Quantitative determination of platelet associated immunoglobulines

Simple method for the flow cytometric determination of platelet associated immunoglobulin for the differential diagnosis of

 

• Idiopathic Thrombocytopenic Purpura (ITP).
• High reliability through polyclonal anti-sera against human IgA, IgG, IgM.
• Improved sensitivty through use of RPE conjugates for detection of autoimmune-globulines.
• Standardized test procedure.

Antibodies and reagents für 25 tests. Test kit contains CD42a FITC conjugate, polyclonal anti-human Immunoglobulins, RPE conjugate; polyclonal anti-human IgA, IgG, IgM, RPE conjugates; polyclonal anti-human rabbit, RPE conjugate,negative control; goat serum and 10 x washing buffer. Evaluation by flow cytometry.

SUMMARY and EXPLANATION

THROMBOCYTEST^® immune allows the flow cytometric quantitation of platelet associated immunoglobulin. The test kit contains various polyclonal antibodies labelled with the fluorescence dye phycoerythrin (PE) as follows: antibodies against total human immunoglobulins, against human IgA, IgM und IgG and polyclonal antibodies against rabbit immunoglobulin as a negative control. The test kit also contains a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody which recognizes a platelet specific antigen and further reagents. The fluorescence 2 intensity values correspond to the amount of platelet associated immunoglobulin.

APPLICATIONS

THROMBOCYTEST^® immune is useful for the evaluation of thrombocytopenia (low platelet count). The main effect of this reduced platelet count is an increased risk of bleeding. There is a high risk of spontaneous bleeding once the platelet count drops below 5,000 to 20,000 platelets per µl. The quantitation of platelet associated immunoglobulin allows the differential diagnosis to distinguish the cause of thrombocytopenia. Thrombocytopenia may result from a failure of platelet production (myelosuppression caused by cancer, drugs such as chemotherapy agents, alcohol or viral infections) or an increased rate of platelet destruction. Increased destruction of platelets can occur due to two different general mechanisms (immunologic and nonimmunologic thrombocytopenia).

Idiopathic thrombocytopenic purpura (Morbus Werlhof, ITP) is the most common acquired thrombocytopenia. ITP is an autoimmune disorder characterized by antibodies directed against platelets. These antibodies bind to various structural platelet antigens and can cause destruction and removal of platelets.

The determination of autoantibodies against thrombocytes helps differentiate immune form nonimmune thrombocytoenia. The direct flow cytometric test allows the quantitation of platelet associated total immunoglobulin as well as the determination of the class (IgA, IgM or IgG) of immunoglobulin.

THROMBOCYTESTâ immune may also be useful for the evaluation of functional defects of thrombocytes. In some cases, the binding of antibodies to platelets may induce platelet dysfunction.

TEST PRINCIPLES

THROMBOCYTEST^® immune allows the flow cytometric quantitation of platelet associated immunoglobulin. The test kit contains various polyclonal antibodies labelled with the fluorescence dye phycoerythrin (PE) as follows: antibodies against total human immunoglobulins, against human IgA, IgM und IgG and polyclonal antibodies against rabbit immunoglobulin as a negative control. The test kit also contains a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody which recognizes a platelet specific antigen and further reagents.

Platelet rich plasma is isolated from EDTA whole blood and incubated over night at 2-8°C. The thrombocytes are stained with PE conjugated polyclonal antibodies against human immunoglobulins, human IgA, IgM and IgG and with a FITC conjugated monoclonal anti-CD42a antibody. Polyclonal antibodies against total rabbit immunoglobulins serve as negative control. The samples are incubated for 20 min on ice, washed and analysed by flow cytometry.

REAGENTS PROVIDED

• CD42a FITC Conjugate
• Polyclonal Anti-Human Immunoglobulins, RPE Conjugate
• Polyclonal Anti-Human IgA, RPE Conjugate
• Polyclonal Anti-Human IgG, RPE Conjugate
• Polyclonal Anti-Human IgM, RPE Conjugate
• Polyclonal Anti-Human Rabbit, RPE Conjugate”, negative control
• Goat Serum
• 10 x WASHING BUFFER