Association of immature platelets with perioperative complications in neurosurgery

Abstract Immature platelets are newly formed platelets with an increased prothrombotic potential. This study evaluates whether immature platelets are associated with relevant complications in neurosurgical patients. Data were obtained in the frame of a prospectively conducted observational study exploring the association between immature platelets and major cardiovascular events after surgery. Immature platelet fraction (IPF) and H-IPF (highly fluorescent immature platelet fraction) were measured preoperatively and postoperatively at the neurosurgical ward (24–72 hours after surgery). Therapy-relevant complications after surgery were stratified using the Clavien-Dindo Grade (CDG >2) as primary outcome. Data were analyzed in 391 neurosurgical patients. While preoperatively there were no differences in IPF or H-IPF, patients with higher therapy-complication grades had higher values post-op compared to patients with lower grade complications (≤2 CDG). Cut-off values identified by receiver operating characteristic curve analysis revealed that there were significantly more patients with H-IPF ≥0.95% in the group with serious complications (CDG >2) [odds ratio OR (95% confidence interval CI) = 2.06 (1.09–3.9), p = .025], whereas this association was not present for the IPF cutoff value. In a multivariate model, H-IPF≥0.95% was independently associated with serious complications after surgery [OR (95% CI) = 1.97 (1.03–3.78), p = .041]. These findings suggest that H-IPF is associated with surgical complications and may improve risk stratification of neurosurgical patients (clinicaltrials.gov: NCT02097602, registration date: 27/03/2014). Plain Language Summary What is the context? Immature platelets are newly formed platelets with a higher thrombotic potential and play an important role in atherothrombotic events. Higher levels of immature platelets were observed in patients with acute coronary syndrome or stroke. Lately, the focus in immature platelet research shifted from observation to outcomes. Immature platelets were identified as independent predictors of major cardiovascular events in cardiologic patients with coronary artery disease. Besides, an association between immature platelets and major cardiovascular events was described in surgical patients after non-cardiac surgery. What is new? This study builds on these findings and extends the focus to perioperative complications after neurosurgery. The data were obtained prospectively in the frame of an observational clinical trial exploring the association of immature platelets and major cardiovascular events in general. Data measured in the neurosurgical cohort of that study (391 neurosurgical patients) were analyzed in the present work. Within the limitations of our study, our analyses suggest that the postoperative IPF (immature platelet fraction) and H-IPF (highly fluorescent immature platelet fraction) values, which were measured at the neurosurgical ward after surgery are both associated with higher therapy-relevant complication grades (>2 according to Clavien-Dindo Grade), whereas preoperatively obtained values were not. What is the impact? This is the first study showing a relationship between immature platelets and therapy-relevant perioperative complications in neurosurgical patients. It could be a pilot trial for varied scientific questions including risk stratification of neurosurgical patients.


Plain Language Summary
What is the context?
• Immature platelets are newly formed platelets with a higher thrombotic potential and play an important role in atherothrombotic events.• Higher levels of immature platelets were observed in patients with acute coronary syndrome or stroke.• Lately, the focus in immature platelet research shifted from observation to outcomes.
Immature platelets were identified as independent predictors of major cardiovascular events in cardiologic patients with coronary artery disease.Besides, an association between immature platelets and major cardiovascular events was described in surgical patients after non-cardiac surgery.
What is new?
• This study builds on these findings and extends the focus to perioperative complications after neurosurgery.• The data were obtained prospectively in the frame of an observational clinical trial exploring the association of immature platelets and major cardiovascular events in general.Data

Introduction
Identifying patients at risk for perioperative complications and reducing postoperative morbidity are an integral part of improving outcome after surgery [1].Recently, immature platelets have been identified as a new biomarker associated with cardiovascular complications after surgery [2,3].Immature or reticulated platelets (RP) are newly formed platelets, analogous to reticulocytes in erythropoiesis, which play an important role in atherothrombotic events [4][5][6][7][8][9].They are larger in size than mature platelets and contain a higher amount of RNA [10][11][12][13].Immature platelets are functionally more active and have a higher prothrombotic potential [11,14,15].They may be associated with higher on-treatment platelet reactivity in patients with acute or chronic coronary syndromes treated with certain antiplatelet drugs [14,16,17].While higher levels of immature platelets were observed in patients with acute coronary syndrome [18], data regarding the profile of RP in stroke are limited [19][20][21][22].The prevalence of high on-treatment platelet reactivity (HTPR) in patients with transient ischemic attack (TIA) and ischemic stroke varies between studies in the literature [23,24], but a clear relationship between the percentage of reticulated platelets and HTPR status has not yet been clearly established in an ischemic cerebrovascular disease patient population.Lately, the focus in immature platelet research has shifted from observation to outcomes [25].In this context, immature platelets were identified as independent predictors of major cardiovascular events not only in cardiology patients with coronary artery disease but also in surgical patients after non-cardiac surgery [2,3,26,27].
While there are hundreds of studies addressing risk calculations in cardiac surgery, there are only few in the neurosurgical research field [28].The aim of this study was to further stratify risk strategies in neurosurgical patients and evaluate the impact of immature platelets on surgical complications.

Study design
Immature platelets were measured prospectively in the frame of an observational clinical study exploring the association of immature platelets and major cardiac and cerebrovascular events after non-cardiac surgery in general [2].Data obtained in the neurosurgical cohort of that study were analyzed in this work.The study protocol was approved by the local ethics committee (Ethics Commission of the Medical faculty, Technical University of Munich) and registered at clinicaltrials.gov(Identifier: NCT02097602, registration date: 27/03/2014).The research was performed in accordance with the Declaration of Helsinki and all patients provided informed written consent before enrollment.In the main study, patients older than 18 years who were undergoing high-risk or intermediate risk elective non-cardiac surgery were eligible to participate.Surgical risk was defined according to the 2007 guidelines of American College of Cardiology (ACC) and American Heart Association (AHA).Procedures with a reported cardiac risk >5% (aortic and other major vascular surgery) were classified as high-risk-surgery, whereas procedures with a cardiac risk ranging from 1 to 5% (for example: intraperitoneal and intrathoracic surgery, head and neck surgery) were defined as intermediate risk surgery [29].Patients were excluded if they were younger than 18 years or undergoing low-risk surgery (superficial procedures according to the 2007 guidelines of American College of Cardiology (ACC) and American Heart Association (AHA)) [29].Further details on the design are described in the initial study [2].The definitions used to assess patient's medical history are provided in the Supplement.
The primary study outcome was the occurrence of therapyrelevant surgical complications stratified by Clavien-Dindo Grade (CDG) [30]: Grade I: any deviation from the normal postoperative course without the need for pharmacological treatment or interventions (Allowed therapeutic regimens are drugs as antiemetics, antipyretics, analgetics, diuretics, electrolytes, and physiotherapy); Grade II: Requiring pharmacological treatment with drugs other than the exceptions listed above, including transfusions and total parenteral nutrition; Grade III: Requiring surgical, endoscopic or radiological intervention; Grade IV: Lifethreatening complication (including central nervous system (CNS) complications) requiring intermediate care (IC) or intensive care unit (ICU) management, Grade V: death.A member of the research team obtained preoperative patient information.Two team members blinded to platelet count results reviewed patients` record (paper as well as electronic chart) for primary outcome measures.As the CDG is therapy-oriented, minor neurological deficits like minimal weakness or tingling might be graded the same way as a major neurological deficit as hemiplegia [31].Therefore, two different approaches were used in assessing CDG.One team member assessed CDG without including minor neurology and the other one factored in any neurological symptoms.Participants or their next relative were contacted by telephone 3 months after enrollment for assessing mortality 90 days after surgery.
Data collection as well as the assessment of mortality after 3 months were planned and obtained prospectively.The initial CDG evaluation was performed prospectively.The CDG scoring taking into account any neurological symptoms, as well as the differentiation of the surgical procedure (tumor-spine-craniotomy), were recorded retrospectively from the electronic patient record (ERP) or hospital notes.

Immature platelet measurement
A fully automated analyzer (Sysmex XE-5000, Sysmex, Kobe, Japan) was used for measuring immature platelets [32].Automated assays were performed on EDTA-whole blood samples.The device applies fluorescent dyes containing polymethine and oxazine, which penetrate the cell membrane and stain the RNA.Stained cells are then sorted and passed through a semiconductor laser beam.A scattergram is provided using a computer algorithm after measurements of cell volume (by using forward scattered light) and RNA content (represented by fluorescence intensity).This system allows differentiation between immature (green dots) and mature platelets (blue dots) and reports the immature platelet fraction (IPF), defined as percentage of platelets (with large size and high fluorescence intensity) of the total optical platelet count [33].Further, the highly fluorescent immature platelet fraction (H-IPF%), a directly measured immature platelet parameter which reports the largest and highest fluorescent percentage within the IPF with a major amount of m-RNA is reported (highly fluorescent immature platelet fraction; H-IPF).The measurements were made in routinely acquired blood samples at admission (pre-operatively) and 24-72 hours after surgery at the neurosurgical ward (post-operatively).

Statistical analysis
Variables are presented as means and standard deviations (SD) or as medians and interquartile ranges, as appropriate.Differences between continuous variables were assessed using a two-sided Student's t-test or a Mann Whitney-U-test, if distributions were skewed.Categorical variables were analyzed using the Chi square test.Time-dependent receiver-operating curve (ROC) analysis was performed in order to determine an IPF or H-IPF level with a prognostic significance for surgical complications.The point maximizing sensitivity and specificity was considered as the optimal cutoff.In order to test whether immature platelets are an independent risk factor for surgical complications, a multivariate regression was performed.Based on the results in the univariate testing and previously published data, age, postoperative hemoglobin (Hb) and patients' comorbidities stratified by the American Society of Anesthesiologists' (ASA) classification system (ASA ≥2) were identified as potential confounders and included in the multivariate analysis [2,34].A p-value <.05 was considered statistically significant.For outcome testing, only the therapy-relevant CDG was used.All statistical tests were performed using SPSS statistics 27 (IBM SPSS, Inc.).

Results
A total of 391 patients were enrolled between February and September 2014.All included neurosurgical patients with an available in-house blood testing at admission were considered.Three-month outcome data were available in 359 patients.32 (8%) patients were not included in the 3-month follow-up since attempts to contact them failed.
The median age was 60 ± 16 years, and 45% of the patients were female.Almost half of the patients had a history of arterial hypertension, and 10% had a history of coronary artery disease.Baseline characteristics are shown in Table 1.241 (62%) patients underwent spine surgery, while a craniotomy was performed in 101 cases (26%).Patients classified within the column "tumor surgery" could have been included in the spine surgery group, the craniotomy group or none of them.11 of the 241 (5%) patients who underwent spine surgery were operated due to a tumor.In the craniotomy group 79 of the 101 patients (78%) underwent surgery due to a tumor disease.
Sixty-six percent of the patients did not develop any complications after surgery (Table 2).In 13% of the cases, a higher complication grade (>2 CDG) occurred.When also taking into account slight neurological complications, further 8% were graded ≥1 CDG.The therapy-relevant CDG score and outcome results are presented in detail in Table 2.At 3 months of follow up, death was reported in 22 patients (6%).

Discussion
This is the first prospective study showing a relationship between immature platelets and therapy-relevant perioperative complications in neurosurgical patients.Postoperatively elevated H-IPF at the surgical ward is independently associated with serious complications after surgery and might help in risk stratification of neurosurgical patients.
Prospective risk assessment studies in neurosurgical patients are scant.Considering elective cranial neurosurgery for example, there are only few prospective studies evaluating factors capable of predicting surgery-related outcomes [28].While there are scores like ASA, Charlson Comorbidity Score or Karnofsky Performance Index which are mainly used in preoperative risk stratification, there is little evidence for factors in the postoperative setting [34][35][36][37].In our study, preoperatively assessed immature platelets did not differ between patients that developed complications and those who did not.Postoperatively, however, there was a difference in H-IPF associated with a higher risk for surgical complications even after adjustment for physical state, age or bleeding (reflected by a drop in postoperative hemoglobin).Events are given as total numbers (n) and percentage (%) of 391 patients.Data are referring to therapy-relevant complications and number of deaths in theperioperative period.A reasonable explanation is that surgical trauma causes a cascade of neuroendocrine, inflammatory and acute-phase responses resulting in enhanced platelet activation and production [1,38].Both, IPF and H-IPF were elevated post-operatively.While the association between IPF and ischemic events is well described, this study adds new evidence that immature platelets may even help identifying patients with a higher risk for surgical complications in the post-op phase [19,27].It also allows new insights by highlighting H-IPF, a parameter which was previously presented by few investigators only [26,39].In a previous study, we were able to show an association between IPF as well as H-IPF with major cardiac and cerebrovascular events [2].In the present work, the observed association between perioperative complications was noticeably higher for the highly fluorescent immature platelet fraction (H-IPF), those reticulated platelets with a major amount of RNA.Therefore, H-IPF may represent even a more precise parameter for a timely detection of complications after neurosurgery [40,41].The identified cutoff (H-IPF≥0.95%)was similar to the one associated with cardiovascular death in patients with acute coronary syndrome (0.9%) [26].While an association between preoperatively assessed reticulated platelets and mortality after 30 days was described by other investigators, we did not observe any in 3 months-mortality, neither preoperatively nor postoperatively [3].This might be due to a different study design with differing surgical disciplines and follow-up periods.
In our study, Clavien Dindo Classification which is widely used in diverse surgery disciplines and therefore allows comparisons was chosen for assessing postoperative complications [42].Interestingly, H-IPF was associated with CDG in the univariate testing, even when minor postoperative neurological deficits not necessarily changing the therapeutic regimen were taken into account, possibly due to an inflammatory reaction or sympathetic stress [43].Further work is required to determine whether patients who exhibit an increase in the IPF/H-IPF post-op compared with their baseline values pre-op might be at higher risk of developing specific complications following neurosurgery and who might warrant even closer observation in the post-op phase.
Finally, in recent years, multivariate risk prediction models have been developed in neurosurgery to personalize therapy and reduce risks of harmful events after surgery [44,45].As an easily obtained and affordable parameter, immature platelets might add to these findings and indicate an emerging subclinical post-op complication in neurosurgical patients.Immature platelets and complications in neurosurgery 5

Study limitations
First, this study does not investigate the mechanisms behind the IPF/H-IPF elevation.Second, the exact threshold values applied by the analyzer defining cells as IPF or H-IPF are not public.Third, for future study designs, a time-scale for relevant pre-op medical conditions as well as post-op complications might help in a differentiated interpretation of elevated levels of immature platelets.Although this wasn't the focus of this particular study, differentiated Stroke-scales like National Institutes of Health Stroke Scale (NIHSS) or modified Rankin Scale might allow better insights from a clinically relevant aspect than CDG in patients developing stroke post-operatively [46,47].Further, sample size limited the number of variables that could be examined to confirm the relationship between H-IPF and adverse events.In order to confirm the cutoff value for H-IPF and to find a possible cutoff value for IPF associated with surgical complications, larger studies are needed in a preferably multicenter setting.Nevertheless, the current study shows that a single postoperative measurement of H-IPF in the surgical ward might improve risk stratification of neurosurgical patients.

Conclusion
In conclusion, we found that higher levels of postoperatively assessed immature platelets (H-IPF≥0.95%)are independently associated with an increased risk for serious surgical complications after neurosurgical procedures (CDG >2).

Table I .
Baseline characteristics.

Table II .
Perioperative complications and length of stay data.

Table III .
Immature platelet fraction and the association with therapy-orientated surgical complications.