New Assays For Hit Based On Heparindependent Antigens

The present understanding of how heparin-dependent antibodies contribute to the development of HIT allowed us to develop new assays for measuring these antibodies, by mimicking closely the conditions thought to occur in vivo. For this approach, functionally available heparin is coated onto a solid surface such as an enzyme-immunoassay (EIA) plate or any other surface. This can be achieved by different means: coating protamine sulfate in the presence of a large excess of heparin; coating streptavidin/biotinylated heparin; or coating heparin covalently bound to a carrier protein (such as albumin) or a polymer. The patient plasma or serum is incubated with this heparinized surface. If chemokines that exhibit heparin affinity are present, they bind to the coated heparin, exposing neoepi-topes, and thereby capturing heparin-dependent antibodies (Fig. 5). In addition, if antigenic heparin-protein complexes are present, they can also directly bind to heparin through the heparin-binding protein. Using an anti-IgG/A/M or an anti-IgG or a combination of anti-IgG, -IgA and -IgM peroxidase conjugates allows measurement of all the antibody isotypes (useful for screening for HIT) or only the IgG isotype (the preferred assay for confirming the diagnosis of HIT) or proceeding to a full isotyping of those antibodies (which remains a convenient tool for research studies). This approach is flexible, very sensitive, and highly specific for heparin-dependent antibodies. It allows identification of the antibody isotypes of clinical relevance.

An interesting improvement consists in supplementing the reaction milieu with platelet lysates or with lysates from leukocyte-platelet concentrates, or, when required, directly with PF4, IL-8, or any other high affinity heparin-binding protein. This provides PF4 or other chemokines in excess for forming the heparin-dependent antigenic target for HIT antibodies. Using platelet lysates, the assay correlates fully with the conventional EIA for measuring anti-PF4-H antibodies of fjfjj ;jufh iCV f V r iĆ¼-^EIA plate

Protamine SO,

SA + UFH-biotin

Specimen

EIA plate EIA plate

UJjJ

Platelet or Leukocyte Lysates

Conjugate TMB

'jEIA plate

EIA plate

Color development

Reading A450 nm

FIGURE 5 (See color insert) Assay for testing heparin-dependent antibodies, associated with HIT, by their binding to functionally available heparin through the heparin cofactor antigen (usually PF4); heparin is coated in a large excess as a complex with aprotinin or biotinylated and reacted with coated streptavidin. Abbreviations: EIA, enzyme-immunoassay; IL-8, interleukin-8; PF4, platelet factor 4; SA, streptavidin; TMB, tetramethyl benzidine; UFH, unfractionated heparin.

IgG isotype using plasmas from patients with clinically-suspected HIT (r2 = 0.89), although some IgG positive patients were negative with the conventional PF4-H EIA, possibly because additional antigenic complexes are only measured with the new assay. When comparing the assay performed with or without platelet lysates, two groups of patients with HIT are identified: those for whom the antibody binding is totally dependent on the presence of platelet lysates and those in whom antibodies in the dilute patient plasma (or serum) bind "directly" to functionally active heparin. At this time, no clinical significance has yet been identified regarding these differences.

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