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It may has up to museums before you goed it. Golovinomyces orontii Wildermuth et al. NahG Arabidopsis groups to G. Phytophthora infestans Halim et al. Glazebrook ; Heck et al. SA for competition of service everyone. SA having in time Spoel et al. Problems associated with these tests include stringent specimen requirements and limited specificity. Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by flow cytometry to make a definitive diagnosis of PNH 29 33 34 In affected patients, two or more populations of erythrocytes can be readily identified, with different degrees of expression of CD55 and CD59 Fig.

Reticulocyte counts are based on identification of residual ribosomes and RNA in immature nonnucleated red blood cells RBCs. Traditionally, a blood smear is stained with a dye that precipitates the nucleic acid, and the cells are counted manually This method is subjective, imprecise, labor-intensive, and tedious. The flow cytometric enumeration of reticulocytes uses fluorescent dyes that bind the residual RNA, such as thiazole orange 37 This method provides excellent discrimination between reticulocytes and mature RBCs, with greater precision, sensitivity, and reproducibility than the traditional method 37 Because the fluorescence intensity is directly proportional to the amount of RNA and related to the immaturity of the RBC, a reticulocyte maturity index has been used clinically to assess bone marrow engraftment and erythropoietic activity and to help classify anemias 34 38 40 Some current automated cell counters use similar technology to estimate reticulocyte counts In the blood bank, flow cytometry can be used as a complementary or replacement test for red cell immunology, including RBC-bound immunoglobulins and red cell antigens Flow cytometry is being used increasingly in the blood bank to assess leukocyte contamination in leukocyte-reduced blood products 45 Immunologic monitoring of HIV-infected patients is a mainstay of the clinical flow cytometry laboratory.

Infected lymphocytes may be lysed when new virions are released or may be removed by the cellular immune system. The absolute CD4 count provides a powerful laboratory measurement for predicting, staging, and monitoring disease progression and response to treatment in HIV-infected individuals.

Quantitative viral load testing is a complementary test for clinical monitoring of disease and is correlated inversely to CD4 counts 47 Perhaps the best example of simultaneous analysis of multiple characteristics by flow cytometry involves the immunophenotyping of leukemias and lymphomas. Immunophenotyping as part of the diagnostic work-up of hematologic malignancies offers a rapid and effective means of providing a diagnosis. The ability to analyze multiple cellular characteristics, along with new antibodies and gating strategies, has substantially enhanced the utility of flow cytometry in the diagnosis of leukemias and lymphomas.

Different leukemias and lymphomas often have subtle differences in their antigen profiles that make them ideal for analysis by flow cytometry. Diagnostic interpretations depend on a combination of antigen patterns and fluorescence intensity. Several recent review articles are available 49 50 51 52 53 54 55 56 57 58 59 Flow cytometry is very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias.

Although most acute myeloid leukemias are difficult to classify by phenotype alone, flow cytometry can be useful in distinguishing certain acute myeloid leukemias, such as acute promyelocytic leukemia 61 Flow cytometry can also be used to identify leukemias that may be resistant to therapy In ALL, phenotype has been shown to correlate strongly with outcome 64 Region A , mature lymphocytes; region B , mature monocytes; region C , mature granulocytes; region D , immature myeloid cells.

Right , the population in region D from the left panel expresses CD13, a myeloid marker, and CD34, a stem cell marker. The co-expression of these two markers in the peripheral blood is highly suggestive of an acute myeloid leukemia. The B-cell lymphoproliferative disorders often have specific antigen patterns. The use of a wide range of antibodies allows clinicians to make specific diagnoses based on patterns of antigen expression. Not only is the presence or absence of antigens useful in making specific diagnoses, the strength of antigen expression can also aid in diagnosis.

One example is the weak expression of CD20 and immunoglobulin light chains commonly seen in chronic lymphocytic leukemia. Flow cytometry is particularly good at identifying clonality in B-cell populations. Although T-cell neoplasms may exhibit a predominance of antigens CD4 or CD8, these antigens should not be considered as surrogate markers of clonality.

The use of antibodies to the T-cell receptor family may occasionally be helpful in a small percentage of cases; however, many reactive processes can show expansion of particular T-cell receptor clones 66 67 68 Antigen deletions are common in T-cell lymphomas and may suggest neoplasia, but the only way to definitively diagnose T-cell clonality is by molecular methods.

Flow cytometry bioinformatics

Flow cytometry can be used for lymphoma phenotyping of fine needle aspirates, and is a powerful adjunct to cytologic diagnosis The high sensitivity and capacity for simultaneous analysis of multiple characteristics make flow cytometry useful for the detection of minimal residual disease, especially if abnormal patterns of antigen expression are present 71 72 73 74 Flow cytometry is not recommended for the diagnosis of Hodgkin lymphoma, chronic myelogenous leukemia, or myelodysplastic syndrome, although disease progression in the latter two conditions can often be monitored.

Neutropenia may be immune or nonimmune in nature. The work-up frequently entails a bone marrow examination. Immune neutropenia may result from granulocyte-specific autoantibodies, granulocyte-specific alloantibodies, or transfusion-related anti-HLA antibodies. Flow cytometry can readily identify anti-neutrophil antibodies that are either bound to granulocytes or free in plasma Autoimmune neutropenias may develop in patients with autoimmune disorders such as Felty syndrome, systemic lupus erythematosus, and Hashimoto thyroiditis.

When immune-related, flow cytometry can detect anti-neutrophil antibodies and confirm the origin of neutropenia, possibly eliminating the need for a bone marrow procedure. Conversely, the absence of anti-neutrophil antibodies narrows the differential diagnosis to nonimmune causes such as bone marrow failure, myelodysplasia, or marrow-infiltrative processes.

Functional deficiencies of leukocytes can be assessed by flow cytometry. Assays for oxidative burst, phagocytosis, opsonization, adhesion, and structure are available One clinical example measures neutrophil adhesion molecules central to a diagnosis of leukocyte adhesion deficiency syndrome type I This syndrome is characterized by an immunodeficiency related to defective neutrophil and monocyte migration to sites of inflammation. This receptor complex binds endothelial cell ligands such as intercellular adhesion molecule-1 CD54 antigen , which is necessary for neutrophil adherence and transendothelial migration 78 The analysis of platelets by flow cytometry is becoming more common in both research and clinical laboratories.

Platelet-associated immunoglobulin assays by flow cytometry can be direct or indirect assays, similar to other platelet-associated immunoglobulin immunoassays.

Content Types

In autoimmune thrombocytopenic purpura, free serum antibodies are not found as frequently as platelet-bound antibodies 80 81 82 In contrast, in cases of alloantibody formation, serum antibodies may be detected without evidence of platelet-associated antibodies Flow cytometry is an excellent method for direct analysis of platelet-bound antibodies, and it has also been shown to be of benefit in detection of free plasma antibodies 81 The use of thiazole orange, a fluorescent dye that binds RNA, allows immature platelets also referred to as reticulated platelets to be quantified 86 87 The reticulated platelet count can be used to determine the rate of thrombopoiesis.

This measurement can separate unexplained thrombocytopenias into those with increased destruction and those with defects in platelet production.


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Flow cytometry is a rapid and useful method of obtaining a diagnosis. Until recently, functional analysis of platelet activation was used primarily in research. Many immunological markers of platelet activation have been described, and the commercial availability of antibodies permits flow cytometric determination of platelet activation 95 96 Platelet activation may be clinically important in stored blood components, after cardiopulmonary bypass and renal dialysis, and in the treatment of patients with myocardial infarction or thrombotic events.

Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. For example, OKT3 is a mouse anti-human antibody useful in treating transplant rejection.

Valery V. Tuchin (Author of Tissue Optics)

Circulating concentrations of OKT3 can be quantified by incubating with normal CD3-positive lymphocytes, followed by a fluorescently labeled anti-mouse antibody Fluorescence values are compared to a calibration curve generated with known amounts of OKT3. Recently, multiplex assays for several antigens have become possible by the use of beads indexed by incorporating two different dyes 99 Flow cytometry is a powerful technique for correlating multiple characteristics on single cells.

This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. Applications in hematology include DNA content analysis, leukemia and lymphoma phenotyping, immunologic monitoring of HIV-infected individuals, and assessment of structural and functional properties of erythrocytes, leukocytes, and platelets.

Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease. Skip to main content. Michael Brown , Carl Wittwer. Published August Michael Brown. Abstract The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. General Principles Flow cytometry measures optical and fluorescence characteristics of single cells or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads.

View this table: View inline View popup. Table 1. Common clinical uses of flow cytometry.

Advanced Optical Flow Cytometry

Figure 1. Schematic of a flow cytometer. Immunophenotyping Applications in Hematology The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Figure 2. Hemoglobin F test for feto-maternal hemorrhage. Figure 3. Diagnosis of PNH. Figure 4.

Introduction

Immunophenotyping of acute myeloid leukemia. Table 2. Common phenotypes of B-cell lymphoproliferative disorders. Quantification of Soluble Molecules Soluble antigens or antibodies can be quantified by flow cytometry if standard cells or beads are used. Summary Flow cytometry is a powerful technique for correlating multiple characteristics on single cells. Flow cytometry and clinical diagnosis. Shapiro H. Practical flow cytometry, 3rd ed. New York: Wiley-Liss, pp.. Flow cytometry: its applications in hematology. Haematologica ; 80 : 69 Recktenwald DJ.

Introduction to flow cytometry: principles, fluorochromes, instrument set-up, calibration. J Hematother ; 2 : Watson JV. The early fluidic and optical physics of cytometry. Cytometry ; 38 : 1 Principles of flow cytometry. Transfus Sci ; 16 : Joensuu H , Klemi PJ. DNA aneuploidy in adenomas of endocrine organs. Am J Pathol ; : DNA aneuploidy in follicular adenomas of the thyroid gland.

DNA cytometry consensus conference. Cytometry ; 14 : Clinical and biological significance of aneuploidy in human tumours. J Clin Pathol ; 37 : Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology. Improved DNA content histograms from formalin-fixed, paraffin-embedded liver tissue by proteinase K digestion.

Prognostic importance of blast cell DNA content in childhood acute lymphoblastic leukemia. Blood ; 65 : DNA flow cytometry in solid tumors: practical aspects and clinical applications. Semin Diagn Pathol ; 6 : 55 Clinical aspects of alveolar rhabdomyosarcoma with translocation t 1;13 p36;q14 and hypotetraploidy. Pathol Oncol Res ; 5 : Cytogenetics of cranial base tumors. J Neurooncol ; 20 : Hartwell EA. American Society of Clinical Pathologists [see comments]. Am J Clin Pathol ; : Evaluation of methods for detection and quantitation of fetal cells and their effect on RhIgG usage.

Am J Clin Pathol ; 76 : Detecting fetomaternal hemorrhage: a comparison of five methods [see comments]. Transfusion ; 31 : Measurement of feto-maternal haemorrhage: a comparative study of three Kleihauer techniques and two flow cytometry methods. Clin Lab Haematol ; 19 : Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry.

Transfusion ; 38 : New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease. Cytometry ; 32 : Detection of hemoglobin variants in erythrocytes by flow cytometry. Cytometry ; 35 : Flow cytometry in diagnosis and management of large fetomaternal haemorrhage [see comments]. J Clin Pathol ; 48 : Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood. Am J Hum Genet ; 63 : Quantitation of fetal-maternal hemorrhage by flow cytometry. A simple and accurate method.

Am J Clin Pathol ; 91 : Flow cytometric analysis of decay-accelerating factor CD55 on neutrophils from aplastic anaemia patients. Br J Haematol ; 90 : The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria.


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Blood ; 87 : Somatic mutations of the PIG-A gene found in Japanese patients with paroxysmal nocturnal hemoglobinuria. Blood ; 85 : Rotoli B , Boccuni P. The PIG-A gene somatic mutation responsible for paroxysmal nocturnal hemoglobinuria. Haematologica ; 80 : Elghetany M , Davey FR. Erythrocytic disorders. Henry JB eds. Clinical diagnosis and management by laboratory methods, 19th ed : WB Saunders Philadelphia. Markedly high population of affected reticulocytes negative for decay-accelerating factor and CD59 in paroxysmal nocturnal hemoglobinuria.

Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. Blood ; 86 : Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: a tool for measuring the extent of the PNH clone. Leukemia ; 10 : Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index.

Arch Pathol Lab Med ; : Flow cytometric reticulocyte maturity index: a useful laboratory parameter of erythropoietic activity in anemia. Cytometry ; 22 : 35 Enumeration of reticulocytes using fluorescence-activated flow cytometry. Pathol Immunopathol Res ; 7 : Kinetics of erythrogenesis after bone marrow transplantation. Am J Clin Pathol ; 97 : Utility of flow cytometric reticulocyte quantification as a predictor of engraftment in autologous bone marrow transplantation.