Pharmacogenomics of Heparin-Induced Thrombocytopenia

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.

Inclusion Criteria:

- 18 years of age or older

- Ability to give informed consent

- Have undergone testing for heparin/PF4 antibody with or without SRA testing for HIT

- Have received treatment with unfractionated heparin or low molecular weight heparin
(enoxaparin, dalteparin, tinzaparin) prior to antibody or SRA testing

Exclusion Criteria:

- Less than 18 years of age

- Pregnancy

- Hemoglobin less than 9 mg/dL (for larger 5 ounce blood draw)

- Known Human Immunodeficiency Virus (HIV) infection

- Inability to give informed consent