Lack of early platelet and leukocyte activation can indicate complications after major burn injury

1. Introduction

Platelets are known traditionally for their predominant physiological functions in haemostasis and thrombosis. The general physiological changes occurring immediately after burn injury are important for the initial survival of the patient. Microthrombi formation within the immediate vicinity of burn injury is essential for maintaining the integrity of the microvasculature surrounding the burn wound. Although this phenomenon may serve as a defence system against bleeding by burn wound vessels, the generalized systemic microthrombi formation may lead to reduction in organ perfusion and create DIC [1, 2]. Burns leads to the disruption of coagulation homeostasis by inducing changes in the balanced processes of clot formation and lysis. An acute burn induced coagulopathy, characterized by endothelial injury, release of acute phase reactants, and impaired coagulation and fibrinolytic pathways [1]. A subsequent, subacute, and a persistent hypercoagulable state follows, independently contributing to increased mortality [3, 4]. Thermal injury is also associated with an initial transient thrombocytopenia followed by normalization of platelet count and eventual reactive thrombocytosis in both human [5, 6] and animal models [7]. Platelet counts after burn injury tend to follow a distinct pattern; falling to a nadir at days 2–5, then rising to a peak value at days 10–18. However, non-haemostatic roles of thrombocytes as sentinels of the innate immune system during infection and inflammation are becoming increasingly recognised [8]. Platelets are now conceded as inherent elements of the innate immune response, monitoring and rapidly responding to harmful signals [9]. After heat trauma the immune system is activated by the tissue damage and this can in turn activate specific types of responses that can enhance innate immune reactivity, control excessive pro-inflammatory responses, and reduce continued tissue damage [10]. The systemic inflammatory response after burns is associated with both cellular and humoral inflammatory responses. During the first step of the cellular response neutrophil leukocytes become activated as a part of a non-specific immune function. This increases the adherence of the neutrophils and their chemostaxis to the interstitium. Furthermore, the leukocytes free radical production and water uptake increase, too. All these changes in leukocyte function can be measured by sophisticated methods. However, the water uptake which reduces their specific gravity can also be easily measured. Previous studies have found that anticoagulated whole blood samples from healthy individuals contained a maximum of 10% increase in leukocyte concentration in the upper half of the blood column (due to up flotation i.e. “antisedimentation”) after one hour of gravity sedimentation. The degree of water uptake by leukocytes associated with their activation leads to further decrease in their specific gravity, therefore, antisedimentation ratio will increase [11, 12]. This ratio is known as the leukocyte antisedimentation rate (LAR) which can be measured by whole blood gravity sedimentation [13–15]. Based on this concept the platelets’ antisedimentation rate (PAR) can be measured, too. Rozanovic and al. found that among patients who admitted to the ICU immediately after polytrauma or severe burn injury and who had a drop in their LAR levels on days 1 and 2 after admission, had a higher chance for septic complications and consequent mortality. Among these patients, the evaluation of the previous and the following 3 days since the patients treated as septic, a sudden drop in the levels of the LAR levels might have been predicted the onset of sepsis earlier than C-reactive protein or procalctionin [16].

The aim of this study was to investigate the time course of PAR and LAR after burn injury, the possible differences of the platelets’ and the leukocytes’ activation among the members of the survivor and the non-survivor groups in the early post-burned period. Moreover, we aimed study the features of PAR and LAR both in non-septic and septic burned patients.

2. Materials and methods

2.2. Inclusion and exclusion criteria

Patients who had 20% or more total burnt surface area (TBSA) were screened to our study. The exclusion criteria were Baux index above 100, patient’s or relative’s refusal, age under 18 years, chronic use of steroid drugs, malignant haematology disease and immune suppressive therapy affecting the normal immune response (chemo-, radiotherapy etc.), New York Heart Association (NYHA) grade IV heart failure, patient with chronic haemodialysis treatment, liver cirrhosis or portal hypertension. Primary and secondary outcomes were intensive care survival and development of septic complications, respectively.

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Fig.1.

Measurement of the platelet and the leukocyte antisedimentation rate (PAR and LAR). See description in the text.

2.5. Statistics

Statistical Package for the Social Sciences (SPSS) statistics software, version 22.0 (IBM Corporation, USA) was used for statistical analysis. The data was expressed as median and inter-quartile range (IQR) as the distribution was not normal according to Kolmogorov-Smirnov test. Kruskal-Wallis test followed by Mann-Whitney U test were used for interday analysis.

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Fig.2.

(a) The kinetics of platelet antisedimentation rate (PAR) levels. Dark columns = all burnt patients. Data are given as median, 25–75% interquartile range and 5–95% confidence interval. (b) The kinetics of leukocyte antisedimentation rate (LAR) levels. Dark columns = all burnt patients. Data are given as median, 25–75% interquartile range and 5–95% confidence interval.

Jonckheere-Terpstra test was used to detect significant trends across the study period. In all cases values of p < 0.05 were considered significant.

4. Discussion

Although LAR was reported in mixed population of burns and polytrauma earlier [16], PAR was examined as an absolute novelty in patients treated in the ICU [16]. Importantly, the kinetics of PAR and LAR were first explored in burn patients here. After the traumatic insult of the host, no matter what the origin is, thrombocytopenia is common and it is associated with poor outcomes [9]. After burns decreasing platelet count occurs with a nadir between days 2 and 5 followed by a peak of thrombocytosis at around days 11–17 [8]. This early stage thrombocytopenia can develop due to several mechanisms: (i) hemodilution by fluid resuscitation during the initial treatment of burn injury; (ii) platelet activation with subsequent peripheral consumption; (iii) by depressed bone marrow production [5]. In 1981, Chotow and Michas first noticed the abnormality of platelet levels after burn injury. In their study, a decrease in platelet levels was observed in all patients after the initial period of burn shock, and it took 7–12 days to return within the normal range [22]. In 1997, Takashima et al. described the blood platelets features in severely injured burned patients [23]. Regarding to Mazur, et al. during the period thrombocytopenia, thermal injury harms the skin’s micro vessels, so the platelets may be trapped in microthrombi or may be mobilized to coagulate as a part of thrombotic process [24]. This results in an increased consumption of platelets and facilitates the bone marrow to begin the synthesis of new platelets. Dysfunction of immature platelet subpopulation was recently published in patients on antiplatelet therapy due to vascular disease [21]. This may require interaction, during the later stages of megakaryocytopoesis and platelet release, with such regulatory factors as erythropoietin, cytokines, interleukins and thrombopoetin [24]. Regarding to Cato et al., it is reasonable to suggest that platelets are being consumed within the burn wound as a result of destruction of the skin’s vessels and the subsequent microthrombi formation. These microthrombi form by 24–48 hours and so this may coincide with the nadir. It is also well documented that the permeability of surrounding vessels increases along with development of widespread vascular hyperpermeability, and this may lead to increased activation of platelets through interaction with tissue factor on the sub-endothelium and activated clotting factors, leading to subsequent aggregation and consumption [8]. In this present study we hypothesized that the platelet’s activation can be described by PAR. Among the patients who survived the ICU treatment an earlier increase of the PAR levels (T2 in survivors and T3 in non-survivors) were seen, and significantly higher PAR levels were found at T2, T4 and T5 comparing to those patients who died in the ICU. As platelets have pivotal role in the immune response too, we believe that an earlier increase of PAR reflects an earlier activation of the innate immune system, and a more effective inflammatory response to the thermal injury [8, 9]. Lack of early platelet activation suggested a dysregulated immune response. In their retrospective study, Cato and his coworkers found that most of the survivors after severe burn injury displayed a much higher platelet count at the nadir with a significantly greater platelet count at day 5. On average, non-survivors, did not display thrombocytosis at any given moment within the 50 days post-injury [8]. We focused more to the activation features and the dynamic changes of the platelets rather than the platelet count itself. Activated platelets may interact with circulating leukocytes, potentiating their ability to extravasation into the site of injury.

LAR was previously found as an indicator of leukocyte activation [13, 16]. The higher the LAR value expressed in percentage, the more increased the number of activated leukocytes. In our study, LAR was used to explore the activation of leukocytes as a response to thermal injury. The kinetics of LAR was very similar to PAR, in patients who survived the ICU treatment elevation started at T2, while in non-survivors at T3, only. After admission to the ICU and on the following 3 days the patients who survived the ICU treatment had significantly higher LAR levels, compared those who died - suggesting that lack of elevation of LAR is associated with poor outcome. Similarly, in acute ischemic stroke Molnar et al. [25] detected an early (within 6 hours from admission) elevation of LAR in patients with favourable stroke outcome, while lack of elevation of LAR at post-stroke 24 hours indicating dysfunction of immune response was explored in patients with poor outcome. In this present study, the first blood sample was taken at the patient’s arrival to our ICU. In this study, the immune-paralysis induced by the thermal injury itself was explored by a missing early elevation of LAR indicating a dysregulated immune response [26].

We assumed two possibilities behind the features of the PAR and LAR kinetics in the SU and the NSU groups. The first one is the possibility of a later or less effective immune response, or an overwhelming counter inflammatory response to the inflammation after burn injury. The PAR and LAR kinetics and their later elevation in the non-survivors after burn injury are identical with the results of Csontos et al. [27], who found an elevated anti-inflammatory cytokine expression profile (elevated IL-10 levels) in non-surviving burned patients compared to survivors. Rozanovic et al. [16] found that in the mixed population of polytrauma and burned patients who failed to increase LAR levels from the next day after ICU admission had higher risk for infectious complications and consequent adverse events. On the other hand, based on previous observations of circulating leukocytes [13–15], it is possible that the activated cells have passed the capillary wall while the less mature cells remain in the circulation –thus we may conclude the inverse of the real processes. This whole process may also be exacerbated by inflammatory cytokines (e.g. IL-6) during the systemic inflammatory response after thermal injury. In this study of the PAR and LAR kinetics of the observed whole burned population in this study are in concordance with the previously mentioned study of Csontos et al. [27] who observed the elevation of inflammatory cytokines (e.g. IL-6, IL-8) from the second day after injury and reaching the peak value on day 4. Since PAR and LAR may mirror the cytokine kinetics, the decreased immune response seems to be more feasible. Higher PAR and LAR values among the survivors suggesting a more effective immune response to thermal injury.

Diagnosis of sepsis is challenging in patients with severe burn injury because the systemic inflammatory response can mask the classical diagnostic criteria. In burned patients the ABA’s 2007 Consensus sepsis trigger criteria [17] are used widely. The recent Sepsis-3 definition [18] criteria has not yet been applied in burn population and warrants evaluation of its discriminatory performance in this setting before it can be applied to the evaluation of potential laboratory diagnostic markers [8]. In 43 critically ill burn patients, Lavrentieva et al. estimated the prognostic value of serum PCT, CRP, white blood cell count and core temperature as markers of sepsis. There was a significant difference in PCT between patients with infected and non-infected systemic inflammatory response syndrome (SIRS). Compared to pre-sepsis period, PCT was significantly higher on first septic day and declined gradually in patients who survived between days 3 and 7 [28]. In a study of 60 burned patients with and without infection, similarly to our study, Barati et al. demonstrated significantly higher PCT levels in the septic group compared to those without sepsis. The serum PCT level was a highly efficient laboratory parameter for the diagnosis of severe infectious complications after burn [29]. In these studies, the white blood cell count, temperature, erythrocyte sedimentation rate (ESR) and CRP levels did not differ significantly in non-infected and infected SIRS [28, 29]. Murray et al. reported that core temperature, white blood cell count, neutrophil percentage failed to predict bloodstream infections [30]. Their retrospective review was based on electronic records of 223 burn patients’ 1063 blood cultures taken between 2001 and 2004. Although, white blood cell count and neutrophil percentage were significantly higher in patients with positive than negative culture results, receiver operating characteristic curve analysis was not convincing enough in prediction of blood stream infection.

Instead of ESR, leukocyte or platelet count, a dynamic approach was applied here using PAR and LAR values as indicators of the activated platelets and leukocytes. Based on LAR values, Bogar et al. could discriminate patients with bacteremic and non-bacteremic fever in the ICU [15]. In our study among the patients who did not have septic complications after burn injury had significantly higher PAR levels at T5 compared to the septic population. We think this may be an adaptive response due to increased “de novo” platelet production. The circulating platelet population is heterogeneous in size and age. This population includes for example the immature platelets, as a marker of platelet turnover, which have the potential association with clinical outcomes in patients with cardiovascular disease [31]. So far, it is still not clear how the function and the activational status of platelets develop after burns. Marck et al. concluded in their study with 6 burned patients with over 15% TBSA that platelets after burn-injury appears to be functional and not overly activated. However, burn patients seem to remain in a procoagulant state for an extensive period, which may impact their pathology [32]. An increasing tendency of LAR was observed from T2 in both NSE and SE groups. An earlier elevation of LAR was also explored among burn victims who did not develop septic complications (at T2 in NSE vs at T3 in SE group). These results are identical with the results of Molnar et al., who have observed the lack of increase in LAR at 24 post-stroke hours in acute ischemic stroke patients with later manifested post-stroke infections [25]. In their study a weak positive correlation was found between the severity of stroke and LAR values. In this regard, both PAR and LAR might have a predictive value [25] regarding ICU outcome. In contrast to PAR, the kinetics of LAR was not differed significantly comparing the NSE and the SE groups suggesting that the immune function of platelets can even better describe the immunopathology of post-thermal injury.

5. Limitations of the study

Ten patients who became septic after burn injury are not enough to observe their kinetics around the development of sepsis –further patients need to be involved. In our case we faced to the problem with anyone who is dealing with sepsis at the time we have no adequate laboratory parameter, radiological finding or any other sign that confirm the presence of infection, so in our practice we can rely on clinical signs only. The microbiological results for declaration of sepsis were not useful too, due to time delay, sample contamination or the possibility of false negative findings that may not be ruled out. Further studies with higher sample size required to confirm our findings and to explore the predictive power of PAR and LAR in the outcome of thermal injury.

Conflict of interest

The authors have read the journal’s policy and declare the following: no potential conflict of interest relevant to this article can be reported.