Article, Pulmonology

The diagnostic value of serum pentraxin 3 levels in pulmonary contusion

a b s t r a c t

Purpose: To investigate the difference in pentraxin 3 (PTX 3) levels between patients with pulmonary contusion and healthy volunteers.

Materials and methods: This study was conducted with a group of 20 trauma patients diagnosed with pulmonary contusion and 30 healthy individuals enrolled as a control group in a Tertiary university hospital.

Results: Median PTX 3 levels were 7.05 (3.29-13.1), ng/ml in the contusion group and 1.03 (0.7-1.58) ng/ml in the control group. PTX 3 titers were significantly higher in patients with pulmonary contusion compared to those of the control group (p b 0.001). An area under the curve (AUC) value of 0.968 investigated using ROC analysis to determine the diagnostic value of the PTX-3 in pulmonary contusion patients was mea- sured. A PTX-3 cut-off value of 2.06 produced 95.5% sensitivity and 86.7% specificity.

Conclusion: PTX 3 levels in pulmonary contusion increased significantly compared to the healthy control group. If supported by wider series, PTX 3 may be expected to be capable of use as a marker in pulmonary contusion.

(C) 2016

Introduction

Pulmonary contusion is a common and significant cause of mortality and morbidity, particularly in cases of blunt Thoracic trauma [1]. In addi- tion to local injury in the lung in pulmonary contusion, systemic inflam- matory response is also triggered. Oxidative injury also accelerates the destructive process [1-3].

Pentraxins are a multi-functional Protein Superfamily involved in in- flammatory response. Circulating levels of pentraxin 3 (PTX 3), one of the main acute phase reactants, can increase up to 3-5 times above basal values in Inflammatory conditions. PTX 3 is produced in the in- flammation site and binds immediately to the endothelium. PTX 3 levels are thought to be an independent marker of Inflammatory processes [4]. Lung injury associated with pulmonary contusion is frequently ob- served in severe traumas, particularly chest traumas. Endothelial dys- function occurs with capillary leakage in the pulmonary parenchyma. Post-traumatic proinflammatory response (such as burns, surgical in- terventions, soft-tissue damage and thoracic injury) increases alongside a decrease in microcirculation. Various cytokines, such as IL-1, IL-6, IL-8

* Corresponding author.

E-mail address: [email protected] (O. Tatli).

and TNF-?, are released in association with trauma, tissue injury and cell death, and these trigger PTX 3 release by stimulating Endothelial cells, muscle cells, fibroblasts, adipocytes and chondrocytes. PTX 3 further increases the release of cytokines such as IL-1, IL-6, IL-8 and TNF-? by stimulating pro-inflammatory cytokines and complement activation (Fig. 1). Respiratory distress and hypoxemia associated with this develop due to pulmonary contusion-induced pulmonary edema. When pulmonary contusion is accompanied by acute respi- ratory distress syndrome, mortality levels reach 56-76% [5-8]. Post-traumatic proinflammatory response increases with a decrease in pulmonary microcirculation.

In the light of this information from the literature, acute inflammato- ry response plays a significant role in the pathophysiology of pulmonary contusion. PTX 3 levels might thus be anticipated to increase, as an in- flammatory marker, in pulmonary contusion, an acute inflammatory process. This study was intended to investigate whether or not PTX 3 in- creases in patients with pulmonary contusion.

Materials and methods

This research was performed as a prospective clinical study. Patients presenting to the Karadeniz Technical University (KTU) Medical Faculty

http://dx.doi.org/10.1016/j.ajem.2016.11.030

0735-6757/(C) 2016

426 O. Tatli et al. / American Journal of Emergency Medicine 35 (2017) 425428

Image of Fig. 1

Fig. 1. A comparison of serum PTX 3 levels in the pulmonary contusion and control groups.

Emergency Medicine Department and diagnosed with pulmonary con- tusion within 6 months of receipt of KTU Medical Faculty Ethical Com- mittee approval (Approval no:2016-122) were included in the study. Twenty patients diagnosed with pulmonary contusion in the emergen- cy department and 30 healthy volunteers were enrolled.

Patients presenting to the emergency department with isolated tho- racic trauma and aged over 18 were included. Subjects with non-thorac- ic trauma, sepsis, vasculitis, burns or a recent history of surgery or refusing to participate were excluded. Various clinical and demographic characteristics of the patients enrolled, such as symptoms, physical ex- amination findings, X-ray and Computerized tomography findings were recorded onto study forms.

As much serum specimen as the vacuum would permit was placed into CBC tubes containing EDTA in order to measure PTX 3 levels at time of presentation. Following centrifugation for 10 min at 3000 rpm, plasma was separated and stored at -80 ?C.

Determination of plasma pentraxin 3 levels

PTX 3/TNF-inducible gene 14 protein (TSG-14) levels in human plas- ma were determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Cat No: DPTX30, Lot: 334734, Minneapolis, USA) in line with the manufacturer’s recommendations. Plasma sam- ples stored at – 80 ?C were brought to Room temperature. Into all streptavidin-coated wells was added 200 mL PTX 3 biotinylated anti- body. This was then incubated for 60 min in a microplate shaker. At the end of this period, in order to ready the plates for use, each plate was washed with 300 mL washing buffer to remove unbound antibod- ies. PTX 3/TSG-14 standards were prepared in line with the kit proce- dures. Standards, controls and specimens were activated with 30-min pretreatment with D solution. Into each plate well was added 100 mL Assay Diluent RD1-56 solution. Next, 20 mL from the pre-treated stan- dards, controls and specimens was added to this solution and incubated for 120 min at room temperature in a microplate shaker. Following in- cubation, the plate was washed four times with plate buffer with a plate washer. Next, 200 uL PTX 3 conjugate was added to each well and incubated for 120 min at room temperature in a microplate shaker. Following incubation, the plate was washed four times with plate buffer with a plate washer. Subsequently, 200 uL of TMB substrate solutions

was added to each well for coloring and left to incubate for 30 min at room temperature. At the end of 30 min, 50 uL of stop solution was added to each well, and the color of the specimens was observed to turn to yellow. Absorbances of specimens were measured at a wavelength of 450 nm on a VERSA (designed by Molecular Devices in California, USA) microplate reader. A standard chart was prepared using absorbance values obtained against standard concentrations (Fig. 2). PTX 3 in specimens were calculated as ng/mL using this

Fig. 2. Chart showing PTX 3 interaction in trauma. IL = interleukin, TNF = tumor necrosis factor, LPS = lipopolysaccharide.

O. Tatli et al. / American Journal of Emergency Medicine 35 (2017) 425428 427

standard chart. The reliability of intra-assay distribution of this ELISA method was 3.6% and that of inter-assay was 4.9%.

Statistical analysis

Statistical analyses were performed on SPSS 23 software. Values for the control and patient groups were expressed as percentages for cate- goric variables, while normally distributed numerical data were expressed as mean plus standard deviation (X +- SD) and non-normally distributed data as median and interquartile percentages. Normality of distribution of parameters was assessed using the Shapiro-Wilk test. Normally distributed parameters were compared using the Student t test and non-normally distributed parameters using the Mann-Whitney U test. Ratios were compared in 2 x 2 tables using Pearson’s chi square and Fisher’s exact tests.

ROC analysis was performed to determine the diagnostic value of the PTX-3 in pulmonary contusion patients.

P values <=.05 were regarded as significant.

Results

Median ages were 41.5 (29.5-58.5) in the control group and 35.5 (23.5-54.5) in the patient group. Women comprised 26.7% and men 73.3% of the control group, while 20% of the patient group were women and 80% men. No significant difference was determined be- tween the groups in terms of sex or age distribution (p N 0.05) (Table 1). When vital findings in the control and patient groups were evaluat- ed, no significant difference was observed between the two groups in

terms of systolic or diastolic blood pressure values (p N 0.05).

In terms of comorbidities other than pulmonary contusion, coronary artery disease (CAD) was determined in 20% of the contusion group and 10% of the control group, hypertension in 30% of the contusion group and 26.7% of the control group, and diabetes mellitus in 10% of the contusion group and 13.3% of the control group. No relationship was determined in terms of CAD, HT or DM between the groups (p N 0.05) (Table 1).

Median PTX-3 level was 1.026 ng/mL (0.7-1.58) in the control group compared to 7.029 ng/mL (3.29-13.1) in pulmonary contusion, and the difference was statistically significant (p b 0.001). (Table 2, Fig. 1).

An area under the curve (AUC) value of 0.968 investigated using ROC analysis to determine the diagnostic value of the PTX-3 in pulmonary contusion patients was measured (Fig. 3). A PTX-3 cut-off value of

2.06 produced 95.5% sensitivity and 86.7% specificity. A PTX-3 cut-off value of 1.46 produced 100% sensitivity and 73.3% specificity. A PTX-3 cut-off value of 3.07 produced 77.3% sensitivity and 100% specificity.

Table 1

Basic demographic and clinical characteristics of the control and patient groups Control (no. = 30) Patient (no. = 20) p

Table 2

Serum PTX 3 levels in the pulmonary contusion and control groups.

Total no.

Median levels ng/ml

P value

Pulmonary contusion

20

7.029 (3.29-13,1)

b 0.001

Control group

30

1.026 (0.7-1.58)

b 0.001

Discussion

Pulmonary contusion in adults generally manifests together with in- jury to other organs, while in the pediatric age group it takes the form of isolated injury [9]. pulmonary contusions are generally seen in areas close to solid structures such as the ribs, vertebrae, liver and heart [10] The pathophysiology of pulmonary contusion is still not completely clear Blood extravasation into the interstitium and alveolar space also occurs as a result of injury to the alveolar-capillary surface. The ventila- tion perfusion ratio is thus compromised, and hypoventilation and hyp- oxemia may occur [11]. Atelectasis and consolidation may sometimes develop. If the traumatized area is extensive, pulmonary function will be severely impaired. Hemoptysis is rare [12]. Pneumatocele may devel- op as a result of alveolar rupture in some patients [13]. Lacerations may occur in the parenchyma and pleura, thus giving rise to pneumothorax and/or hemothorax.

Diagnosis is made clinically; abnormal auscultation findings, dys- pnea, tachypnea, rales and a decrease in respiratory sounds may be ob- served. Hemoptysis, cyanosis and hypotension are rarely seen [12]. Severe contusions generally manifest within 24 h. Radiologically, non-anatomical areas of consolidation may be seen in single or multiple pulmonary regions [11]. The majority of clinically evident pulmonary contusions appear on pulmonary X-rays and are most commonly confused with aspiration. IntraPulmonary hemorrhage generally reaches a peak level in the first 6 h. Computerized tomography is therefore more definitive in showing pulmonary contusion. Thoracoscopy can be used as an alternative diagnostic technique, particularly in children [14]. Pulmonary contusion generally returns to normal within 4-6 days. If the contusion is severe, or if accompanying events such as aspiration, infections or ARDS develop, then these

ROC Curve

1,0

0,8

0,6

Sensitivity

0,4

0,2

0,0

No. (mean) (median)

% (+-SD)

(IQR)

No. (mean) (median)

% (+-SD)

(IQR)

Age Sex

Female

41.5

8

(29.5-58.5)

26.7

35.5

4

(23.5-54.5)

20

N 0.05

N 0.05

Male Vital signs

22

73.3

16

80

Systolic BP

125.00

105-160

130.00

100-190

N 0.05

Diastolic BP

85.00

70-105

80

50-110

N 0.05

Smokers

14

46.7

4

20

b0.05

CAD

3

10

4

20

N 0.05

HT

8

26.7

6

30

N 0.05

DM

4

13.3

2

10

N 0.05

0,0

0,2

0,4

0,6

0,8

1,0

1 – Specificity

IQR: Interquartile range. Fig. 3. ROC curve analyses of the diagnostic capability of PTX-3 for pulmonary contusion.

428 O. Tatli et al. / American Journal of Emergency Medicine 35 (2017) 425428

patients must be monitored in hospital since their conditions may be- come critical very rapidly. respiratory support is the most important component of treatment. The question of fluid restriction is still contro- versial [15]. Patients’ hemodynamic status must be closely monitored. Active chest physiotherapy can be used to prevent atelectasis. Pain must be controlled. If the patient is not well ventilated, mechanical ven- tilation support must be provided with intubation. Prophylactic antibi- otic use is questionable [11,16]. Early treatment with high-dose corticosteroids is recommended. The radiological appearance in pulmo- nary contusion improves in a few days with appropriate treatment and Gas exchange [17].

Several studies have been performed concerning early identification of pulmonary contusion, but none have investigated the use of PTX 3, an inflammatory marker, in determining pulmonary contusion.

PTX 3 measurement is one of the diagnostic tests for several dis- eases, from Ovarian torsion to pleural fluid effusion, and particularly car- diovascular diseases. Immunohistochemical studies have reported high PTX 3 levels in subjects with atherosclerotic lesions, cardiovascular dis- eases and myocardial infarction Plasma PTX 3 levels have therefore been investigated in these patient groups [17-20].

Studies involving PTX 3 reveal that levels increase in some inflam- matory diseases serum. In a study of patients with Takayasu arteritis, Ishihara et al. observed significantly higher serum PTX 3 levels during disease activation compared to non-activated periods and compared to a healthy control group. They also reported that PTX 3 was more spe- cific than CRP in showing arterial inflammation [21].

Diagnosis of pulmonary contusion may be missed for various rea-

sons. Easily available Diagnostic biomarkers giving rapid results are therefore needed. In the light of the findings from this study levels of the inflammatory marker PTX 3 are significantly higher in patients with pulmonary contusion compared to healthy individuals. If support- ed by wider series studies, PTX 3 may be of use as a diagnostic marker in patients with pulmonary contusion.

Limitations

The first limitation of this study is the relatively small patient num- ber involved. In addition, determination of serum PTX 3 levels in pa- tients with contusion was performed at time of presentation to the emergency department, rather than at time of trauma, and the relation between serum PTX 3 levels and severity of contusion was also not evaluated.

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