Immunologic characterization of T-lymphoproliferative disorders by monoclonal antibodies

A series of 11 mouse monoclonal antibodies reactive with human T lymphoid cells at different stages of differentiation was used for immunological classification of leukaemic cells of 16 patients with T cell lymphoproliferative disorders by using a fluorescence assay. The majority of T-ALL cells had an immature or early thymic phenotype and T lymphoblastic lymphoma had phenotypes corresponding to different levels of more mature stages of T cell differentiation, Two cases of T-CLL and one adult patient with mycosis fungoides had mature T cell phenotypes being T-3+, T-4-, T-8+, cytotoxic/suppressor cell types and one case of T-CLL had T-3+, T-4+, T-8-, "helper/inducer" cell type, too. These results suggested that surface marker analysis in T cell lymphoproliferative disorders may be used as a highly reproducible immunological classification system that will provide additional information about phenotypes of leukaemic cells in connection with morphological analysis and clinical diagnosis.     

Detection of bladder cancers using a SAMBA 200 cell image processor

The cell image analysis of urinary sediments was performed using a SAMBA 200 system. Cell profiles were created using 18 parameters related to size, shape, densitometry and chromatin texture. Learning sets of about 50 cell images per class were constructed for bening, degenerated benign, atypical, malignant and degenerated malignant urothelial cell types as well as for squamous epithelial and white blood cell types. A four-level hierarchic decision tree involving a discriminant analysis at each node was designed and then evaluated against a test set of 700 cells from the various classes. All of the cell images involved in this study were acquired from Papanicolaou-stained specimens obtained for routine screening. In spite of some misclassification errors, the analysis of the occurrence of cells in the various classes, especially the percentage of cells classified as suspicious (both atypical and malignant cells), by the SAMBA 200 system resulted in the separate clustering of the positive specimens (49 carcinomas grade II and higher) and the negative ones (26 benign samples). The preliminary results suggest that the cell population features (occurrence rate of cells in the various classes and mean cell profile within a class) may be of diagnostic value in designing a classifier dedicated to the prescreening of urinary sediments for the detection of bladder cancers.     

An automated method of differential red blood cell classification with application to the diagnosis of anemia.

A method of automated red cell analysis suitable for the rapid classification of large numbers of red cells from individual blood specimens has been developed, and preliminarily tested on normal bloods and clinically proven cases of anemias and red cell disorders. According to this method digital image processing techniques provide several features relating to shape and internal central pallor configurations of red cells. These features are used with a fully automated decision logic to rapidly provide a quantitative "red cell differential" analysis, a report of the percentage subpopulations of recognized categories of red cells. For each subpopulation, measurements of mean cell area, mean cell hemoglobin content and mean cell hemoglobin density are provided. The nine types of red cell disorders studied with this method were: (a) iron deficiency anemia, (b) the anemia of chronic disease, (c) beta-thalassemia trait, (d) sickle cell anemia, (e) hemoglobin C disease, (f) intravascular hemolysis, (g) hereditary elliptocytosis, (h) hereditary spherocytosis, and (i) megaloblastic anemia due to folic acid deficiency. Preliminary indications are that the red cell differential is useful in distinguishing between these conditions.     

Cellular automaton simulation of tumour growth -- equivocal relationships between simulation parameters and morphologic pattern features.

OBJECTIVE: To develop an interpretation procedure which estimates simulation parameters (tumour cell motility, tumour cell adhesion, autocrine and paracrine growth control, stroma destruction) of simulated patterns solely based on morphometric features of the morphologic pattern. METHODS: A cellular automaton computer simulation program was developed which produces morphologic patterns by growth of a seed of tumour cells. At the beginning of each simulation run certain simulation parameters are assigned to the tumour cells. After the run has been completed, the resulting pattern is evaluated by a set of morphometric features. Simulation parameters and resulting morphometric features of 27,800 simulations were stored in a database and were used for the evaluation of potential relationships. RESULTS: Correlation analysis showed highly significant correlations between morphometric features on the one hand and the preset simulation parameters (tumour cell motility, tumour cell adhesion, autocrine and paracrine growth control, stroma destruction) on the other. Correlation coefficients, however, varied from 0.72 to 0.99. When only one simulation parameter varied while all others were kept constant, morphometric features yielded a highly reliable estimate of the particular simulation parameter. When variability was extended to 4 simulation parameters, morphometric features were less effective in estimating the setting of the parameters. Though in all patterns tested several possible simulation parameter constellations could be ruled out, morphometric features were usually compatible with more than one set of simulation parameters thus preventing a straightforward interpretation. CONCLUSIONS: Though simulation parameters significantly and reproducibly influence the resulting morphologic pattern as characterized by morphometric features, estimates of the simulation parameters based on morphometric features yield equivocal results.     

Infrared spectroscopy of exfoliated cervical cell specimens. Proceed with caution

OBJECTIVE: To determine whether diagnostic information may be recovered from the infrared spectra of exfoliated cell specimens by using a novel spectral feature extraction method, in conjunction with linear and quadratic discriminant analysis, for spectral classification. STUDY DESIGN: Over 800 infrared spectra were included in the study, with corresponding clinical diagnoses based upon cytology and, when available, histology reports. Three sets of classification trials were carried out with the aim of distinguishing the spectra corresponding to normal specimens from CIN 1, 2 and 3. For each of these three cases, the procedure was to: (1) develop a set of provisional classification models using only a "training" subset of the spectra, and (2) test each provisional model by its ability to correctly predict the diagnoses on the basis of the remaining spectra. RESULTS: For optimal classification trials, training set classification accuracies were 68% for normal/CIN 1, 73% for normal/CIN 2 and 81% for normal/CIN 3; for the corresponding test sets the classification accuracies were 60%, 60% and 67%, respectively. CONCLUSION: The infrared spectra of exfoliated cervical cells carry information regarding the presence or absence of dysplasia, and that information is recoverable--albeit imperfectly at this stage--from the spectra of "real life" cell preparations.     

基于对象特征的山东省丘陵地区多时相遥感土地覆被自动分类

对于基于像元的土地覆被分类来说,植被的分类是难点。使用多时相面向对象分类方法可以较好的解决这个问题。以山东省烟台市丘陵地区为研究区,采用Landsat TM(Landsat Thematic Mapper remotely sensed imagery)、DEM(Digital Elevation Model)、坡度、坡位、坡向等多种数据,利用基于对象特征的多时相分类方法对研究区进行土地覆盖自动分类。首先对影像进行多尺度分割并检验分割结果选取合适的分割尺度,然后分析对象的光谱、纹理、形状特征。根据各类地物的光谱特征、地理相关性、形状、空间分布等特征,明确类别之间的差异。建立决策树使用隶属度函数进行模糊分类,借助支持向量机提高分类精度。研究结果表明,通过使用多时相影像采用面向对象分类方法,相对于传统的基于像素的分类可以明显提高分类精度,尤其是解决了乔灌草的区分问题。     

T cell lymphoproliferative disorders associated with anti-tumor necrosis factor alpha antibody therapy for ulcerative colitis: literature summary

The enhanced risk of development of lymphoproliferative disorders in patients with inflammatory bowel disease has been attributed to immunosuppressive/immunomodulatory therapies. Infliximab is a chimeric monoclonal immunoglobulin G1 antibody directed against tumor necrosis factor alpha (TNF-α) that was approved by the Food and Drug Administration (FDA) in 1998 as an effective therapeutic agent against inflammatory bowel disease. Malignant lymphomas of both B and T cell lineage have been described in patients undergoing therapy involving TNF-α blockade. To date, eight cases of Epstein–Barr virus (EBV)-negative hepatosplenic T cell lymphoma associated with infliximab have been reported to the FDA’s Adverse Event Reporting System, as well as several other T cell lymphoproliferative disorders with aggressive clinical outcomes. We present the histologic, immunophenotypic, and molecular features of a T cell lymphoproliferative disorder involving the axillary lymph node of a 33-year-old male following infliximab treatment for ulcerative colitis. These EBV-negative lymphomas suggest that lymphoproliferative disorders following infliximab treatment for inflammatory bowel disease may involve EBV-independent immune dysregulation. The spectrum of lymphoproliferative disorders associated with infliximab and the potential mechanisms by which they occur are discussed.     

Combined analysis of tumor growth pattern and expression of endogenous lectins as a prognostic tool in primary testicular cancer and its lung metastases

The aim of this study was to glyco- and immunohistochemically analyze expression of distinct growth/adhesion-related markers of primary testicular carcinomas and their lung metastases in relation to the risk of developing lung metastases and survival of patients, and to correlate immunohistochemical staining profile and syntactic structure analysis in order to delineate new prognostic parameters for this tumor type. Clinical features of 50 patients with primary testicular carcinomas and their corresponding lung metastases were evaluated and compared to those of a control cohort of 25 cases. The set of eight probes including labeled galectins-1 and -3, specific non-cross-reactive antibodies against galectins-1, -3, and -8 as well as anti-Ki-67, anti-bcl-2, and anti-p53 was applied to formalin-fixed, paraffin-embedded tumor sections of both primary and metastatic lesions. Syntactic structure analysis computed staining intensities and structural features of the tumor cells. These parameters were set into relation separately and in combination to clinical data including tumor stages, smoking habits, applied cytostatic therapy, disease-free interval, and survival. The risk of testis cancer patients to develop lung metastases depends in descending order on the tumor cell type (non-seminoma versus seminoma), tumor cell heterogeneity (mixed versus monomorphous cell type), age of patients, and pT stage. The extent of differential expression of galectin-related features between primary and secondary lesions was pronounced. Prognostic correlations for distinct galectin-related features were delineated in combination with data from syntactic structure analysis, for example cluster radius of galectin-3-positive tumor cells and post-surgical and total survival. Lengths of disease-free interval and total survival of patients were also correlated to characteristics obtained by syntactic structure analysis and their combination with galectin data in the first place, then to smoking habits, percentage of proliferating cells in the primary and secondary tumors, and finally to expression of certain galectins and of p53. Patients with non-seminoma testicular cancer should be thoroughly controlled for lung metastases. Regarding marker selection, our study underscores that further investigation of the growth-regulatory network of galectins is clearly warranted.     

Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

CD16+ NK lymphoproliferative disorders: cellular and molecular characterization

We studied the mononuclear cells obtained from 2 patients with CD16+ lymphoproliferative disorders. In both subjects, over 80% of the circulating peripheral blood mononuclear cells were CD16+, CD2+, CD7+, CD3-, CD4-, and CD8-. In 1 patient, greater than 60% of the cells expressed HLA-DR and HLA-DQ gene products. Functional analysis of the natural killer (NK) cell activity of cells from this patient demonstrated 76% killing of K562 targets at ratios as low as 1:1 effector:targets. Karyotype analysis demonstrated a deletion on the long arm of chromosome 6, supporting the contention that the lymphocytosis in this patient was due to a clonally expanded population of cells. In additional studies, Southern blot analysis of DNA extracted from cells of this patient revealed that the beta-chain of the T cell receptor was of germ line configuration. This information supports the hypothesis that the clonally expanded NK population in this patient is of a lineage distinct from T cells and represents a true NK leukemia.     

Comparison of pmoA PCR Primer Sets as Tools for Investigating Methanotroph Diversity in Three Danish Soils

Three particulate methane monooxygenase PCR primer sets (A189-A682, A189-A650, and A189-mb661) were investigated for their ability to assess methanotroph diversity in soils from three sites, i.e., heath, oak, and sitka, each of which was capable of oxidizing atmospheric concentrations of methane. Each PCR primer set was used to construct a library containing 50 clones from each soil type. The clones from each library were grouped by restriction fragment length polymorphism, and representatives from each group were sequenced and analyzed. Libraries constructed with the A189-A682 PCR primer set were dominated by amoA-related sequences or nonspecific PCR products with nonsense open reading frames. The primer set could not be used to assess methanotroph diversity in these soils. A new pmoA-specific primer, A650, was designed in this study. The A189-A650 primer set demonstrated distinct biases both in clone library analysis and when incorporated into denaturing gradient gel electrophoresis analysis. The A189-mb661 PCR primer set demonstrated the largest retrieval of methanotroph diversity of all of the primer sets. However, this primer set did not retrieve sequences linked with novel high-affinity methane oxidizers from the soil libraries, which were detected using the A189-A650 primer set. A combination of all three primer sets appears to be required to examine both methanotroph diversity and the presence of novel methane monooxygenase sequences.     

Nucleolar organizer region activity in lymphoid cells of human lymphatic nodes in the norm

Comprehensive approach to study nucleolar organizer region activity rating in lymphoid cells of 87 human normal lymphatic nodes was proposed. Possibility of visualization of basic morphofunctional nucleolar types and different subtypes of nucleolonemal nucleoluses was demonstrated at light microscopy level basing on 7387 prolymphocyte nucleus research of argentophile granule allocation features and dimensions. Distribution index of extranucleolar argentophile granules was estimated. The complex of parameters of lymphoid cell nucleolar set can be used as control for study of different lymphoproliferative processes.     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

Cytologic findings in posttransplant lymphoproliferative disease

Transplant patients are at an increased risk of developing lymphoproliferative disorders (LPDs). To examine the role of cytology in diagnosing LPDs, the cytologic reports on all transplant patients seen at the University of Iowa from January 1983 to July 1988 were reviewed. Thirteen of 649 transplant patients developed LPD; 10 of those 13 patients had a total of 25 cytologic specimens obtained within two months of the diagnosis of LPD available for review. Ten specimens (four cerebrospinal fluids [CSFs], four effusions, one respiratory specimen and one liver aspirate) from six patients were positive for LPD. Immunophenotypic or immunogenotypic studies on cytologic specimens from four patients showed a clonal B-cell process. The cytologic features ranged from those of a plasmacytoid LPD to those of an immunoblastic or large-cell lymphoma. An additional seven specimens (five CSFs, one effusion and one liver aspirate) from four patients were suspicious for LPD, exhibiting rare atypical cells or cells with plasmacytoid features. Ten patients died with LPD within 12 months; three are alive. Cytologic specimens, especially body fluids, are frequently positive in LPD and may be useful diagnostically. Since the differential diagnosis includes reactive lymphocytosis, confirmatory immunophenotypic or immunogenotypic studies are recommended.     

Gray zones around diffuse large B cell lymphoma. Conclusions based on the workshop of the XIV meeting of the European Association for Hematopathology and the Society of Hematopathology in Bordeaux, France

The term “gray-zone” lymphoma has been used to denote a group of lymphomas with overlapping histological, biological, and clinical features between various types of lymphomas. It has been used in the context of Hodgkin lymphomas (HL) and non-Hodgkin lymphomas (NHL), including classical HL (CHL), and primary mediastinal large B cell lymphoma, cases with overlapping features between nodular lymphocyte predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B cell lymphoma, CHL, and Epstein–Barr-virus-positive lymphoproliferative disorders, and peripheral T cell lymphomas simulating CHL. A second group of gray-zone lymphomas includes B cell NHL with intermediate features between diffuse large B cell lymphoma and classical Burkitt lymphoma. In order to review controversial issues in gray-zone lymphomas, a joint Workshop of the European Association for Hematopathology and the Society for Hematopathology was held in Bordeaux, France, in September 2008. The panel members reviewed and discussed 145 submitted cases and reached consensus diagnoses. This Workshop summary is focused on the most controversial aspects of gray-zone lymphomas and describes the panel’s proposals regarding diagnostic criteria, terminology, and new prognostic and diagnostic parameters.     

Determination of residual T- and B-cell content after immunomagnetic depletion: proposal for flow cytometric analysis and results from 103 separations

BACKGROUND: T- and B-cell depletion of apheresis products is an attractive alternative to standard stem cell enrichment in haplo-identical transplantation. Thorough T- and B-cell depletion is necessary for prevention of acute GvHD and T-cell depletion-associated lymphoproliferative disorders. However, the large number of non-T and -B cells in the graft requires special protocols for the determination of extremely low frequencies of residual T cells. METHODS: Apheresis products from healthy donors were T- and B-cell depleted by the CliniMACS system using CD3 and CD19 Ab reagents and the LS tubing set. The recovery of cells and degree of depletion were determined. A four-color multigating strategy was used for enumeration of residual T and B cells. RESULTS: One-hundred and three separations were performed, with a mean cell recovery of 38+/-12%, CD34 recovery of 61+/-16% and CD56 recovery of 63+/-33%. T and B cells were depleted by log 4.15+/-0.46 and log 3.64+/-0.63, respectively. Four-color multigating flow cytometry allowed the detection of single T cells. DISCUSSION: Combined T- and B-cell depletion is a feasible method for obtaining stem cell grafts with acceptable stem cell recovery, profound T- and B-cell depletion and a very high amount of NK cells and monocytes. However, analysis of residual T cells is challenging and requires special protocols.     

FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line

We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs.We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2″-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug.     

FTIR imaging of wheat endosperm cell walls in situ reveals compositional and architectural heterogeneity related to grain hardness.

Endosperm cell walls of cultivars of wheat (Triticum aestivum L.) selected for their endosperm texture (two soft and two hard) were analysed in situ by Fourier transform infrared (FTIR) microspectroscopy. FTIR imaging coupled with statistical analysis was used to map the compositional and structural heterogeneity within transverse sections from which cell contents had been removed by sonication. In the majority of grains analysed, two distinct populations of endosperm cells could be identified by spectral features that were related to cell morphology and age, regardless of cultivar. The main cell-wall component responsible for these differences was the polysaccharide arabinoxylan. In a few samples, this heterogeneity was absent, for reasons that are not understood, but this was not correlated to endosperm texture or growth conditions. Within the same population of endosperm cells, cell walls of hard endosperm could be distinguished from those of soft endosperm by their spectral features. Compared to hard cultivars, the peripheral endosperm of soft cultivars was characterised by a higher amount of polymer, whose spectral feature was similar to water-extractable arabinoxylan. In contrast, no specific compound has been identified in the central endosperm: structural differences within the polysaccharides probably contribute to the distinction between hard and soft cultivars. In developing grain, a clear difference in the composition of the endosperm cell walls of hard and soft wheat cultivars was observed as early as 15 days after anthesis.     

Infrared spectroscopy as a tool for discrimination between sensitive and multiresistant K562 cells.

Fourier transform infrared spectroscopy was performed on human leukemic daunorubicin-sensitive K562 cells and their multiresistant counterpart derived by selection. Statistical analysis, including variable reduction and linear discriminant analysis was performed on sensitive and multiresistant cells spectra in order to establish a diagnostic tool for multiresistant pattern. For each of the two methods of data reduction tested [genetic algorithm or principal component analysis (PCA)] discrimination between the two cell lines was found to be possible. The best results, obtained with PCA-reduction, showed an accuracy of 93% on a distinct test set of spectra. These results demonstrate the efficiency of Fourier transform infrared spectroscopy for classification. Further analysis of the spectral differences indicated that discrimination between resistant and sensitive cells was based on variations in all cellular contents. Lipid and nucleic acid decreased, relatively, while the protein content increased.