexity-concavity (C-C) and slimness (SL), were assessed to determine their suitability as shape descriptors. To achieve this, the contours of lymphoid cells in cytologic preparations of NHL were quantitated by means of semiautomated image analysis. C-C was able to identify nuclear clefts, with the values obtained by this index reflecting the number and depth of invaginations present; this index was unaffected by ellipsoid shapes. SL was able to describe ellipsoid profiles, irrespective of the presence of nuclear clefts; the index also characterized indentations, although precise identification of this form of invagination was difficult. This study confirms the usefulness of these indices in the characterization of nuclear shape; utilization of these shape descriptors enhances the accuracy of more-objective approaches to the morphologic classification of lymphoid lesions.

Lymph node cellular morphology: comparative study of imprints and cytocentrifuge smears.

To assess the usefulness of the lymph node imprint technique in quantitating cell types a comparative study using cytocentrifuge smears as controls was undertaken. The cell dimensions and distribution of cell types were compared in 12 patients with non-Hodgkin's lymphoma (NHL) using semi-automated image analysis. The results show that the imprint procedure introduces selective bias in favour of certain cell types; moreover, certain cell measurements, such as nuclear shape, are easier to see on cytocentrifuge smears. The imprint technique compares unfavourably with cytocentrifuge smears when precise quantitation of cell types is required.

A review of toxicology studies on cyanurate and its chlorinated derivatives.

Chlorinated cyanurates are added to swimming pools as disinfectants. In the presence of water, these materials hydrolyze to yield cyanurate and hypochlorous acid. To evaluate the safety of exposure to these materials, a comprehensive testing program was undertaken. This review summarizes the results of acute and subchronic tests on chlorinated isocyanurates. Findings from acute, subchronic, reproduction, metabolism, mutagenicity, and chronic/carcinogenicity tests on cyanurate are also summarized. Results from these tests indicate that chlorinated isocyanurates are safe for use in swimming pools.

The immunogold staining technique for the measurement of lymphocyte subpopulations in bronchoalveolar lavage fluid.

The immunogold staining technique was evaluated for use in the identification of lymphocyte subsets in bronchoalveolar lavage fluid. The results obtained compared favorably to the identification of lymphocytes by the E-rosette method, the acid alpha-naphthyl-acetate esterase (ANAE) stain and immunofluorescence microscopy. The main advantages of the immunogold staining method include improved cellular detail and the attainment of permanent preparations, allowing for reassessment, intralaboratory comparison and the performance of further histochemical techniques if desired.