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J S Ploem

Publications and source records attributed to J S Ploem.

At least 19 recordsLinked to original sources
PubMed1992

Preparation and microscopic visualization of multicolor luminescent immunophosphors.

The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.

Adsorption↗
PubMed1992

The prognostic value of DNA content measured by image cytometry in soft tissue sarcomas.

Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a significant prognostic value and correlates well with histological grading (p = 0.01). Tumour volume was found to be an independent prognostic factor (p = greater than 0.1) [chi 2 test]. The DNA content of the primary tumour and of multiple local recurrences remained similar.

Adolescent↗
PubMed1990

Multiple fluorescence in situ hybridization.

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.

2-Acetylaminofluorene↗
PubMed1990

Symptomatic cytomegalovirus (CMV) infections identified by image cytometry and other parameters for CMV infection.

Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptomatic CMV infection. All symptomatic patients had one or more positive urine cultures or a positive IEA assay. However, 15 patients with positive urine cultures and 12 patients with a positive IEA assay lacked any signs of symptomatic CMV disease. Moreover, 6 out of 15 patients with positive buffy coat cultures for CMV did not have symptomatic CMV disease. Using a computerized system to quantify IEA-positive granulocytes, we show that the absolute number of positive cells per million correlates very well with the occurrence of symptomatic CMV disease.

Antibodies, Viral↗
PubMed1990

Interobserver variability in the cytological diagnosis of 1500 Papanicolaou stained cervical monolayer specimens.

Cervical specimens from 1500 patients were prepared by means of a centrifugation procedure to obtain monolayer specimens suitable for automated screening using a machine. After staining according to Papanicolaou, each specimen was diagnosed by four independent cytologists from two different institutes. Within each institute, noncorresponding screening results were discussed to arrive at a conclusion diagnosis. After discussion of the discrepancies between the two centers, the conclusion diagnoses were combined to one final cytological diagnosis for each specimen. This final diagnosis is to be used as a reference diagnosis to evaluate machine classification as obtained by the AUTOPLAN/MIAC system. This system is presently being tested both in Leiden and in Frankfurt for its accuracy of detecting abnormal lesions in cervical specimens. The used diagnostic procedure resulted in a negative reference diagnosis for 1217 of the 1500 specimens; 170 specimens were diagnosed CIN I or II (mild or moderate dysplasia) and 113 specimens had a positive reference diagnosis (CIN III or invasive carcinoma). Based on these three diagnostic classes, the agreement between the four independent cytologists and the reference diagnosis varied between 93.60% and 96.60%, whereas 95.33% of all 6000 diagnoses correlated with the reference diagnosis.

Female↗
PubMed1990

The present state of the automated micronucleus test for lymphocytes.

This minireview presents the state of the art with respect to automated detection of micronuclei (MN) in binucleated lymphocytes. Emphasis is on an image analysis technique, based on the principles of mathematical morphology (pattern recognition), which combines a personal computer with an image processing board and a board for microscope control. The basic idea behind this procedure is that nuclei plus MN and cytoplasms are analysed separately and sequentially by capturing images from gallocyanin-stained nuclei plus MN and naphthol yellow-S stained cytoplasms from one microscope field by using different filters. Major steps in the identification of nuclei and MN are separation of nuclei and MN from background by determination of periphery of the nuclei and MN, and artefact rejection procedures. After changing the filter, a binary image is constructed from cytoplasms and artefacts. Finally, stored information from selected binucleated objects with/without MN is combined with the cytoplasm image to check whether selected objects belong to the same cytoplasm. The procedure described above allows automated detection of binucleated lymphocytes with or without MN. The current capacity to detect 63% of binucleated cells and 57% of the MN within them is quite acceptable. To avoid false positives, artefact rejection procedures need to be improved before the method can be used routinely.

Humans↗
PubMed1990

Automated image cytometry in cytopathology.

Image cytometry is used more and more for the study of clinical cytology, notably for the determination of morphometrical and densitometrical values, the quantification of monoclonal antibody labelling and the detection of DNA probes after in situ hybridisation. Aspects of automated and interactive image cytometry are discussed, including a brief evaluation of limitations and advantages of the image technique in connection to flow cytometry. Some new technologies such as a sampling technique for paraffin embedded tissue and a new automated microscope, which are of special interest to the pathologist, are described in more detail. Applications in image cytometry include diagnostic and prognostic studies. Examples of diagnostic studies are the automated screening for cervical cancer and the detection of rare remaining cancer cells (minimal residual disease) in the peripheral blood. The use of archival material in image cytometry allows interesting retrospective studies with regard to the relation of the course of the disease with the ploidy characteristics of the tumor.

Autoanalysis↗
PubMed1989

Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy.

Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible. In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.

3,3'-Diaminobenzidine↗
PubMed1989

Automated cell analysis for DNA studies of large cell populations using the LEYTAS image cytometry system.

Image cytometry by means of LEYTAS features analysis of both fresh and archival cellular material. Although not as accurate in ploidy determination as flow cytometry, LEYTAS cytometry incorporates extensive artefact rejection algorithms, thereby allowing detection of low frequency cells. This feature is very useful for the search of rare cells, as e.g. in cervical screening, or for the quantitation of the number of high DNA content cells in the total cell sample. LEYTAS main components are an automated microscope (Autoplan) and a Modular Image Analysis Computer (MIAC), both from Wild Leitz (W-Germany). This paper discusses LEYTAS instrumentation and cell analysis by means of programs especially written for LEYTAS.

Algorithms↗
PubMed1989

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

Aneuploidy↗
PubMed1988

Automated screening for micrometastases in bone marrow smears.

It is possible to detect micrometastases in primary breast cancer using immunocytochemical staining of bone marrow smears. However, using the light microscope the procedure is time-consuming and laborious because such cells occur rarely (less than 1 in 10,000). Using an image analysis system, the Leytas machine, and a specially prepared reproducible slide it has been possible to automate the technique. A 100% concordance was found between the machine and the light microscope in the identification of slides containing moderate to high numbers of tumour cells in bone marrow, and in those containing no tumour cells. However, in those slides containing low numbers of tumour cells (1-10 tumour cells/10(6) normal bone marrow cells) the sensitivity was decreased to 91%. In the presence of non-specific staining the false positive rate was increased from 0% to 22%. This method represents a potential improvement in the assessment of an important clinical staging procedure.

Bone Marrow↗
PubMed1988

Reflection contrast microscopy. Visualization of (peroxidase-generated) diaminobenzidine polymer products and its underlying optical phenomena.

Reflection contrast microscopy (RCM) has proven to be a useful tool for the study of living cells (Ploem 1975). Due to the effective suppression of aspecific reflected light by polarization optics combined with a quarter lambda plate at the front lens of the objective, low intensity reflection signals originating from minor amounts of precipitated diaminobenzidine (DABox) in immunocytochemically stained specimens, can be made visible. RCM has been successfully applied in demonstrating single copy nucleic acid sequences using in situ hybridization procedures (Landegent et al. 1984). We have systematically studied the aspects of image formation of DABox by RCM by using a model system consisting of glass slides coated with peroxidase containing protein layers to determine the conditions for optimal sensitivity of this detection method. Moreover, investigations were performed to study the relationship between the amount of reflected light and DABox depending on the thickness of the object. Both theoretical and practical evidence is obtained to show that DABox detection by RCM is based on interference phenomena occurring in the layer of DABox, and less on selective reflection. This restricts the type of specimen which can be used for sensitive detection of DABox by RCM. Consequently, in ultrathin (40 nm) sections osmificated DABox was visualized in peroxidatic positive cell organelles with high contrast and resolution. Similar results were obtained with immunoperoxidase stained material embedded in Lowicryl under conditions that did not allow visualization of the staining product by bright field microscopy.

3,3'-Diaminobenzidine↗
PubMed1988

[Importance of the DNA content for the prognosis of invasive carcinoma of the bladder. Determination by photocytometry].

In a retrospective study, a DNA histogram was established from specimens obtained by cystectomy in 65 carcinomas of the bladder (stages pT1 to pT4, pN0, pN1 and pN2). Flow cytometry and automated photocytometry were systematically compared. Automated photocytometry makes it possible to differentiate between diploid, polyploid and aneuploid tumors. Among aneuploid tumors, the DNA content of tumor stem cells make it possible to identify another subgroup. The analysis of various tumor sections showed that the DNA histogram obtained by photocytometry was a stable and reproducible characteristic of the tumor. The subgroups of carcinoma of the bladder, determined by topology, have markedly different long-term prognoses. In contrast to automated photocytometry, flow cytometry did not allow for reproducible determination of the biologic characteristics of carcinoma of the bladder.

Cytophotometry↗
PubMed1988

Modern image analysis methods in hematology.

Image analysis instrumentation presently includes multiparameter microscopy and fast processing. One type of image analysis microscope uses only 1 objective and 2 TV cameras, thereby obtaining 2 magnifications simultaneously: a low magnification for cell selection and a high magnification for further cell analysis. Image analysis is very capable of detecting rare cells among a large population of normal cells. This recognition of very rare events becomes increasingly important in biomedical applications. Investigations such as the detection of mutant cells in the peripheral blood and the detection of rare cancer cells in bone marrow as a sign of early metastases, are described to illustrate the potentialities of image analysis in hematology.

Bone Marrow↗
PubMed1987

Use of DNA image cytometry in addition to flow cytometry for the study of patients with advanced ovarian cancer.

Forty-five patients with advanced ovarian cancer were studied with both DNA flow cytometry (FCM) and automatic DNA image cytometry carried out with the Leiden Television Analysis System (Leytas). There was a significant difference in survival between the diploid and nondiploid cases as determined by FCM. Furthermore, the presence of nuclei with a high DNA content (defined as a DNA content higher than 5C) as determined by Leytas indicated a poor prognosis. When the combined results of FCM and Leytas were taken into account, three different groups of patients could be distinguished. The group of patients with a diploid malignancy (n = 12) had a median survival of more than 60 months. The group of patients (n = 11) with a nondiploid tumor having fewer than 100 nuclei with a high DNA content per 1600 microscope fields formed an intermediate group (median survival, 42 months), whereas the median survival of the remaining patients (n = 22), who had a nondiploid malignancy combined with more than 100 of these nuclei per 1600 microscope fields, was only 15 months. In addition, comparison of the clinical parameters by means of a multivariate analysis (Cox regression model) showed that the combined results of FCM and DNA image cytometry had the largest influence on survival. It is concluded that DNA image cytometry appears to be supplementary to FCM for the study of DNA ploidy abnormalities and that the combined results of these methods have a major influence on the clinical outcome.

Aneuploidy↗