Table 3.
Functional and immunologic assays: end points and methodological principles
| Functional | ||
|---|---|---|
| Methodology | End point | |
| Serotonin release-based assays | ||
| 14C SRA | β counter | Detects % release of 14C labeled serotonin release from activated platelets |
| EIA SRA (type of functionalized immunoassay) | ELISA-based | Serotonin release from activated platelets |
| HPLC SRA | HPLC | Serotonin release from activated platelets |
| Flow cytometry–based assays | ||
| FCA-intraplatelet serotonin assay | Flow cytometry | Detecting loss of intraplatelet serotonin stores within activated platelets |
| FCA-membrane GP | Flow cytometry | Detecting increased surface expression of activation markers like P-selectin or anionic phospholipids |
| PMPGA | Flow cytometry | Detection of PMPs |
| Platelet aggregation detection–based assays | ||
| HIPA | Visual assessment | Visual detection of platelet aggregation |
| PAT | Using platelet aggregometer | Detecting change in light transmittance due to aggregation of activated platelets |
| HIMEA | Multiple-electrode aggregometry | Detecting change in impedance due to aggregation of activated platelets on electrodes |
| HIT-mimicking monoclonal antibody–based assays | ||
| 5B9, IgG1 antibody | — | Formation of a 5B9–PF4 complex |
| HemosIL HIT-Ab (PF4-H) assay using KKO, an IgG2bκ antibody | Latex particle–enhanced immunoturbidimetry | Inhibition of agglutination of KKO-coated latex beads to PF4–polyvinylsulfonate complexes in the presence of human anti-PF4/heparin antibodies |
| Miscellaneous | ||
| ATP-release assay | Lumiaggregometery | Detecting ATP release from activated platelets |
| TGA | Fluorometer | Detecting fluorescence or light emitted from generation of thrombin |
| FcγRIIa-proteolysis assay | Western blot/densitometry | Detecting proteolysis of FcγRIIa |
| LIA | Turbidimetry | Opposite to conventional immunoassays in that presence of HIT antibodies results in inhibition of platelet agglutination through competition with an HIT-mimicking monoclonal antibody |
| DT40-Luc, a cell-based functional assay | Luciferase assay luminometry | Detecting luciferase activity when human FcγRIIa-transfected chicken cell line coupled to a luciferase reporter is activated by HIT antibodies |
| Immunologic assays | ||
| Immunologic assay | Methodology | End point |
| Optical density-based assay | ||
| ELISA | OD | Semiquantitative test, assesses the intensity of color change by measuring OD, which is proportional to the concentration of bound antibodies |
| Visual assessment–based assay | ||
| PaGIA | Visual assessment | Visual assessment of agglutination |
| Particle-immunofiltration assay (PIFA) | Size-dependent filtration | Inability of matrixed microparticles (formed as a result of antigen–antibody interaction) to move through the filter, with resulting color change indicating a positive test |
| Lateral-flow immunoassay (STiC) | Immunochromatography/lateral flow | Based on capillary action, which induces a flow of the test sample (patient serum) along a solid phase (gold nanoparticles coated with an antiligand); visible line upon addition of buffer containing biotinylated human PF4–polyanion (PA) complex indicates a positive reaction |
| Miscellaneous | ||
| Chemiluminescent immunoassay (AcuStar HIT-IgG) | Detection of emitted light produced by the reaction utilizing H202 and catalyst | The tracer (isoluminol-labeled antibody) binds specifically to the PF4–antibody complex and subsequently emits light when an oxidizer and a catalyst are added, indicating positive result |
| Latex-agglutination assay | Agglutination/clumping of latex beads | Patient sample mixed with latex beads coated with a PF4 antigen; clumping of latex beads (agglutination) indicates the presence of HIT antibodies |
Abbreviations: ATP, adenosine triphosphate; 14C, carbon 14; EIA, enzyme immunoassay; DT40-Luc, DT40 luciferase; FCA, flow-cytometry assay; GP, glycoprotein; HPLC, high-pressure liquid chromatography; HIT, heparin-induced thrombocytopenia; HIMEA, heparin‐induced multielectrode aggregometry; HIPA, heparin-induced platelet activation; KKO, murine HIT–like monoclonal antibody; LIA, latex immunoturbidimetric assay; OD, optical density; PF4, platelet factor 4; PAT, platelet-aggregation assay; PMPGA, platelet microparticle–generation assay; PRP, platelet-rich plasma; SRA, serotonin-release assay; TGA, thrombin-generation assay; PaGIA, particle-gel immunoassay.