Jwa-Kyung Kim and Sung Gyun Kim contributed equally to this study.
Anti-heparin/platelet factor 4 (PF4) antibodies may trigger severe thrombotic complications in hemodialysis (HD) patients. Tetrameric PF4 has a high affinity for extracellular DNA, which is a key component of neutrophil extracellular traps (NETs); therefore, the interactions between anti-heparin/PF4 antibodies and NETs can contribute to prothrombotic events.
Anti-heparin/PF4 antibody levels were measured by enzyme-linked immunosorbent assay and an optical density > 1.8 was regarded as clinically significant. We additionally measured serum nucleosome levels as representative markers of NETs, and the contributions of anti-heparin/PF4 and increased serum nucleosome levels to the primary functional patency loss of vascular access was assessed.
The frequency of anti-heparin/PF4 antibodies was significantly higher in incident HD patients compared to prevalent HD patients (23.6% vs. 7.7%). Serum nucleosome levels, as well as the white blood cell counts, neutrophil counts, and high- sensitivity C-reactive protein levels, were significantly higher in anti-heparin/PF4 antibody-positive patients compared to the control. Platelet counts tended to be lower in the patients with anti-heparin/PF4 of >1.8 than in the controls. Relative risk calculations showed that the presence of anti-heparin/PF4 antibodies increased the risk of primary functional patency failure by 4.28-fold, and this risk increased further with higher nucleosome levels. Furthermore, in the anti-heparin/PF4 antibody-positive group, the time to first vascular intervention was much shorter, and the risk of repeated intervention was higher, compared to the controls.
In incident HD patients, the presence of anti-heparin/PF4 antibodies was associated with increased NET formation; this could be a strong predictor of vascular access complications
Heparin-induced thrombocytopenia (HIT) is a serious immune reaction to heparin often characterized by severe thrombosis; the morbidity and mortality rates of HIT patients are high [
Patients undergoing continuous renal replacement therapy or intermittent hemodialysis (HD) typically require anticoagulation treatment to prevent the clotting of extracorporeal circuits. Heparin is the drug most commonly used for this purpose [
There are several phases of HIT, including an acute phase and various recovery phases [
Unlike most other forms of drug-induced immune thrombocytopenia, HIT is associated with vascular thrombosis, with the primary mechanism of platelet activation and resultant injury to endothelial cells [
Given that vascular access complications are largely due to the thrombotic occlusion of a stenotic fistula or graft [
This observational, single-center study included incident HD patients, who had recently started dialysis treatment, and MHD patients, who underwent regular HD (three times per week), between January 2016 and June 2017. Of the 105 incident HD patients, 31 were excluded for the following reasons; presence of an acute infection at the time of starting HD (n = 3), decompensated liver cirrhosis (n = 2), decompensated heart failure with ejection fraction of <30% (n = 6), hematologic diseases (n = 1), nonachievement of long-term vascular access (n = 10), death or recovery of renal funcion within 3 months (n = 3), or loss to follow up or transfer to another clinic (n = 6). Of the 169 MHD patients, 26 were excluded due to the presence of hematologic diseases (n = 3), decompensated liver cirrhosis (n = 2), decompensated heart failure with ejection fraction of <30% (n = 4), nonachievement of long-term vascular access (n = 12), or nonuse of heparin due to increased bleeding tendency (n = 5). In total, 74 incident HD patients and 143 MHD patients were analyzed. Among the 74 incident HD patients, 55 used unfractionated heparin for anticoagulation, and 19 did not use heparin due to recent gastrointestinal bleeding (n = 5), a large hematoma or bruise associated with vascular access (n = 4), or some other reason (n = 10). The study protocol was approved by the Institutional Review Board of Hallym University Sacred Heart Hospital (No. 2016-I067), and informed consent was obtained from all patients.
Baseline demographic data were obtained, including age, sex, and comorbidities, along with clinical data regarding the underlying cause of renal disease. Venous blood samples were collected into K2-ethylenediaminetetraacetic acid (EDTA)-coated tubes and 0.109 M trisodium citrate-coated tubes (Becton Dickinson, Franklin Lakes, NJ, USA). For the isolation of plasma, the blood was separated by centrifugation at 1,500 ×g for 20 minutes at 4°C. Isolated plasma samples were aliquoted and stored at –80°C until analysis.
For MHD patients, sampling was performed immediately before the HD session in the middle of the week. Biochemical analyses of white blood cells (WBCs), neutrophils, lymphocytes, platelets, hemoglobin, serum albumin, cholesterol, blood urea nitrogen, and creatinine were performed. The neutrophil to lymphocyte ratio was also calculated. Levels of the high-sensitivity C-reactive protein (hs-CRP) inflammatory cytokine were also measured.
The anti-heparin/PF4 antibodies were detected using a commercial heparin/PF4 enzyme-linked immunosorbent assay (ELISA) kit (PF4 IgG, HAT 45G; Immucor GTI Diagnostics, Waukesha, WI, USA) [
To evaluate NET formation, histone-DNA complex (nucleosome) NET biomarkers were measured using a Cell Death Detection ELISAPLUS Kit (Roche, Mannheim, Germany). According to the manufacturer’s instructions, a mixture containing anti-histone-biotin antibody, anti-DNA-peroxidase antibody, and EDTA-plasma was placed in streptavidin-coated wells. After incubation for 2 hours, the wells were washed and reacted with a substrate for 20 minutes. The reaction was stopped using a stop solution. OD was calculated in the same way as in the heparin-PF4-IgG antibody assay. As we reported previously, the highest quartile of serum nucleosome (Q4) was used as the cutoff value for significant increase of NETs [
The primary outcomes of this study were primary functional patency loss, defined as thrombotic occlusion of vascular access (including abandonment of the access site) requiring surgical or percutaneous endovascular intervention (percutaneous transluminal angioplasty [PTA] or thrombectomy) following initial cannulation, and achievement of adequate dialysis within 6 months of the first dialysis session [
The secondary outcomes were as follows; the time taken for successful cannulation in three consecutive HD sessions, the time to the first intervention and frequency of first interventions within 3 months, and the time between the first intervention and reintervention. We also analyzed the abandonment rate of initial vascular access during follow-up.
As an exploratory study, the sample size of the study was planned based on the expected mean differences between the incident HD, MHD, and control populations (non-heparin users) of 20% of the anti-PF4 antibody level. Using nonparametric sample size estimation, the sample size calculation for the incident HD patients resulted in a sample size of 50 to 60 patients and control groups who were eligible for the study in the same study period to achieve a power of at least 80%. Variables with normal distributions are expressed as the means ± standard deviations, and the Kolmogorov-Smirnov test was used to analyze the normality of the distribution of each parameter. Categorical variables are expressed as percentages and were compared using the chi-square test. Cumulative survival curves were derived using the Kaplan-Meier method, and differences between survival curves were compared using the log-rank test. Pearson’s correlation analysis was used to clarify the relation ship between the number of immune cells and anti-PF4 antibody levels. A Cox proportional hazards model was used to identify factors independently associated with vascular access abandonment, based on hazard ratios (HRs) with 95% confidence intervals (CIs). A p-value less than 0.05 was regarded as significant. All statistical analyses were performed using IBM SPSS version 22.0 (IBM Corp., Armonk, NY, USA).
During the 18-month period from January 2016 to June 2017, a total of 105 incident HD and 169 MHD patients were enrolled in this study, and a total of 74 and 143 patients were evaluated. The median dialysis duration for MHD patients was 39.7 months.
We divided the patients into three groups; incident HD patients with heparin free, incident HD patients with heparin use, and MHD patients. We then compared the anti-heparin/PF4 antibody levels among the three groups. As expected, anti-heparin/PF4 antibodies were rarely detected in non-heparinized HD patients (mean OD, 0.39 ± 0.11). Notably, the mean anti-heparin/PF4 antibody OD value was significantly higher in incident HD patients compared to MHD patients, even though both HD patient groups were regularly exposed to heparin (1.34 ± 0.65 vs. 0.93 ± 0.47, p < 0.001) (
Focusing on the incident HD group, we examined the relationships between clinical and biochemical parameters and clinically significant anti-heparin/PF4 antibody levels (
Anti-heparin/PF4 antibodies are associated with platelet activation and endothelial cell damage; therefore, we conducted thorough follow-up examinations to evaluate the HD vascular outcomes of all patients. Vascular complications are largely due to thrombosis and stenosis following endothelial damage and inflammation. Our data showed that patients with strong positive anti-heparin/PF4 antibodies had significantly higher rates of primary functional patency loss within 6 months of the first HD compared to the control group (53.8% vs. 21.4%, p = 0.03) (
We then examined the relationships between the presence of clinically significant anti-heparin/PF4 antibody levels and the times of first cannulation and first intervention. There were no cases of primary maturation failure up to 3 months after the creation. However, the time taken for successful cannulation in three consecutive HD sessions was significantly longer in the strongly positive anti-heparin/PF4 antibody group compared to the control group; this was likely due to the higher vascular intervention rate in the strong positive group compared to the controls (72.7 ± 34.3 days vs. 55.5 ± 16.7 days, p = 0.03). Supporting this, patients who had clinically significant anti-heparin/PF4 antibody levels experienced earlier vascular intervention compared to control patients (
Additionally, we compared the rates of access abandonment between the strongly positive anti-heparin/PF4 antibody group and the control group (
Due to the close association between strong anti-heparin/PF4 antibody positivity and high WBC and neutrophil counts in peripheral blood, we evaluated the relationship between anti-heparin/PF4 antibody and serum nucleosome levels. Consistent with our previous findings, the serum nucleosome levels varied significantly among the HD patients. The median serum nucleosome OD value was 0.16, and the cutoff value for the highest quartile (Q4) was 0.52. Serum nucleosome levels exhibited a close relationship with anti-heparin/PF4 antibody levels (r = 0.309; p = 0.03) (
In this study, we investigated the potential impact of anti-heparin/PF4 antibodies on HD vascular access outcomes and their relationship with NET levels. There were several strengths to our study, the first being that we measured anti-heparin/PF4 antibody levels in both incident HD and MHD patients, which enabled direct comparison of the two groups. Given our results, we suggest that timing is an important factor when testing for anti-heparin/PF4 antibodies. Another strength of our study was that we only focused on vascular access. The causes of cardiovascular and cerebrovascular events in HD patients are complex and multifactorial; although the causes are similarly complex for vascular outcomes, the main pathogenesis of vascular complications is thrombotic occlusion with stenosis. Furthermore, we measured serum nucleosome levels alongside performance of the anti-heparin/PF4 antibody test since neutrophil dysregulation and increased NET formation have been reported to contribute to thrombosis in uremic conditions. Platelet-neutrophil interactions could be key in the formation of vascular access clots.
According to previous HIT studies, anti-heparin/PF4 antibody positivity rates were within the range of 2.3% to 10.3% in HD patients. The presence of HIT antibodies was not found to be associated with adverse clinical outcomes; however, previous studies only examined MHD patients, without considering incident HD patients [
In this report, we focused on the incident HD group and further evaluated the prognostic role of anti-heparin/PF4 antibodies in vascular outcomes. Notably, patients with clinically significant antibody levels had higher WBC and neutrophil counts than those without. Consistent with this finding, serum nucleosome levels were much higher in the antibody-positive group. Taken together, this suggests that the presence of anti-heparin/PF4 antibodies was accompanied by neutrophil activation and increased NET formation
We evaluated the value of anti-heparin/PF4 antibodies for predicting functional vascular access (defined as successfully matured and used for dialysis) and the subsequent loss of primary functional patency within 6 months of its first use. Because the main objective of the study was to investigate the thrombotic occlusion associated with anti-heparin/PF4 antibodies, patients with decompensated heart failure or decompensated liver cirrhosis were specifically excluded to rule out potential inflow problems with respect to vascular access. An interval of <3 months between vascular access creation and first use likely indicates that any subsequent loss of primary functional patency is not due to anatomical problems or low blood flow. Our results showed that the presence of anti-heparin/PF4 antibodies was a powerful predictor of vascular access failure (odds ratio, 7.3; p = 0.02), even after adjustment for diabetes and inflammatory conditions. In addition, the risks of vascular intervention within 3 and 6 months were 3.1 and 6.3 times higher, respectively, in the positive anti-heparin/PF4 antibody group than in the control group. Positive HIT antibodies were linked to a higher incidence of repeated vascular intervention within 3 months of the first vascular intervention and to the abandonment of vascular access. Consistent with our data, O’shea et al. [
Another notable finding in this study was that patients with positive anti-heparin/PF4 antibodies had higher serum nucleosome levels than the control group, and the vascular outcomes were worse in patients with both higher nucleosome levels and strongly positive antibodies. Given the strong association of the anti-heparin/PF4 antibodies with serum neutrophil and nucleosome levels, it is reasonable to suggest that an interaction between the anti-heparin/PF4 antibodies and excessive NET could be a risk factor for vascular thrombosis. Supporting our data, Perdomo et al. [
A limitation of this study was that a relatively small number of patients were analyzed, all of whom were from a single center. We tried to include more patients by extending the study period, but the availability of anti-heparin/PF4 antibody commercial kits was limited due to their infrequent use in clinical practice. Therefore, the results are not representative. However, our data are nonetheless valuable; by comparing the antibody levels between MHD and incident HD patients, we discovered a relationship between anti-heparin/PF4 antibody levels and the time of heparin exposure. Relatively few studies including incident HD patients have been published. Another limitation of this study was that although we measured serum nucleosome levels, we were unable to directly visualize NETs
In conclusion, the frequency of anti-heparin/PF4 antibodies was significantly higher in incident HD patients compared to MHD patients. This likely explains why previous studies involving MHD patients failed to show any significant prognostic role of anti-heparin/PF4 antibodies. In incident HD patients, we found that the presence of anti-heparin/PF4 antibodies was associated with increased NET formation and that this could be a strong predictor for subsequent vascular access complications. This provides evidence for a pathological interaction between platelet activation, increased NET formation, and endothelial damage
All authors have no conflicts of interest to declare.
This research was supported by a National Research Foundation grant funded by the Korean government (2020R1A2C110138611) and the Hallym University Research Fund.
Conceptualization, Funding acquisition: JKK, SGK
Data curation: JNA, HSL
Formal analysis: JKK, SGK, JNA, HSL
Investigation: HWL, JKK, YRS, HJK
Methodology: YRS, HJK
Writing–original draft: HWL, JKK
Writing–review & editing: YRS, HJK
All authors read and approved the final manuscript.
In patients without heparin use (heparin-free), the antibody level was very low (negative control). In patients with heparin use, the level was significantly higher in incident HD patients compared to maintenance HD patients.
HD, hemodialysis; PF4, platelet factor 4.
(A, B) The Ab level had a positive correlation with WBC and neutrophil counts but had a negative relationship with platelet count. Also, the levels of hs-CRP, a serum inflammatory marker, were significantly higher in patients with strongly positive Ab levels (B).
Ab, antibody; hs-CRP, high-sensitivity C-reactive protein; OD, optical density; PF4, platelet factor 4; WBC, white blood cell.
(A) Patients with anti-heparin/PF4 Abs underwent more frequent and earlier vascular interventions compared to the control group. Therefore, they had a much higher incidence of vascular access failure. (B, C) The abandonment risk was also significantly higher in patients with Abs.
Ab, antibody; PF4, platelet factor 4.
(A) Patients with strongly positive anti-heparin/PF4 Abs had significantly higher levels of nucleosomes. (B, C) When divided into four groups according to the nucleosome highest quartile and an OD level of 1.8, the highest rates of primary patency loss were observed in patients with an Ab OD > 1.8 and nucleosome OD > 0.52 (Q4).
Ab, antibody; OD, optical density; PF4, platelet factor 4.
Baseline characteristics of all patients
Variable | Heparin-free | Heparin use |
p-value |
|
---|---|---|---|---|
Incident HD | Maintenance HD | |||
No. of patients | 19 | 55 | 143 | |
Age (yr) | 70.6 ± 11.2 | 70.9 ± 11.3 | 64.9 ± 12.5 | 0.001 |
Male sex | 17 (63.2) | 28 (50.9) | 75 (52.4) | 0.46 |
Body mass index (kg/m2) | 22.6 ± 4.6 | 24.1 ± 4.3 | 23.6 ± 3.5 | 0.34 |
Diabetes mellitus | 19 (68.5) | 23 (41.8) | 86 (60.1) | 0.19 |
Previous CVD | 3 (10.7) | 5 (9.4) | 28 (19.7) | 0.04 |
Systolic BP (mmHg) | 137 ± 25 | 142 ± 17 | 140 ± 20 | 0.57 |
Diastolic BP (mmHg) | 77 ± 20 | 76 ± 12 | 74 ± 12 | 0.43 |
HD duration (mo) | 1.1 (0.2–1.7) | 1.0 (0.3–1.8) | 39.7 (16–80) | - |
Anti-platelet agent use | 3 (15.7) | 23 (41.8) | 81 (56.6) | 0.26 |
Vascular access type | 0.01 | |||
AVF | 6 (31.6) | 36 (68.5) | 118 (82.5) | |
AVG | 5 (26.3) | 17 (31.5) | 25 (17.5) | |
Anti-PF4 antibody, OD | 0.39 ± 0.19 | 1.34 ± 0.65 | 0.93 ± 0.47 | 0.001 |
>1.8, strong positive | NA | 13 (23.6) | 11 (7.7) | |
1.0–1.8, weak positive | NA | 19 (34.5) | 32 (22.3) | |
0.4–1.0, intermediate | NA | 23 (41.8) | 93 (65.0) | |
<0.4, negative | NA | 0 (0) | 7 (4.9) |
Number (%) only, mean ± standard deviation, or median (interquartile range).
AVF, arteriovenous fistula; AVG, arteriovenous graft; BP, blood pressure; CVD, cardiovascular disease; HD, hemodialysis; NA, not applicable; OD, optical density; PF4, platelet factor 4.
p-value for comparison between incident HD and maintenance HD.
Comparison of biochemical data and vascular outcomes according to PF4 antibody level
Variable | Incident HD with heparin use (n = 55) |
p-value | |
---|---|---|---|
OD ≤ 1.8 (n = 42) | OD > 1.8 (n = 13) | ||
Age (yr) | 70.8 ± 11.2 | 69.1 ± 11.7 | 0.93 |
Male sex | 21 (50.0) | 5 (38.5) | 0.22 |
Diabetes mellitus | 24 (57.1) | 6 (46.1) | 0.47 |
Previous CVD | 3 (7.1) | 2 (15.3) | 0.30 |
Systolic BP (mmHg) | 139.6 ± 19.0 | 148.5 ± 16.4 | 0.08 |
Diastolic BP (mmHg) | 74.0 ± 13.0 | 80.1 ± 9.9 | 0.09 |
Vascular access type | 0.27 | ||
AVF | 29 (69.0) | 8 (61.5) | |
AVG | 13 (31.0) | 5 (38.5) | |
Biochemical parameter | |||
WBC count | 6,427 ± 1,960 | 9,205 ± 3,580 | 0.001 |
Hemoglobin (g/dL) | 10.5 ± 1.3 | 10.9 ± 1.3 | 0.71 |
Neutrophil count | 4,350 ± 1,509 | 6,435 ± 3,116 | 0.004 |
Neutrophil/lymphocyte ratio | 4.0 ± 2.9 | 4.3 ± 2.6 | 0.77 |
Platelet (103/mL) | 200 ± 40 | 168 ± 70 | 0.07 |
<100 × 103/mL | 2 (4.7) | 2 (15.4) | |
BUN (mg/dL) | 57.6 ± 17.6 | 54.4 ± 22.6 | 0.58 |
Creatinine (mg/dL) | 6.8 ± 1.9 | 6.3 ± 1.9 | 0.46 |
Albumin (g/dL) | 3.6 ± 0.6 | 3.6 ± 0.3 | 0.90 |
Total cholesterol (mg/dL) | 154.1 ± 29.3 | 148.6 ± 36.9 | 0.56 |
hs-CRP | 1.10 ± 1.04 | 3.50 ± 2.70 | 0.006 |
Vascular outcome | |||
Primary outcome | |||
Loss of primary patency in 6 mo | 9 (21.4) | 7 (53.8) | 0.03 |
Abandonment in 6 mo | 0 (0) | 4 (30.7) | |
First intervention in 6 mo | 9 (21.4) | 6 (46.2) | |
Secondary outcome | |||
Time to first needling (day) | 55.5 ± 16.7 | 72.7 ± 34.3 | 0.03 |
Time to first intervention (mo) | 5.2 ± 4.0 | 13.4 ± 6.9 | 0.07 |
First intervention in 3 mo | 6 (14.3) | 6 (46.2) | 0.02 |
Time to reintervention | |||
Re-PTA in 3 mo | 4 (9.5) | 5 (38.4) | 0.02 |
Abandonment during follow-up | 3 (7.1) | 6 (46.1) | 0.003 |
Data are expressed as mean ± standard deviation or number (%).
AVF, arteriovenous fistula; AVG, arteriovenous graft; BP, blood pressure; BUN, blood urea nitrogen; CVD, cardiovascular disease; HD, hemodialysis; hs-CRP, high-sensitivity C-reactive protein; OD, optical density; PTA, percutaneous transluminal angioplasty; WBC, white blood cell.
Correlation analysis of Anti-PF4 antibody with nucleosome level and blood cell variables
Variable | WBC | Neutrophil | Platelet | Nucleosome level | ||||
---|---|---|---|---|---|---|---|---|
r | p | r | p | r | p | r | p | |
Anti-PF4 antibody | 0.397 | 0.003 | 0.37 | 0.006 | –0.37 | 0.005 | 0.31 | 0.03 |
WBC | - | 0.96 | <0.001 | 0.31 | 0.02 | 0.25 | 0.08 | |
Neutrophil count | - | - | - | - | 0.29 | 0.03 | 0.35 | 0.04 |
PF4, platelet factor 4; WBC, white blood cell.
Cox proportional HRs for predicting abandonment of vascular access
Variable | HR (95% CI) | p-value |
---|---|---|
Univariate analysis | ||
Anti-PF4/heparin antibody, >1.8 | 7.29 (1.79–25.65) | 0.004 |
Multivariate analysis | ||
Age, per 1 yr | 1.14 (0.96–1.34) | 0.13 |
Diabetes mellitus, presence | 1.06 (0.09–2.27) | 0.34 |
Vascular access type (AVG) | 3.10 (0.66–14.46) | 0.15 |
hs-CRP (mg/L), >1.0 | 3.07 (0.32–15.11) | 0.19 |
Anti-PF4/heparin antibody, >1.8 | 7.32 (1.31–40.88) | 0.02 |
AVG, arteriovenous graft; CI, confidence interval; hs-CRP, high-sensitivity C-reactive protein; HR, hazard ratio; PF4, platelet factor 4.