Method of detection of platelet-activating antibodies that cause heparin-induced thrombocytopenia/thrombosis

Inventors

Aster, Richard H. • BOUGIE, Daniel W. • JONES, Curtis Gerald • PADMANABHAN, Anand

Assignees

Versiti Blood Research Institute Foundation Inc • Blood Center Research Foundation Inc

Abstract

The present invention provides a method of detecting platelet activation in a patient, the method comprising the steps of a) obtaining a blood sample from a patient suspected of having heparin-induced thrombocytopenia (HIT); b) incubating an effective amount of platelet factor 4 (PF4) with a sample of platelets to yield a sample of PF4-treated platelets; c) contacting the patient blood sample with the PF4-treated platelets; and d) measuring the extent of platelet activation, wherein an increase in platelet activation compared with results obtained using a normal blood sample is indicative of the patient having HIT.

Core Innovation

A method is provided for detecting platelet-activating pathogenic HIT antibodies as opposed to non-activating non-pathogenic HIT antibodies in a patient suspected of having heparin-induced thrombocytopenia (HIT). The method obtains a patient sample selected from whole blood, plasma, and serum, incubates platelet factor 4 (PF4) with isolated normal platelets to yield PF4-treated platelets, and contacts the patient sample with the PF4-treated platelets to create a combined sample. The method measures activation of the PF4-treated platelets in the combined sample, where an increase in platelet activation relative to platelet activation when a normal whole blood, plasma, or serum sample is substituted is indicative of platelet-activating pathogenic HIT antibodies.

In one embodiment, PF4 is incubated with isolated normal platelets in the absence of heparin to yield PF4-treated platelets, and the assay measures the amount of antibodies bound to platelets in the combined sample. Increases in platelet-bound antibodies in the combined sample, relative to the amount of antibodies bound when a normal whole blood, plasma, or serum sample is substituted, indicate that the patient has platelet-activating pathogenic HIT antibodies. The method may specify that the PF4-incubated platelets are isolated normal platelets or washed normal donor platelets, and may further limit that the patient has received heparin.

The disclosed assay framework is also used to detect platelet-activating serotonin release assay positive (SRA(+)) HIT antibodies as opposed to SRA(−) HIT antibodies in a patient who has received heparin. After obtaining a patient sample from whole blood, plasma, or serum, PF4-treated platelets are generated by incubating PF4 with isolated normal platelets, the patient sample is contacted with the PF4-treated platelets to create a combined sample, and activation of the PF4-treated platelets is measured. An increase in platelet activation in the combined sample, compared to platelet activation obtained when a normal whole blood, plasma, or serum sample is substituted, is indicative that the patient has platelet-activating SRA(+) HIT antibodies.

Claims Coverage

The independent claims are 1, 9, 12, and 15. Across these independent claims, the core inventive features focus on generating PF4-treated platelets from isolated normal platelets, contacting the PF4-treated platelets with a patient sample, and differentiating pathogenic platelet-activating HIT antibodies, including SRA(+) antibodies, by measuring either platelet activation or platelet-bound antibody amount relative to a substituted normal sample. A total of four independent claims define these core elements, with additional limitations specifying assay context and distinct readouts.

Across the independent claims, the patent covers methods that form PF4-treated platelets with isolated normal platelets, expose them to a patient sample, and use a relative comparison to a substituted normal whole blood, plasma, or serum sample to indicate platelet-activating pathogenic HIT antibodies. Claim coverage includes activation-based readouts and an alternative readout of measuring the amount of antibodies bound to platelets, with contextual limitations such as PF4 incubation in the absence of heparin and patient heparin receipt.

Stated Advantages

Documented Applications