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.