Cancer and Blood Diseases Institute

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency disorder (PID), characterized by defective lymphocyte homeostasis. Its main clinical manifestations are lymphoproliferation, causing lymphadenopathy, [hepato]splenomegaly and hypersplenism, autoimmunity (autoimmune cytopenias and other autoimmune disorders) and a highly increased, life-long, risk of lymphoma. From a laboratory standpoint, there is defective Fas-mediated apoptosis, the immunophenotypic presence of T-cells that lack CD4 or CD8 expression, so-called ab-double negative T cells (ab-DNTC), other lymphocyte-specific immunophenotypic changes, hyperimmunoglobulinemia and the presence of auto-antibodies. ALPS is linked to genetic defects in the gene encoding Fas (CD95) in approximately 75 percent of patients (classified as ALPS type Ia). In several other ALPS patients, defects have been found in the gene encoding FasL (ALPS type Ib), caspase-10 (ALPS type IIa) or caspase-8 (ALPS type IIb). ALPS patients without a genetic diagnosis are currently classified as ALPS type III (~20-25 percent of patients).

Current diagnostic criteria, as proposed by the National Institutes of Health (NIH) ALPS Study Group are: chronic non-malignant lymphadenopathy or splenomegaly, increased circulating ab-DNTCs (≥1 percent of total lymphocytes or >20 cells / mcl) and defective Fas-mediated lymphocyte apoptosis in vitro.

This flow cytometric assay, using a combination of conjugated monoclonal antibodies, is intended to screen for the presence of ALPS. Together with clinical and laboratory data of the patient, the ALPS panel will help in making a preliminary determination whether the patient has ALPS. Further testing, including genetic testing and demonstration of defective in vitro Fas-mediated apoptosis, may be required to finalize the diagnosis of ALPS.

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• Issue 1: New Assays
• Issue 5: Immunophenotyping 101
• Issue 6: Pattern Recognition

B-cell activating factor (BAFF), also known as B Lys, TALL-1, and THANK, is a TNF superfamily member (TNFSF13B) known for its role in the survival and maturation of B cells. The BAFF gene encodes a putative 285 amino acid type II transmembrane protein. A 152 amino acid form can also be shed from the membrane by proteolytic cleavage and this soluble form is detectable in human serum. BAFF is produced by several cell types and tissues including monocytes, macrophages, neutrophils, dendritic cells, T lymphocytes, spleen, lymph node, and bone marrow. Its expression in resting monocytes is up-regulated by IFN-α, IFN-β, LPS and IL-10. It is thought to exist as a homotrimer, but it may also exist as a heteromer in association with related TNFSF member APRIL.

BAFF is a ligand for at least three TNF receptor superfamily (TNFRSF) members: B-cell maturation antigen (BCMA / TNFRSF17), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI / TNFRSF13B), and BAFF receptor (BAFF R / BR3 / TNFRSF13C). TACI and BAFF R are receptors for both BAFF and APRIL; however BAFF R selectively binds BAFF. All three receptors are primarily expressed by B cells. BAFF appears to be necessary for the proper transition from T1 to T2 phases of the B cell maturation pathway. Mechanisms underlying BAFF effects on B cell survival may include the up regulation or down regulation of anti- or pro-apoptotic members of the Bcl-2 family, respectively. Consistent with a role in human autoimmune disorders, BAFF is elevated in the serum of patients with SLE and Sjogren’s syndrome. It is also produced locally in the joints of patients with inflammatory arthritis and serum levels correlate with antibody titers in arthritis and Sjogren’s syndrome.

Principle of the assay: The Quantikine Human BAFF Immunoassay is a solid-phase ELISA designed to measure human BAFF levels in plasma and can be used to determine relative mass values for naturally occurring human BAFF. The assay employs the quantitative sandwich enzyme immunoassay technique.

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• Issue 5: Monitoring B-cell Depletion Therapy (part 2)

Although many aspects of B-cell biology take place in compartments other than peripheral blood, valuable information regarding the B-cell system can be obtained by B-cell immunophenotyping. The first steps of B-cell development and maturation take place in the bone marrow, independent of interactions between B cells and antigen. Upon completion of these first steps, the recent bone marrow emigrants migrate to specialized zones in the spleen and to other secondary lymphoid tissues. Through several transitional phases, these immature B cells differentiate into mature, naive, B cells. Mature B cells become either circulating or non-circulating (lymphoid organ resident) B cells. Following engagement with their antigens, B cells undergo several processes in secondary lymphoid organs (e.g. lymph nodes) that collectively are referred to as the germinal center reaction. Upon completion of these processes, B cells differentiate into plasma cells through several intermediate stages, memory B cells, or are deleted. Peripheral blood memory B cells, as well as plasma-blasts and early (circulating) plasma cells can be immunophenotypically identified in peripheral blood on the basis of surface expression of certain markers.

This flow cytometric B-cell panel, using a combination of conjugated monoclonal antibodies, is intended to provide a global overview of B-cell development and differentiation. Using specific combinations of surface markers, its goal is to detect defects in the normal sequence of maturation and differentiation through the absence of certain subpopulations, and / or the presence of unusual populations (e.g. transitional B cells), not normally found in peripheral blood. In addition, aberrant B-cell function may be detected by alterations in the normal distribution of B-cell populations, including plasma-blasts. In conjunction with other laboratory data, as well as clinical information, this assay can assist in providing a more detailed picture of the B-cell compartment and its context with the overall immune system.

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• Issue 2: New Assays
• Issue 4: Monitoring B-cell Depletion Therapy (part 1)
• Issue 6: Patient Vignette

The CD40 ligand (CD40L), also referred to as gp39, TRAP and CD154, is a tumor necrosis factor (TNF) family member glycoprotein present on the surface membrane of activated CD4⁺ T cells and a small subset of activated CD8⁺ T cells. The CD40L expression on activated T cells plays a pivotal role in B cell activation, proliferation and differentiation. Mutations in the CD40L gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM).

ICOS (inducible costimulator) is a human T-cell specific molecule, structurally and functionally closely related to CD28, and present on the surface membrane of activated (CD4⁺) T cells. ICOS-expression is found on T cells in germinal centers of lymphoid tissue where it interacts with counter-receptors on B cells that are undergoing the germinal center reaction. Thus, ICOS plays a crucial role in T-cell dependent B-cell activation, differentiation and memory formation. This is underscored by the discovery that in certain patients with common variable immunodeficiency (CVID), ICOS is not expressed on activated T cells as a result of mutations in the ICOS gene.

This procedure is a flow cytometric assay that uses whole blood to screen for abnormalities in the expression of CD40L and ICOS on the surface of in vitro activated CD4⁺ T cells. It has also been observed that both gene and surface expression of CD40L by activated T cells is depressed in a subgroup of common variable immunodeficiency while the lack of ICOS up-regulation on activated T cells would suggest the possibility of a genetic defect in the ICOS gene, underlying CVID.

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• Issue 2: New Assays
• Issue 3: Patient Vignette
• Issue 4: Patient Vignette
• Issue 6: Patient Vignette

Naïve T lymphocytes are mature resting T cells that have not yet encountered antigen. Memory T cells are long-lived antigen-specific T cells that have the capacity to quickly differentiate to an end stage effector cells upon re-exposure to antigen. Naïve and memory cells are present in both the CD4 and the CD8 subsets of T cells. Two isoforms of the CD45 molecule, CD45RA and CD45RO, are expressed on the surface of naïve and memory cells, respectively. Using four-color flow cytometry, monoclonal antibodies against these antigens are used to quantitate the relative proportions of each subset in the peripheral blood.

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• Issue 3: Immunophenotyping 101
• Issue 9: Focus Assay - REVISED RARO Panel
  

The use of therapeutic monoclonal antibodies for the treatment of cancer has become a promising approach.  Campath-IH (anti-CD52) targets the CD52 antigen present on the surface of most normal human monocytes, lymphocytes, as well as some malignant T cells and B cell lymphomas.  Once the monoclonal antibody binds with the CD52 antigen, it initiates antibody-dependent cellular toxicity and complement binding, which leads to apoptosis and activation of normal T cell cytotoxicity against the malignant cells.  Campath-1H is currently approved for the treatment of patients with relapsed/refractory chronic lymphocytic leukemia. CD52 expression can be found in the vast majority of low-grade B cell lymphoproliferative disorders such as follicular lymphoma, lymphoplasmactyic lymphoma, hairy cell leukemia and mucosa-associated lymphoid tissue lymphomas and may prove useful in treatment. Campath therapy has also been used to prevent graft versus host disease following allogeneic bone marrow transplantation by inducing lymphopenia.

CD55 is included in the panel to monitor the development of paroxysmal nocturnal hemoglobinuria (PNH)-like symptoms in a small group of patients receiving Campath with complications of hemolysis and thrombosis. Both CD52 and CD55 are antibodies against the  glycosylphosphatidylinositol (GPI) anchor on the cell membrane.  If a small clone of T cells are negative for CD52 and CD55, they will not be targeted with Campath and will survive and multiply after treatment.  This development of GPI deficient T cells has been noted in patients with chronic lymphocytic leukemia, patients following allogeneic stem cell transplants, and patients with aplastic anemia.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder arising by somatic mutation in the pluripotent hematopoietic stem cell. A close relationship between PNH and aplastic anemia and other myelodysplastic disorders have been reported. PNH is characterized by the deficiency, absolute or partial, of all proteins anchored to membrane by the glycosylphosphatidylinositol (GPI) anchor. At least 15 of these proteins have been shown to be lacking on the abnormal blood cells of these patients. The most prominent clinical feature in PNH is intravascular hemolysis due to increased susceptibility of red blood cells to complement-mediated lysis. This defect in PNH is caused by the lack of GPI-anchored proteins on the red cell surface, a consequence of a block in the biosynthesis of the GPI molecule where N-acetylglucosamine is transferred to the phosphoinositol molecule. Not only is this defect seen in erythrocytes, but is also shared by WBC’s and platelets of affected individuals.

Flow cytometric analysis easily permits testing for the GPI-anchor expression by immunophenotyping erythrocytes and WBC’s with anti-CD59 (termed: membrane attack complex inhibitory factor, [MACIF], membrane inhibitor of reactive lysis [MIRL] and prolactin) and FLAER, an inactive variant of aerolysin.

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• Issue 3: A FLAER for PNH

The Leuko64 kit is designed for use on flow cytometers. The kit is composed of a mixture of monoclonal antibodies with specificities to CD64 (FITC conjugated) and CD163 (phycoerythrin conjugated) and a proprietary fluorescent bead suspension used for instrument calibration and standardization of the leukocyte CD64 and CD163 expression on human blood leukocytes. Automated software for flow cytometric data analysis using iterative cluster finding algorithms is included in the assay kit. The Leuko64 assay is intended for use in the measurement of leukocyte neutrophil CD64 levels, which increase in response to infection, sepsis and tissue injury. It is also documented that neutrophil CD64 expression rapidly increases within hours by mediators of inflammation, such as interferon-gamma and G-CSF.

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• Issue 1: New Assays

CD107a, also known as LAMP-1 (Lysosome-associated membrane protein – 1), is normally expressed on the membranes of lysosomes (lytic granules) found in the cytoplasm of cytotoxic cells. These lysosomes contain perforin and granzymes and, when the cell is presented with a target cell, are mobilized to move to the surface of the effector cell, fuse with the target cell membrane and release their contents. The perforin inserts into the plasma membrane of the target cells forming pores that allow destruction of the target cell both by osmotic lysis and by allowing entry of apoptosis-inducing granzymes. The CD107a is then transiently expressed on the effector cell surface during this degranulation process. The other cells that express CD107a are degranulated platelets, PHA-activated T cells, TNF-a activated endothelium and FMLP activated neutrophils.

MUNC 13-4 is involved in vesicle priming and has been described as a positive regulator of secretory lysosome exocytosis.² It has been observed that mutations in the gene regulating MUNC 13-4 results in defective cytolytic granule exocytosis, despite polarization of the lytic granules and docking with the plasma membrane.² Our studies indicate that lack of CD107a after presentation with a target cell results in statistically significant differences in patients with MUNC 13-4 mutations as compared to other patients with HLH and no MUNC 13-4 mutations.

In this assay, mononuclear cells from peripheral blood are stimulated with K562, target cells, which induces degranulation. Tagged anti-CD107a is present with the cells during the stimulation period. After the six-hour incubation at 37o, the cells are surface stained with markers to allow the analysis of just NK cells. If the NK cells have been degranulated, the CD107a will be expressed on the effector surface and will be detected by flow cytometry.

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• Issue 4: News From the Genetics Lab
• Issue 5: Down the Pipeline

X-SCID is characterized by profound impairment of both cellular and humoral immunity due to the absence or markedly diminished number of T cells and NK cells together with abnormal B cell function. This lethal disorder of the immune system is caused by defects in the common gamma chain (gc) gene, a subunit of the IL-receptors of IL-2, IL-4, IL-7, IL-9 and IL-15, all of which are necessary for lymphocyte development and function. The gamma chain receptor is a glycoprotein expressed by most peripheral blood T and B lymphocytes, NK cells, monocytes and granulocytes. Interleukin-7 (IL-7) was originally discovered to be a pre-B cell growth factor. Soon thereafter, a broader role for IL-7 in leukocyte development and function began to be identified. IL-7 has now been shown to be a critical cytokine for normal T and B lymphopoiesis and a mobilizer of pluripotent stem cells and myeloid progenitors. Gene mutations of a survival signal through the IL7 R alpha chain has been shown to be important for T cell development and contributing to the SCID phenotype. CD127 is the alpha chain of the IL-7 receptor. It is a 75-80 kDa transmembrane molecule that associates with CD132 (IL-2R gamma chain) to form the high affinity IL-7R. The expression of CD132 and CD127 can be easily demonstrated on normal peripheral blood lymphocytes by direct immunofluorescent staining and flow cytometric analysis to assist in the rapid diagnosis of these two forms of SCID phenotypes and to monitor post bone marrow transplant lineage specific engraftment.

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• Issue 4: Patient Vignette

Cytotoxic T lymphocytes (CTL) are a subset of lymphocytes that have the ability to lyse target cells bearing specific antigens. To assess the functionality of CTL’s, target cells previously primed to contain radioactive chromium within the cell membrane, are incubated with patient or control lymphocytes. Lysis of the target cells by the CTL’s is measured by the amount of chromium released into the supernatant.

Various diseases show disturbances in the proportion of circulating Th1 and Th2 CD4 cells, and in overall cytokine production by T-cells. By observing intracellular IFN-g, the Th1 CD4 cells can be enumerated and by looking at IL-4, the Th2 CD4 cells can be quantitated. If overproduction of cytokines is suspected, IFN-g, IL4 and TNF-a can be analyzed in CD4, CD8, NKT and NK cells.

Cytokine production is stimulated by incubating whole blood or mononuclear cells with Phorbol 12-Myristate 13 Acetate and Ionomycin. Activation is carried out in the presence of Brefeldin A, which inhibits intracellular transport, causing all cytokines produced during the activation to be retained inside the cell. The activated cells are stained with surface monoclonal antibodies for phenotyping, then fixed, permeabilized and stained with antibodies to the cytokines IFN-g, TNF-a and IL4. The cells are then analyzed by flow cytometry.

Cytokines are small secreted proteins that mediate and regulate immunity, inflammation and hematopoiesis. Cytokines are produced de novo in response to an immune stimulus. They generally (although not always) act over short distances and short time spans and at very low concentrations. They act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter its behavior. Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation and secretion of effector molecules.

It is common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types (pleiotropy). Cytokines are redundant in their activity, meaning similar functions can be stimulated by different cytokines. Cytokines are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. Cytokines can also act synergistically (two or more cytokines acting together) or antagonistically (cytokines causing opposing activities). Cytokine short half life, low plasma concentrations, pleiotropy, and redundancy all complicate their isolation and characterization.

Cytokine levels in plasma and other biological fluids are now recognized as potential and useful markers of ongoing clinical disorders. The measurement of the levels of cytokines and / or soluble markers of immune activation can provide reliable information regarding the disease diagnosis, disease stage, prognosis, and the evaluation of therapy.

This assay uses spectrally encoded antibody-conjugated beads to measure the following cytokines: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-g, TNF-a, and GM-CSF.

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• Issue 1: Down the Pipeline

FoxP3, also known as FORKHEAD BOX P3, SCURFIN, and JM2, is a 49-55 kDa protein and a member of the forkhead or winged helix family of transcription factors. It is constitutively expressed in regulatory T cells (Treg), a subset of CD4+ T cells that are responsible for suppressing a variety of physiological and pathological immune responses, primarily through the elimination of self-reactive lymphocytes. Treg cells can be identified by their high expression of CD25, the IL-2 receptor alpha chain. Defects of Treg cells are thought to play a role in autoimmune and inflammatory diseases. Mutations of the FoxP3 gene have been described in patients with Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome (IPEX). This assay uses flow cytometry to determine the proportion and intensity of intracellular FoxP3 protein expression in the subset of CD4+ T cells that are CD25 bright and CD127 negative. The PCH101 antibody reacts with the amino terminus of human foxp3 protein.

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• Issue 2: Down the Pipeline

Flow cytometric measures of lymphocyte activation has clinical application in the following areas: evaluation of cellular immune response, assessment of the activity of autoimmune disease processes, and monitoring of pre- and post-transplant for early signs of graft rejection, to name a few. Detection of activated T-cells is perceived as providing a more precise indication of the dynamics of immune function than could be obtained simply from counting absolute or relative numbers of different lymphocyte types in the blood. Quiescent lymphocytes, i.e. not motivated by an antigenic stimulus, express little if any surface appearance of the activation antigens (CD69, CD25, CD154, CD134, CD95, CD71, HLA-DR) and only upon stimulation will these antigens up-regulate or over-express on the surface of lymphocytes.

Lymphocytes are a subpopulation of white blood cells, bone marrow-derived cells that can be differentiated into subsets of T cells, B cells, and Natural Killer cells by expression of distinguishing cell surface molecules. T cells can be further differentiated into CD4 and CD8 cells which each have unique roles in the immune response. Defects in one or more lymphocyte lineages can indicate the presence of an immune deficiency. This assay uses MultiSet antibodies and software to detect the following lymphocyte subsets: T cells (CD3, CD4 and CD8); B cells (CD19); and NK cells (CD16 and CD56). If results of a CBC / Diff are available, absolute numbers of these subsets can be determined.

The bare lymphocyte syndrome (BLS) is a rare immunodeficiency disorder caused by or associated with the failure of expression of cell surface antigens encoded for by the major histocompatibility complex (MHC).   It is now apparent that this syndrome is heterogeneous with regards to defective cell surface antigen expression.   In some patients with this disorder, there is defective expression of only class l MHC antigens encoded for by the HLA-A, B and C genetic loci, while in other patients class ll MHC antigens (HLA-Dr, DQ and DP) are not expressed.  Patients who fail to express both class l and class ll MHC antigens have also been identified.  Clinically, this syndrome is manifested as a combined immunodeficiency presenting early in life, and affected individuals are susceptible to a number of severe and/or opportunistic infections by a wide variety of pathogens.  MHC Class I and Class II epitope expression on peripheral blood lymphocytes and monocytes are performed by multiparameter flow cytometry.

The complex cascade of events that comprise the cellular portion of the immune response include the proliferation of lymphocytes in response to upstream events (activation, Ca++ flux, etc.) This assay tests the capacity of lymphocytes to proliferate in response to exposure to mitosis-inducing agents (mitogens). Specifically, peripheral blood mononuclear cells are exposed to phytohemagglutinin (PHA), concanavalin A (ConA), and pokeweed mitogen (PWM), and cultured for several days. An excess of radio-labeled thymidine is made available to the cells during the later part of the culture period. As the stimulated cells enter the S phase of the cell cycle, they synthesize DNA, incorporating the radio-labeled thymidine which is then quantitated using a microplate scintillation counter. Results are expressed as counts per minute (cpm).

Neopterin biosynthesis is closely associated with cellular immune system activation. Increased levels of neopterin have been measured in patients with viral infections, suggesting that the increased concentrations may originate from the patient’s immune response against the virally infected cells. Antigenic stimulation of human peripheral blood mononuclear cells has been shown to lead to neopterin release in culture medium and human macrophages produce neopterin in vitro when stimulated with interferon gamma. Therefore, determining neopterin levels in human body fluids offers a beneficial and innovative tool in monitoring diseases associated with cell-mediated immunity activation.

Principle of the assay: The assay is a solid phase competitive ELISA. The intensity of color that develops is inversely proportional to the amount of antigen in the sample. Final concentration is determined directly using the standard curve. 

Recruitment of neutrophils from the blood stream to extravascular sites of inflammation is a critical event in host defense against bacterial infection and in the repair of tissue damage. In response to extravascular stimuli such as bacterial-derived chemoattractants, or endogenous lipid and peptide mediators generated at sites of infection or tissue damage, leukocytes are first observed to “roll” along the vessel wall adjacent to the site of inflammation. Some of the rolling cells subsequently adhere firmly or “stick,” then diapedese between endothelial cells and migrate through the subendothelial matrix to the site of inflammation. CD11b / CD18 and its endothelial cell ligands including intracellular adhesion molecule-1 (ICAM-1, CD54) are necessary for firm neutrophil adherence and transendothelial migration. Leukocyte adhesion deficiency syndrome type 1 (LAD-1) results from a congenital deficiency of the leukocyte b₂ intergrin receptor complex CD11 / CD18 on the cell surface.

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• Issue 5: Patient Vignette 

Leukocytes have the ability to respond to chemotactic stimuli that are attractant factors derived from several sources, the chief being the complement proteins of plasma. Chemotaxis of white blood cells (leukotaxis) is the unidirectional migratory response of attraction of the cells to an increasing chemical gradient. Leukotaxis assays are performed to determine the extent to which leukocytes can respond to chemotactic stimuli. Leukotaxis disorders are found most commonly in patients who present histories of chronic, recurrent bacterial infections. Disorders may also be due to interval abnormalities in leukocytes, or they may be caused by defects in the plasma substrate system (complement) that generate chemoattractants.

In Chronic Granulomatous Disease (CGD), microbial killing is defective due to a mutation in one of four known components of the NADPH oxidase system. This prevents the generation of an oxidative burst and results in an inability to generate toxic oxygen radicals, which seriously compromises the patient’s ability to kill phagocytosed microorganisms.

A flow cytometric whole blood assay determines the ability of polymorphonuclear (PMNs) cells to produce an oxidative burst. This is accomplished by indirectly measuring the increase in fluorescence generated by the oxidation (by O2-) of a laser sensitive dye, dihydrorhodamine (DHR) 123. DHR is an uncharged and nonfluorescent reduction product of the mitochondrion-selective dye rhodamine 123. When incubated with PMNs, DHR diffuses into the cells and localizes in the mitochondria. When the cells are stimulated with phorbol-12-myristate-13 acetate (PMA) the DHR is oxidized to the highly fluorescent rhodamine 123. The level of fluorescence is proportional to the amount of dye oxidized. CGD patients’ PMNs remain non-fluorescent while the PMNs obtained from female carriers with the X-linked form of GCD have two populations of cells; one fluorescent (normal), and one nonfluorescent (abnormal active X chromosome).

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• Issue 4: Patient Vignette

Neutrophils are expert phagocytes by virtue of their expression of a number of specialized receptors. The opsonic attachment of microorganisms to neutrophils occurs via the Fc-gamma receptors as well as receptors for the complement-derived opsonins C3b and iC3b. After opsonized microbes attach to the neutrophil membrane via these receptors, the cell surrounds it forming a phagosome. Now isolated to a small microenvironment, the organism becomes susceptible to the highly concentrated nonoxidative and oxidative products that the neutrophil spills into the phagosome as it forms a phagolysosome.

The ability of neutrophils to phagocytize and kill bacteria is assessed by a phagocytic microbicidal assay using acridine orange (AO). AO has the property to bind to nucleic acids with different affinities. This binding to DNA produces a green fluorescence while the binding to denatured DNA or RNA produces a red fluorescence. Based on the definition of bacterial death as denatured DNA, the property of AO is utilized to study bacterial-cell relationships.

Natural Killer (NK) cells are a subset of cytotoxic lymphocytes that have the ability to induce cell death in tumor cells or in virally infected cells. NK cells play a crucial role in the homeostasis of the immune system. They are typically characterized by the expression of CD16 and CD56 on their cell surface. This assay makes use of the fact that NK cells will lyse the human erthroleukemia cell line, K562, in vitro. K562 cells are loaded with radioactive chromium and incubated with various ratios of a mononuclear cell preparation. Lysis of the K562 targets by NK cells contained in the cell preparation is measured by the amount of chromium released into the supernatant.

Perforin is a 70kD protein with cytolytic functions. It is expressed in the cytoplasmic granules of cytotoxic T lymphocytes (CTLs) and Natural Killer (NK) cells. Cytolytic cells release the contents of their granules, including perforin in response to recognition of their target cell. In the presence of calcium, perforin forms transmembrane pores in the membrane of the target cell, facilitating cell death. Mutations in the perforin gene have been associated with primary Hemophagocytic Lymphohistiocytosis (HLH). The absence or otherwise atypical staining pattern of cytoplasmic perforin therefore can indicate a disease state. In this assay, peripheral blood is stained with both surface and intracellular monoclonal antibodies and analyzed using four-color flow cytometry.

Granzyme B is a serine protease which is involved in apoptotic cell death. Granzyme B, along with other enzymes, is contained in cytoplasmic lytic granules and is released primarily by Cytotoxic T Lymphocytes and Natural Killer cells. When Granzyme B is released from cytotoxic cells, it enters the target cell through the non-classical receptor-mediated entry pathway. Intracellularly it is enabled by perforin to convert to cytosolic Granzyme B, which then activates Executioner Caspase causing cell death. If perforin is not present there is a break in the apoptosis pathway and cell death does not occur. Levels of Granzyme B, when compared with perforin levels, can be indicative of differing clinical states.

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• Issue 2: Patient Vignette
• Issue 4: Patient Vignette
• Issue 4: News From the Genetics Lab

Phosphorylation is a mechanism of cell signaling involved in many functional pathways including: differentiation, activation, proliferation and apoptosis. Detection of the phosphorylation of STAT5 Tyrosine is important in evaluating the IL-2 signal transduction pathway. Patients having decreased phosphorylation of STAT5 may have pathways that are non-functional or compromised. Patients with Severe Combined Immune Deficiency (SCID) attributed to either JAK3 deficiency or defects of the common gamma chain shared by many cytokine receptors, namely IL-2, will have decreased phosphorylation of STAT5.

Principle of the assay: Following stimulation of whole blood with IL-2, phosphorylation of STAT5 is measured in CD4 T cells using monoclonal antibodies and flow cytometry. The amount of phosphorylation of STAT5 through the IL-2 pathway is age dependent in healthy individuals, with younger children having less pSTAT5 than older children and adults.

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• Issue 6: New Assays

SLAM-Associated Protein (SAP) is a small lymphocyte-specific signaling molecule that is defective or absent in patients with X-linked LymphoProliferative Disease (XLP). Mutations in the SAP gene (SH2D1A) have been described in a majority of patients with the clinical syndrome of XLP. XLP is a primary immunodeficiency, which is characterized by an extreme susceptibility to EBV and should always be considered in males with EBV associated HLH. Half of XLP patients can have a fatal outcome with EBV infection because of explosive activation and proliferation of lymphocytes in many organs, which leads to fulminant hepatitis and bone marrow failure with hemophagoctosis. This assay is a rapid screening test for the presence of the SAP protein.

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• Issue 1: Patient Vignette
• Issue 4: News From the Genetics Lab

The hemoglobin-haptoglobin scavenger receptor (CD163 / HbSR) is a monocyte / macrophage-restricted transmembrane protein of the scavenger receptor cysteine-rich family. Antigen expression is related to monocyte / macrophage differentiation, with weak expression on peripheral blood monocytes and abundant expression on the majority of tissue macrophages. Monocytes and macrophages play a central role in host response to infection. They synthesize and secrete a variety of inflammatory mediators. It has become increasingly clear that the pro-inflammatory process is balanced by associated anti-inflammatory mechanisms that result in monocyte deactivation, characterized by a decrease in HLA-DR expression and the release of anti-inflammatory cytokines such as IL-10.

It has been shown that the extracellular portion of CD163 is shed from the cell surface in the form of soluble CD163 when the cells are appropriately stimulated. Serum levels of sCD163 have been shown to be associated with levels of CRP. It has also been shown that sCD163 acts as a cytokine with modulatory effects on other cells. sCD163 may be a valuable marker in diseases with macrophage / monocyte involvement, particularly in infectious, inflammatory and myeloproliferative diseases. This assay uses a non-competitive sandwich ELISA technique to measure soluble CD163 in plasma.

The interleukin-2 (IL-2) receptor complex is a trimer, in which all three chains are in contact with the ligand. The alpha subunit of this complex, IL-2R (also known at Tac antigen and as CD25) is a 55 kDa transmembrane glycoprotein with only 13 amino acids of 351 located on the cytoplasmic side of the membrane.A soluble form of IL-2R appears in serum and plasma, concomitant with its increased expression on cells.Increased levels of the soluble IL-2R in biological fluids correlate with activation of T and / or B cells. Results of a number of studies suggest a correlation of levels of IL-2R in serum with disease activity in autoimmune and infectious disorders as well as in transplantation rejection. Markedly increased IL-2sR levels have been associated with hematologic malignancies. Levels of IL-2R correlate with tumor burden and response to therapy in numerous malignancies. IL-2R levels can be used to predict the long-term prognosis in non-Hodgkin’s lymphoma patients and to assess the status of patients with HIV and acquired immunodeficiency.

Principle of the assay: The IMMULITE 1000 sIL2R is a solid-phase, two-site chemiluminescent immunometric assay. Test units containing a bead coated with a murine monoclonal anti-IL2R antibody have sample added by the machine. Liquid reagent containing alkaline phosphatase conjugated to rabbit polyclonal anti-IL2R antibody is then added. The test unit incubates a defined time period by following a route on the Incubation Carousel. The machine washes the beads twice with water and Chemiluminescent Substrate is added. The substrate contains Adamantlyl Dioxetane Phosphate. The alkaline phosphatase portion of the antibody (which has sandwiched the patient’s sIL2R on the bead) will cleave the dioxetane portion and produce light. The machine’s Luminometer reads the light produced, converts it to CPS (counts per second) and, using the standard curve recorded in the machine, converts to U / ml.

The T cell receptor (TCR) is a molecule on the surface of T lymphocytes and is the primary receptor responsible for recognizing MHC bound antigens on antigen presenting cells. It is a heterodimer typically comprised of an alpha chain and a beta chain (>90 percent of T cells), but occasionally comprised of a gamma chain and a delta chain (<10 percent of T cells). TCR gamma / delta cells arise early in development and although their role in the immune response is not fully understood, they may play a regulatory role in bacterial infections. Increased circulating TCR gamma / delta cells may indicate certain disease states. This test uses monoclonal antibodies against alpha-beta and gamma-delta antigens and flow cytometry to quantitate the relative proportions of each subset in the peripheral blood.

TCR is a molecular complex that comprises two units. A recognition unit composed of either ab or gd heterodimers, located on the cell surface and a transducing unit, the CD3 complex, common to ab and gd heterodimers, that triggers the T cell when the recognition unit is engaged with the antigen. There are four TCR gene loci (a, b, g and d). Each locus is composed of several V (variable) segments, a short D (diversity) segment (b and d only), a short J (joining) segment and one or two C (constant) region exons. During T cell ontogeny, a T cell “chooses” at random one V, D, (if any), J and C segment. The D and J regions are short but very diverse due to an additional process that adds or deletes nucleotides at random, when the V, D, and J segments are linked together. The consequence of this process is that a given T cell displays a single and unique combination on its cell surface. There are in fact 65 Vb segments in the b locus that are grouped into 25 subfamilies (22 functional families).

This test is designed to quantitate 24 antigens of the TCR Vb repertoire of human T lymphocytes in eight test tubes using a flow cytometric procedure (immuno-stain, lyse, wash and fix). The assay is achieved by combining three TCR Vb specific antibodies in a single test but with only two colors. One TCR Vb antibody is conjugated to FITC, another to PE, and the third to both FITC and PE. In this way, the third Vb stained population shows up in quadrant 2 in an FL1vs FL2 histogram (see figure 1). Results will distinguish polyclonal from oligoclonal or monoclonal T cell proliferation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by the triad of thrombocytopenia, eczema and immune deficiency. The gene responsible for WAS is located on the short arm of the X chromosome at Xp11.22. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Detection of WASP is possible by permeabilizing peripheral blood lymphocytes, monocytes and neutrophils and staining intracellularly with mouse anti-human WASP antibody then analyzing on a flow cytometer. The absence or otherwise atypical staining pattern of intracellular WASP therefore can indicate the disease, carrier state, or mixed chimerism after BMT.

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• Issue 4: Patient Vignette
• Issue 6: Pattern Recognition

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by the triad of thrombocytopenia, eczema, and immune deficiency.  The gene responsible for WAS is located on the short arm of the X chromosome at Xp11.22. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Detection of WASP is possible by permeablizing peripheral blood leukocytes and staining intracellularly with mouse anti-human WASP antibody then analyzing on a flow cytometer.  The absence or otherwise atypical staining pattern of intracellular WASP therefore can indicate the disease, carrier state, or mixed chimerism after Bone Marrow Transplantation (BMT).  Success of engraftment after BMT can be evaluated by performing multi-color flow cytometric analysis using monoclonal antibodies to cell surface antigens to characterize the hematopoetic cell populations of interest before the intracellular staining for WASP.

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• Issue 9: Patient Vignette
  

Deficiency of X-linked Inhibitor of Apoptosis (XIAP), caused by mutations in the BIRC4 gene, is the second most common cause of X-Linked Lymphoproliferative Syndrome (XLP). The most common cause is deficiency of SLAM Associated Protein (SAP) caused by mutations in the SH2D1a gene. XLP due to BIRC4 mutation is associated with the development of HLH and other lymphoproliferative disorders, sometimes in association with EBV.

XIAP is an intracellular protein expressed in many tissues. In order to rapidly screen patients for this disorder, patient and normal sample lymphocytes are fixed, permeabilized and stained with a mouse monoclonal antibody against XIAP, followed by secondary PE-conjugated anti-mouse antibody staining. Following intracellular staining, residual PE conjugated anti-mouse antibody is blocked, followed by lymphocyte surface marker staining. Samples are analyzed by five-color flow cytometry, and XIAP expression is measured in the CD4+ and CD8+ T cells, NK cells, and B cells.

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• Issue 7: New Assay Development

Children with Severe Combined Immunodeficiency (SCID) are prone to repeated and persistent infections that can be very serious or life-threatening. Infants with SCID typically experience pneumonia, chronic diarrhea, and widespread skin rashes. If not treated in a way that restores immune function, children with SCID usually live only a year or two. ZAP-70related SCID is one of several forms of severe combined immunodeficiency, a group of disorders with several genetic causes.ZAP-70 is an intracellular tyrosine kinase that is recruited in the CD3 T cell receptor (TCR) complex and is required for T cell activation following TCR engagement.  ZAP-70 deficiency is a rare autosomal recessive form of SCID characterized by a lack of CD8+ T cells and presence of circulating CD4+ T cells. Most individuals with ZAP-70related SCID are diagnosed in the first 6 months of life. ZAP-70SCID is a rare disorder where only about 15 affected individuals have been identified.

A permeabilization reagent renders intracellular antigens accessible and allows cytoplasmic and nuclear immunophenotyping of leukocytes.  It creates apertures in the membrane without affecting the gross morphology of the cell and therefore preserves its flow cytometric light scattering characteristics.  This permeabilization reagent allows one to perform simultaneous surface and intracellular antigen staining.  A “Fix and Perm Cell Permeabilization Kit” standardizes the procedure that consists of, first fixing cells with reagent A, and second, permeabilizing the leukocytes and lysing red cells using reagent B.