Patients and study design
This prospective observational study was multicentered and was conducted in Tohoku University Hospital (Sendai, Japan), Hokkaido University Hospital (Hokkaido, Japan), Japan Red Cross Maebashi Hospital (Gunma, Japan), Tokyo Saiseikai Central Hospital (Tokyo, Japan), and Kagawa University Hospital (Kagawa, Japan). The ethical committees of each of the hospitals approved the protocol of this study. All of this research and protocol were approved by Ethics Committee of Tohoku University Graduate School of Medicine (2018-1-156).
All patients gave written informed consent and the study was conducted in accordance with the Declaration of Helsinki.
We prospectively enrolled trauma patients from March 2018 to February 2019. The inclusion criteria were age ≥ 18 years and Injury Severity Score (ISS) ≥ 9. ISS was calculated based on the Abbreviated Injury Scale (AIS) 90, using radiology reports and clinical findings. Anatomical injury sites were divided into head and neck, face, chest, abdomen, pelvis and extremities, and skin. Trauma was classified into blunt, penetrating, or other. This study included patients who did not receive any heparin, as well as those who received it as only flushes and as treatment.
During hospitalization, clinical information including changes in the platelet count and timing of heparin administration were collected. The blood samples were drawn at day 0 (on admission), day 5 – 6, day 7 – 9, day 10 – 12, day 13 – 15, and day 30 or at the time of transfer to another department or hospital, whichever came first. If the patient was transferred or died within 30 days, the cases were treated as censoring events. The patients’ sera drawn at the above timing were shipped with dry ice to the reference laboratory (Division of Transfusion Medicine, National Cerebral and Cardiovascular Center) for the assessment of HIT antibodies and were stored at -70℃ until use.
Seroconversion and serological assays for HIT antibodies
Antigen immunoassay was performed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit to detect anti-PF4/heparin IgG antibodies (PF4-IgG, Genetic Testing Institute, Waukesha, WI, USA). The ELISA results were expressed as optical density (OD), and the cut-off value was set at 0.4 OD, as per the manufacturer’s instructions. We defined seroconversion as testing negatively on day 0 followed by a positive test afterwards. If the patient had already tested positive on day 0, then seroconversion was defined as an OD increase by more than 30% or by at least 0.4 OD, according to a previous study. (13)
The washed platelet activation assay was performed as described in detail elsewhere. (2,14) In brief, washed platelets, prepared from HIT antibody-sensitive healthy volunteers, were used. Platelet microparticles, activated by HIT antibodies in a heat-inactivated serum, were quantified by flow cytometry. Positivity was defined as washed platelet activation in the presence of a therapeutic concentration of UFH (0.1 U/mL) or low molecular weight heparin (0.3 U/mL), but not at very high concentrations (100 U/mL), in addition to activation inhibition by the addition of an anti-FcγRII antibody (IV.3, Stemcell Technologies, Vancouver, Canada). If the serum did not activate the washed platelets in the presence of a therapeutic concentration of UFH or low molecular weight heparin, but the control tested positive, then it was defined as negative.
Since the washed platelet activation assay is demanding and labor-intensive, (2) and HIT induced by antibodies with platelet-activating properties is exclusively caused by anti-PF4/heparin antibodies of IgG class, (15) we first performed the assay for the sample that tested ELISA positive and demonstrated the highest OD in the time-series samples of each patient. If the sample tested negative, we defined the patient as a non-seroconverter in the washed platelet activation assay. If the sample tested positive, then the washed platelet activation assay was performed for all time-series samples.
Seroconversion rate differences
To clarify the seroconversion rate difference of anti-PF4/heparin IgG in ELISA and HIT antibodies, assessed by washed platelet activation assay, patients were divided based on the trauma severity into mild (9 ≤ ISS ≤ 15), moderate (16 ≤ ISS ≤ 24), and severe injury groups (25 ≤ ISS), according to a previous study. (16)
In addition, we investigated the influence of heparin administration on seroconversion rates. For this purpose, patients were divided based on the purpose of heparin administration into no heparin (patients did not receive any heparin during the study period), heparin flush (patients only received heparin flushes for catheter patency), and heparin treatment groups (patients received heparin for anticoagulation during invasive treatments including surgery, catheter embolization, and thromboprophylaxis). Only UFH was used in this study.
Time to seroconversion and subsequent disappearance
We investigated the period of time (days) required to achieve seroconversion of anti-PF4/heparin IgG in ELISA or HIT antibodies, detected by the washed platelet activation assay, after the onset of trauma, and the subsequent disappearance rate on day 30.
Continuous variables are presented as medians and quartiles, and categorical variables as frequencies and percentages. The Fisher’s exact test was used to compare categorical data among the groups, whereas numerical data were compared using the Kruskal-Wallis test. All p-values from paired comparisons were adjusted with the Bonferroni correction for multiple comparisons.
All statistical analyses were performed with EZR statistical software (Saitama Medical Center, Jichi Medical University, Saitama, Japan, available at http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html), (17) which is a modified version of the R commander (R Foundation for Statistical Computing, Vienna, Austria). A p-value < 0.05 indicated statistical significance.