Flow cytometric diagnosis of myelodysplasia and myeloproliferative disorders

The current diagnosis of both myelodysplastic syndromes and myeloproliferative disorders relies in large part on subtle and subjective morphologic findings and the presence of cytogenetic abnormalities. Consequently, diagnosis of these disorders is often difficult and tentative with diagnosis at early stages representing a particular challenge. There is a need for new diagnostic techniques to allow a more definitive and objective diagnosis for these diseases. The published literature relating to the potential diagnostic utility of flow cytometric immunophenotyping in the diagnosis of myelodysplastic syndromes and myeloproliferative disorders is reviewed, and the increasingly important contribution of this technique to the diagnosis of these disorders emphasized.     

Monoclonal antibody panels for acute leukemia and myelodysplastic syndrome diagnosis. Results of a co-operative quality control group

The need for standardization criteria and result reproducibility in immunophenotyping hematological diseases has increased along with their clinical importance. Our group "Policentric Study Group on Immunological Markers", is composed of 40 laboratories. Its aim, over recent years, has been to find a standardized way of immunophenotypic analysis applicable to various hematological diseases. The objective of this study is to contribute to the debate concerning standardization of monoclonal antibody panels and immunophenotypic analysis procedures in acute leukemia (AL) and myelodysplastic syndrome (MDS), with the following targets: to improve interlaboratory reproducibility of the immunophenotyping data, and interpretative results; to study, with improved feasibility, correlation between immunophenotype and clinical or biological findings on a large number of AL and MDS cases; to verify the utility of the proposed monoclonal antibody panels for proper AL and MDS classification, and to detect minimal residual disease. In the field of AL and MDS our experience is based on about 1800 and 700 cases respectively analyzed over the last five years. Starting from these experiences and data of the literature we have elaborated the proposed panels of monoclonal antibodies and the methods of analysis. We have suggested a standardized immunophenotypic approach to study AL and MDS. In particular our work has focused on the gating strategy. This aims at drawing a gate of analysis having high purity and recovery, and on the choice of monoclonal antibody combinations for multiparametric analysis, particularly the normal antigen expression on each step of lineage differentiation or their clinically relevant aberrant expressions. A standardized criteria has become a necessary starting point in any kind of analytical process. In the field of acute leukemias and myelodysplastic syndromes the work of this polycentric group has focused on the pre-analytical and analytical steps to be taken in cytometric evaluation of hematological malignancies. The results obtained may contribute to reaching intra and inter-laboratory reproducibility.     

Routine immunophenotyping of acute leukaemias

Recent progress in immunophenotyping includes the availability of monoclonal antibodies (MAbs), knowledge of specificity and reactivity patterns of these reagents, and the technical improvements and standardization of immunofluorescence and immunocytology staining procedures, including flow cytometry. These advances have contributed significantly to the establishment of immunophenotyping as an essential diagnostic tool in the differential diagnosis of types of acute leukaemia. Immunophenotyping allows for the objective and reproducible distinction of acute lymphoblastic leukaemia (ALL) from acute myeloblastic leukaemia (AML) and of T-lineage from B-lineage ALL. Immunologically defined ALL and AML subtypes have been found to convey prognostic significance. Using cell lineage-specific and differentiation stage-specific MAbs, cases of T- and B-lineage ALL and of AML can be further classified into a number of different subtypes. Routine immunophenotyping concentrates on the diagnostic enquiry into a few major, clinically relevant subtypes; only a limited number of crucial reagents are employed that are commercially available. The simplification and standardization of discriminatory immunomarker panels make immunophenotyping a reliable diagnostic instrument for the provision of critical data to make a differential diagnosis. An effort to identify the nature and origin of the blast cells precisely, immunological typing definitely plays an important part in the multiple-marker analysis of acute leukaemia (morphology, cytochemistry, karyotyping, genotyping) for applied diagnostic and fundamental research purposes.     

Combined cytomorphologic and immunophenotypic analysis in the diagnostic workup of lymphomatous effusions

OBJECTIVE: To analyze the results of cytomorphology and immunophenotyping in 54 patients with lymphomatous effusions. STUDY DESIGN: We report the results of cytomorphology and immunophenotyping in 54 patients with lymphomatous effusions. Twenty-three of the 54 had a previous diagnosis of NHL. In the remaining 31 patients, lymphomatous involvement was clinically suspected. RESULTS: Thirty-three lymphomatous effusions were positive for involvement by NHL. Twenty-one of these 33 patients (64%) had a previous diagnosis of NHL. Of the remaining 12 patients with newly diagnosed NHL, 11 had high grade lymphoma, and one had follicular center lymphoma. Twenty effusions were considered to be reactive; only two of these patients had NHL. One effusion revealed involvement by a previously unknown carcinoma. We observed seven false negative results if only one of both methods was considered. A high grade NHL was not diagnosed by immunophenotyping in one case, and six cases of low grade NHL could not be detected by cytomorphology. The combined strategy of cytomorphology and immunophenotyping had a sensitivity of 100% and specificity of 100% in our study, confirmed by follow-up studies. CONCLUSION: Both methods have shown difficulties in the examination of lymphomatous effusions. Cytomorphology has problems distinguishing reactive effusions from low grade NHL. The detection of high grade NHL by immunophenotyping is difficult. However, both methods together offer the advantage of dual staining ability and are most helpful in distinguishing clonal lymphomatous from reactive effusions.     

Past and present concepts in flow cytometry: a European perspective

The development of flow cytometric instrumentation, methods and research concepts in Europe has been a continuous driving force for the general scientific advancement in this area over the years. This review addresses early European concepts of continuing interest with regard to instrumentation, data analysis, clinical and eperimental DNA analysis, cell function and microbiology at their worldwide first appearence while flow cytometric immunology and immunophenotyping will be covered separately. Flow cytometry represents an efficient approach to the enormous complexity of molecular cell architecture and cell function by the analysis of apparent molecular cell phenotypes in heterogeneous cell samples. The present merger of flow and image cytometry into the method independent cytomics discipline increases the potential of cell analysis very significantly. It opens the way for predictive medicine as well as for predictive cytopathology and predictive cytology in everyday clinical and medical practice. Current progress is driven by joint advances in molecular fluorescence technologies and instrument development. This complements the analysis of genome sequence information in an efficient way.     

Development of quartz-crystal-microbalance-based immunosensor array for clinical immunophenotyping of acute leukemias

An integrated piezoelectric immunosensor array has been developed to immunophenotype acute leukemias in clinic. Each quartz crystal microbalance (QCM) was fabricated with plasma-polymerized film of n-butylamine, nanogold particles, and protein A (PA) to be used to immobilize antibodies in orientation. Leukemic lineage-associated monoclonal antibodies were separately immobilized onto the nanogold-PA-modified surface of the crystals, which were constructed by a 2 x 2 type of probes forming a QCM-based immunosensor array. The main detection conditions were investigated, including the immobilization amount of antibodies, pH, immunoreaction time, sample dilution ratio, etc. The immunophenotyping feasibility of the new technique was investigated through simultaneously analyzing Jurkat cells by the immunosensor array method, immunohistochemistry, and flow cytometry. It was found that the developed technique could readily identify leukemia samples in 5 min and might monitor dynamically the immunoreaction processes. Moreover, comparison studies were carried out for CD antigens expressed on the nucleated cells isolated from 96 acute leukemic patients and 24 normal subjects using the QCM-based immunosensor method and the fluoroimmunoassay. Results obtained by immunophenotyping patients' samples with the immunosensor-based method achieved the rate of 88.93% in 768 groups of numerical data, where no significant statistical difference was observed between the two methods when checked by chi2 analysis (chi2 = 3.4, p > 0.05). This new immunosensor array showed the merits of high sensitivity, high specificity, good reproducibility, easy operation, and low cost. The results of specimen evaluation indicated that it might be clinically suitable for quantifying human differentiated leukocytes and immunophenotyping of acute leukemias.     

Genome-wide identification studies – A primer to explore new genes in plant species

Genome data have accumulated rapidly in recent years, doubling roughly after every 6 months due to the influx of next-generation sequencing technologies. A plethora of plant genomes are available in comprehensive public databases. This easy access to data provides an opportunity to explore genome datasets and recruit new genes in various plant species not possible a decade ago. In the past few years, many gene families have been published using these public datasets. These genome-wide studies identify and characterize gene members, gene structures, evolutionary relationships, expression patterns, protein interactions and gene ontologies, and predict putative gene functions using various computational tools. Such studies provide meaningful information and an initial framework for further functional elucidation. This review provides a concise layout of approaches used in these gene family studies and demonstrates an outline for employing various plant genome datasets in future studies.     

Reviewing population studies for forensic purposes: Dog?mitochondrial DNA

The identification of dog hair through mtDNA analysis has become increasingly important in the last 15 years, as it can provide associative evidence connecting victims and suspects. The evidential value of an mtDNA match between dog hair and its potential donor is determined by the random match probability of the haplotype. This probability is based on the haplotype’s population frequency estimate. Consequently, implementing a population study representative of the population relevant to the forensic case is vital to the correct evaluation of the evidence. This paper reviews numerous published dog mtDNA studies and shows that many of these studies vary widely in sampling strategies and data quality. Therefore, several features influencing the representativeness of a population sample are discussed. Moreover, recommendations are provided on how to set up a dog mtDNA population study and how to decide whether or not to include published data. This review emphasizes the need for improved dog mtDNA population data for forensic purposes, including targeting the entire mitochondrial genome. In particular, the creation of a publicly available database of qualitative dog mtDNA population studies would improve the genetic analysis of dog traces in forensic casework.