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Children's Cincinnati, #30 Plasma Cytokine Plasma
Test CodeNMCP017
Preferred Specimen
Sodium Heparin blood
Minimum Volume
5-10 mL
Instructions
"1. 5-10ml Sodium Heparin blood per test should be adequate for most patients unless they are lymphopenic. If you have volume constraints and/or an absolute lymphocyte count (ALC) please see the Customized Volume Sheet on our website (www.cchmc.org/DIL) or call for adjusted volume requirements for the following tests: Antigen or Mitogen Stimulation, CTL Function, NK Function or CD107a.
2. Results of a same day CBC/Diff must accompany the sample where indicated (used to calculate absolute cell counts).
3. CSF samples should be shipped at 2-8°C if they will be received within 48 hours, or frozen for delivery beyond 48 hours of collection.
4. EDTA plasma samples (BAFF, Plasma Cytokines, Neopterin, sCD163, SIL-2R) can be shipped unspun, ambient, for next day delivery. Spin/separate plasma from cells, store at –20°C and ship on dry ice for delivery beyond 24 hours of collection.
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2. Results of a same day CBC/Diff must accompany the sample where indicated (used to calculate absolute cell counts).
3. CSF samples should be shipped at 2-8°C if they will be received within 48 hours, or frozen for delivery beyond 48 hours of collection.
4. EDTA plasma samples (BAFF, Plasma Cytokines, Neopterin, sCD163, SIL-2R) can be shipped unspun, ambient, for next day delivery. Spin/separate plasma from cells, store at –20°C and ship on dry ice for delivery beyond 24 hours of collection.
"
Transport Temperature
room temp, Overnight shipping is strongly recommended
Methodology
ELISA
Setup Schedule
" TESTS MUST BE RECEIVED MONDAY – FRIDAY WITHIN 24 HOURS OF BEING DRAWN The laboratory operates Monday through Friday, 8:00 am to 5:30 pm (Eastern Standard Time).
We cannot accept deliveries on Saturdays/Sundays and certain holidays.
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We cannot accept deliveries on Saturdays/Sundays and certain holidays.
"
Clinical Significance
"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
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.
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). Cytokines’ short half-life, low plasma concentrations, pleiotropy and redundancy all complicate their isolation and characterization.
"
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
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.
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). Cytokines’ short half-life, low plasma concentrations, pleiotropy and redundancy all complicate their isolation and characterization.
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