Immunophenotyping Research Papers - Academia.edu (original) (raw)

This is a literature review about large granular lymphocyte leukemia (LGLL), a rare and misdiagnosed oncohematological disease, characterized by a clonal expansion of T-cells (T-LGLL) or NK-cells (NK-LGLL) in the bone marrow and/or peripheral blood. The clinical features of LGLL include cytopenias (anemia, neutropenia and thrombocytopenia), lymphocytosis (usually discrete), lymphadenopathy, hepatomegaly, splenomegaly, immune abnormalities and constitutional symptoms (fever, night sweats and weight loss). The diagnosis is based on the confirmation of the clonality of T-cells or NK-cells (polymerase chain reaction and Southern blot are the two methods most commonly used) and typical findings of the immunophenotypic analysis of peripheral blood lymphocytes (flow cytometry analyses for specific surface antigens). In contrast to the chronic and indolent course of T-LGLL, NK-LGLL has an acute presentation and poor clinical outcome. There are different current treatment options, depending on clinical presentation.

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- Algorithms, Adolescent, Stem Cell Transplantation, Child

Rare tetraploidy with large 5q deletion in acute myeloid leukemia with myelodysplasia-related changes (AML-MRC)

- by K. Indrak
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- Leukemia, Acute Myeloid Leukemia, Fluorescence in situ hybridization, Clinical Sciences
- by Jesús Miguel
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- Genetics, Cytogenetics, Lymphoma, Bone marrow

We report six patients with acute leukemia characterized by the presence of a t(lo;ll) (pl l-p15;q13-q23), either as sole cytogenetic abnormality (three patients) or as part of a complex abnormal karyotype. The morphologic and cytochemical features of four patients were consistent with FAB-M5A, while two patients presented with FAB-Ll characteristics. By immunophenotyping, myeloid leukemia

- by Peter Wiernik
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- Stem Cell, Acute Myeloid Leukemia, Trisomy 21, Immunophenotyping

Immunophenotyping, as suggested by WHO, may improve diagnosis of childhood leukemia since it offers a better classification of the hematopoietic lineage of malignant cells as compared to morphology. Therefore, we aimed to determine the proportion of the immunophenotypic subtypes of acute leukemia in Indonesian children. Samples were obtained from patients (0-14 years of age) in 4 hospitals in Indonesia. We analyzed 541 suspected leukemia samples presented over a 4-year period (March 2006 - July 2010) by flow cytometry. Immunophenotyping allowed classification into acute myeloid leukemia (AML) and ALL (B-lineage and T-lineage ALL). Of 541 samples, 136 were tested using a single color method and 405 with a three-color method. Concordance with morphology was very good (?=0.82) using the three-color method with a panel of 15 monoclonal antibodies (n=387). A relatively high percentage of acute leukemia was classified as AML (23%). Of the ALL samples 83% were B-lineage ALL and 17% T- line...

- by Eddy Supriyadi
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- Flow Cytometry, Indonesia, Adolescent, Acute Myeloid Leukemia

22 Protective antibodies in Plasmodium falciparum malaria are only acquired after years of 23 repeated infections. Chronic malaria exposure is associated with a large increase in 24 atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent 25 viral infections. Understanding the function of atypical MBCs and their relationship to 26 classical MBCs will be critical to developing effective vaccines for malaria and other 27 chronic infections. We show that VH gene repertoires and somatic hypermutation rates of 28 atypical and classical MBCs are indistinguishable indicating a common developmental 29 history. Atypical MBCs express an array of inhibitory receptors and B cell receptor 30 (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses 31 including proliferation, cytokine production and antibody secretion. Thus, in response to 32 chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs 33 becoming refractory to BCR-mediated activation and potentially interfering with the 34 acquisition of malaria immunity. 35 3 Introduction 36

- by Kassoum Kayentao
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- Malaria, Adolescent, Gene expression, Plasmodium

Loss of genomic material from chromosomal band 13q14.3 is the most common genetic imbalance in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, pointing to the involvement of this region in a tumor suppressor mechanism. From the minimally deleted region, 3 candidate genes have been isolated, RFP2, BCMS, and BCMSUN. DNA sequence analyses have failed to detect small mutations in any of these genes, suggesting a different pathomechanism, most likely haploinsufficiency. We, therefore , tested B-CLL patients for epigenetic aberrations by measuring expression of genes from 13q14.3 and methylation of their promotor region. RB1, CLLD7, KPNA3, CLLD6, and RFP2 were downregulated in B-CLL patients as compared with B cells of healthy donors, with RFP2 showing the most pronounced loss of expression. To test whether this loss of gene expression is associated with methylation of CpG islands in the respective promotor regions, we performed methylation-sensitive quantitative polymerase chain reaction analyses and bisulfite sequencing on DNA from B-CLL patients. No difference in the methylation patterns could be detected in any CpG island of the minimally deleted region. Downregulation of genes within chromosomal band 13q14.3 in B-CLL is in line with the concept of haploinsufficiency, but this tumor-specific phenomenon is not associated with DNA methylation. (Blood. 2002; 99:4116-4121)

- by Infinity Valencia
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- Oncology, Molecular Medicine, Cancer, Biology

Occupational exposure to asbestos is strongly associated with pulmonary diseases, cancer and immunotoxic effects. Both systemic and local immunity may play an important role in the pathogenesis of these events. Immune cells appear to be influenced by asbestos exposure, either through direct effects or as a result of the host's protective response to exposure. In this study several immune system parameters were assessed in workers (n = 61) with at least 5 years' exposure to asbestos at an industrial plant. Workers exposed to asbestos fibres had significantly increased levels of immunoglobulin E and concentrations of interleukin-6 and-8 in comparison with two sets of controls (in-plant and town control groups). The levels of soluble adhesion molecule ICAM-1 were higher in the exposed group compared to the town control group. Significantly increased levels of IgA were found in asbestos-exposed group in comparison to the town control. Evaluation of the expression of adhesion molecules on lymphocytes, monocytes and granulocytes by flow cytometry showed significant increases in the class of selectins CD62L on monocytes and granulocytes. Moreover, significantly increased expression of markers CD69 and CD66b on eosinophils was found among workers exposed to asbestos. In conclusion, exposure to asbestos fibres was found to have several effects on immune system. Alterations of these immune parameters may indicate hypersensitivity (increased levels of IgE, increased expression of activation markers CD66b and CD69 on eosinophils) and an elevated inflammatory status (increased levels of interleukins-IL-6, IL-8) in exposed workers.

- by Ladislava Wsolova
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- Toxicology, Flow Cytometry, Asbestosis, Asbestos

Several single nucleotide polymorphisms (SNPs) have been associated with susceptibility to autoimmune diseases, but the mechanisms responsible for the associations are poorly understood. To test the hypothesis that the variation of the basal levels of the gene products is significantly influenced by genetic polymorphism, we investigated whether SNPs in genes CD40, CD28, CTLA4, CD80, CD86, BAFF and IL6 are affecting mRNA or protein expression. The surface expression of the proteins on unstimulated monocytes,

- by Maria-Luiza Petzl-Erler
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- Genetics, Gene expression, Immunogenetics, Autoimmune diseases

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- by Giuseppe Leone
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- Chronic Disease, Immunophenotyping, B Lymphocytes, Biochemistry and cell biology

Flow cytometry biomarkers distinguish DOCK8 deficiency from severe atopic dermatitis, Clinical Immunology (2013), ABSTRACT DOCK8 deficiency is a primary immunodeficiency characterized by recurrent sinopulmonary infections, dermatitis with cutaneous infections, elevated serum IgE levels, eosinophilia, and a high incidence of food allergy. Given the seriousness of DOCK8 deficiency, it is important to recognize it early and initiate appropriate therapy. Diagnosis relies on examining DOCK8 protein expression and sequencing of the 48 exons in the DOCK8 gene, but these assays are not always readily available. A major problem facing clinicians is that DOCK8 deficiency shares many clinical and laboratory features with severe atopic dermatitis. Here, we have identified biomarkers routinely measured by flow cytometry on whole blood in clinical immunology laboratories that may be used in distinguishing DOCK8 deficiency from severe atopic dermatitis. The use of these biomarkers may help the clinician identify those patients who are most likely to have DOCK8 mutations and would benefit from further specialized diagnostic testing.

- by Andre Megarbane
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- Immunology, Clinical immunology, Mutation, Differential Diagnosis
- by M. Dominis
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- Clinical Pathology, Biopsy, Clinical, Clinical Sciences
- by Alejandra Zárate-Osorno and +1
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- Cancer, Immunohistochemistry, Follow-up studies, Risk factors

The Cytometry Part B: Clinical Cytometry supplement (72B, Supplement 1, 2007) titled '2006 Bethesda International Consensus Conference on Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasia' is sponsored by the... more

- by Maria Arroz
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- Flow Cytometry, Medicine, Quality Control, Immunophenotyping

In the absence of a donor alternative a stem cell transplantation consisting of two cord blood components originating from the haploidentical brother was performed in a 2-year-old girl with c-ALL, early CNS relapse and 7% of blast cells in the BM 14 days before transplantation. Because of various ongoing infectious complications at that time, 1/8 of the immunogenetically acceptable sibling

- by Tim Niehues and +1
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- Hematopoietic Stem Cells, X Rays, Cell therapy, Haplotypes

Common variable immunode®ciency (CVID) is a primary immunode®ciency disease characterized by low serum immunoglobulins IgG, IgA, and usually IgM. The central immune de®ciency is impaired secretion of immunoglobulins and lack of antibody production; however, T cell dysfunction and a variety of in¯ammatory complications suggest global immune dysregulation. A number of reports have documented the association of primary immunode®ciency diseases with the development of non-Hodgkin's lymphoma (NHL). In CVID, the risk has been estimated to lie between 1.4% and 7%. As for NHL arising in other immunode®ciency states, the lymphomas in CVID are extranodal and are usually B cell in type. Of 22 B cell lymphomas that have appeared over a period of 25 years in a cohort of subjects with CVID, ®ve lymphomas, appearing in more recently studied subjects, that arose in mucosal sites would be classi®ed as mucosaassociated lymphoid tissue (MALT) lymphomas. MALT lymphomas are low-grade B cell lymphomas that result from a proliferation of neoplastic marginal-zone related cells of lymphoid tissue and tend to occur in organs that have acquired lymphoid tissue due to long-term infectious or autoimmune stimulation. Lymphomas of this kind have not been described in patients with congenital immunode®ciency, although chronic mucosal antigen stimulation is an integral part of these immune de®ciency states. Am.

- by Charlotte Cunningham-rundles
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- Adolescent, Comorbidity, Follow-up studies, Diagnostic Imaging

In the present study we compared the immunophenotypic subtypes of breast ductal invasive carcinomas with their ipsilateral, axillary lymph node metastasis. The ER (estrogen receptor), PR (progesterone receptor), Her2 (human epidermal growth factor receptor 2), and CK5 (cytokeratin 5) status and the proliferation marker Ki-67 were determined by immunohistochemistry on specimens from 43 women. All selected cases were diagnosed as invasive breast carcinomas, of no special type (NST), G2 grade of differentiation. The most frequently encountered subtype at both sites was luminal B. We determined that tumor profile evaluated by surrogate markers is not stable during the metastatic process. The total rate of shifted cases was 23.26% (10 cases), and the highest rate of shifting (6.98%) was encountered from luminal B/Ki-67 to luminal A subtype. In five cases, the subtype shifted to a poorer one according to prognosis. These data support the hypothesis that breast cancer is a heterogeneous disease, with substantial variability of cellular components within each category, a statement applicable to invasive breast carcinomas of NST type too. The receptor profile of this carcinoma, indicated by surrogate markers, is not stable throughout the metastatic process.

- by Veaceslav Fulga
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- Immunohistochemistry, Biopsy, Cell Differentiation, Phenotype

An immunohistochemical study on a case of lupus erythematosus panniculitis (LEP), without discoid lupus erythematosus (DLE) or systemic lupus erythematosus (SLE) signs, showed that the cells in skin infiltrates were immunologically committed lymphocytes (OKT4, OKT8, OKTll and HLA-DR positive cells) and elements of the monocyte-macrophage lineage (Leu M3 and Leu M5 positive). No immunophenotypically identifiable B-lymphocytes were seen. Immunofluorescent IgG, IgM, C3 and C4 deposits were found in blood vessel walls of the deep dermis. These findings, similar to that described in the skin changes of SLE and DLE, suggest that immunological mechanisms are operative in localized LEP, where the dermal lesions are the only expression of the disease.

Interest in hepatic ductular reactions (DRs) has risen in recent years because of a greater appreciation of their potential roles in regeneration, fibrogenesis, and carcinogenesis. However, confusion exists because there is significant, but often unappreciated diversity at the tissue, cellular, and subcellular levels in DRs of different diseases and stages of disease. DRs are encountered in virtually all liver disorders in which there is organ-wide liver damage and cell loss, but are also present in focal lesions such as focal nodular hyperplasia and adenoma. Moreover, diverse DR phenotypes can be present within any single disease entity, and are shaped by the etiology and evolution of the disease. Although much remains to be clarified, recent studies suggest that the diversity of appearances of the DRs are likely to reflect the differing signals at the anatomic, cellular, and molecular levels driving the proliferative response. These appear to determine the relative proportions of transit-amplifying cells, the degree of hepatocytic or cholangiocytic differentiation, and their relationships with stromal, vascular, and inflammatory components. The molecular signaling pathways governing these regenerative fate decisions closely replicate those found in human and other vertebrate embryos and more generally in stem cell niches throughout the body. Like the latter, complex interactions with matrix as well as mesenchymal and inflammatory cells, vessels, and innervation are likely to be of fundamental importance. Embracing systems/tissue biological approaches to exploring DRs, in addition to more traditional cellular and molecular biological techniques, will further enhance our understanding and, thereby, we believe potentiate new therapeutic possibilities.

- by Neil Theise
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- Hepatology, Liver diseases, Clinical Sciences, Hepatocytes

Immune responses arise from a wide variety of cells expressing unique combinations of multiple cell-surface proteins. Detailed characterization is hampered, however, by limitations in available probes and instrumentation. Here, we use the unique spectral properties of semiconductor nanocrystals (quantum dots) to extend the capabilities of polychromatic flow cytometry to resolve 17 fluorescence emissions. We show the need for this power by analyzing, in detail, the phenotype of multiple antigen-specific T-cell populations, revealing variations within complex phenotypic patterns that would otherwise remain obscure. For example, T cells specific for distinct epitopes from one pathogen, and even those specific for the same epitope, can have markedly different phenotypes. The technology we describe, encompassing the detection of eight quantum dots in conjunction with conventional fluorophores, should expand the horizons of flow cytometry, as well as our ability to characterize the intricacies of both adaptive and innate cellular immune responses.

- by David Price
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- Immune response, Flow Cytometry, Semiconductors, Nanotechnology

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (alphaCD3/alphaCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or alphaCD3/alphaCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or alphaCD3/alphaCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3+CD4+ or CD3+CD8+ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time. In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or alphaCD3/alphaCD28 as positive control.

- by Prescilla Jeurink
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- Cryobiology, Kinetics, Cytokines, Cryopreservation

We investigated the expression of novel anti-inflammatory interleukin (IL)-38 and regulatory T (Treg) lymphocytes in childhood asthma patients. The protein and mRNA expression level of IL-38, periostin, peripheral CD4⁺CD25⁺CD134⁺ T lymphocytes as well as CD4⁺CD25(high)FoxP3⁺ and CD4⁺CD25(high)CD127(-) Treg lymphocytes from 40 asthmatic patients and 20 normal control (NC) subjects were studied using ELISA, qPCR and flow cytometry. Serum and supernatant cytokines/chemokines were determined by multiplex assay. Serum IL-38, IL-5, IL-17, IL-6, interferon-γ, periostin, IL-1β and IL-13 concentrations were significantly higher in asthmatic patients with or without steroid treatment than those in controls (all p < 0.05). The percentages of both CD4⁺CD25(high)FoxP3⁺ and CD4⁺CD25(high)CD127(-) Treg lymphocytes were markedly decreased in asthmatic patients with and without steroid treatment than those in controls (all p < 0.05). The elevated IL-38 concentration negatively correlated with ...

- by Chun Wong
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- Organic Chemistry, Biomarkers, Asthma, Gene expression

Interphase fluorescence in situ hybridization (FISH) is an alternative to conventional chromosome analysis of chronic lymphocytic leukemia (CLL) cells. We analyzed 172 samples from 136 possible CLL cases using a FISH panel. Reflex testing with probes to CCND1, BCL2, BCL3, BCL11A, c-MYC, MALT1, and a break-apart immunoglobulin heavy chain (IGH) probe was done if more than 2 signals for 14q32 occurred. For 111 cases, there were sufficient data for analysis. Of 111 cases, 81 (72.9%) had 1 or more genetic abnormalities. The most frequent abnormality was 13q-, followed by trisomy 12, 11q-, and 17p-. In 13 cases, there were IGH abnormalities. Two cases with CCND1/IGH fusion were reclassified as mantle cell lymphoma. Four CLL cases had IGH fusion with BCL2, BCL3 (2 cases), and BCL11A; no fusion partner was detected in 7 cases. Morphologic features were atypical for CLL in 2 cases with IGH fusion (BCL11A and BCL3). The FISH CLL panel is useful to identify prognostic aberrations and to clarify diagnosis in cases with unusual morphologic features.

- by Rohit Gupta
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- FISH, Chronic Lymphocytic Leukemia, Aged, Prognosis

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cellrich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

The complexity of the lymphostromal interplay that is essential to abT-cell development is reflected by the heterogeneity of both lymphocytes and thymic stromal cells. While panels of monoclonal antibodies have described many of the cellular components of these microenvironments, the means to quantify stromal cell subsets using flow cytometry remains poorly defined. This study refines and compares various stromal cell isolation procedures and determines the effects of various digestion enzymes on important surface molecules. Three-and four-color flow cytometry is used to correlate established and novel stromal cell markers to define thymic fibroblasts, epithelium and a unique subset of thymic endothelium that express MHC class II. This work provides a basis for the purification of thymic stromal cells for further phenotypic, functional and genetic analysis. D : S 0 0 2 2 -1 7 5 9 ( 0 1 ) 0 0 4 9 3 -8

- by Ann Chidgey
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- Immunology, Flow Cytometry, Cell separation, Mice

consultancy fees from Biotest and LFB. M. T. de la Morena has received consultancy fees from the Atlantic Research Group. F. J. Espinosa-Rosalesis on the Baxter board; has received lecture fees from Grifols and CSL Behring; and has received travel support from Grifols, CSL Behring, and Octapharma. I. Quinti is on the Baxter board; has received consultancy fees from FKB; has received research support from the Plasma Protein Therapeutics Association; has received lecture fees from Baxter and Kedrion; and has received travel support from Baxter. J. M. Routes has received research support from the National Institutes of Health/ National Institute of Allergy and Infectious Diseases (granulomatous lymphocytic interstitial lung disease in common variable immunodeficiency grant-R34 planning grant). M. L. K. Tang is on the Nestle Nutrition Institute Medical Advisory Board Oceania; is on the Danone Nutritia Medical Advisory Board Australia New Zealand and Global Scientific Advisory Board; has received consultancy fees from GLG Consultants and Deerfield Institute; has received lecture fees from Danone Nutricia and Health Ed; has a patent on a method for inducing tolerance; received royalties from Wiley as a coauthor of Kids Food Allergies for Dummies; and has received payment for developing an educational module for MD Linx. K. Warnatz has received lecture fees from Baxter, GlaxoSmithKline, CSL Behring, Pfizer,

Background:Pathogen infection is a complex process in which several pathogen-recognition receptor (PRR) pathways are activated to induce proinflammatory mediators. The activation of multiple PRRs suggests an interaction between Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptor (NOD) signaling pathways.Pathogen infection is a complex process in which several pathogen-recognition receptor (PRR) pathways are activated to induce proinflammatory mediators. The activation of multiple PRRs suggests an interaction between Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptor (NOD) signaling pathways.Methods:To understand the modulation induced by NOD2 signals on successive responses to pathogen-associated molecular patterns (PAMPs), we examined how muramyl dipeptide (MDP) pretreatment reprograms the MDP+LPS (lipopolysaccharide) response of monocytes from human peripheral blood.To understand the modulation induced by NOD2 signals on successive responses to pathogen-associated molecular patterns (PAMPs), we examined how muramyl dipeptide (MDP) pretreatment reprograms the MDP+LPS (lipopolysaccharide) response of monocytes from human peripheral blood.Results:Preexposure to bacterial MDP components induced selective tolerance to a subsequent NOD2+TLR4 stimulation. MDP pretreatment inhibited the production of tumor necrosis factor alpha (TNFα) and interleuken 10 (IL10), whereas IL6 and IL8 remained unaffected. MDP-induced tolerance was independent of receptor downregulation but was associated with reduced levels of phosphorylated TAK1 and abrogated phosphorylation of the downstream MAPK.Since Nod2 mutations have been associated with susceptibility to develop Crohn's disease (CD), we compared the MDP-induced tolerance in healthy donors and CD patients with compound heterozygous Nod2 mutations (Mut-Nod2) expressing variant NOD2 proteins. MDP-induced tolerance in Mut-Nod2 patients reduced IL10 but not TNFα production. In contrast with healthy donors, a p38-independent TNFα production was observed during the kinetics of the MDP+LPS response in Mut-Nod2 patients.Preexposure to bacterial MDP components induced selective tolerance to a subsequent NOD2+TLR4 stimulation. MDP pretreatment inhibited the production of tumor necrosis factor alpha (TNFα) and interleuken 10 (IL10), whereas IL6 and IL8 remained unaffected. MDP-induced tolerance was independent of receptor downregulation but was associated with reduced levels of phosphorylated TAK1 and abrogated phosphorylation of the downstream MAPK.Since Nod2 mutations have been associated with susceptibility to develop Crohn's disease (CD), we compared the MDP-induced tolerance in healthy donors and CD patients with compound heterozygous Nod2 mutations (Mut-Nod2) expressing variant NOD2 proteins. MDP-induced tolerance in Mut-Nod2 patients reduced IL10 but not TNFα production. In contrast with healthy donors, a p38-independent TNFα production was observed during the kinetics of the MDP+LPS response in Mut-Nod2 patients.Conclusions:Our findings suggest that the selective tolerance induced by MDP in healthy donors was related to the modulation of a convergent nub of NOD2 and TLR4 signaling pathways. This MDP-induced tolerance was impaired in Mut-Nod2 CD patients, resulting in a p38-independent TNFα production and an imbalance between pro- and antiinflammatory cytokines that could be partly responsible for the pathogenesis of CD. Inflamm Bowel Dis 2009Our findings suggest that the selective tolerance induced by MDP in healthy donors was related to the modulation of a convergent nub of NOD2 and TLR4 signaling pathways. This MDP-induced tolerance was impaired in Mut-Nod2 CD patients, resulting in a p38-independent TNFα production and an imbalance between pro- and antiinflammatory cytokines that could be partly responsible for the pathogenesis of CD. Inflamm Bowel Dis 2009

We report 3 cases of lymphoid neoplasms with mixed lineage features of T-, NK-, or B-cell marker expression and clonal gene rearrangement for both T-cell receptor and immunoglobulin light chain IgK. A characteristic of our cases was the lack of expression of the specific B-cell transcription factor, Pax5, which is essential for maintaining the identity and function of mature B cells during late B lymphopoiesis. In the absence of Pax5, B cells in vitro can differentiate into macrophages, dendritic cells, granulocytes, and T/NK cells. Methylation analysis of the Pax5 gene in our cases suggests that its inactivation by this epigenetic event in a committed or mature B cell, before plasma cell differentiation, may well be a common pathogenetic mechanism in mature lymphoid neoplasms with expression of multilineage antigens. In particular, case 1 may represent a mixed NK-and B-cell lineage; and cases 2 and 3 may represent mixed T and B-cell lineage, respectively. Aberrations in the DNA methylation patterns are currently recognized as a hallmark of human cancer. Cases with aberrant phenotypes require molecular analysis for lineage assignment. Studies of such cases may be helpful to better elucidate whether they represent a distinct entity with clinical, immunophenotypic, and molecular characteristics or an incidental phenomenon during malignant transformation. Interestingly, these cases were all characterized by poor clinical outcome.

- by Stefano Pileri
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- Immunohistochemistry, Lymphoma, Natural Killer cells, DNA methylation

The phenotype of normal, reactive and malignant plasma cells. Identification of "many and multiple myelomas" and of new targets for myeloma therapy P henotype establishment is used for the diagnosis of several hematopoietic malignancies such as leukemia, but not for multiple myeloma (MM). However, phenotyping is a very sensitive and powerful approach, useful for both diagnosis and clinical follow-up. Establishing the phenotype of myeloma cells and its difference from that of normal PC has proven highly useful for (i): clearly identifying and characterizing malignant cells; 1-3 (ii) identifying prognostic markers; 4-7 (iii) preventing a false diagnosis of MM ; 8 (iv) evaluating minimal residual disease; 9 (v) suggesting antibody-based targeted therapies; 6,10-17 (vi) defining targeted downstream signaling therapies; 18-19 and (vii) computerizing the myeloma cell growth and differentiation through CD45 intraclonal hierarchy. 20 This review presents data on the phenotype from normal and reactive PC, as well as from primary and immortalized myeloma cells, in order to summarize how the study of the myeloma cell phenotype has brought up new concepts by identifying different patient entities as well as by establishing an intraclonal hierarchy. This approach lays the bases for new phenotype-dependent treatments for patients with MM.

- by Martine Amiot and +1
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- Multiple Myeloma, Drug Delivery Systems, Immunophenotyping

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigenspecific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigenspecific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-c secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigenexpressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCRexpressing effector cells after co-culture with DC.

- by Tim Greten
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- Immunology, Flow Cytometry, Dendritic Cells, Cancer Immunology

Guidelines for the use of flow cytometry and cell sorting in immunological studies

- by Francesco Liotta
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- Immunology, Research Design, RNA, Flow Cytometry

We describe our experience in the identification of 1 9 cases of AML-MO categorized among 200 consecutive AML cases. Leukaemic cells from our cases were morphologically marked by agranular basophilic cytoplasm, finely dispersed chromatin and prominent nucleoli. In two cases heavily vacuolated and monocytoid-shaped blasts were also observed. Cytochemistry (MPO, SBB, aANAE, aNBE, NASDCAE, AP, PAS) was negative in 14 cases, five cases expressing a very faint cytoplasmic positivity for aNBE (not exceeding 30% of the blasts) and aANAE (not exceeding 41%) which was sodium fluoride resistant. In these five cases other monocytic markers (e.g. CD14) were not in favour of myelomonocytic differentiation. All the cases were anti-MPO positive at frequency > 10%. Phenotypic analysis also revealed myeloid features with all the patients having at least one myeloid antigen (CD13, CD33. CD15), Tdt was expressed in nine cases and CD7 in six cases. All cases but one were positive for CD34. Cytogenetic analysis, performed in 16 cases, showed no adequate growth in two cases and no consistent abnormality in four; among the remaining 10 cases no consistent abnormality was observed, the most common finding was trisomy 8 (two cases) and 4 (two cases) and aberrations of chromosomes 2 , 3 , 5 , 7 , 9 , 1 2 and 21. No cases of (t9;22), Ph chromosome were observed. Interestingly three out of five patients with faint aNBE/aANAE positivity relapsed as typical M 4 (one case) or M5a (two cases).

- by Francesco Buccisano and +2
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- British, Acute Myeloid Leukemia, Light, Monoclonal Antibodies

Flow cytometry is now accepted as an ideal technology to reveal changes in immune cell composition and function. However, it is also an error-prone and variable technology, which makes it difficult to reproduce findings across laboratories. We have recently developed a strategy to standardize whole blood flow cytometry. The performance of our protocols was challenged here by profiling samples from healthy volunteers to reveal age- and gender-dependent differences and to establish a standardized reference cohort for use in clinical trials. Whole blood samples from two different cohorts were analyzed (first cohort: n = 52, second cohort: n = 46, both 20-84 years with equal gender distribution). The second cohort was run as a validation cohort by a different operator. The "ONE Study" panels were applied to analyze expression of >30 different surface markers to enumerate proportional and absolute numbers of >50 leucocyte subsets. Indeed, analysis of the first cohort reve...

- by Birgit Sawitzki
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- Flow Cytometry, Multidisciplinary, Aged, Sex Factors

This session of the Society for Hematopathology/European Association for Haematopathology Workshop focused on acute leukemias of ambiguous origin. We provide an overview of mixed-phenotype acute leukemia (MPAL) as recognized in the current World Health Organization classification and summarize diagnostic criteria for major categories of MPAL: B/myeloid, T/myeloid, B/T, and B/T/myeloid. Most MPAL cases submitted were B/myeloid and T/myeloid MPAL, the most frequent types, but three cases of B/T MPAL were also submitted, and examples of all categories are illustrated. We emphasize that a comprehensive approach to immunophenotyping is required to accurately establish the diagnosis of MPAL. Flow cytometry immunophenotyping using a large panel of antibodies is needed as well as confirmatory immunohistochemical analysis and cytochemistry studies for myeloperoxidase and nonspecific esterase. We discuss technical issues in determining blast lineage and possible pitfalls in MPAL diagnosis. In...

- by marie c bene
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- Leukemia, World Health Organization, Immunophenotyping, B Lymphocytes

Lymphoplasmacytic lymphoma involving the bone marrow can be difficult to diagnose, and pathological features that predict the presence of associated Waldenströ m's macroglobulinemia have yet to be identified. To address these issues, marrow histology, immunohistochemistry, and flow cytometry were studied from 35 lymphoplasmacytic lymphoma cases that had comprehensive clinical assessment for Waldenströ m's macroglobulinemia. In all cases, the plasma cells were analyzed by a novel 6-color flow method. Both immunohistochemistry and flow cytometry were useful in identifying the lymphoid and plasmacytic disease components. In 19 cases, immunohistochemistry revealed an earlier unrecognized pattern of plasma cell infiltration in which they were physically separate from the lymphoid infiltrates. B-cell flow cytometry revealed monotypic cells in 96% of the cases. Approximately half of these were CD5 and/or CD23 positive, although none had features of chronic lymphocytic leukemia, and none of the B cells had flow cytometric features suggesting plasmacytic differentiation. In contrast, highly sensitive 6-color plasma cell flow cytometry revealed monotypic cells in 32 of the 35 cases; in 20 cases, the pattern of CD38 and CD138 coexpression detected was identical to that seen in plasma cell malignancies such as multiple myeloma. In 18 of these 20 lymphoplasmacytic lymphoma cases, these plasma cells were CD19 positive, distinguishing them from those of true plasma cell neoplasms, which are CD19 negative. It is interesting that the two lymphoplasmacytic lymphoma cases with CD19-negative plasma cells had an IgG isotype serum paraprotein. Apart from this, no other pathological correlates of the clinical or laboratory features of symptomatic Waldenströ m's macroglobulinemia were identified.

- by Ellen McPhail
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- Flow Cytometry, Histology, Immunohistochemistry, Clinical Biochemistry

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Bot...

- by M. Kusche-Gullberg
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- Immunology, Flow Cytometry, Mast Cells, Histamine

Incidence and outcomes of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) are not well established at the population level, especially since the widespread use of immunophenotyping. We studied the epidemiology of CLL in Manitoba (Canada) by combining data from a centralized flow cytometry facility and the provincial cancer registry for the period 1998-2003. Of 616 cases identified, 27% of patients identified by flow cytometry were not on the cancer registry. The age-adjusted incidence of 7.99/100,000 is substantially higher than the reported incidence in registry reports. We also noted differences in relative survival based on age and gender.

- by Matthew Seftel and +1
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- Flow Cytometry, Survival Analysis, Leukemia, Chronic Lymphocytic Leukemia
- by Michael Poidinger
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- Immunology, Flow Cytometry, TB, Optometry and Ophthalmology
- by Katalin Goda
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- Chemistry, Flow Cytometry, Fluorescent Dyes and Reagents, Medicine

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.

- by Carl Wittwer
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- Hematology, Flow Cytometry, Clinical Chemistry, DNA

Swine skin is one of the best structural models for human skin, widely used to probe drug transcutaneous passage and to test new skin vaccination devices. However, little is known about its composition in immune cells, and among them dendritic cells (DC), that are essential in the initiation of the immune response. After a first seminal work describing four different DC subpopulations in pig skin, we hereafter deepen the characterization of these cells, showing the similarities between swine DC subsets and their human counterparts. Using comparative transcriptomic study, classical phenotyping as well as in vivo and in vitro functional studies, we show that swine CD163(pos) dermal DC (DDC) are transcriptomically similar to the human CD14(pos) DDC. CD163(pos) DDC are recruited in inflamed skin, they migrate in inflamed lymph but they are not attracted toward CCL21, and they modestly activate allogeneic CD8 T cells. We also show that CD163(low) DDC are transcriptomically similar to the...

- by Michel Bonneau and +1
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- Immunology, Cytokines, Inflammation, Macrophages
- by Maurizio Martelli
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- Cancer, Treatment Outcome, Adolescent, Weight Loss

The techniques of flow cytometry are becoming more and more important for the clinical hematology laboratory. No longer a novelty confined to a few specialized institutions as it was 10 years ago, flow cytometry has blossomed into a mature discipline. The methodology is well-known, the mechanical apparatus is readily available, and the role it plays in clinical hematology is increasingly appreciated. The burgeoning number of scientific articles devoted to this topic attests to the interest it has aroused as a tool for both medical research and patient care. In fact, more than a thousand such papers are now published each year and it would be impossible to deal with all the methodologies and applications of FCM currently utilized or under development. Throughout this paper four relevant hematologic fields are briefly discussed, in which FCM appears to be of great help at present: the immunophenotyping of leukemias and lymphomas, the measurement of proliferative activity and DNA ploid...

- by Alberto Orfao
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- Hematology, Flow Cytometry, Leukemia, Lymphoma

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (∼0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450–antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450–antibody conjugates are functional and stable.

Abstract: Immunophenotyping of hematologic malignancies represents one of the most relevant clinical applications of flow cytometry. Classically, leukemic/lymphomatous cells have been considered to reflect the immunophenotypic characteristics of different precursors and mature healthy cells blocked at certain differentiation stages. Recently, accumulating evidence has shown that neoplastic cells display several aberrant phenotypic patterns. These aberrant phenotypes are believed to reflect genetic abnormalities present in pathologic cells, and recent data have shown that at least in acute leukemias, myelodysplastic syndromes, chronic lymphoproliferative disorders, and plasma cell dyscrasias, they may be present in almost all patients. The aim of this work is to review recent advances in flow cytometry and the role of gating strategies more useful in the identification and characterization of neoplastic cells of different hematologic malignancies.

- by Francesco Gervasi
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- Flow Cytometry, Multidisciplinary, Phenotype, Immunophenotyping

Neuroblastoma is the most common extracranial solid tumor in early childhood with a pluripotent neural cell origin. The disease often infiltrates the bone marrow, and these cases are candidates for flow cytometric diagnosis and disease follow-up, more so in atypical cases that are diagnostically challenging by other methods. Here, we report two neuroblastoma cases, one with characteristic neuroblastoma cells forming Homer-Wright rosettes found in the bone marrow sample by histological examination. This case showed the classical immunophenotype with CD81/CD56/CD117 positive and CD45 negative labeling and 80% bone marrow infiltration. Minimal residual disease measurement was performed in a regular fashion, and flow cytometry results showed good correlation with other disease markers. Analyzing 300,000 events provided a sensitivity level of 0.01%. The second case showed atypical cell morphology on histological examination, after which flow cytometric analysis was initiated. Atypical cells displayed CD81 and bright CD56 but CD117 negative immunophenotype, with a 28% bone marrow infiltration. In most cases the primary tumor can be identified, but in this particular case only bone marrow involvement could be detected using flow cytometry, as such making it a powerful tool for diagnosis and disease monitoring.

- by Csongor Kiss
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- Flow Cytometry, Neuroblastoma, Immunophenotyping, Part

Prebiotics may increase Zn absorption, a mineral known to play a central role in the immune system. Zn-deficient states are characterised by suppressed immune function, while prebiotics may improve both gut and cell-mediated immunity. Our objective was to determine if inulin alters the number and proportion of immune cells in the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP), ex vivo cytokine secretion, intestinal permeability and Zn status in healthy as well as Zn-deficient rats. Weanling female rats were fed diets supplemented with 5 % cellulose (CEL) or 5 % inulin (PRE) for 4 weeks. The rats received the CEL or PRE diet ad libitum (ZN) or in restricted amounts (DR), or deficient in Zn (ZD) for another 4 weeks. The PRE-fed rats had a higher number and proportion of dendritic cells in PP, and greater ex vivo secretion of IL-2, IL-10 and interferon-g from spleen and MLN cells compared with CEL-fed rats. PRE reduced the number and proportion of T cell receptor (TCR)-ab þ CD8 þ cells in spleen and CD45RA þ cells in MLN compared with CEL. ZD rats had lower serum IgG2a and T cell numbers in MLN compared with ZN and DR rats. TCRgd þ cell numbers in PP were higher in ZD-PRE rats compared with ZD-CEL rats. Femur Zn concentrations of DR-PRE rats were higher than those of DR-CEL rats. Intestinal permeability was unchanged. The higher proportion and number of dendritic cells in the PP of inulin-fed rats indicates a need for further research on how prebiotics and their metabolites affect immune function possibly through intestinal dendritic cells.

- by Carla Taylor
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- Nutrition and Dietetics, Cytokines, Animal Production, Dendritic Cells