Heparin-Induced Thrombocytopenia Functional Assay
- The HITAlert™ Kit is used for the detection of heparin complex specific antibodies, which are capable of activation of thrombocytes (platelets) and may lead to the development of immune heparin induced thrombocytopenia (HIT).
- FOR RESEARCH USE ONLY, NOT FOR DIAGNOSTIC USE.
- Detection of heparin complexes specific antibodies
- Reactivity independent of PF4
- Kit also detects IL-8/heparin and NAP-2/heparin complex antibodies
- Complete kit with ready-to-use components
- Functional Assay
- Non-radioactive method
- Standard equipment and methodology (flow cytometer)
- Rapid results (<2 hr)
- Patented method
Heparin-induced thrombocytopenia (HIT) is an acute, immune-mediated process that may result in life-threatening thrombosis. HIT is caused by platelet-activating, heparin-dependent antibodies, that form complexes of heparin together with either platelet factor-4 (PF-4), interleukin-8 (IL-8) or neutrophil-activating peptide 2 (NAP-2). Detection of these antibodies is an important laboratory function.
The antibodies are initially formed after 5 or more days when a subject has been on heparin therapy. An immune response to a heparin dose may be observed within minutes or hours if the subject has had previous exposure to heparin and antibodies are already circulating.
Researching HIT is difficult because its signs are non-specific and due to the above mentioned structure it is very important to look for the presence and function of the complex specific antibodies.
For detection of HIT, two different kinds of assays can be used, the immunological and functional assay. The functional assays reproduce the in-vivo pathophysiology. This is in contrast to the immune-detection antigen-based assays, which measure antibodies reactive to the heparin platelet-factor 4 (PF4) complexes. Pathogenic antibodies may also react with other heparin complexes, such as heparin-interleukin-8, or heparin-neutrophil activating peptide 2, thus, not being detected by those assays.
An immunological assay can be the Enzyme-Linked Immunosorbent Assay (ELISA) or the Gel Test (PaGIA), which is similar to ELISA. The disadvantage of these tests is the fact that they are non-functional assays, they give more than 10% false negative and a 20% disagreement with SRA.
A functional assay is the Serotonin-release assay (SRA), which is sensitive and specific, but non-feasible because of the use of radio isotypes. Another functional assay is the Aggregation Assay but it has a low sensitivity and a long time to perform.
For the HITAlert™ Kit donor platelets (PRP) are used, which are incubated in the presence of test serum and in the presence or absence of heparin. When pathogenic antibodies are present, the activation of the donor platelets is shown using a platelet activation marker. By incubating the samples with an antibody against platelets and the activation marker this reaction can be visualized using flow cytometry.
Figure: Results of HITAlert™ Kit: (A) dot plot of PE positive platelets, (B) negative subject, (C) positive subject.
Format: Functional based flow cytometry assay, each kit contains sufficient reagents to test 6 subjects
Use: To detect heparin-dependent platelet microparticle formation in research applications.
Manufactured by: IQ Products, The Netherlands
Reagent A Assay buffer 5 ml
Reagent B Heparin 150 µl
Reagent C Platelet Activator (Ca-Ionophore) 1 vial
Reagent D Staining buffer 20 ml
Reagent E Platelet marker (Monoclonal antibody) 200 µl
Reagent F Platelet Activation marker (Recombinant Protein) 200 µl
Reagent G Heparin 1000 U/ml 150 µl
2.2 ml PP vials used for the sample incubation 30
Laboratory material required but not included:
- Flow cytometer
- citrate blood collection tube, for instance Greiner Vacuette 454382
- Tubes fitting the flow cytometer
- 70% or 96% ethanol
- Shaker (for instance ELISA plate shaker or platelet agitator)
- Laboratory centrifuge
- Adjustable micropipettes and tips