Comparison of automated and manual methods of estimation of methylmalonic acid.

An automated method for estimation of methylmalonic acid in urine is described that compares well with results obtained by a manual method. The temperature at which color is developed can be set anywhere between ambient and 90°C without loss of linearity over a certain range of concentrations. The higher temperatures have greater sensitivity but a smaller linear range than do lower incubation-temperatures.     

A new,liquid-based cytology technique

OBJECTIVE: To evaluate a new liquid-based cytology technique, Papspin (Thermo Shandon, Pittsburgh, Pennsylvania, U.S.A.). STUDY DESIGN: Three thousand cervical samples were examined. Each cervix was sampled with a Cervex Brush (Roche, Oss, the Netherlands), used first for a Pap smear and afterwards for a Papspin. One cytospin was prepared from each vial. RESULTS: An identical rate of epithelial cell abnormalities (3.8%) was detected with the two methods. Diagnostic concordance was observed in 86% of the 114 cases. Differences in diagnoses occurred in 168 of 3,000 cases (5.6%) concerning fungal infection (22 cases), epithelial cell abnormalities (24 cases) and minimal differences within the nonneoplastic Bethesda category (122 cases). Endocervical cells were absent from 158 Papspins (5.3%) and 66 Pap smears (2.2%), while they were present in the respective Pap smear or Papspin. Seven Papspins were considered "satisfactory, but limited by ..." (SBLB) as compared to 33 Pap smears given the absence of endocervical cells. CONCLUSION: Discordances concerning epithelial cell abnormalities were observed in 24 of 3,000 cases (0.8%). Fungal infections were more easily diagnosed on Papspin. The absence of endocervical cells in 5.3% of Papspins is due to a bias of methodology. Quality improvement was evident on Papspin for SBLB specimens. HPV testing could be performed with good results.     

Concordance between original screening and final diagnosis using imager vs. manual screen of cervical liquid-based cytology slides

OBJECTIVE: To compare agreement of pathologists and cytotechnologists and technologist productivity before and after implementation of the Cytyc ThinPrep Imager. STUDY DESIGN: Using the Cytyc ThinPrep Imaging System, a retrospective analysis was conducted, from the first 6 months in 2004 and in 2005. Total cases in January through June were 79,791 in 2004 and 76,887 in 2005. Data on the number and type of changes from one impression to another were collected in a "confusion matrix". The chi2 test with 1 degree of freedom was used to calculate the significance of the difference between the groups. RESULTS: Changes in diagnosis were most frequently seen in negative for intraepithelial lesion, atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion. Interobserver agreement before the imager (weighted kappa) was 0.74 and after was 0.73. CONCLUSIONS: The number of high-grade lesions detected increased with the imager (p < 0.01). Technologist productivity increased by an average of 2.2 slides/hour.     

Comparison of gas accumulation profiles of several feeds using manual or automated gas production methods

The relationship of metabolizable energy (ME) content of the diet to gas production measured by the Hohenheim gas test (HGT) has been studied intensively. However, the HGT is being replaced by automatic systems like the automated pressure evaluation system (APES) and comparison with the HGT method is required before ME estimation can be automated. This study compared the two different gas production methods (HGT and the APES) with regard to the cumulative gas profile. With the APES method, the release of gas may occur at any time, assuming fixed amounts of gas being released for each venting, after reaching fixed values of pressure. With the HGT method manual readings are performed at defined time points. For comparison purposes, gas production was calculated on the basis of ml/200 mg dry matter (DM), as usual in the HGT method. For 11 feeds analyzed (grass silage, meadow hay, fresh red clover, fresh birdsfoot trefoil, whole-crop oat silage, maize stover and ear maize, dairy compound feed and soybean meal) the APES method produced on average 5.5 ml (range 1.1–8.3 ml/200 mg DM) less gas on average compared to the HGT method (0–120 h incubation time). Reasons for the differences may be related to the measurement conditions of each method itself. The ratio of sample size to rumen fluid (mg/ml) is 20:1 in the HGT method and 100:1 in the APES method, which may have influenced the colonization rate and contributed to a larger lag phase in APES. The estimates are based on two runs, which were performed on different days, with the APES method generating a large run effect. In conclusion, the amount of gas produced using the APES method deviated consistently from the amounts of gas produced by the HGT method in a large range of gas production (30–72 ml/200 mg DM). Using the laboratory protocol as proposed by each method, the suggested mathematical correction of the gas measured through APES appears applicable for gas production after 24 h, needing a larger number of samples to prove its efficacy.     

Analysis of the reliability of manual and automated immunohistochemical staining procedures. A pilot study

OBJECTIVE: To study the variation in the number of stained cells and staining intensity comparing 2 immunostainers and manual staining for estrogen receptor (ER) expression in breast carcinoma. STUDY DESIGN: In 5 cases, 15 consecutive paraffin sections were investigated after simultaneous immunohistochemical ER staining. The slides were evaluated using a CM-2 TV image analysis system (Hund, Wetzlar, Germany). One viewing field, identified around a histologic structure present on all 15 sections, was analyzed. The percentage of immunoreactive cells (PP), mean grey values of the immunopositive (GVpos.) and immunonegative nuclei (GVneg.), and immunohistochemical staining intensity (SI, defined as GVneg.-GVpos.) were calculated. RESULTS: The mean PP values were higher for immunostainers A (70.2%) and B (53.8%) than for manual staining (40.8%). The results were significantly different comparing the 2 immunostainers (P = .0143) or immunostainer A and manual staining (P < .0001). Also, the mean SI values were higher for immunostainers A (24.5 +/- 2.8% [CV]) and B (18.5 +/- 31.1%) than for manual staining (10.8 +/- 33.8%). These differences revealed statistical significance comparing the immunostainers with manual staining (.0001 < P = .0048). CONCLUSION: Our results underline the higher staining quality using immunostainers in comparison with manual staining.     

Comparison of conventional and liquid-based cytology, and human papillomavirus testing using SurePath preparation in Japan

We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.     

A generalized machine for automated flow cytology system design.

A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.     

Significance of atypia in conventional Papanicolaou smears and liquid-based cytology: a follow-up study

The diagnosis of atypical squamous epithelial cells, borderline nuclear changes, is associated with some controversy, as it encompasses benign, reactive, as well as possible neoplastic conditions. The aim of this study was to evaluate the follow-up diagnoses of cytological atypia in conventional Papanicolaou smears (CP) and liquid-based samples by the ThinPrep Pap Test (TP). A total of 1607 CP smears from 1 January 2000 to 31 December 2000 and 798 TP samples from 1 January 2002 to 31 December 2002 diagnosed as atypia were included. The results show that the detection rate of atypia in cervical cytological samples was reduced by 41.3% (P < 0.001) in TP compared with CP. Cytological and histological follow-up data showed the presence of neoplastic lesions in 34.7% of patients screened by TP versus 22.3% of patients screened by CP, corresponding to a 55.6% increase in TP (P < 0.001). Follow-up diagnosis of mild dysplasia was seen more than twice as often in TP than in CP (12.8% versus 5.0%, P < 0.001). The prevalence of moderate and severe dysplasia was significantly increased with 26.7% in TP compared with CP (21.9% versus 17.2%, P < 0.01). In conclusion, the ThinPrep Pap Test yielded a significant decrease in atypia rates compared with the conventional Papanicolaou test. In subsequent follow-up the percentage of neoplastic lesions was significantly increased in the ThinPrep Pap Test samples.     

Simplified methods for automated ion-exchange separation of peptides and accelerated manual Edman degradations

An apparatus for the automated separation of peptides and one for the manual Edman degradation of peptides is presented. Both are composed of relatively simple and inexpensive items and are capable of being constructed in most laboratories. Each may also serve as an important laboratory teaching and demonstration device. As many as five peptides may be simultaneously degraded with the manual Edman apparatus and as many as six degradations may be performed daily on each peptide.     

Comparison of autofocus methods for automated microscopy

Traditional autofocus methods were designed for microscopes driven by single processor computers. As computers are developed that exploit massive parallelism when acquiring and analyzing images, parallel cellular logic techniques became available to focus automatically. This paper introduces the reader to both cellular logic techniques for autofocus and a new spectral moment autofocus measure. It then compares these methods with more traditional autofocus methods. It is shown that traditional methods based on measurements of image power-give the best results when tested on one set of real images and two sets of synthetic images. The next best methods are the cellular logic and spectral moment techniques, while the worst are those based on the image probability density function or histogram.     

Comparison of manual and automated methods for identifying target sounds in audio recordings of Pileated, Pale-billed, and putative Ivory-billed woodpeckers

ABSTRACT.   Although offering many benefits over manual recording and survey techniques for avian field studies, automated sound recording systems produce large datasets that must be carefully examined to locate sounds of interest. We compared two methods for locating target sounds in continuous sound recordings: (1) a manual method using computer software to provide a visual representation of the recording as a sound spectrogram and (2) an automated method using sound analysis software preprogrammed to identify specific target sounds. For both methods, we examined the time required to process a 24-h recording, scanning accuracy, and scanning comprehensiveness using four different target sounds of Pileated Woodpeckers ( Dryocopus pileatus ), Pale-billed Woodpeckers ( Campephilus guatemalensis ), and putative Ivory-billed Woodpeckers ( Campehilus principalis ). We collected recordings from the bottomland forests of Florida and the Neotropical dry forests of Costa Rica, and compared manual versus automated cross-correlation scanning techniques. The automated scanning method required less time to process sound recordings, but made more false positive identifications and was less comprehensive than the manual method, identifying significantly fewer target sounds. Although the automated scanning method offers a fast and economic alternative to traditional manual efforts, our results indicate that manual scanning is best for studies requiring an accurate account of temporal patterns in call frequency and for those involving birds with low vocalization rates.     

Outcomes of cervical liquid-based cytology suggesting a glandular abnormality

Objective: To ascertain the positive predictive value of both ?glandular neoplasia (national standard code 6) and borderline change (national standard code 8) in glandular cells in liquid‐based cervical cytology specimens in Cardiff and Vale NHS Trust and to outline the histological outcomes of these cases. Method: Eighty‐nine liquid‐based (Surepath?) cervical cytology cases were retrospectively identified from a 2‐year period (January 2005 to December 2006) and correlated with histopathological diagnoses. Results: Initial punch biopsy histology revealed 18 cases (21%) of cervical glandular intraepithelial neoplasia (CGIN). A further nine cases (10%) of CGIN were identified following local excision or hysterectomy. Ten cases of invasive malignancy were identified: four endocervical adenocarcinomas (all node negative, TNM stage T1b1), five endometrial adenocarcinomas and one squamous cell carcinoma. There were 10 with high‐grade cervical intraepithelial neoplasia (CIN) alone. Women diagnosed with endometrial malignancy presented later with an average age of 64.6 years compared with 34.9 years for endocervical lesions. Taking high‐grade CIN or worse as a positive outcome, the overall positive predictive value (PPV) of glandular abnormalities on cytology (both code 6 and 8) was 58.1% [95% confidence interval (CI) 47.8, 68.4]. PPV for borderline change in glandular cells alone was 24.1% (95% CI 8.5, 39.6) and for ?glandular neoplasia alone 75.4% (95% CI 64.3, 86.5). Conclusion: With our interpretation of the classification, women with cytological diagnoses of glandular neoplasia of the cervix should initially be investigated by local resection rather than punch biopsy, and those with borderline change in glandular cells with repeat cytology.     

Comparison of the conventional cervical smear and liquid-based cytology: results of a controlled, prospective study in the Abruzzo Region of Italy

OBJECTIVE: To compare conventional cervical testing (CCT) and liquid-based cytology (LBC) within a randomized trial performed during 2001-2002 in the Abruzzo Region of Italy, including a cost-outcome comparative analysis. STUDY DESIGN: Study subjects were recruited in the framework of a controlled, randomized study organized in the Abruzzo Region. Women aged 2 6-64 years were randomized to an active arm (LBC) or control arm (CC1). The particip ating laboratories had no previous ex perience with LBC. RESULTS: The inadequacy rate was 4.3% in CCT and 1.3% in the LBC arm (D < 0.001). Atypical squamous cells of undetermined sign ifi cance and atypical glands of undetermined significance reports were more frequent at CCT vs. LBC. A small, insignificant excess of low grade squamous intraepithelial lesions or high grade squamous epithelial lesions+ reports was observed in the LBC arm. The cervical intraepithelial neoplasia 2+ (CIN2+) detection rate was not statistically different in the 2 arms (CCT=0.54%, LBC= 0.66%, p = 0.28). In the overall series positive predictive value was slightly but not significantly higher in the LBC arm. LBC increased costs by 4.2% per both screened women and CIN2+ detected. CONCLUSION: The study reflects the introductory phase of LBC in laboratories without prior LBC experience. In this setting LBC reduced the inadequacy rate and decreased reading and was at least as sensitive as and more specific than CCT. Utilization of LBC in organized screening programs will be based on local feasibility, considering that the high cost of LBC is only partially compensated for by other benefits, such as residual cellular material, available for molecular testing, including human papillomavirus testing.     

Impact of liquid-based gynecologic cytology on an HIV-positive population

OBJECTIVE: To examine the imprint of liquid-based technologies for cervicovaginal cytology on HIV-positive women, who are at high risk for cervical intraepithelial neoplasia. STUDY DESIGN: We performed a retrospective search of the cytopathology files of Johns Hopkins Hospital for the cervicovaginal cytology of HIV-positive women to examine the effect of liquid-based technology on this population. RESULTS: Significant intraepithelial lesions (SILs) (low grade SIL or greater) were identified in 24% of the conventional smears and 23% of the liquid-based cytology. Atypical squamous cells of undetermined significance (ASCUS)/atypical glandular cells of undetermined significance was diagnosed in 15% of the conventional smears and 9% of the liquid-based preparations (P = .02). In patients with ASCUS diagnoses and tissue follow-up within 7 months, significant SILs were identified in 29% with conventional smears and in 65% with liquid-based cytology. CONCLUSION: There was no statistically significant difference in the rate of SILs between conventional smears and liquid-based cervicovaginal preparations in HIV-positive women. The diagnosis of ASCUS on liquid-based cytology may have an increased likelihood of representing a significant SIL in comparison to conventional smears. For the high-risk, HIV-positive population, immediate colposcopy and biopsy may be warranted following ASCUS diagnoses on liquid-based cytology.     

Comparison of three different methods for automated classification of cervical cells.

Over 4600 exfoliated squamous cervical cells taken from appropriate Papanicolaou samples were classified as normal, mildly dysplastic, moderately dysplastic and severely dysplastic by an experienced cytopathologist. The slides were de-stained and subsequently re-stained with Feulgen Thionin-SO2 stain. Images of the nuclei were then captured, recorded and processed employing an image cytometry device. Automated classification of the cells was carried out using three different methods--discriminant function analysis, a decision tree classifier and a neutral network classifier. The discriminant function analysis method, which combined all dysplastic cells into an abnormal group, achieved a combined error rate of less than 0.4% for moderate and severe dysplastic cells, and less than 40% for mildly dysplastic cells. All three methods yielded comparable results, which approached those of human performance.     

P16INK4A as an adjunct test in liquid-based cytology

OBJECTIVE: To assess the utility of P16INK4A as an adjunct test in liquid-based cytology in cases with equivocal morphologic changes of high grade squamous intraepithelial lesion (HSIL). STUDY DESIGN: P16INK4A immunoreactivity was investigated in residual ThinPrep material (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) from 30 cases with equivocal diagnoses of HSIL that had corresponding follow-up biopsies. Two control ThinPrep cases were included: 1 HSIL with biopsy-confirmed cervical intraepithelial neoplasia (CIN) 3 and a negative specimen with a corresponding biopsy of squamous metaplasia. The expression of P16INK4A in ThinPrep specimens and corresponding biopsies was scored as previously described. A ThinPrep case was scored positive if it contained > 10 abnormal cells with nuclear and cytoplasmic immunocytochemical staining. Corresponding biopsies were scored as having negative, sporadic, focal or diffuse staining. RESULTS: The P16INK4A antibody assay was positive in 19 of 30 ThinPrep cases (63.3%). Seventeen of the 19 (89.4%) biopsies corresponding to the positively stained ThinPreps also were positive, with a score of at least focal positivity in the dysplastic regions (2 CIN 1, 4 CIN 2, 11 CIN 3; 2 lesions lost in the tissue recut). The assay was negative in 11 ThinPreps (36.6%) and 10 biopsies (33.3%) with tissue confirmation of chronic cervicitis (5), squamous metaplasia (2), CIN 1 (3) and 1 lesion lost in the tissue recut. Seventeen of 18 (94.4%) ThinPreps confirmed as high grade lesions upon biopsy showed P16INK4A positivity. The control HSIL case with a CIN 3 biopsy was diffusely positive for P16INK4A, and the control negative case with biopsy diagnosis of squamous metaplasia was negative. Nondysplastic squamous and metaplastic epithelium in 7 biopsies and nondysplastic squamous or metaplastic cells in ThinPrep cases were negative. Sporadic staining of bacteria, inflammatory cells and endocervical cells was noted. CONCLUSION: ThinPrep cases in the equivocal cytologic category with the corresponding tissue biopsy assayed for P16INK4A expression showed that there was utility for this type of testing. A larger series comparing corresponding ThinPrep and tissue biopsies will be undertaken. The role of HPV infection in these cases will also be explored.