Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study.

BACKGROUND: Delta neutrophil index (DNI) can be used as a biomarker for infection to predict patient outcomes. We aimed to investigate the relationship between DNI and clinical outcomes in trauma patients who underwent abdominal surgery.
MATERIALS AND METHODS: We retrospectively analyzed injured patients who underwent emergent abdominal surgery in the regional trauma center of Wonju Severance Christian Hospital between March 2016 and May 2018. Patient characteristics, operation type, preoperative and postoperative laboratory findings, and clinical outcomes were evaluated. Logistic regression analysis was performed for risk factors associated with mortality.
RESULTS: Overall, 169 patients (mean age, 53.8 years; 66.3% male) were enrolled in this study, of which 19 (11.2%) died. The median injury severity score (ISS) was 12. The non-survivors had a significantly higher ISS [25(9-50) vs. 10(1-50), p<0.001] and serum lactate level (9.00±4.10 vs. 3.04±2.23, p<0.001) and more frequent shock (63.2% vs 23.3%, p<0.001) and solid organ injury (52.6% vs. 25.3%, p = 0.013) than the survivors. There were significant differences in postoperative DNI between the two groups (p<0.009 immediate post-operation, p = 0.001 on postoperative day 1 [POD1], and p = 0.013 on POD2). Logistic regression analysis showed that the independent factors associated with mortality were postoperative lactate level (odds ratio [OR] 1.926, 95% confidence interval [CI] 1.101-3.089, p = 0.007), postoperative sequential organ failure assessment score (OR 1.593, 95% CI 1.160-2.187, p = 0.004), and DNI on POD1 (OR 1.118, 95% CI 1.028-1.215, p = 0.009). The receiver operating characteristics curve demonstrated that the area under the curve of DNI on POD1 was 0.887 (cut-off level: 7.1%, sensitivity 85.7%, and specificity 84.4%).
CONCLUSIONS: Postoperative DNI may be a useful biomarker to predict mortality in trauma patients who underwent emergent abdominal surgery.

1. Introduction

The current biomarkers for diagnosis of sepsis or infections include white blood cell (WBC) count, lactic acid, procalcitonin, and C-reactive protein (CRP) [1-3]. The release of immature neutrophils into the bloodstream during infection or sepsis leads to an elevation of the immature/total granulocyte ratio which is defined as neutrophil ‘left-shift’. This granulocytic ‘left-shift’ or increase in immature granulocyte (IG) rate is commonly used as a diagnostic marker of infection or sepsis in the clinical setting. However, it is difficult to accurately measure IG using a microscopic examination of blood smears, and its diagnostic value remains controversial [4, 5]. Technological advances in an automated cell analyzer have enabled the acquisition of the delta neutrophil index (DNI) using leukocyte differentials obtained from two independent channels–the myeloperoxidase channel and the lobularity/nuclear density channel. The DNI is calculated as the difference between leukocyte differentials measured in these two channels, which reflects the proportion of circulating IG [6]. Several studies reported that DNI was associated with disease severities of sepsis or septic shock and mortality in patients with various infectious conditions such as bacteremia, pneumonia, and peritonitis [7-11]. Moreover, recent studies showed that DNI was associated with the severity and prognosis of non-infectious inflammation-related diseases, such as acute myocardial infarction, pulmonary embolism, upper gastrointestinal hemorrhage, and cardiac arrest [12-15]. However, there are few studies about the use of DNI in trauma patients. Therefore, the aim of this study was to evaluate the usefulness of DNI as a predictor of mortality in trauma patients who underwent emergent abdominal surgery.

2. Patients and methods

2.1 Patient selection and data collection

The study was approved by the institutional review board of Wonju Severance Christian Hospital (IRB no. CR319077). All data were fully anonymized before access and IRB waived the requirement for informed consent. Among 6291 injured patients who were admitted in the regional trauma center of a tertiary university hospital between March 2016 and May 2018, 173 patients who underwent emergent abdominal surgery were enrolled in this study. After exclusion of four patients who died within six hours of admission, the final study population was 169 (Fig 1).

Fig 1

Patient flow chart.

The primary end-point was to evaluate the effectiveness of DNI to predict postoperative mortality in injured patients who underwent emergent abdominal surgery. The secondary end-point was to compare DNI with other biomarkers for prediction of mortality and to access the cut-off level of DNI. The demographic and clinical characteristics of the patients such as age, sex, injury mechanism, injury severity score (ISS), associated injury (abbreviated injury scale, AIS ≥3), initial shock, diagnosis, gastrointestinal (GI) perforation, solid organ injury, serum lactate, and sequential organ failure assessment (SOFA) score on intensive care unit (ICU) admission were retrospectively reviewed.

2.2 DNI and laboratory tests

We reviewed the results of laboratory tests such as DNI, WBC count, and C-reactive protein (CRP), at four time points (initially in the emergency room [ER], immediate postoperative, postoperative day (POD) 1, POD2). In our institution, DNI is presented along with complete blood count tests without extra charge. A specific type of automatic cell analyzer (ADVIA 120/212; Siemens, Tarrytown, NY, USA) was used for the calculation of DNI. This flow cytometry-based hematologic analyzer uses two independent WBC counting methods–a myeloperoxidase (MPO) channel and a lobularity/nuclear density channel. DNI value was calculated using the following formula: DNI = (leukocyte subfraction assayed using the MPO channel of a cytochemical reaction)–(leukocyte subfraction assayed using the nuclear lobularity channel based on reflected light beam measurements) [6].

2.3 Statistical analysis

Continuous variables were presented as the mean±standard deviation or the median values (ranges), and comparative analysis was performed using a Student’s t-test. Categorical variables were analyzed by the Chi-square test and Fisher’s exact test. To identify the independent risk factors for mortality, a multivariate analysis was performed using logistic regression. A receiver operating characteristics (ROC) curve was constructed, and the Youden Index method was used to find the optimal cut-off values for lactate, DNI, and SOFA score to predict mortality. All statistical analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA). Statistical significance was accepted for p <0.05.

3. Results

3.1 Baseline clinical characteristics

The study enrolled 169 consecutive injured patients who underwent emergent abdominal surgery during the study period (27 months). The mean age was 53.8±17.1 years, and 112 (66.3%) patients were men. Most common injury mechanism was road traffic collision, and 57 patients (33.7%) had associated injuries (AIS ≥3). The ISS was greater than 15 in 78 (46.2%) patients, and 47 (27.8%) patients initially had shock status. The mean level of serum lactate was 3.73±3.14 mmol/L, and the median SOFA score at the time of ICU admission was 4 (0–18). Nineteen (11.2%) patients died, and the most common cause of death was sepsis, followed by hemorrhage and multiple organ failure. Thirteen patients (68.4%) died within 7 days, and 4 patients (26.3%) died between 7 and 28 days. The other one died 28 days later (Table 1). The most common injury site was the small bowel (40.8%) followed by liver (11.8%), spleen (9.5%), and abdominal wall (5.9%). GI perforations were identified in 62 (36.7%) patients (Table 2).

Table 1

Patient characteristics.

Variable	N = 169 (%)
Age	53.8±17.1
Sex (male)	112 (66.3)
Injury severity score	13 (1–50)
Injury severity score > 15	78 (46.2)
Associated injury (AIS ≥3)	57 (33.7)
Injury mechanism
Road traffic collision	104 (61.5)
Penetrating trauma	31 (18.3)
Hit & crush	19 (11.2)
Fall	6 (3.6)
Slip down	5 (3.0)
Others	4 (2.4)
Initial shock	47 (27.8)
Initial WBC	12064±5904
Initial DNI (%)	0.6 (0–52.8)
Initial CRP (mg/dL)	0.29 (0–30.10)
Initial serum lactate	3.73±3.14
Postoperative WBC	10674±5226
Postoperative DNI	3.6 (0–48.8)
Postoperative CRP	0.29 (0–26.40)
Postoperative lactate	3.43±2.60
SOFA score on ICU admission	4 (0–18)
Mortality	19 (11.2)
Sepsis	7 (36.8)
Hemorrhage	6 (31.6)
MODS	2 (10.5)
Others	4 (21.1)

AIS, abbreviated injury scale; WBC, white blood cell; DNI, delta neutrophil index; CRP, C-reactive protein; SOFA, sequential organ failure assessment; MODS, multiple organ dysfunction syndrome.

Table 2

Patient diagnosis.

Diagnosis	N = 169
Small bowel injury	69 (40.8%)
Liver injury	20 (11.8%)
Colorectal injury	18 (10.7%)
Spleen injury	16 (9.5%)
Abdominal wall injury	10 (5.9%)
Major vascular injury	9 (5.3%)
Pancreatic injury	5 (3.0%)
Stomach injury	4 (2.4%)
Other	12 (7.1%)
Multi-organ injury	6 (3.6%)
GI perforation	62 (36.7%)

GI, gastrointestinal.

Other; 5 omental injuries, 3 retroperitoneal hemorrhages, 1 gallbladder injury, 1 teratoma rupture, 1 renal injury, 1 none.

3.2 Comparison between survivors and non-survivors

ISS [25(9–50) vs 10(1–50), p <0.001] and SOFA score [9.5 (6–18) vs 3 (0–15), p <0.001] on ICU admission were significantly higher in non-survivors than in survivors. Moreover, non-survivors had significantly more frequent associated injury (63.2 vs. 30%, p = 0.004), shock in ER (63.2 vs. 23.3%, p <0.001), and solid organ injury (52.6 vs. 25.3%, p = 0.013) than survivors. Serum lactate was significantly higher in non-survivors than in survivors on initial measurement in ER (9.00±4.10 mmol/L vs. 3.04±2.23 mmol/L, p <0.001) and immediate post-operation (7.64±3.54 mmol/L vs. 2.82±1.73 mmol/L, p <0.001) (Table 3).

Table 3

Comparison between survivors and non-survivors.

	Survivor (n = 150)	Non-survivor (n = 19)	P-value
Age (year)	53.9±16.8	53.6±19.6	0.944
Sex (male)	98 (65.3%)	14 (73.7%)	0.468
Injury severity score (ISS)	10 (1–50)	25 (9–50)	<0.001
Injury severity score (ISS) > 15	62 (41.3%)	16 (84.2%)	<0.001
Associated injury (AIS ≥3)	45 (30%)	12 (63.2%)	0.004
Shock	35 (23.3%)	12 (63.2%)	<0.001
Initial WBC (/mm3)	12254±5670	10558±7514	0.353
Initial DNI (%)	0.45 (0–44.7)	3.3 (0–52.8)	0.053
Initial CRP (mg/dL)	0.29 (0–30.10)	0.29 (0.29–23.80)	0.215
Initial Lactate (mmol/L)	3.04±2.23	9.00±4.10	<0.001
GI perforation	57 (38.0%)	5 (26.3%)	0.319
Solid organ injury	38 (25.3%)	10 (52.6%)	0.013
SOFA score on ICU admission	3 (0–15)	9.5 (6–18)	<0.001
Postoperative shock	12 (8.0%)	18 (94.7%)	<0.001
Postoperative WBC (/mm3)	11096±5091	7161±5139	0.002
Postoperative DNI (%)	3.3 (0–41.2)	9.0 (2.3–48.8)	0.009
Postoperative CRP (mg/dL)	0.32 (0–26.40)	0.29 (0–13.70)	0.921
Postoperative lactate (mmol/L)	2.82±1.73	7.64±3.54	<0.001
POD1 WBC (/mm3) (n = 163)	9846±3620	7017±3949	0.005
POD1 DNI (%) (n = 162)	1.5 (0–35.9)	17.0 (1.4–57.0)	0.001
POD1 CRP (mg/dL) (n = 153)	10.40 (0–35.80)	2.80 (0–17.30)	0.014
POD2 WBC (/mm3) (n = 158)	8695±3080	5561±3972	0.001
POD2 DNI (%) (n = 157)	0.8 (0–52.9)	27.9 (0–62.1)	0.004
POD2 CRP (mg/dL) (n = 145)	15.20 (0–35.10)	7.32 (0–32.1)	0.013
ICU stay (day)	4 (1–90)	3 (1–58)	0.450
Duration of hospitalization (day)	21 (2–697)	3 (1–58)	<0.001

AIS, abbreviated injury scale; WBC, white blood cell; DNI, delta neutrophil index; CRP, C-reactive protein; SOFA, sequential organ failure assessment; POD, postoperative day; ICU, intensive care unit.

WBC counts were significantly higher in survivors than in non-survivors on immediate post-operation (11096±5091 vs. 7161±5139, p = 0.002), POD1 (9846±3620 vs. 7017±3949, p = 0.005), and POD2 (8695±3080 vs. 5561±3972, p = 0.001) [Fig 2A]. In addition, CRP was significantly higher in survivors than in non-survivors on POD1 [10.40 mg/dL (0–35.80) vs. 2.80 mg/dL (0–17.30), p = 0.014] and POD 2 [15.20 mg/dL (0–35.10) vs. 7.32 mg/dL (0–32.10), p = 0.013] [Fig 2B]. DNI in non-survivor was significantly higher than in survivors on immediate post-operation [9.0 (2.3–48.8) vs. 3.3% (0–41.2), p = 0.009], POD1 [17.0 (1.4–57) vs. 1.5% (0–35.8), p = 0.001], and POD 2 [27.9 (0–62.1) vs. 0.8% (0–52.9), p = 0.004] [Fig 2C].

Fig 2

(A) White blood cell (WBC) count during the perioperative period. The mean WBC counts of survivors were significantly higher than that of the non-survivors on the immediate postoperative day, postoperative day 1, and postoperative day 2. (B) C-reactive protein (CRP) during the perioperative period. The mean CRP levels of the survivors were significantly higher than that of the non-survivors on postoperative day 1 and postoperative day 2. (C) Delta neutrophil index (DNI) during the perioperative period. The mean DNI of the non-survivors was significantly higher than that of the survivors on the immediate postoperative day, postoperative day 1, and postoperative day 2.

3.3 Independent risk factors for mortality in critically ill and injured patients who underwent emergent abdominal surgery

The logistic regression model using variables that were noted in the univariate analysis showed that immediate postoperative lactate [odds ratio (OR) 1.926 [95% confidence interval (CI) 1.201–3.089], p = 0.007], SOFA score on ICU admission [OR 1.593 (95% CI 1.160–2.187), p = 0.004], and DNI on POD1 [OR 1.118 (95% CI 1.028–1.215), p = 0.009] were independent risk factors associated with mortality (Table 4).

Table 4

Independent risk factors for postoperative mortality.

Variable	Risk factors for mortality
	Odd ratio (95% CI)	P-value
Initial shock	0.096 (0.007–1.311)	0.079
Postoperative lactate level (mmol/L)	1.926 (1.201–3.089)	0.007
SOFA score on ICU admission	1.593 (1.160–2.187)	0.004
POD1 DNI (%)	1.118 (1.028–1.215)	0.009

Injury severity score>15, solid organ injury, combined injury, initial shock, postoperative lactate level, SOFA score on ICU admission, and DNI on POD1 were used as variables for multivariate analysis. CI, confidence interval; DNI, delta neutrophil index; SOFA, sequential organ failure assessment; POD1, postoperative day 1; ICU, intensive care unit.

3.4 Performance of DNI and other laboratory markers in critically ill and injured patients who underwent emergent abdominal surgery

When the ROC curves of the postoperative lactate, SOFA score on ICU admission, and DNI on POD1 were conducted to predict mortality, area under curve (AUC) of the immediate postoperative lactate, SOFA score on ICU admission, and DNI on POD1 were 0.874 (95% CI, 0.773–0.975, p <0.001), 0.941 (95% CI, 0.898–0.984, p <0.001), and 0.887 (95% CI 0.798–0.976, p <0.001), respectively. The optimal cut-off points for the postoperative lactate, SOFA score on the ICU admission, and DNI on the POD1 were 5.105 mmol/L (sensitivity: 71.4%, specificity: 92.6%), 6.5 (sensitivity: 92.9%, specificity: 84.4%), and 7.1% (sensitivity: 85.7%, specificity: 84.4%), respectively (Fig 3).

Fig 3

Receiver operating characteristics (ROC) curves for the delta neutrophil index (DNI) (postoperative day 1, POD1), lactate (immediate postoperative, PostOP), and sequential organ failure assessment (SOFA) score (PostOP) between the survivors and non-survivors.

The area under the ROC curve was 0.887 (95% confidence interval, 0.798–0.976) for the DNI (POD1).

DNI POD1 (AUC = 0.887) (95% CI 0.798–0.976, p <0.001)

cut-off level: 7.1%

sensitivity: 85.7%, specificity: 84.4%

PostOP Lactate (AUC = 0.874) (95% CI, 0.773–0.975, p <0.001)

cut-off level: 5.105

sensitivity: 71.4%, specificity: 92.6%

PostOP SOFA (AUC = 0.941) (95% CI, 0.898–0.984, p <0.001)

cut-off level: 6.5

sensitivity: 92.9%, specificity: 84.4%

4. Discussion

This study showed that DNI on the POD1 was an independent risk factor to predict the mortality in critically ill and injured patients who underwent emergent abdominal surgery. Moreover, the ROC curve for the DNI on POD1 confirmed that the optimal cut-off for predicting mortality was 7.1%, and the sensitivity (85.7%) and specificity (84.4%) were high with an AUC of 0.887. Mean DNI had a different pattern throughout the study period compared with other biomarkers such as WBC count and CRP. The mean DNI in survivors decreased after initial elevation whereas it continuously increased in non-survivors. Additionally, a recent study on DNI in patients with sepsis caused by peritonitis reported that DNI on POD3 was an independent risk factor for postoperative mortality, and the patterns of mean DNI were different between survivors and non-survivors. Moreover, the AUC for DNI was 0.88, and the optimal cut-off value was 7.8%, with a sensitivity of 77.3% and specificity of 95.9% [7]. This result is quite similar to that of our study. Indeed, in our hospital, it was possible to recognize the high probability of death in patients with elevated DNI level above 7.1% the day after surgery. DNI helped surgeons explain the patient’s condition earlier to the caregiver and determine further evaluation and general ward transfer.

Previous studies about surgical and medical patients showed that DNI was a useful biomarker to predict disease severity or prognosis in patients with various infections or sepsis [7, 10, 16, 17]. However, studies about usefulness of DNI in patients who had tissue injury or hemorrhage due to trauma are limited [18]. Systematic inflammatory response syndrome (SIRS) is initiated within 30 minutes after severe injury, which is associated with an inflammatory response to hemorrhage or tissue damage rather than infections. Damage-associated molecular patterns (DAMPs) are released into the extracellular space by tissue damage that triggers an inflammatory response without infection. DAMPs activate the innate immune systems such as neutrophils, monocytes, and complements. This change can result in endothelial and organ damage and immunoparesis. As a result, a vicious cycle is triggered due to continuous inflammation and immune activation. In this process, neutrophils are activated initially by SIRS, but their bactericidal function is markedly impaired. This functional impairment is compensated by the release of immature banded neutrophils [19]. In the present study, DNI appears to be correlated with the severity of SIRS induced by tissue damage and hemorrhage in patients with severe trauma. In other words, increase in the value of DNI in non-survivors indicated severe SIRS, which may result in poor clinical outcomes as a result of progressive persistent inflammation, immunosuppression, and catabolism syndrome [20]. A recent study on trauma patients who were admitted in ICU reported that high DNI values at 12 and 24 hours from admission were strong independent predictors of multiple organ dysfunction syndrome (MODS). Increased DNI at 12 hours was a strong independent predictor of short-term mortality. In addition, the cut-off value of DNI at 12 hours to predict 30-day mortality was 5.3% in that study [18], which was similar to the result in our study.

In this study, we found that although 62 (36.7%) patients had GI perforation, there was no significant difference in the mortality rate according to GI perforation (patients with GI perforation 8.1% vs. patients without GI perforation 13.1%, p = 0.319). This result has several implications. First, most patients with abdominal injuries underwent early surgery if necessary, and therefore, many patients with GI perforation might not progress to septic shock. Second, there are causes other than infection that exacerbate the condition of a trauma patient. As described above, SIRS due to tissue damage and hemorrhage was a major deterioration factor in the early stage of major trauma and was closely associated with patient severity and prognosis. Severe SIRS seemed to cause MODS and eventually lead to death [19, 20].

Several studies suggested that WBC count was only a nonspecific indicator of stress such as infection, inflammation, tissue necrosis, and hemorrhage in injured patients [21, 22]. Another study on patients with abdominal injury showed that the WBC count within 24 hours after admission had limited diagnostic value for predicting hollow viscus injury [23]. Similarly, the present study showed that the WBC count had poor predictability in distinguishing survivors from non-survivors over time after surgery. Although there was a significant difference in the CRP level between the two groups on POD1 and POD2, it is difficult to use CRP as a predictor of mortality, because the survivor group had higher CRP level than the non-survivor group, and CRP had a relatively slow changing pattern. A study performed in patients with multiple trauma showed that CRP increased easily and had very slow kinetics compared to other biomarkers [24]. In contrast, several studies have shown that initial lactate and lactate clearance are clinically useful in predicting mortality of trauma patients [25-27]. In our study, we found that postoperative lactate level was an independent risk factor for postoperative mortality and had a high predictability of mortality with an optimal cut-off level of 5.105 mmol/L (AUC 0.874, sensitivity 71.4%, and specificity: 92.6%). Taken together, DNI on POD1, postoperative SOFA score, and postoperative lactate may be used complementarily to predict patient death.

There are some limitations to our study. First, it is hard to confirm the usefulness of DNI, because of the small sample size and single institutional study. Second, data of the present study did not include serial levels of procalcitonin which is currently an important biomarker for diagnosis of infection and sepsis, because reimbursement for procalcitonin is limited within twice a week in the Korean national medical insurance. Third, there may be a selection bias in the present study, because of its retrospective nature. Despite these limitations, our study may be meaningful in that it is the first study to evaluate the usefulness of DNI to predict clinical outcome in abdominal trauma patients who underwent emergency surgery. In the future, large-scale prospective studies will be needed to confirm the results of our study.

Comparison of other variables between survivors and non-survivors.

(DOCX)

Click here for additional data file.

(XLSX)

Click here for additional data file.

10 Feb 2020

PONE-D-19-35229

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

PLOS ONE

Dear Dr. Jang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Mar 26 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Itamar Ashkenazi

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This research received no specific grants from funding agencies in the public, commercial, or not-for-profit sectors."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

3. Thank you for stating the following in the Competing Interests section:

"The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: Saidabad Clinic

a.     Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article by Jang explores mortality prediction in trauma patients undergoing emergent abdominal surgery.

The DNI is the immature granulocyte fraction determined by subtracting the fraction of mature PMN leukocytes from the sum of myeloperoxidase-reactive cells and reflects the number of immature neutrophils. It has been used in sepsis and several other conditions.

Several questions: 1. What were the inclusion/exclusion criteria?

2. Was the study exploring in hospital deaths only or was there an attempt to examine 28 days mortality or longer? How does the death distribution look like?

3. In Table 1&3, is this the mean?

4. Can a predictor based on DNI difference and the other idependent variable identified and POD be presented in an aggregate score so that it would have clinical utility for the practicing surgeon?

5. Could the author add a sentence about the scientific rationale why increased DNI is associated with mortality?

Reviewer #2: The manuscript looks interesting, well written and intelligible.

The methodology of the study looks appropriate and rigorous.

The work seems to add something new to the field of trauma research.

My recommendation is to accept.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Roberto Faccincani

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

13 Feb 2020

Reviewer #1: The article by Jang explores mortality prediction in trauma patients undergoing emergent abdominal surgery.

The DNI is the immature granulocyte fraction determined by subtracting the fraction of mature PMN leukocytes from the sum of myeloperoxidase-reactive cells and reflects the number of immature neutrophils. It has been used in sepsis and several other conditions.

Several questions: 1. What were the inclusion/exclusion criteria?

�--> To clarify the inclusion and exclusion criteria, we added a patient flow chart to Figure 1.

2. Was the study exploring in hospital deaths only or was there an attempt to examine 28 days mortality or longer? How does the death distribution look like?

�--> Twelve (63.2%) patients died within 7 days, and 4 (21.1%) died between 7 and 28 days. The other one died of sepsis after 28 days.

3. In Table 1&3, is this the mean?

�--> As we described in ‘statistical analysis’ section of patients and methods, continuous variables were mainly presented as the mean±standard deviation. When no following a normal distribution, it was expressed as median(range).

4. Can a predictor based on DNI difference and the other idependent variable identified and POD be presented in an aggregate score so that it would have clinical utility for the practicing surgeon?

� --> In multivariated analysis, POD1 DNI, postoperative lactate, and postoperative SOFA score were identified as independent risk factors related to mortality. Authors confirmed the AUC using the ROC curve of these variables, and confirmed that the cut-off value of POD1 DNI was the criterion having the highest sensitivity and specificity at 7.1%. The postoperative lactate and SOFA scores were also analyzed in the same way, indicating cut-off levels of 5.1 and 6.5.

Indeed, in our hospital, it was possible to recognize the high probability of death in patients with elevated DNI levels above 7.1% the day after surgery. Doctors were able to explain the patient’s condition earlier to the caregiver and helped determine further evaluation and general ward transfer. However, we did not calculate a score that combines these three scores.

5. Could the author add a sentence about the scientific rationale why increased DNI is associated with mortality?

� --> DNI can be seen as a value representing the proportion of immature granulocytes. As SIRS caused by tissue damage increases the release of damage-associated molecular patterns (DAMPs), SIRS becomes more severe and the multiple organ failure of the patient worsens. In addition, when neutrophils are functionally impaired by SIRS, the release of immature banded neutrophils increases to compensate for this, which is indicated by an increase in DNI. This is described in the discussion section (243-249th line).

Reviewer #2: The manuscript looks interesting, well written and intelligible.

The methodology of the study looks appropriate and rigorous.

The work seems to add something new to the field of trauma research.

My recommendation is to accept.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

17 Feb 2020

PONE-D-19-35229R1

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

PLOS ONE

Dear Dr. Jang,

I am returning the revised manuscript since I need some clarifications.

You respond to each of the first reviewer's questions but except in one case I do not see in your response letter a description of what changes did you make within the manuscript. Associated to this issue, when I examined the marked copy, I did not always understand why certain places were highlighted.

I suggest the following. Under each of the reviewer's questions introduce two subheadings: authors' response, desciption of changes made. Thus for each of the questions we will have your answer followed by an explanation if changes were made in the manuscript and their location. If no changes were made, just write that no changes were made.

Thank you,

Itamar Ashkenazi M.D.

Academic Editor

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Feb 2020

Dear Academic Editor

As you mentioned, we answered reviewer’s questions point-by-point and clearly stated whether the manuscript was corrected or not.

Also we highlighted again in the only changed points of the manuscript.

Thank you for your comments.

Ji Young Jang M.D.

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article by Jang explores mortality prediction in trauma patients undergoing emergent abdominal surgery.

The DNI is the immature granulocyte fraction determined by subtracting the fraction of mature PMN leukocytes from the sum of myeloperoxidase-reactive cells and reflects the number of immature neutrophils. It has been used in sepsis and several other conditions.

Several questions:

1. What were the inclusion/exclusion criteria?

�--> To clarify the inclusion and exclusion criteria

We added a patient flow chart as Figure 1(line 87-88).

2. Was the study exploring in hospital deaths only or was there an attempt to examine 28 days mortality or longer? How does the death distribution look like?

� --> Thirteen (68.4%) patients died within 7 days, and 5 (26.3%) died between 7 and 28 days. The other one died of sepsis after 28 days.

We added this contents to the Results section (line 140-141).

3. In Table 1&3, is this the mean?

� --> As we described in ‘statistical analysis’ section of patients and methods, continuous variables were mainly presented as the mean±standard deviation. When no following a normal distribution, it was expressed as median(range).

4. Can a predictor based on DNI difference and the other idependent variable identified and POD be presented in an aggregate score so that it would have clinical utility for the practicing surgeon?

�--> In multivariated analysis, POD1 DNI, postoperative lactate, and postoperative SOFA score were identified as independent risk factors related to mortality. Authors confirmed the AUC using the ROC curve of these variables, and confirmed that the cut-off value of POD1 DNI was the criterion having the highest sensitivity and specificity at 7.1%. The postoperative lactate and SOFA scores were also analyzed in the same way, indicating cut-off levels of 5.1 and 6.5.

Indeed, in our hospital, it was possible to recognize the high probability of death in patients with elevated DNI levels above 7.1% the day after surgery. DNI helped surgeons explain the patient’s condition early to the caregiver and determine further evaluation and general ward transfer. However, we did not calculate a score that combines these three scores.

Authors added this content to the discussion section (line 232-235).

5. Could the author add a sentence about the scientific rationale why increased DNI is associated with mortality?

�--> DNI can be seen as a value representing the proportion of immature granulocytes. As SIRS caused by tissue damage increases the release of damage-associated molecular patterns (DAMPs), SIRS becomes more severe and the multiple organ failure of the patient worsens. In addition, when neutrophils are functionally impaired by SIRS, the release of immature banded neutrophils increases to compensate for this, which is indicated by an increase in DNI.

Since this content is already described in the discussion section, authors did not make any additional descriptions (239-248th line).

Reviewer #2: The manuscript looks interesting, well written and intelligible.

The methodology of the study looks appropriate and rigorous.

The work seems to add something new to the field of trauma research.

My recommendation is to accept.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Feb 2020

PONE-D-19-35229R2

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

PLOS ONE

Dear Dr. Jang,

Thank you for submitting your manuscript to PLOS ONE. Thank you also for clarifying the reviewers' comments and making the appropriate changes. I want to aplogize but there is one more issue that needs to be dealt with before considerning this manuscript suitable for publication.

ISS and SOFA are scores based on several components.  Considering these as continuous variables is not appropriate.  There is no ISS 15 for example.  ISS 16 is not twice as bad as ISS 8...

I recommend either presenting these as medians and ranges, or presenting proportion of patients above a certain threshold. For example, proportion of patients with ISS of 16 or higher.

Please introduce these before I hand in the final decision.

We would appreciate receiving your revised manuscript by Apr 05 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Itamar Ashkenazi

Academic Editor

PLOS ONE

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Feb 2020

PONE-D-19-35229R2

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

PLOS ONE

Dear Dr. Jang,

Thank you for submitting your manuscript to PLOS ONE. Thank you also for clarifying the reviewers' comments and making the appropriate changes. I want to aplogize but there is one more issue that needs to be dealt with before considerning this manuscript suitable for publication.

ISS and SOFA are scores based on several components. Considering these as continuous variables is not appropriate. There is no ISS 15 for example. ISS 16 is not twice as bad as ISS 8...

I recommend either presenting these as medians and ranges, or presenting proportion of patients above a certain threshold. For example, proportion of patients with ISS of 16 or higher.

Please introduce these before I hand in the final decision.

--> Thank you for your important comment.

The authors discussed the reviewer’s opinion and decided to present proportion (ISS>15) and median (range) for the ISS. However, the SOFA score on ICU admission was linearly correlated with the mortality rate of patients, we decided to keep it as a continuous variable. Since the standard deviation of SOFA score is relatively large, we modified it to median (range).

The multivariate analysis was re-run using the modified ISS>15 and confirmed no change in the main results. In addition, tables and manuscript were revised.

Reference

Ferreira et al. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA.2001 Oct 10;286(14):1754-8

Thank you gain.

Ji Young Jang M.D.

Submitted filename: Response to reviewers 2.docx

Click here for additional data file.

24 Feb 2020

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

PONE-D-19-35229R3

Dear Dr. Jang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Itamar Ashkenazi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

6 Mar 2020

PONE-D-19-35229R3

Delta neutrophil index for predicting mortality in trauma patients who underwent emergent abdominal surgery: A case controlled study

Dear Dr. Jang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Itamar Ashkenazi

Academic Editor

PLOS ONE