Pharmacogenomics of Heparin-Induced Thrombocytopenia (PHIT)

Description

Inclusion Criteria: 18 years of age or older; Participants with heparin/PF4 antibody and SRA testing for HIT, including:

participants with negative heparin/PF4 antibodies and negative SRA (controls), participants with positive heparin/PF4 antibodies and negative SRA(seroconversion cases), participants with positive heparin/PF4 antibodies and positive SRA (HIT cases); Treatment with unfractionated heparin or low molecular weight heparin (enoxaparin, dalteparin, tinzaparin) within 7 days of blood draw Exclusion Criteria: Less than 18 years of age; Inability to give informed consent There is no randomization involved in this study. Participants will be enrolled in equal numbers from three categories: (1) participants without HIT testing negative for heparin/PF4 antibodies (controls); (2) participants without HIT testing positive for heparin/PF4 antibodies (seroconversion cases); (3) participants with HIT testing positive for both heparin/PF4 antibodies (HIT cases).

Recruitment will be facilitated through collaborators in the Coagulation Laboratory and through key study personnel in the Banner Univerisyt Medical Center - Tucson. Patients with positive and negative heparin/PF4 antibody and serotonin release assay results will be identified in the Coagulation Laboratory and the patient's provider will be approached about potential participation in research studies. If the potential participant is interested, they will be approached by study personnel for study description and possible consent. Alternatively, clinical collaborators who are consulted and participating directly in a patients care may contact a potential participant directly for willingness to participate in the study.

Patients consenting to the study will be asked to provide a one blood sample of approximately 150 milliliters. Peripheral blood mononuclear cells (PBMCs) will be isolated from this sample and stored at -80C before use in proposed experiments. Genomic DNA will also be isolated from the sample to perform HLA sequencing.

HIT will be confirmed with a functional serotonin release assay and HIT likelihood based on clinical course. Detailed data will be collected regarding participant's hospitalization, heparin dose and duration, platelet counts, surgical history, and co-morbidities. After consent of participants, a peripheral blood sample will be acquired, CD positive T cells sorted using flow cytometry, and genomic DNA isolated as previously described.

The TCR repertoire and the proportion of specific TCR variants for a particular sample will be determined using a combination of novel technologies available in the investigator's laboratory and core facilities. The TCR repertoire of a sample will be determined using next-generation sequencing technology. The new Adaptive Technologies ImmunoSEQ TCR kit is likely to be utilized as this kit has been optimized to obtain an unbiased quantitative profile of rearranged TCR alleles from a genomic DNA sample. Currently this kit is in the beta test phase and VANTAGE is one of the test sites.

TCR sequencing will be performed using multiplex PCR system to amplify rearranged TCRbeta DNA using primers specific to a functional TCR Vbeta segment as previously described. Genomic templates will be amplified and observed relative abundance of Vbeta 5.1 family TCR clonotypes will be inferred from sequence data using an expectation maximization algorithm. CDR3 motifs as defined by the international ImMunoGeneTics information system (IMGT) will be predicted based on DNA sequences. For every read, best sequence alignment against reference sequences will be computed and reads with low quality scores will be discarded. To exclude unspecific T cells due to sorting impurities and/or stimulation background, antigen-specific clones with frequencies below 1 percent will be neglected.

TCR alleles known to be important in specific DHS will be targeted using droplet digital PCR (ddPCR). The ddPCR is a new technology that provides absolute quantification (real-time PCR provides relative quantification) of target template based on oil-emulsion droplet PCR reactions. The investigator's laboratory has experience with this new technology and assays for a number of important TCR alleles have already been optimized on the ddPCR in the investigator's laboratory.

To minimize risk extra blood will be collected for this study at the same time as primary care labs are drawn whenever possible. Participants will be asked to provide 150ml of blood in order to test qualitative immune responses. The risks associated with participating in this study relate to uncommon complications of venipuncture and could include discomfort, bruising, infection, and/or blood clot formation. Patients will be monitored for safety and comfort by trained clinical personnel.

Subjects will be monitored during the course of the study for any adverse events. Serious adverse events will be reported to the IRB within 10 days of the PI's notification of the event. Non-serious adverse events or instances of noncompliance with the protocol will be reported at the time of continuing review. The trial will be monitored in an ongoing fashion. There is no DSMB nor are any interim analyses planned.

Details