Editor ’ s Choice – Sex Differences in Response to Administration of Heparin During Non-Cardiac Arterial Procedures

62; 200 e 250 seconds: 41% vs. 53%, p ¼ .058; 251 e 280 seconds, 26% vs. 15%, p ¼ .030). The mean ACT after 100 IU/kg heparin was 233 seconds (95% con ﬁ dence interval [CI] 224 e 243) for females and 226 seconds (95% CI 221 e 231) for males ( p ¼ .057). After a bolus of 5 000 IU of heparin ( n ¼ 411), females reached signi ﬁ cantly higher levels of anticoagulation than males (mean ACT 204 seconds vs. 190 seconds: p (cid:2) .001; ACT < 200 seconds: 44% vs. 66%; p < .001; ACT 200 e 250 seconds: 47% vs. 30%, p ¼ .001; ACT 251 e 280 seconds: 7.8% vs. 2.3%, p ¼ .009). Thirty minutes after heparin administration, 58% of all patients had an ACT < 200 seconds. ATECs did not differ between females and males (6.9% vs. 5.1%, p ¼ .33) but bleeding complications were higher in females (27% vs. 16%, p ¼ .001). Conclusion: Heparin leads to signi ﬁ cantly longer ACT in females during NCAP. Further research is needed to investigate whether individually based heparin protocols lead to fewer bleeding complications and lower incidence of ATECs.


INTRODUCTION
Until now, no distinction has been made between female and male patients during invasive treatment for vascular disease.
Increasing evidence, however, shows that treatment outcomes and particularly complications are different between females and males. 1e3 A recent cohort study showed that females developed more bleeding complications after invasive treatment for peripheral artery disease. 4 Over the past decades it has become clear that there are differences in pharmacokinetics and pharmacodynamics between females and males and that female sex is associated with a higher risk of developing adverse drug reactions. 5e8 In open and endovascular non-cardiac arterial procedures (NCAPs) unfractionated heparin (further heparin) is used to prevent arterial thromboembolic complications (ATECs). 9,10

EJVES Open Access
Although good studies on heparin use and its benefits or risks are sparse, some studies have shown that myocardial infarction was found to be significantly more frequent in non-heparinised patients than in heparinised patients. 9,11 Besides heparin's many benefits, including immediate effect, low cost, and the possibility to quickly reverse its effect, a major disadvantage is the unpredictable effect in the individual patient. 12e16 The activated clotting time (ACT) can be used to monitor the patient's peri-procedural (anti) coagulation. Heparin dosing can be adjusted based on ACT measurements to reach a desired level of anticoagulation. Although the optimal target ACT during NCAP has not been determined yet, previous investigations suggest an ACT between 200 and 250 seconds may be preferable. 17 Little is known about differences in the effect of heparin in males and females during NCAPs. The aim of this study was to investigate whether there is a difference in the effect of heparin in males and females during NCAPs. The hypothesis was that females might be more sensitive to heparin.

Study design
The current study is part of a prospective multicentre cohort study designed to investigate the optimal heparin dose protocol and optimal ACT values during NCAPs. Patients older than 18 years were eligible for inclusion. Exclusion criteria were patients requiring acute intervention, allergy to heparin, history of heparin induced thrombocytopenia, patients who received heparin prior to surgery, a known history of coagulation disorders, or an estimated glomerular filtration rate < 30 mL/min (based on Kidney Disease: Improving Global Outcomes guidelines, 2012). 18 From January 2016 onwards, this database is being collected for the MANCO trial registered at clinicaltrials.gov (identifier: NCT03426293) and the Dutch Trial Registry (identifier: NTR6973). Collected data are stored in an electronic database using Castor EDC. A case report form was used to gather patient data. The protocol from the MANCO trial was evaluated and approved by the Medical Ethics Committee Noord-Holland. Data were collected from Dijklander hospital, Hoorn; Amsterdam UMC, Amsterdam location VU Medisch Centrum; and Isala hospital, Zwolle. For the current study, patient data were recorded until March 2020. Earlier results of the MANCO study have been published. 19

Heparin protocol
The effect of a single dose of heparin on the ACT was investigated using two dosing protocols: a standardised bolus of 5 000 IU or 100 IU/kg bodyweight.

Activated clotting time
In all participating centres, ACT was measured using the Hemostasis Management System Plus from Medtronic (Medtronic, Minneapolis, MN, USA). High range cartridges were used during all procedures. For every ACT measurement, 3 mL of arterial blood was drawn from the patient.
Beforehand, 5 mL of blood was discarded of to prevent contamination of heparin residues. ACT was measured at baseline, five minutes after heparin administration, and every 30 minutes thereafter until the procedure was finished. At the end of surgery, ACT was measured, and protamine was administered at the surgeon's discretion.

Heparin effect over time
To investigate the ongoing effect of heparin in the current study, ACTs five and 30 minutes after heparin administration were compared. For each patient, the absolute difference in seconds between the five minute and 30 minute ACT was calculated, which was defined as ACT drop. The ACT drop might help by predicting whether a patient remains within the therapeutic anticoagulation range after 30 minutes. The median of the ACT drop was reported for the total cohort and per protocol. Based on previous research, an ACT < 200 seconds was defined as underdosed, an ACT between 200 and 250 seconds on target, an ACT between 251 and 280 seconds moderately overdosed, and an ACT > 280 second overdosed. The mean ACT was calculated for both dosing protocols and thereafter compared between males and females.

The effect of different doses of heparin
The effect of body weight and body surface area (BSA, IU/ m 2 ) on ACT was investigated using the ACT after the heparin dose. 20 Patients were grouped together based on body weight or BSA, and the dose per bodyweight or BSA was calculated for every patient. Consequently, it was evaluated at which initial heparin dose the majority of patients in each group reached the preferred ACT of 200 e 250 seconds. These analyses were performed in the search for the optimal dosage of heparin for males and females.

Arterial thromboembolic complications and bleeding complications
The number of ATECs and bleeding complications were registered up to 30 days after the primary procedure or during the same hospital admission. ATECs included myocardial infarction, cerebrovascular accident, deep venous thrombosis, pulmonary embolism, peripheral embolism, bowel ischaemia, thromboembolic renal insufficiency, athero-embolism, spinal cord ischaemia, and graft thrombosis.
To determine the incidence and severity of bleeding complications, the European Multicentre Study on Coronary Artery Bypass Grafting (E-CABG) registry was used to score and grade intra-and post-operative bleeding. 21

Statistical analyses
Statistical analyses were performed using (SPSS version 25 (Armonk, NY: IBM Corp.). A normality test on eligible data was performed using the ShapiroeWilk test. Continuous normally distributed variables were expressed as mean AE standard deviation. Skewed variables were expressed as median with interquartile range (IQRs). Categorical variables were expressed as counts and percentage. The independent t test was used to test normally distributed data between groups. The ManneWhitney U test was used to test skewed data. Binary data were tested using the chi square test. Multivariable analysis was performed using logistic regression with the variables that had p < .20 in the univariable analyses. Test results were reported using the p value. A p value less than .050 was considered statistically significant. Subjects with missing data were excluded per analysis.
Prior to the retrospective analysis, a power analysis was performed to calculate the required number of patients in each of the groups. The difference between females and males who reach a target ACT of > 200 seconds was set at 15%. Using an alpha of .05 and a power of .80, the minimum number of patients required per group was 169 (total of 338 patients).

RESULTS
Seven hundred and seventy-eight patients were included in the current study. The cohort consisted of 574 (73%) males, and 204 (26%) females. Patient characteristics and the type of procedures are detailed in Table 1. Females were significantly shorter, weighed less, had a lower body mass index (BMI) and BSA. They underwent open procedures significantly more often. Males underwent an aortic procedure more often, particularly endovascular aneurysm repair. All patients underwent surgery under general anaesthesia. Two hundred and eighteen (28%) patients received a bolus of protamine.

The initial effect of heparin
The results of the measured ACTs are displayed in Figure 1 and Table 2. Seven hundred and eleven patients were enrolled in the 5 000 IU or in the 100 IU/kg dosage group.
Sixty-seven patients received a different dose or had no ACT recorded at five minutes. For both male and female patients, less than half reached an ACT of 200 e 250 seconds (39% and 44% respectively, p ¼ .23). Females less often reached an ACT < 200 seconds (34% vs. 49%, p < .001) and more often an ACT of 251 e 280 seconds (16% vs. 7.5%, p < .001). No difference between females and males was found for an ACT of > 280 seconds, p ¼ .49).
In the 100 IU/kg group 113 of 214 (53%) males vs. 35 of 86 (41%) females reached an ACT of 200 e 250 seconds (p ¼ .058). Females more often reached an ACT of 251 e 280 seconds (26% vs. 15%, p ¼ .030). Females in this group were more likely to have an ACT > 250 seconds if they were taller (p ¼ .037), had a higher weight (p ¼ .043), and higher BSA (p ¼ .031). A wide variation in ACT values was found for both females and males.
Four hundred and eleven patients received 5 000 IU of heparin and had their ACT measured five minutes after heparin administration. Females reached significantly higher levels of anticoagulation. No difference between females and males was found for patients exceeding an ACT of 280 seconds (p ¼ .52). A significant difference was found in mean ACT between females and males (204 seconds, 95% CI 198 e 210, vs. 190 seconds, 95% CI 186 e 193, p ¼ <.001). males), no difference was found between females (19 seconds, IQR 31) and males (19 seconds, IQR 29). Five minutes after heparin administration, a total of 161 (36%) patients had an ACT below the therapeutic range (< 200 seconds). After 30 minutes 260 (58%) patients had an ACT < 200 seconds ( Table 3).

The effect of different doses of heparin
The results of the effect of heparin dose on the five minute ACT are shown in Tables 4 and 5 No reliable results can be calculated for heparin doses > 110 IU/kg and for doses > 5 000 IU/m 2 because of sheer patient numbers. No weight based, BSA based, or BMI based dosage protocol could be found where the vast majority of patients reached the target ACT after the initial bolus of heparin.

Arterial thromboembolic complications and bleeding complications
The combined results of the incidence of ATECs and bleeding complications are displayed in Table 6. No difference was found in the incidence of ATECs between males and females (5.1% vs. 6.9%, p ¼ .33). Because of the low absolute number of ATECs, multivariable analysis was not performed. A total of 143 (18%) bleeding complications occurred in the cohort. When combining all bleeding complications, females developed more bleeding complications than males (27% and 16%, respectively, p ¼ .001), mainly grade 1. Of the patients who developed a bleeding complication, 39% of females and 38% of males had an ACT > 250 seconds, measured at any time during the procedure. Fifty-one (36%) of the patients with a bleeding complication had received protamine. Fifteen (10%) patients underwent re-operation due to bleeding.
Univariable analyses were performed on variables associated with bleeding complications, showing a considerable effect for sex, protocol 5 000 IU, protocol 100 IU/kg, ACT > 250 seconds, ACT > 200 seconds, carotid intervention, open aortic procedure, endovascular aortic procedure,  femorodistal intervention, and protamine. Multivariable analysis of the total patient group showed that when corrected for influencing factors, patients who underwent an open aortic intervention, or who received protamine, had a significantly greater probability of bleeding complications. Patients after carotid intervention or after endovascular aortic intervention had a lower probability of bleeding complications. No significant effects for sex, heparin dosing protocol, or ACT values was found (Supplementary Table S1).

DISCUSSION
Female patients reach significantly longer ACTs than male patients after heparin administration in NCAPs. This effect was found for both the weight based dosing protocol of 100 IU/kg heparin and for a standardised bolus of 5 000 IU of heparin. These findings expand on a previous smaller study showing that females reached higher levels of activated partial thromboplastin time after a fixed dose of heparin without correcting for weight. 22 Analysis of the effect of the heparin dose on the ACT showed that there is no dose at which the majority of the patients reached a target ACT range of 200 e 250 seconds. A study on percutaneous coronary interventions aiming at an ACT of 300 seconds also found a correlation between body weight and BSA on heparin response but found no added benefit of weight based heparin dosing. 20 In the present cohort, heparin dose between 80 and 109 IU/kg for males resulted in target ACT in just over half of the patients. For females, no specific weight based dose could be identified at which the majority reached a target ACT range of 200 e 250 seconds. Using a BSA based heparin protocol, 56% of males reached an ACT of 200 e 250 seconds after a dose of between 4 000 and 4 999 IU/m 2 . For females, 2 000 e 2 999 IU/m 2 led to 46% of them reaching an ACT of 200 e 250 seconds. However, it needs to be noted that patient groups were small in this analysis.
Because of the high variance in the elimination of heparin in both females and males, repeated monitoring of the ACT at least every 30 minutes seems necessary. This can be performed at low cost (cartridges are less than V4 in The Netherlands).
Elimination of heparin is thought to be subject to saturable and non-saturable mechanisms. 23,24 At low doses, the highly efficient saturable mechanism is mainly responsible for the elimination of heparin. The role of the non-saturable mechanism becomes more relevant when the dose of heparin increases (> 100 IU/kg), which leads to an increased heparin half life. 25e27 In the current study, patients with a higher dose of heparin showed a greater difference in ACT between the five and 30 minute mark. The increase in the ACT drop can be explained by the fact that doses of up to 100 IU/kg are still effectively cleared by both elimination mechanisms. Based on the results of the current study, it seems unlikely that there is a considerable difference in elimination of heparin between males and females, since the ACT drop was equal (19 seconds, IQR 29, and 19 seconds, IQR 31). However, in future research this should be analysed using measurements of plasma heparin levels. Sex differences in response to heparin might be possible, as previous research shows a higher risk of heparin induced thrombocytopenia in females than in males. 28 However, in the current study, no patients developed heparin induced thrombocytopenia. In addition, heparin resistance, the failure to achieve a specified anticoagulation level despite the use of what is considered to be an adequate dose of heparin, is reported in the literature. Until now it has not been known whether heparin resistance is more common in females or males. 29,30 Moreover, hormonal factors could influence the effect of heparin, as sex related differences in haemostasis have been reported in previous research. 31,32 Due to the heterogeneous procedures included in the study, only exploratory analysis was performed on clinical complications. ATECs occurred in 5.1% of males and 6.9% of females. Females suffered from more bleeding complications than males (27% vs. 16%). This difference remained after sub-analysis for open procedures but was not significant after multivariable analysis. Larger studies, containing more homogeneous surgical procedures, need to be performed to investigate the association between sex and complications.
A strength of the current study is that data were collected prospectively, which results in a limited number of missing values. In addition, the data were collected in two general hospitals and one university hospital, so data from a large cohort could be analysed.
A natural limitation of the ACT is that it may be affected by many variables such as hypothermia, platelet count, and Data are presented as n (%). ACT ¼ activated clotting time. * 51 patients had no dose recorded, did not have an ACT measurement after five minutes, or the height or weight was unknown. function, ATIII deficiency and time from blood collection to analysis. 33,34 To reduce the influence of these factors on the ACT, the temperature of patients was maintained as constant as possible during surgery using a convection temperature management system, patients with a low platelet count or platelet dysfunction were excluded from the study, and the time between blood collection and analysis was kept as short as possible.

Conclusion
Heparin administration during NCAPs leads to longer ACTs in females than in males. The anticoagulatory effect of heparin proved unpredictable in both males and females, even after body weight or BSA based dosing. Therefore, monitoring of coagulation with ACT seems crucial to ensure the patient has safe and tailor made peri-procedural anticoagulation. Likewise, repeated monitoring of the ACT at least every 30 minutes is necessary because of the large variation in heparin elimination. Future research needs to investigate whether a specific heparin protocol for females leads to safer anticoagulation, and possibly fewer complications.

CONFLICT OF INTEREST
None.

FUNDING
This research was supported by a grant from Medtronic (reference number: A1395092 / SH-3950). Medtronic had no involvement in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.