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W E Mesker

Publications and source records attributed to W E Mesker.

At least 19 recordsLinked to original sources
PubMed2006

Prognostic relevance of occult tumour cells in lymph nodes in colorectal cancer.

AIMS: Presently, in Europe the treatment of node-negative colorectal cancer (CRC) patients consists of surgical resection of the primary tumour without adjuvant systemic therapy. However, up to 30% of these patients will develop disease recurrence. These high-risk patients are possibly identified by occult tumour cell (OTC) assessment in lymph nodes. In this paper, studies on the clinical relevance of OTC in lymph nodes are reviewed. METHODS: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer, lymph node, polymerase chain reaction, mRNA, immunohistochemistry, micrometastases and isolated tumour cells. Additional articles were identified by cross-referencing from papers retrieved in the initial search. RESULTS: The upstaging percentages through OTC assessment and the prognostic relevance of OTC in lymph nodes vary among studies, which is related to differences in techniques used to detect OTC. CONCLUSIONS: We conclude that OTC examination techniques should be standardized to illuminate whether OTC in lymph nodes can reliably identify high-risk node-negative patients.

Antibodies, Neoplasm↗
PubMed2005

The feasibility and reliability of sentinel node mapping in colorectal cancer.

AIMS: Sentinel node mapping (SNM) has been introduced in colorectal cancer (CRC) to improve staging by facilitating occult tumour cell (OTC) assessment in lymph nodes that are most likely to be tumour-positive. In this paper, studies on the feasibility and reliability of SNM in CRC are reviewed. METHODS: A literature search was conducted in the National Library of Medicine by using the keywords colonic, rectal, colorectal, neoplasm, adenocarcinoma, cancer and sentinel. Additional articles were identified by cross-referencing from papers retrieved in the initial search. RESULTS: There is a large variation in identification rates and false-negative rates mainly due to the learning curve effect, differences in SNM technique and tumour stage. CONCLUSIONS: We conclude that SNM in CRC is technically feasible. Standardization of SNM procedures is mandatory to resolve the debate on the reliability of sentinel node status for predicting the tumour status of all lymph nodes. Only then can adjuvant treatment of patients upstaged by OTC detection in sentinel nodes be justified.

Colonic Neoplasms↗
PubMed2004

Supervised automated microscopy increases sensitivity and efficiency of detection of sentinel node micrometastases in patients with breast cancer.

AIMS: To investigate the practicality and sensitivity of supervised automated microscopy (AM) for the detection of micrometastasis in sentinel lymph nodes (SLNs) from patients with breast carcinoma. METHODS: In total, 440 SLN slides (immunohistochemically stained for cytokeratin) from 86 patients were obtained from two hospitals. Samples were selected on the basis of: (1) a pathology report mentioning micrometastases or isolated tumour cells (ITCs) and (2) reported as negative nodes (N0). RESULTS: From a test set of 29 slides (12 SLN positive patients, including positive and negative nodes), 18 slides were scored positive by supervised AM and 11 were negative. Routine examination revealed 17 positive slides and 12 negative. Subsequently, automated reanalysis of 187 slides (34 patients; institute I) and 216 slides (40 patients; institute II) from reported node negative (N0) patients showed that two and seven slides (from two and five patients, respectively) contained ITCs, respectively, all confirmed by the pathologists, corresponding to 5.9% and 12.5% missed patients. In four of the seven missed cases from institute II, AM also detected clusters of four to 30 cells, but all with a size < or = 0.2 mm. CONCLUSIONS: Supervised AM is a more sensitive method for detecting immunohistochemically stained micrometastasis and ITCs in SLNs than routine pathology. However, the clinical relevance of detecting cytokeratin positive cells in SLNs of patients with breast cancer is still an unresolved issue and is at the moment being validated in larger clinical trials.

Automation↗
PubMed2002

Insights into the sites of X ray and neutron induced chromosomal aberrations in human lymphocytes using COBRA-MFISH.

A multi-colour fluorescence in situ hybridisation (MFISH) assay has been developed, for simultaneous visualisation of all human chromosomes in 24 different colours. This assay is based on the simultaneous use of combinatorial labelling and ratio labelling, the so called combined binary ratio labelling (COBRA). This technique is used to study the spectra of chromosomal exchanges induced by X ray and neutrons in human lymphocytes. With X rays the dose-effect relationships for both dicentrics and translocations were linear-quadratic, whereas with neutrons these were linear. Among aberrant cells, average estimates of the minimum number of breaks was higher for neutrons than for X rays. Moreover, the induced chromosomal exchange patterns were more complex following neutron irradiation in comparison with X rays. COBRA-MFISH was found to have a greater resolving power over partial labelling for the accurate detection of complex translocations and insertions. With neutrons the frequencies of both were higher than those induced by X rays, and their relative proportions to the total frequencies were independent of dose. These data suggest insertions can be used as the 'signature' of high LET radiation.

Cells, Cultured↗
PubMed1998

Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis.

In order to detect fetal nucleated red blood cells (NRBCs) in maternal blood, a protocol was developed which aimed at producing a reliable staining method for combined immunocytochemical and FISH analysis. The technique had to be suitable for eventual automated screening of slides. Chorionic villi washings, cord blood, and maternal blood samples were used for this study. After a density gradient separation and centrifugal cytology, slides were stained either with 3,3-diaminobenzidin (DAB), a marker for heme, or with antibodies against the gamma-chain of fetal hemoglobin (HbF). FISH analysis for both X- and Y-chromosomes was performed on the same slides. Cytocentrifugation provided a controlled cell density on the slides with good cell morphology. Both the DAB and HbF staining were suitable for manual screening of large numbers of slides. The HbF staining, although supposed to be more specific for fetal NRBCs, appeared to be more sensitive to minor changes in preparation. We were eventually able to combine HbF staining with FISH analysis, and produced a detection efficiency of >85% for both X- and Y-chromosome signals. This preparation protocol simplifies the detection of NRBCs in maternal blood. Immunocytochemical staining and FISH analysis can be performed on the same cell with good image contrast, thus facilitating both manual and automated image analysis. This will facilitate the use of this approach for prenatal diagnosis.

3,3'-Diaminobenzidine↗
PubMed1998

Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis.

A protocol to detect fetal nucleated red blood cells (NRBCs) was tested in 217 pregnant women and in 19 nonpregnant controls. All the pregnant women were sampled after chorionic villus sampling (CVS); 20 were also sampled pre-CVS. NRBC recognition was based upon morphology by using staining of hemoglobin with 3,3-diaminobenzidin (DAB) or by immunocytochemical staining for fetal hemoglobin (HbF). This was combined with FISH analysis for both the X- and Y-chromosomes on the same cells. Progressive refinement of the methods increased the number of cases where NRBCs were detected from 53% (DAB) to 75% and 78% for DAB and HbF staining, respectively, with on average 43 NRBCs (range, 1-220). DAB gave a slightly higher yield than HbF in the lower cell count range (<25). In 6 out of 18 controls, NRBCs were detected with DAB, vs. 1 out of 19 (5%) with HbF. FISH analysis in 41 cases resulted in correct sex prediction in 80% (DAB) and 89% (HbF), respectively. Our data demonstrated an increase of cases with NRBCs (30% to 75%), as well as a rise of the mean number of NRBCs (6 to 29 cells), after CVS. We conclude that DAB staining is a straightforward way to screen for the presence of NRBCs in maternal blood, but is not specific for NRBCs of fetal origin. HbF immunophenotyping is a reliable marker for fetal NRBCs, which detected slightly fewer NRBCs than DAB-staining, but improved sex prediction and significantly reduced false-positive results. CVS at 10-13 weeks of gestation causes a significant increase of NRBCs in maternal blood. These data indicate that further refinement of NRBC detection is needed before application of noninvasive prenatal diagnosis using maternal blood is feasible.

Chorionic Villi Sampling↗
PubMed1998

Two-colour immunocytochemical staining of gamma (gamma) and epsilon (epsilon) type haemoglobin in fetal red cells.

We have developed a two-colour immunocytochemical staining method for the detection of fetal and embryonic haemoglobin in erythroid cells. The method was applied to study these haemoglobin types in fetal red cells. Specimens from fetal blood (10 weeks), cord blood and fetal liver (14 weeks) as well as chorionic villus samples (10-13 weeks) were stained for gamma and epsilon chains using CY3 and FITC labelled antibodies. Morphometric analysis was applied to determine cell size. Samples from organs involved in early embryonic development contained relatively large erythroblasts expressing the epsilon globin chain (megaloblasts); later in gestation the gamma chain was co-expressed by the same cells which ultimately became smaller and contained HbF (alpha 2 gamma 2) only. This phenomenon was confirmed in CVS samples in which all cell types were abundantly present. Since fetal erythroblasts are considered candidate cells for non-invasive prenatal diagnosis using FISH, we studied the phenotype of erythroblasts circulating in the maternal blood. The majority of erythroblasts in maternal blood appeared to be of the relatively small gamma globin-containing cell type. However, careful screening of the same maternal blood samples also revealed erythroblasts expressing epsilon or epsilon and gamma globins simultaneously, although at low frequency. Control specimens from non-pregnant women did not show nucleated red cells expressing either of the haemoglobin types. These observations may contribute to the better recognition of fetal cells in the maternal blood for prenatal diagnosis.

Chorionic Villi Sampling↗
PubMed1998

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.

Automation↗
PubMed1998

Prenatal diagnosis of trisomy 13 on fetal cells obtained from maternal blood after minor enrichment.

In a pilot study to establish fetal nucleated red blood cell (NRBC) detection in maternal blood, trisomy 13 was diagnosed by FISH analysis at 11 weeks' gestation. The NRBCs were detected after a single-step ficoll density gradient enrichment. In blood samples taken both before and after CVS, 52 and 80 NRBCs, respectively, were found to be positive for fetal haemoglobin. In 47 per cent of these cells, FISH analysis for X and Y chromosomes confirmed the fetal sex. Moreover, 48 per cent of these NRBCs showed three fluorescent signals for a chromosome 13 probe, which confirmed the diagnosis of trisomy 13, previously detected at CVS karyotyping. This is the first report of non-invasive prenatal diagnosis of trisomy 13, i.e., pre-CVS, in the first trimester. The high number of fetal NRBCs detected indicates a connection with aneuploidy, probably due to early impairment of the feto-maternal barrier.

Adult↗
PubMed1996

Detection of 'rare event' fetal erythroblasts in maternal blood using automated microscopy.

This paper describes the use of automated microscopy to detect fetal erythroblasts in maternal blood. The technology is based on the following approach: (1) the use of centrifugal cytology for the preparation of monolayers; (2) simultaneous staining of fetal hemoglobin (immunoalkaline phosphatase) and chromosome sequences (FISH); (3) multi-mode microscopy to detect rare events; (4) visual evaluation of image memories containing detected objects. Model systems show that fetal cells in frequencies as low as 1 in a million cells can be detected easily (manually or by automated microscopy). Algorithms for automated cell selection were developed for a test set of 6 patients. Optimization of hardware and software routines will make analysis of several million cells in approximately 1 h feasible.

Autoanalysis↗
PubMed1996

Automated assessment of numerical chromosomal aberrations in paraffin embedded prostate tumor cells stained by in situ hybridization.

We investigated the feasibility of automated counting of in situ hybridization signals (ISH) in interphase cells isolated from paraffin embedded prostate tissue. In total, 34 specimens from 7 patients with prostate cancer were stained with probes specific for the centromeric regions of chromosomes Y, 1, 7, 8, 10, and 15, using an immunoperoxidase based technique suitable for bright-field microscopy. Enumeration of the number of ISH spots of 500 nuclei per specimen was performed (1) using an automatic system developed without any human intervention and (2) using the same system, but including verification of the counts based on visual inspection of the stored images. As reference from each specimen, 200 cell nuclei were evaluated manually, using conventional microscopy. A typical analysis procedure (including user verification) took 35 min. The difference (root mean error) between the automated counting and the counting after visual interaction was relatively small (15%). The percentage of cells with incorrect counts by automated analysis was 20.2%, a number that could easily be improved by user interaction. Detection of cells with aneusomy proved to be more sensitive compared to the routine manual counting, in cases where aberrant frequencies were low. Automated counting of samples with low frequencies (< 10%) resulted in a higher frequency of aberrant cells in 9 of 11 cases, probably due to the fact that an unbiased cell selection is guaranteed. Automated assessment of ISH signals is considered useful for the evaluation of chromosomal aberrations in prostate tumor cells, provided that the counts are visually confirmed.

Aged↗
PubMed1996

Automation of spot counting in interphase cytogenetics using brightfield microscopy.

In situ hybridization techniques allow the enumeration of chromosomal abnormalities and form a great potential for many clinical applications. Although the use of fluorescent labels is preferable regarding sensitivity and colormultiplicity, chromogenic labels can provide an excellent alternative in relatively simple situations, e.g., where it is sufficient to use a centromere specific probe to detect abnormalities of one specific chromosome. When the frequency of chromosomal aberrations is low, several hundreds or even thousands of cells have to be evaluated to achieve sufficient statistical confidence. Since manual counting is tedious, fatiguing, and time consuming, automation can assist to process the slides more efficiently. Therefore, a system has been developed for automated spot counting using brightfield microscopy. This paper addresses both the hardware system aspects and the software image analysis algorithms for nuclei and spot detection. As a result of the automated slide analysis the system provides the frequency spot distribution of the selected cells. The automatic classification can, however, be overruled by human interaction, since each individual cell is stored in a gallery and can be relocated for visual inspection. With this system a thousand cells can be automatically analyzed in approximately 10 min, while an extra 5-10 min is necessary for visual evaluation. The performance of the system was analyzed using a model system for trisomy consisting of a mixture of male and female lymphocytes hybridized with probes for chromosomes 7 and Y. The sensitivity for trisomy detection in the seeding experiment was such that a frequency of 3% trisomic cells could be picked up automatically as being abnormal according to the multiple proportion test, while trisomy as low as 1.5% could be detected after interaction.

Algorithms↗
PubMed1995

Detection improvement of cytomegalovirus antigen in human peripheral blood using monoclonal antibodies and automated reading of cell preparations.

One of the major drawbacks in cytomegalovirus (CMV)-antigenaemia detection for diagnosis of active CMV infection is the low number of CMV-antigen positive cells present in peripheral blood. It is therefore necessary to screen large numbers of peripheral blood granulocytes to find only a few antigen-positive cells. We have optimized this detection by testing several monoclonal antibodies (mAb) to CMV-antigens (mAbs C10/C11, C12, BM222, E13 and SL20). In total 550 blood samples from 40 patients were investigated. More blood samples were found positive with mAb C12 than with the other mAbs. Also the average number of positive cells per slide was highest for mAb C12. Furthermore, duplicate slides were examined automatically using an image analysis system (LEYTAS) and compared to visual detection (cytospin slides). The detection sensitivity of both screening methods was compared for mAb C12. In total 360 slides were analysed, from positive as well as negative blood samples. The sensitivity of the automated screening was 93% and for the visual evaluation of the cytospin slides 73%. In conclusion, mAb C12 was the most suitable of the mAbs tested for detection of antigenaemia, and automatic detection of CMV antigenaemia with image analysis of slides is a sensitive method due to the large numbers of cells that can be screened.

Antibodies, Monoclonal↗
PubMed1995

Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic staining methods with respect to colour segmentation.

The detection of human cytomegalovirus (HCMV) infected poly-morphonuclear leukocytes (PMNLs) for early finding of the pp65 antigen using automated image analysis has been improved. The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate. The number of automatically detected false positive objects due to incomplete inactivation of endogenous peroxidase and strong variations in counterstain intensity could be reduced by 81% using this AP staining method. The number of detected truly positive cells with both staining methods was not significantly different. Furthermore, a new image analysis system providing processing of colour images was evaluated. Since plain differences in absorption wavelength are required for colour segmentation, the red immuno-staining was combined with a green counterstain using methyl green. Screening of AP- instead of PO-stained slides in combination with colour segmentation resulted in a further reduction of the number of falsely detected alarms from 61% to 11%. Consequently, the sensitivity of the automated detection was improved. For AP staining detection of cells in frequencies of approximately one to one million was demonstrated. Screening for CMV-positive, alkaline phosphatase labelled cells using an image analysis system with colour segmentation resulted in a reduced false alarm rate, a better visual interpretation of the images and subsequently an increase in the sensitivity of the automated screening process.

Cytomegalovirus↗
PubMed1994

Detection of immunocytochemically stained rare events using image analysis.

The detection of rare-event cells circulating in peripheral blood using automated image analysis was evaluated using a model system consisting of cells from a breast cancer cell line (SKBR3) seeded in a mononuclear cell suspension. Slides of cells with optimal morphology were prepared according to an optimized preparation procedure based on centrifugal cytology in combination with formalin fixation. SKBR3 cells were immunocytochemically stained for cytokeratin using the cam 5.2 monoclonal antibody and labelled with alkaline phosphatase using CAS-red as substrate. Because, for optimal segmentation of cell images, plain differences in absorption wavelength are required, the red immunostaining was combined with a green nuclear counter-staining based on ethyl green. Slides were automatically screened for cytokeratin-positive SKBR3 cells resulting in a lowest detectable frequency of one positive cell per 1.87 x 10(6) negative cells. A comparison between manual screening and automated screening for cytokeratin-positive cells showed a high level of correlation (0.9998). For the definition of the total number of objects per slide, two counting procedures were evaluated. Results were close to the visual score with a coefficient of variation of 0.47% for the counting procedure used in this study. It is concluded that optimization of preparation and staining procedures for the detection of rare-event cells using automated image analysis results in optimal image contrast and, consequently, in an increase in sensitivity for detecting rare events.

Breast Neoplasms↗
PubMed1994

Quantification of low frequency white blood cells expressing human cytomegalovirus antigen by image cytometry.

Human cytomegalovirus (HCMV) infections cause considerable morbidity and mortality in immunocompromised patients. During HCMV infection, leukocytes appear in the circulation in low frequencies that express the HCMV pp65 protein antigen. Since there is evidence that changes in the frequency of antigen-positive cells in the early phase of the infection have prognostic value, we applied automated image cytometry to quantify these antigen-positive cells. For this purpose weekly peripheral blood leukocyte samples of 80 kidney transplant recipients were visually examined for the presence of antigen-positive cells using an immunocytochemical detection method. Seventeen patients, who reacted positive with this assay, were identified. Next, automated image cytometry was applied to quantitate the frequency of antigen-positive cells in sequential blood samples from the 17 patients. Patients who developed a period of HCMV viremia had a significantly longer antigenemic period and a significantly higher frequency of antigen-positive cells than patients with a HCMV infection who remained nonviremic. Therefore, automated image immunocytometry based screening can be used to distinguish patients at risk for the development of a HCMV viremia. Moreover, automated quantitation reveals prognostic information about the HCMV infection at a more sensitive level than other HCMV detection techniques.

Antigens, Viral↗
PubMed1994

Automated image cytometry for detection of rare, viral antigen-positive cells in peripheral blood.

A cell detection method based upon automated screening is described for recognition of low frequencies (1 in 100,000) of immuno-enzymatically labelled white blood cells in human peripheral blood. The used image cytometry instrumentation (LEYTAS) includes a wide-field, fully automated microscope (Autoplan) and a modular image analysis computer (MIAC), both from Leica, Wetzlar, Germany. The MIAC contains image boards for optimum use of mathematical morphology algorithms. Communication with the MIAC is via a personal computer. Programs for automated cell analysis have been written in C language. Main features of the system are fast analysis of large microscope fields including a count of all cells, selection of objects of interest (alarms), and display of digitally stored images of these alarms. We tested this system for the detection of white blood cells expressing antigen of cytomegalovirus (pp65) in 50 human blood smears from kidney transplant recipients. Immuno-enzymatic (peroxidase) staining was performed with DAB and counterstaining with hematoxylin. For determination of the sensitivity, a series of dilutions of a positive sample with a negative sample was performed. The lowest frequency detected was 1 antigen-positive cell/3 x 10(5) antigen-negative cells. Screening time was about 60 min for one million cells.

Algorithms↗