CXCL9, also known as monokine induced by gamma interferon (MIG), is a member of the CXC chemokine subfamily. CXC chemokines are known to recruit activated lymphocytes and hinder angiogenesis via CXCR3. CXCR3 is a transmembrane protein expressed on activated Th1 lymphocytes. It has been observed in monocytes, endothelial cells, eosinophils, malignant B cells, melanoma cells, CD34+ hematopoietic progenitors and neurons. Expression of CXCL9 is induced by the Th1 cytokine, IFN-gamma. This is dramatically enhanced by the addition of TNF-alpha. CXCL9 production has been proven to be a sensitive measure of antigen-specific IFN-gamma production. CXCL9 has been implicated in pathologies characterized by the accumulation of activated Th1 lymphocytes. These include acute allograft rejection, glomerulonephritis, autoimmunity, rheumatoid arthritis, atherosclerosis, psoriasis and allergic contact dermatitis.
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 erythroleukemia 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.
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.
We use a solid-phase, two-site chemiluminescent immunometric assay, and results are reported in units/ml (U/ml).
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.
This flow cytometry assay is intended to be used as a screening test. Screening tests are neither 100 percent sensitive nor specific, and a normal result should not preclude molecular sequencing if a patient’s clinical presentation suggests that the probability of a diagnosis is high.
Additional information regarding the accuracy of this flow cytometry screening assay is available.[1]
- Abdalgani, M; Filipovich, AH; Choo, S; Zhang, K; Gifford, CE; Villanueva, J; Bleesing, JJ; Marsh, RA. Accuracy of flow cytometric perforin screening for detecting patients with FHL due to PRF1 mutations. Blood 126(15)
Also known as LAMP-1 (lysosome-associated membrane protein – 1), CD107a 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. 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. 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 a six-hour incubation, 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.
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.
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.
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 hemophagocytosis. This assay is a rapid flow cytometry screening test for the presence of the SAP protein.
This flow cytometry assay is intended to be used as a screening test. Screening tests are neither 100 percent sensitive nor specific, and a normal result should not preclude molecular sequencing if a patient’s clinical presentation suggests that the probability of a diagnosis is high. Additional information regarding the accuracy of this flow cytometry screening assay is available.[1]
1. Gifford, CE; Weingartner, E; Villanueva, J; Johnson, J; Zhang, K; Filipovich AH; Bleesing, J; Marsh, RA. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations. Cytometry B Clin Cytom. v. 86 p. 263-271.
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. 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.
This flow cytometry assay is intended to be used as a screening test. Screening tests are neither 100 percent sensitive nor specific, and a normal result should not preclude molecular sequencing if a patient’s clinical presentation suggests that the probability of a diagnosis is high.
Evaluation of degranulation via measurement of CD107a following various stimuli can be used as a diagnostic screening test for the causes of familial hemophagocytic lymphohistiocytosis due to mutations in Munc 13-4, STX11 and STXBP2. A large prospective study revealed that over 95 percent of patients with these forms of HLH can be detected via screening CD107a assays utilizing measurement of CD107a on resting NK cells following exposure to the K562 cell line (Bryceson et al, 2012). We have since adopted this method as our CD107a Mobilization (NK Cell Degranulation assay) and have confirmed it to be useful (unpublished data). However, assays are not always able to be interpreted and reported, due to the fact that patient samples sometimes have too few NK cells.
Because degranulation can also be measured in T cells, we have developed a CD107a degranulation assay utilizing T cells. In this assay, mononuclear cells from peripheral blood samples are exposed to P815 cells coated with anti-CD3 (the P815 cell line is a murine mastocytoma which readily binds the Fc portions of the anti-CD3 antibody), which stimulates degranulation. PE-conjugated anti-CD107a is present with the cells during the stimulation period. After the two-hour incubation at 37°C, the cells are surface stained with markers to allow the analysis of T cells. If the T cells have degranulated, the CD107a will be expressed on the effector cell surface and will be detected by flow cytometry.