ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE...

143
ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE

Transcript of ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE...

Page 1: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

ACUTE SYSTEMIC INFLAMMATION

IN

HEALTH AND DISEASE

Page 2: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de
Page 3: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Cover: Changes of TNF, IL-6, PCT, SAA, CRP, α1AT, WBC, PC, IgM and IgG respectivelyafter an acute inflammatory event. Design by E. Nijsten based on an idea of T.E. Feltkamp

Electronic ISBN 90-367-1453-2 Printed ISBN 90-367-1454-0© M.Nijsten 2001

Page 4: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

2

RIJKSUNIVERSITEIT GRONINGEN

ACUTE SYSTEMIC INFLAMMATION

IN

HEALTH AND DISEASE

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningenop gezag van de

Rector Magnificus, dr. D.F.J. Bosscher,in het openbaar te verdedigen opwoensdag 19 september 2001

om 16.00 uur

door

Maarten Willem Nicolaas Nijstengeboren op 2 april 1960

te Scheveningen

Page 5: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Promotores Prof. Dr. H.J. ten DuisProf. Dr. T.H. The

Referent Dr. R.J. Porte

Beoordelingscommissie Prof. Dr. R.J. GorisProf. Dr. R. van SchilfgaardeProf. Dr. L.G. Thijs

Page 6: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de
Page 7: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Paranymfen H. MakkingaDr. B. de Smet

Aan Anja,

Susan en Peter

Page 8: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de
Page 9: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Abbreviations:

α1AT α1-antitrypsin, acute phase proteinα1AP α1-antiprotease inhibitor, acute phase protein, identical with α1ATAPACHE-II acute physiological and health evaluation score, ICU scoreAPP acute phase proteinC3a activation product of complement factor 3 C3dg rest product after activation of C3C4 complement factor 4CD-14 lipopolysaccharide receptorCRP C-reactive protein, acute phase proteinDIC disseminated intravascular coagulationEDTA ethylenediamine tetraacetic acid, an anticoagulantELISA enzyme linked immuno sorbent assayESR erythrocyte sedimentation rateEURICUS-II European ICU studiesFES fat embolism syndromeGP IIb/IIIa glycoprotein IIb/IIIa receptor, mediates platelet adhesion and aggregationHGF hybridoma growth factor (identical with IL-6)ICU intensive care unitIgG, IgM immunoglobulin G, immunoglobulin MIL-1 interleukin-1, cytokine(rh)IL-6 (recombinant human) interleukin-6, cytokineISS injury severity scoreKD kilodaltonLPS lipopolysaccharide, major bacterial endotoxin componentMODS multiple organ dysfunction score, ICU scoreMPS methyl prednisolone sodium succinateNASCIS national acute spinal cord injury studiesPC platelet countPC2 platelet count at admission day 2 ∆PC/∆t daily rate of change in platelet count∆PC/∆t0→2 daily rate of change in platelet count between admission day 0 and 2∆PC/∆t2→10 daily rate of change in platelet count between admission day 2 and 10PCT procalcitoninPFO patent (or persisting) foramen ovaleSAA serum amyloid A, acute phase proteinSIRS systemic inflammatory response syndromeSOFA sequential organ failure score, ICU scoreTBSA total body surface area(rh)TNF (recombinant human) tumor necrosis factorWBC white blood cell count

Page 10: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de
Page 11: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 1

INTRODUCTION AND SCOPE OF THIS THESIS

Page 12: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

2

INTRODUCTION

Wounds, healing and inflammationWhen the skin is damaged, for example by a small cut, a blood clot will start forming within seconds,and within minutes bleeding will stop. We should realize that the process of wound repair has thenalready started. Under normal circumstances, healing will be the end result of overlapping processes:inflammation, tissue formation and tissue remodelling [1]. Inflammation is a response of the immunesystem, since the immune system guards the individual against infection. Inflammation is defined inWebster's Medical Dictionary as "A local response to cellular injury that is marked by capillarydilatation, leukocyte infiltration, redness, heat, pain, swelling, and often loss of function and thatserves as a mechanism initiating the elimination of noxious agents and damaged tissue[2]."

Since minor wounds are sustained very regularly, the local inflammatory responses initiated by theimmune system are as crucial part of body homeostasis as is feeding or sleeping. Inflammation in anextremely complex homeostatic response that involves neutrophils, platelets, macrophages,endothelial cells and the coagulation and complement systems. The immune system can be dividedinto the innate immune system and the adaptive immune system. Furthermore, the system has beendivided into a humoral and a cellular system. Combined, these two divisions result in the four majorparts (Table 1.1) of the immune system.

This thesis focuses on a number of aspects of the innate humoral immune response. The earlyinflammatory response is part of the innate immune system. In contrast to the adaptive immunesystem, which provides improved responses upon repeated infection, the innate response is not basedon immune memory, but on evolutionary highly preserved mechanisms of pattern recognition ofpotential pathogens. For example, bacterial endotoxin (lipopolysaccharide; LPS) is recognized bythe innate immune system in even very low concentrations, and thus serves as a very strong stimulusof subsequent responses. Acting together, CD-14 and so-called Toll-like receptors (e.g. TLR-4)present on myeloid cells, are believed to form a single pathway common to all mammals totransduct the LPS-signal [3]. The importance of these Toll-like receptors is underscored by thediscovery that the receptors are coupled to a pathway that activates genes mediating innateimmune defenses in mammals, insects, and even plants [4,5].

Table 1.1. Major divisions in the immune system

Innate immune system Adaptive immune system

Humoral Cytokines, complement,

acute phase proteins

Immunoglobulins

Cellular Phagocytes, Natural killer

cells

T-lymphocytes

Page 13: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

Whereas local inflammation inat a certain size of the wounds,

Clinical signs of systemic inflamThe systemic manifestations oadmission are the subject of cause systemic effects (Table will develop. SIRS has been dmanifestations listed in Table intensive care unit nearly all pa

Grades and definitions of syste

Figure 1.1. To preserve a stabimmune system reacts to disturesponses is to eliminate the ditissue damage or infection (thesignals that direct the response

III

Cellula

Hu

C

II

Amplification

I

Induction

Homeostasis

disturbancerecovery

stimulus

response

Immune response phases

r

III

moral

ellular

3

the setting of tiny wounds may not have significant systemic effects, systemic effects will become evident.

mation f the innate immune response that result after trauma, burns or ICU-this thesis. When an inflammatory stimulus is sufficiently strong to1.2), the so-called systemic inflammatory response syndrome (SIRS)efined by a consensus conference [6] as present if two of the clinical1.3 are observed. At the ward many patients may have SIRS, at thetients have SIRS.

mic inflammation

le healthy situation (homeostasis), like other physiologic systems, therbances such as a trauma with phased responses. The purpose of thesesturbance and restore homeostasis. The induction phase the presence of stimulus) is recognized and transducted into cytokine or chemokine

that eliminates the injury. (Adapted from T.H.The et al. 1995)

Page 14: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

4

The American consensus statement contained, apart from the definition of SIRS three additionaldefinitions. The main purpose of these definitions has been to illuminate the distinction betweeninflammation and infection:

SIRS= fever + leukocytosis; Sepsis= SIRS + infection; Severe Sepsis= Sepsis + multiorgan dysfunction; Septic shock= Severe Sepsis + refractory hypotension [6].

Table 1.3. SIRS (Systemic Inflammatory Response Syndrome) criteria[6]

Temperature >380 or <360 CHeart rate > 90 /minRespiratory rate > 20

Hyperventilation (PaCO2 < 4.3 kPa)Leukocytes > 12 109/l or < 4 109/l

Immature neutrophils > 10%

Organ failureMultiple organ failure has been defined in 1985 by Goris as 'generalized, autodestructive inflam-mation' [7]. As more organs are affected by the systemic inflammatory process, the chances ofsurvival decrease.Two examples of how organ failure has been graded are shown in table 1.4, a minimal total score hasa high probability of survival, a maximal score has a very low probability of survival.

Biochemical signs of systemic inflammation - acute phase proteinsAlthough not strictly defined, the acute phase response can be considered to consist of the initialsystemic clinical and biochemical responses that follow for trauma or infection. Fever is the clinicalhallmark of the acute phase response, and it is usually accompanied by tachycardia [8]. Theseclinical signs are paralleled by many biochemical changes. Many of the proteins induced by theinnate immune response are called acute phase proteins.An elevated erythrocyte sedimentation rate (ESR) [9] was the first widely used laboratory

Table 1.4. Examples of two multiple organ failure scoresOrgan/system Goris’ Multiple organ failure

score [7]Sequential organ failureassessment (SOFA) [52]

Cardiovascular 0 – 2 0 – 4Pulmonary 0 – 2 0 – 4Renal 0 – 2 0 – 4Neurologic 0 – 2 0 – 4Liver 0 – 2 0 – 4\aHematologic/ coagulation 0 – 2 0 - 4Gastrointestinal 0 – 2Total score 0 – 14 0 - 24

Page 15: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

5

parameter of inflammation. Increased levels of the acute phase protein fibrinogen are a maindeterminant of an elevated ESR [10]. A large number of proteins have turned out to be acute phaseproteins (APP). What constitutes an acute phase protein has not clearly been defined. Anincreased levels after inflammation and some sort of effector function (as opposed to signallingcytokines) are the most important characteristics of acute phase proteins. Kushner [11] somewhatsweepingly defined APP as those proteins whose plasma concentration rises 25% or morefollowing an inflammatory stimulus. Albumin, the most abundant protein in the circulation, issometimes called a negative acute phase protein since it is down-regulated during inflammation.The metabolic effects and the reasons why albumin levels are decreased, are only partlyunderstood [12]. Although not subject of this thesis, decreased albumin levels are an importantillustration of the relation between inflammation and metabolism.

Nowadays, C-reactive protein (CRP) is the most widely directly measured acute phase protein. Inhealth it is hardly detectable in plasma, but with inflammation it can rise rapidly to 10- or 100-foldlevels. Although CRP’s function is not clearly identified, CRP is probably involved in early non-specific antibacterial defences [13]. Thanks to reliable assays and CRP’s half-life which is in theorder of one day, CRP has gained wide popularity for monitoring inflammation in many diseasestates. On the basis of the rapidity and magnitude of increase in concentration during the acute phaseresponse, acute phase proteins can be divided into 3 groups (Table 1.5).

Source of acute phase proteinsThe liver is quantitatively and qualitatively the major source of acute phase proteins. In severalhepatocyte models for different species, the induction of many acute phase protein-production hasbeen demonstrated. For example in 1966 Hurlimann et al. already performed in vitro studies thatshowed that hepatocytes produce CRP [15].

Table 1.5. Classification of some acute phase proteins

Increase by factor 1.5 Increase by factor 2 to 4 Increase by up to factor 1000Slow response Intermediate response Rapid response

Antitrombin III α1-proteinase inhibitor C-reactive protein (CRP)Complement C3, C4 α1 acid glycoprotein Serum Amyloid A (SAA)Ceruloplasmin α1 anti-chymotrypsinC1-inhibitor Haptoglobinα2-antiplasmin FibrinogenAdapted from Heinrich et al. [14]

Page 16: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

6

Function of acute phase proteinsA close look at the functions - as far as understood - of the acute phase proteins illustrates thepurpose of the acute phase response: provide the organism with those proteins that are necessary tokeep up both an adequate and a restrained inflammatory response. Table 1.5 shows that proteins forthe complement system, the coagulation system and the fibrinolytic system are provided by the acutephase response. Most acute phase proteins have larger molecular weights (>50,000 Dalton) andhigher concentrations (mg/L to g/L range) when compared to cytokines which have a lowermolecular weight and much lower concentrations. This is due to fact that acute phase proteins areoften effector molecules (e.g. thrombus formation, protease inhibitor, bactericidal factor) asopposed to cytokines that are hormone-like proteins. Understandably, effector functions requiremuch higher concentrations than necessary for signalling functions.

Function unknown or no functionCurrently it is possible to detect many biological substances and study their interactions with manycell types. This results in a huge number of possible relations that can be studied. It is helpful to tryto understand these mechanisms in a teleological manner. Teleology is the use of design or purposeas an explanation of natural phenomena [2]. In understanding inflammation this implies thatevolution should have provided the organism with defenses that can plausibly by expected to help theorganism survive. A mammal will certainly have a survival benefit from adequate protection againstminor trauma, since every animal will continuously sustain such traumas during its life. But ananimal cannot logically be expected to have an elaborate defense against the results of septic shock.An animal with septic shock or severe trauma will in all probability not survive anyway, so evolutionhas no interest in providing for defenses in ‘lost cases’. Of course the animal (or man) with septicshock may survive with medical intervention, but at this stage we cannot rely on inflammationresponses behaving in a ‘logical’ way. Thus, we may expect every protein found near a smalluncomplicated wound to have a relevant function, and search aggressively for this function if we donot know it yet. We also may understand why TNFα levels are very high in septic patients, but weshould not expect that these high levels have a specific function. In fact in these patients manymolecules with elevated or depressed levels will not have a function, may have lost their function ormay even function inappropriately.That a distinct degree of systemic inflammation might be beneficial to the host, was already believedcenturies ago [16,17], when patients who did not properly recover from wounds or infection weretreated by inducing additional inflammation. The widely practiced cauterization of wounds with hotirons or hot oil was also performed on healthy skin to enhance recovery of wounds elsewhere (seeillustration). This practice of counter-irritation was also performed in the 20th century, albeit in theslightly more humane form of turpentine abscesses. In fact turpentine in still used in animal models toinduce a sterile acute phase response [18].

Page 17: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

7

History of endogenous pyrogen and IL-6The genesis of fever has long been a subject of investigation. Decades ago it became apparent that an'endogenous pyrogen' had to exist. This substance is made by the body in response to tissue damageor infection and it induces fever by changing the temperature setpoint in the hypothalamus - a processthat can be inhibited by prostaglandin inhibitors like aspirin. With modern molecular biologicaltechnology is became clear that several cytokines not only induce fever but also induce acute phaseproteins. They can thus be considered not only endogenous pyrogens but turned out to be true

hormones of inflammation.In the 1980’s interleukin-1 (IL-1) became the first contender for the role of endogenous pyrogen.Experiments by Aarden et al. showed that hybridoma growth factor (HGF) was active in the IL-1

Figure 1.2. Patient who is cauterized after injury. Drawing by JohannesWechtlin in Feldtbuch der Wundarznei by Hans von Gersdorff, 1540.

Page 18: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

8

assay [19]. A very sensitive bio-assay for HGF was developed, and this assay did not respond to IL-1,indicating that HGF had to be another cytokine. After cloning of cDNA, HGF was discovered to beidentical with a new cytokine named interleukin-6 [20]. In many in vitro and in vivo studies thatfollowed, the B9.9 bioassay for HGF/IL-6 has been very important in defining the pleiotropic role ofIL-6, to be later replaced by ELISAs [21].The cytokines TNFα, IL-6 and IL-1 are elevated within hours after a major inflammatory stimulus[22]. TNFα peaks earlier than IL-6 and also induces IL-6. For example van Gameren andcolleagues [23] showed that administration of IL-6 to patients induced most of the responses listed inTables 1.2 and 1.3. Several studies have assessed the predictive value of cytokine levels for clinicaloutcome. In general, cytokine levels in patients with sepsis are much higher than in trauma patients[24]. In addition it was found by Hack et al. that higher IL-6 levels were associated with mortality insepsis patients [25]. Partly due to the fact that cytokine levels can rapidly change, and due todifficulty in routinely assaying cytokines, these measurements are not used in clinical practice. IL-6levels measured at the bedside (positive at > 1000 pg/ml) have been used to triage patients for anti-TNFα sepsis trials [26].

Methodological advantages of studying patients with mechanical trauma or burns The pathophysiology of inflammation has received intensive attention in patients that are criticallyill, but it is useful as well to study the physiology of inflammation in patients that are not critically ill,e.g. in healthy persons that sustain a nonlethal trauma. Trauma patients usually are healthy before theinjury and show a responses roughly proportional to the severity of the injury [27]. Since the time ofthe injury is a well-defined moment as opposed to sepsis, the timing and order of the elicitedresponses can be studied in more detail. Concerning inflammatory responses in trauma patients, patients with burns represent a special group.Thermal injury is a one-dimensional trauma, with the percentage of burned total body surface(TBSA) being the key parameter. With only the age of the patient and TBSA it is possible to make agood prediction of mortality and hospital stay [28]. Severely burned patients can have extensivetissue damage without accompanying organ damage. Early after sustaining burn injury, patientsshow a pronounced inflammatory response. This occasionally extreme response has made burnspatients a subject of many studies on virtually all known aspects of host responses.

Differentiating infectious from non-infectious causes of inflammation.Both trauma and infection can induce an acute phase reaction, clinically characterized by SIRS.When SIRS persists for a number of days after admission to the hospital, this can be due to thetrauma itself or secondary infection. In the first case a wait-and see policy is often justified. In thesecond case it may be crucial to search for a focus, or to start antibiotics and even perform anoperation. Since a majority of trauma patients at the ICU develop fever, with only proveninfection in half the these patients [29] - it is very useful to possess additional tools to recognizebacterial infection. Especially in immune suppressed patients (e.g. after transplantation) it wouldbe very useful to possess a rapid assay to detect bacterial infection, since in these patients themargins of error are small. Now that many inflammatory markers can be measured in thecirculation, researchers have looked for those markers that can discriminate bacterial infection

Page 19: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

9

from other causes of systemic inflammation. But discriminating causes of inflammation onpatterns of cytokine or acute phase protein responses have proven an elusive goal. Measuring thebacterial product endotoxin itself would seem to be an obvious solution to identify bacteremia andendotoxemia early [30]. Unfortunately circulating endotoxin assays are difficult to use andreproduce [31].In accordance with the concept of the spectrum of increasing systemic inflammatory responseswith increasing infection: SIRS → sepsis → severe sepsis → septic shock, systemic inflammationwith infection is associated with higher cytokine levels and higher levels of CRP. Thus, serialquantitative measurements of the extent of the acute phase response, usually in the form of CRPhave been a major clinical tool in detecting infection. The acute phase response appears tomodulate mainly in intensity and duration, not in the relative enhancement or inhibition ofcomponents of the response. This is of course in agreement with the non-specific nature of theacute phase reaction.

Procalcitonin to differentiate infection and non-infection?The recent introduction of a convenient assay for procalcitonin (PCT) and the first experienceswith measurements of circulating PCT have been promising. Some authors have proposed that apractical parameter that differentiates bacterial infection from other causes of inflammation is nowat hand. Although PCT is biochemically a precursor of calcitonin (a hormone involved in calciumhomeostasis) it is functionally not related to calcitonin. Both the cellular origin and function ofPCT are unknown. Nevertheless, circulating PCT measurements in many patients groups [32]have shown that:- Circulating PCT is elevated proportional to the inflammatory stimulus. - PCT can be induced by infusing endotoxin in volunteers. Elevated PCT-levels are detectable

at 6 hours and disappear with a half-life of about a day [33].- The dynamic range of PCT is very large: PCT rises at least a factor 400 within 6 hours in

volunteers receiving endotoxin. Where one report [34] claims that PCT levels are <0.01 ug/l innormal persons, PCT can reach 1000 ug/l in sepsis [35]. Thus PCT's dynamic range may beeven greater than that of CRP or SAA.

The most interesting aspect of PCT is the evidence that PCT compared to CRP is superior indiscriminating bacterial from non-bacterial inflammation. De Werra [36] compared patients withseptic shock, cardiogenic shock, and bacterial pneumonia. The best predictive value for septicshock was found for PCT in septic shock, with PCT varying from 72 to 135 ng/mL, comparedwith approximately 1 ng/mL in the other groups. The monograph on PCT by Meisner [32]contains a number of early studies that indicate that PCT is better than CRP in discriminatingserious infection from other causes of inflammation. More recently Gendrel et al. reported thatPCT was more specific and sensitive than CRP for the differentiation of bacterial and viralinfections in children [37]. In 236 children with viral infections they found a median PCT of 0.2ug/l and a median CRP of 10 mg/l. In 46 children with bacterial sepsis or meningitis PCT was 18ug/l and CRP 144 mg/l. Thus PCT is about a 100-fold higher in sepsis compared to viral infection,whereas CRP is 'only' 15 times higher.

Page 20: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

10

Still the literature is not conclusive on this very important issue. Those disease categories wheredetermining PCT may not be reliable need to be better defined. For example de Bont [38] foundthat in neutropenic patients PCT responses are much lower than would be expected on the basis ofother signs and elevated CRP-levels. The various results that suggest that preferential induction ofPCT by bacterial infection exists, have led some authors to hypothesize that endotoxin can inducePCT directly. A logical question is whether cytokines like TNFα or IL-6 are necessary andsufficient for the induction of PCT. And although increased levels of IL-6 and TNFα were alsoobserved after endotoxin infusion, this does not prove that TNFα and IL-6 are necessary for theinduction of PCT synthesis, as they are for the acute phase protein induction. Does PCT originatein the liver? Or more general, should PCT be viewed as an acute phase protein?

Platelets and endothelium in inflammation Platelets can be considered inflammatory agents as much as coagulatory agents. In systemicinflammation platelets first disappear from the circulation (thrombocytopenia), and as the patientrecovers uneventfully platelets will reappear in increased amounts [39] (thrombocytosis). Severalcytokines can induce this sequence. After administration to humans, IL-6 will induce a completeacute phase response, including the characteristic sequence of a nadir platelet count at day 2 or 3and a maximal platelet count at day 12 [23]. Concerning the causes of premature disappearance ofplatelets from the circulation, two important possibilities exist. The platelet end up as part of a clotor the platelet can (temporarily) adhere to the endothelium. In both cases platelets can amplifyinflammation through the release of powerful mediators. Consumption of platelets is an integral part of the syndrome of diffuse intravascular coagulation(DIC). The reverse, that DIC always accompanies platelet consumption, is not true. DIC ischaracterized by extensive intravascular formation of fibrin, in severe cases resulting in vascularocclusion and organ failure. DIC occurs secondary to diverse range of serious diseases, like sepsisor trauma [40]. It is important to note that significant decreases in platelet count are observed invirtually all patients with trauma or sepsis. Although a significant proportion of these patients mayhave indicators of 'low-grade' DIC, many do not have DIC, indicating that platelet sequestrationand DIC are not the same [41]. Endothelium, the single cell layer that separates the blood from the organs and tissues, is a veryactive cell system. Endothelium regulates hemodynamics and the transport of molecules and cellsbetween blood and the tissues. Activation of endothelium occurs early and universally [42] in theprocess of inflammation. Endothelial cell activation [43] is characterized by the loss of vascularintegrity, expression of adhesion molecules, transition from an antithrombotic to a prothromboticstate and cytokine production. Exposure of the subendothelium with tissue factor and vonWillebrand Factor and rapid expression of P-selectin can initiate the binding of platelets as well asthe adhesion molecules such as GPIIb/IIIa, P-selectin, CD-31, LFA-1, CD-36 [44] and CD-87(urokinase plasminogen activator receptor; uPAR) [45]. Cardiovascular research has producedextensive evidence of the importance of platelet-endothelium interaction. This is underscored bythe clinical effectiveness of aspirin and the recently introduced GPIIb/IIIa-inhibitors in limiting orpreventing coronary thrombosis [46]. Despite the evidence of the importance of the platelet-endothelial interaction in inflammation, direct studies on this interaction are methodologically

Page 21: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

11

very difficult. Ex vivo, endothelial cells and platelets are not the same as in vivo. Whereas theerythrocyte has a circulating precursor that can be clearly identified and quantified (thereticulocyte), no such equivalent exists for the circulating platelet. Therefore, in thrombocytopeniait is difficult to decide if decreased synthesis or increased aggregation or adhesion is present, evenwhen one resorts to a bone marrow biopsy or radioactively labeled platelet studies [47]. Re-injected radioactively labeled autologous platelets are not identical to native circulating platelets.

Serial platelet counts as an indicator of endothelial activationInstead of performing highly specialized platelet studies in a limited set of patients, in this thesisis was preferred to study serial platelet counts and study them in larger patient groups. Theassumption was that changes in the platelet count are to a large extent correlated to the magnitudeof endothelial activation, which in turn is related to systemic inflammation. A low admissionplatelet count is known to be a relatively strong predictor of adverse outcome in a variety ofdisease states. In meningococcal sepsis [48], after ruptured aortic aneurysm [49], at admission tothe intensive care [50] the early platelet count is one of the strongest predictors of outcome. As aresult the platelet count has been introduced in several intensive care scoring systems, while theleukocyte count has been eliminated from some scoring systems. The multi-organ dysfunctionscore (MODS) [51], the sequential organ failure assessment score (SOFA) [52] and the pediatricrisk of mortality score (PRISM-III) [53] use the platelet count and not the leukocyte count as oneof its component parameters. In contrast to the well-described importance of initial plateletcounts, the relevance of subsequent changes has received little attention. Within the healthyindividual the platelet count is quite stable with an intra-individual variation that is only 20% to30% (or 60 ·109/L) of the inter-individual variation [54,55]. Thus serial platelet counts might inprinciple provide additional information. As long as the platelet count is within the normal range(i.e. 150 to 350 ·109/L) it has been implicitly assumed that such a count is normal. Yet, in patientswith an uneventful clinical recovery after a moderate trauma, thrombocytosis normally developsduring the second week. Thrombopoietin has been identified as a major regulator ofthrombopoiesis [56]. After trauma IL-6 is released, which is also capable of inducingthrombocytosis [23]. In critically ill patients thrombocytosis is often not present, although suchpatients typically have cytokine levels (including IL-6) much higher than those observed afteruncomplicated trauma. Thus despite the fact that many ICU patients are in a more advanced stateof inflammation (ranging from SIRS to sepsis, severe sepsis or even septic shock) compared touncomplicated trauma patients (usually SIRS), they have lower platelet counts. Since in manyinstances these decreased platelet counts are still in the 'normal range' these changes have receivedlittle attention. That platelet counts decrease as a result of increased endothelial activation may bethe explanation of the inverse relation between inflammation and platelet count in critically illpatients.

Page 22: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

12

Fat embolism syndromeThis thesis originated in studies on the incidence and causes of the fat embolism syndrome (FES).FES is typically seen in young patients who sustain isolated long bone fractures [57]. After aninterval of 8 hours to 2 days the three cardinal symptoms of the syndrome appear: • petechiae with a typical upper-body distribution, unlike that seen in severe thrombocytopenia• respiratory distress• cerebral disturbances

As the name of the syndrome denotes, fat is involved in these three cardinal symptoms, since fatemboli of bone marrow origin can be recovered from skin lesions, the lung and the brain duringpathological examination. In the process of embolization, the venous fat apparently easily(by)passes the lungs. Aggressive, early operative stabilization of fractures, and the improvedlevel of supportive care are assumed to have contributed to the decrease in FES-incidence.Although FES in seen less frequently than for example 25 years ago, both its causes and thereasons for its decreased incidence are unclear. Fever and tachycardia are among the 'minorsymptoms' described for FES [57], indicating a link of FES with inflammatory responses. Insteadof being a result of FES, systemic inflammation might be a factor in inducing FES. CRP canagglutinate fat globules into emboli [58] - a property of CRP that has even led to the developmentof a bed-side CRP-test based on fat-agglutination [59]. Here we looked at the relation of earlyinflammatory signs with the subsequent development of FES. Prolonged increases in pressures in (closed) wounds around the fractured bone may assist theprocess of fat intravasation and subsequent embolization. Early operation may decompress thefractured bone and prevent further embolization of fat. The potential detrimental effect of delayedtreatment on the incidence of FES, and the relevance of a persisting open foramen ovale [60] as ashunt for fat globules are explored in this thesis.

Page 23: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

13

OUTLINE AND AIMS OF THIS THESISIn this thesis the systemic counterparts of acute local inflammation were studied. As a part of theinnate immune response, systemic manifestations of inflammation were primarily studied intrauma patients HypothesisThe various stages of the systemic inflammatory response are expressed by distinct markers (pro-inflammatory cytokines as well as acute phase proteins and platelet counts) in a typical timesequence. They reflect the underlying pathophysiological mechanism and may offer possibilitiesfor monitoring (new) intervention strategies by providing better clinical prognostic and diagnostictests. The clinical model used to study these marker kinetics were trauma patients since this patientcategory allows to note the starting point of the (pathophysiologic) chain of events.Trauma patients are young and in general have no comorbity. By a better understanding of thetime sequence of the marker responses in trauma patients with single injuries physician maysubsequently better interpret responses in critically ill patients, most of whom have preexistent co-morbidity.

Aims of this thesisTo study the various stages of the systemic inflammatory response, we selected a number of(circulating) markers that we assumed would adequately reflect and be correlated with(patho)physiological signs and symptoms. The protein responses of IL-6, PCT, CRP, SAA andα1-antiproteinase as well as the platelet count and leukocyte count and physiological parametersas temperature or heart rate were studied.The first studies concern:- Fat embolism syndrome (chapters 2 and 3) The subsequent chapters are ordered according to the sequence of inflammatory events: - Interleukin-6 (chapters 4 and 5)- Procalcitonin (chapter 6)- Platelet counts (chapters 7, 8 and 9)

Two studies (chapters 7 and 9) were an intervention studies; all studies were retrospective indesign, although in chapters 4, 5, 6 and 9 some samples and data were prospectively collected.Not studied in this thesis were: animals, local responses, adaptive immune responses.

Fat embolism syndromeChapter 2 is a retrospective study of the incidence of FES in 172 patients with an isolated fractureof the femoral shaft. The goal was to find associations of the incidence of FES with the type offracture, the timing of operation and an early inflammatory response.Chapter 3 verifies if a right-to-left shunt in the heart has a causal role in FES. Such a shunt couldexplain the transit of large fat globules from the venous to the arterial circulation.

Page 24: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

14

Interleukin-6 and acute phase responsesChapter 4 describes IL-6 levels in patients with burns at a time that IL-6 had never beenmeasured in patients with acute systemic inflammation. The aim was to correlate IL-6 levels withbasic acute phase responses: i.e. fever, CRP-levels and α1-antiproteinase levels, and address thequestion if IL-6 was the long sought endogenous pyrogen. Chapter 5 examines IL-6 and acute phase responses in burns patients in detail. The goal was tostudy time dependent changes of phenomena in which IL-6 could play a causal role. Alsocorrelations of these parameters with IL-6 and the potential causal relations on the basis ofpublished evidence (as it was published at that time) are formulated.

ProcalcitoninChapter 6 it is assumed that endotoxin is not necessary for the induction of PCT. Thus, the directeffects of TNFα and IL-6 on the expression of PCT, SAA and CRP were measured. In vitro,human liver slices were used. In vivo, two groups of cancer patients, treated with TNFα and IL-6respectively, were studied.

Primary and secondary thrombocytopenia Early and late decreased platelet counts in trauma and ICU patients may reflect increasedsequestration due to systemic endothelial activation.The aim in chapter 7 was to examine early changes in platelet count as they occur during the first 2days after trauma. We also investigated if high-dose methylprednisolone administered shortly afterthe injury affected platelet consumption. This may indicate if the pleiotropic effects steroids are ableto affect inflammation related platelet sequestration.In chapter 8 the aim was relate late changes (i.e. > 2 days) in platelet count with mortality, sincepersisting systemic inflammation is known to be associated both with platelet sequestration andmortality. Patients from one surgical ICU were studied. The value of platelet counts was alsocompared to leukocyte counts.The goal in Chapter 9 was to generalize the observations of the previous study and address changesin platelet counts and outcome in a very large heterogeneous European multi-center population ofICU patients. The predictive power for mortality of early and late changes in platelet countrespectively, was investigated. For this purpose a simple mathematical model to describe time-dependent changes in platelet counts was used. We also investigated the behavior of platelet countsaccording to admission groups (medical, surgery scheduled or unscheduled.)

Finally Chapter 10 attempts to integrate the results and give suggestions for further research. Inparticular the timing of all responses observed in the various studies is combined into one scheme.The question what would constitute an ideal inflammatory marker is discussed, as well if such amarker exists.

Page 25: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

15

REFERENCES

1. Singer AJ, Clark RA. Cutaneous wound healing. N Eng J Med 1999;341:738-746.2. Webster's Medical Desk Dictionary. Merrian-Webster. 1986.3. Hirschfeld M, Ma Y, Weis JH, Vogel SN, Weis JJ. Cutting edge: Repurification of

lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. JImmunol 2000 165:618-622.

4. Means TK, Golenbock DT, Fenton MJ. The biology of Toll-like receptors. Cytokine GrowthFactor Rev 2000;11:219-32.

5. MacKay I, Rosen FS. Innate immunity. N Eng J Med 2000;338-344.6. American College of Chest Physicians/Society of Critical Care Medicine consensus conference:

definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.Crit Care Med 1992;20:864-74.

7. Goris RJ, te Boekhorst TP, Nuytinck JK, Gimbrere JS. Multiple-organ failure. Generalizedautodestructive inflammation? Arch Surg 1985;120:1109-1115.

8. Karjalainen J, Viitasalo M. Fever and cardiac rhythm. Arch Intern Med 1986;146:1169-719. Brigden M. The erythrocyte sedimentation rate. Still a helpful test when used judiciously. Postgrad

Med 1998;103:257-274.10. Bedell SE, Bush BT. Erythrocyte sedimentation rate. From folklore to facts. Am J Med

1985;78:1001-1009. 11. Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci 1982;389:39-48.12. Haupt W, Holzheimer RG, Riese J, Klein P, Hohenberger W. Association of low preoperative

serum albumin concentrations and the acute phase response. Eur J Surg 1999;165:307-13.13. Szalai AJ, Agrawal A, Greenhough TJ, Volanakis JE. C-reactive protein: structural biology and

host defense function. Clin Chem Lab Med 1999;37:265-270.14. Heinrich PC, Castell JV, Andus T.Interleukin-6 and the acute phase response. Biochem J 1990

265:621-636.15. Hurlimann J, Thorbecke GJ, Hochwald GM. The liver as the site of C-reactive protein formation. J

Exp Med 1966 123;1966:365-378.16. Gillies HC. Theory and practice of counter-irritation. MacMillan Oxford 1896.17. van Gool J. Farewell speech. May 18 1990. University of Amsterdam.18. Leon LR, Kozak W, Peschon J, Kluger MJ Exacerbated febrile responses to LPS, but not

turpentine, in TNF double receptor-knockout mice. Am J Physiol 1997;272:R563-9 19. Helle M, Boeije L, Aarden LA IL-6 is an intermediate in IL-1-induced thymocyte proliferation. J

Immunol 1989;142:4335-4338 .20. Brakenhoff JP, de Groot ER, Evers RF, Pannekoek H, Aarden LA. Molecular cloning and

expression of hybridoma growth factor in Escherichia coli. J Immunol 1987;139:4116-4121.21. Helle M, Boeije L, de Groot E, de Vos A, Aarden L. Sensitive ELISA for interleukin-6. Detection

of IL-6 in biological fluids: synovial fluids and sera. J Immunol Methods 1991;138:47-5622. Hack CE, Aarden LA, Thijs LG Role of cytokines in sepsis. Adv Immunol 1997;66:101-95

Page 26: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

16

23. van Gameren MM, Willemse PHB, Mulder NH, et al.:Effects of recombinant human interleukin-6in cancer patients: a phase I-II study. Blood 1994; 84:1434-1441.

24. Martin C, Boisson C, Haccoun M, Thomachot L, Mege JL. Patterns of cytokine evolution (tumornecrosis factor-alpha and interleukin-6) after septic shock, hemorrhagic shock, and severe trauma.Crit Care Med 1997;25:1813-1819.

25. Hack CE, De Groot ER, Felt-Bersma RJ, Nuijens JH, Strack Van Schijndel RJ, Eerenberg-BelmerAJ, Thijs LG, Aarden LA. Increased plasma levels of interleukin-6 in sepsis. Blood 1989;74:1704-1710.

26. Abraham E. Why immunomodulatory therapies have not worked in sepsis. Intensive Care Med1999;25:556-566.

27. Giannoudis PV, Smith MR, Evans RT, Bellamy MC, Guillou PJ. Serum CRP and IL-6 levels aftertrauma. Not predictive of septic complications in 31 patients. Acta Orthop Scand 1998;69:184-188

28. Vico P, Papillon J. Factors involved in burn mortality: a multivariate statistical approach based ondiscriminant analysis. Burns 1992;18:212-215.

29. Hene IZ, Bendien SA, Nijsten MWN, ten Duis HJ, Zwaveling JH. Fever and severe trauma. IntensiveCare Med 1998;24 Suppl1:S11.

30. van Deventer SJ, Buller HR, ten Cate JW, Sturk A, Pauw W Endotoxaemia: an early predictor ofsepticaemia in febrile patients. Lancet 1988;i (8586):605-9.

31. Novitsky TJ. Limitations of the Limulus amebocyte lysate test in demonstrating circulatinglipopolysaccharides. Ann N Y Acad Sci 1998;851:416-21

32. Meisner M. Procalcitonin. A new innovative infection parameter. Biochemical and clinical aspects.Berlin: Brahms Diagnostica 1996. ISBN 3-00-000803-9

33. Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, Bohuon C. Procalcitonin increaseafter endotoxin injection in normal subjects. J Clin Endocrinol Metab 1994;79:1605-1608.

34. Snider RH Jr, Nylen ES, Becker KL. Procalcitonin and its component peptides in systemicinflammation: immunochemical characterisation. J Investig Med 1997; 45:552-560.

35. Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C. High serum procalcitoninconcentrations in patients with sepsis and infection. Lancet 1993; 341:515-518.

36. de Werra I, Jaccard C, Corradin SB, Chiolero R, Yersin B, Gallati H, Assicot M, Bohuon C,Baumgartner JD, Glauser MP, Heumann D. Cytokines, nitrite/nitrate, soluble tumor necrosis factorreceptors, and procalcitonin concentrations: comparisons in patients with septic shock, cardiogenicshock, and bacterial pneumonia. Crit Care Med 1997;25:607-613.

37. Gendrel D, Raymond J, Coste J, Moulin F, Lorrot M, Guerin S, Ravilly S, Lefevre H, Royer C,Lacombe C, Palmer P, Bohuon C. Comparison of procalcitonin with C-reactive protein, interleukin6 and interferon-alpha for differentiation of bacterial vs. viral infections. Pediatr Infect Dis J1999;18:875-881.

38. de Bont E, Vellenga E, Swaanenburg J, Visser-van Brummen P, Kamps W. Procalcitonin used as amarker of bacterial infection in neutropenic cancer patients with fever. Infection 2000;28:398-400.

39. Griesshammer M, Bangerter M, Sauer T, Wennauer R, Bergmann L, Heimpel H. Aetiology andclinical significance of thrombocytosis: analysis of 732 patients with an elevated platelet count. JInt Med 1999;245:295-300.

40. Levi M, ten Cate H. Disseminated Intravascular coagulation. N Eng J Med 1999;341: 586-92.

Page 27: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 1

17

41. Neame PB, Kelton JG, Walker IR, Stewart IO, Nossel HL, Hirsch J. Thrombocytopenia insepticemia: the role of disseminated intravascular coagulation. Blood 1980;56:88-92.

42. Lentsch AB, Ward PA Regulation of inflammatory vascular damage. J Pathol 2000;190:343-348.43. Hunt BJ, Jurd KM. Endothelial cell activation. A central pathophysiological process. BMJ

1998;316:1328-132944. Mannel DN, Grau GE. Role of platelet adhesion in homeostasis and immunopathology. Mol Path

1997;50:175-185.45. Piguet PF, Vesin C, Donati Y, Tacchin-Cottier F, Belin D, Barazzone C. Urokinase receptor

(uPAR,CD-87) is a platelet receptor important for TNF-induced endothelial adhesion in mice.Circulation 1999;99:3315-3321.

46. Cannon CP. Incorporating platelet glycoprotein IIb/IIIa inhibition in critical pathways: unstableangina/non-ST-segment elevation myocardial infarction. Clin Cardiol 1999;22(Suppl):IV30-36.

47. Sigurdsson GH, Christenson JT, el-Rakshy MB, et al: Intestinal platelet trapping after traumatic andseptic shock. An early sign of sepsis and multiorgan failure in critically ill patients? Crit Care Med1992;20:458-467.

48. van Deuren M, Neeleman C, van 't Hek LGFM, van der Meer JWM. A normal platelet count atadmission in acute meningococcal disease does not exclude a fulminant course. Intensive Care Med1998;24:157-161.

49. Bradbury AW, Bachoo P, Milne AA, Duncan JL. Platelet count and the outcome of operation forruptured abdominal aortic aneurysm. J Vasc Surg 1995;21:484-491.

50. Bonfiglio MF, Traeger SM, Kier KL, Martin BR, Hulisz DT, Verbeck SR. Thrombocytopenia inintensive care patients: a comprehensive analysis of risk factors in 314 patients. Ann Pharmacother1995;29:835-842.

51. Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. Multiple organdysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med1995;23:1638-1652.

52. Vincent JL, de Mendonca A, Cantraine F, et al. Use of the SOFA score to assess the incidence oforgan dysfunction/failure in intensive care units: results of a multicenter prospective study. Crit CareMed 1998; 26:1793-1800.

53. Pollack MM, Patel KM, Ruttimann UE. PRISM III: An updated pediatric risk of mortality score.Crit Care Med 1996;24:743-752.

54. Buckley MF, James JW, Brown DE, Whyte GS, Dean MG, Chesterman CN, Donald JA. A novelapproach to the assessment of variations in the human platelet count. Thromb Haemost2000;83:480-484.

55. Ross DW, Ayscue LH, Watson J, et al: Stability of hematologic parameters in healthy subjects.Intraindividual versus interindividual variation. Am J Clin Pathol 1988;90:262-267.

56. Kaushansky K. Thrombopoietin. N Engl J Med 1998;339:746-754.57. ten Duis HJ. Fat embolism syndrome. Injury 1997;28:77-85.58. Rowe IR, Soutar AK, Pepys MB. Agglutination of intravenous lipid emulsion ('Intralipid') and

plasma lipoproteins by C-reactive protein. Clin Exp Immunol 1986;66:241-247.

Page 28: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Introduction

18

59. Richter D, Rohricht AM, Nurnberger W, Wahn V, Schroten H. The fat emulsion agglutination test:a reliable and cost effective alternative to the latex agglutination test for rapid bedside CRPmeasurement. Clin Chim Acta 1997; 261:141-148.

60. Webster MW, Chancellor AM, Smith HJ, Swift DL, Sharpe DN, Bass NM, Glasgow GL. Patentforamen ovale in young stroke patients. Lancet 1988;ii:11-12.

Page 29: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 2

FAT EMBOLISM IN PATIENTS WITHAN ISOLATED FRACTURE OF THE FEMORAL SHAFT

H.J. ten Duis, M.W.N. Nijsten, H.J. Klasen, B. Binnendijk

J Trauma 1988; 28: 383-390

Page 30: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism in patients with an isolated fracture of the femoral shaft

20

ABSTRACT

Analysis of basic pathophysiologic variables in fat embolism patients is often restricted by thecomplexity of the different injuries present in each individual patient. To avoid this problem weinvestigated the presence of the fat embolism syndrome in patients with an 'isolated' fracture of thefemoral shaft. Two groups were distinguished: those who had an open fracture or a closed fracturetreated operatively within 24 hours after the accident (decompression group), and those who weretreated initially conservatively (non-decompressed group). Clinical fat embolism occurred only inpatients in the non-decompressed group (3.5%). They showed significantly higher temperatures,lower pulse rates, a progressive hemoglobin decrease, and a fracture localization more proximal(p<0.025) than the other patients in the non-decompressed group; they also showed pathophysiologicpatterns significantly different from the patients in the decompressed group. Although thepathophysiologic mechanism of the onset of clinical fat embolism remains unclear, initialtemperature elevations in combination with 'typical' fracture localization and fracture type appear tohave a predictive value.

INTRODUCTION

The fat embolism syndrome is considered to be a symptom complex of acute respiratory distress,cerebral disturbances, and petechiae after one or more long bone fractures [1]. Many researchers haveinvestigated the pathophysiology of this syndrome, though essential etiologic items remain unclear.Neither quantitative nor qualitative studies on fat embolization have been able to indicate consistentrisk factors for the fat embolism syndrome. The clinical picture of full-blown fat embolism maydevelop after a single long bone fracture, although the incidence increases with the number offractures. The investigation of a category of patients with single injuries enabled us to perform ananalysis of possible etiologic factors without being influenced by accompanying injuries. We studied a group of patients with an isolated fracture of the femoral shaft to determine whichfactors might predispose to the development of the fat embolism syndrome. Especially fracture site,fracture type and degree of comminution were analyzed and also minor fat embolism features likepyrexia, tachycardia, and thrombocytopenia.

Page 31: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 2

21

MATERIALS AND METHODS

In the period 1967 through 1985, 172 consecutive patients, varying from 16 and 65 years of age, withan 'isolated' fracture of the femoral shaft, were studied from the moment they arrived at the

Department of Traumatology at the University Hos-pital Groningen. Individuals with moderate andsevere accompanying injuries were excluded, whilepatients with one minor additional injury (patellafracture, fracture of the clavicle or less) were includedin the study. The investigation was performed onpatients with a closed as well as an open fracture ofthe femoral shaft. Patients with a pathological fractureof the femoral shaft were excluded from thisinvestigation.

Each patient was examined daily for the symptoms offat embolism. Records of temperature, pulse andblood pressure were maintained as well as water and

salt balance. The following investigations were performed daily for 6 days: supine chest X-ray,arterial blood gas analysis, hemoglobin, hematocrit, platelet- and white blood count, coagulograms,fibrinogen concentration levels, and serum elektrolyte and triglyceride determinations. The X-rays ofthe femur, taken in the emergency room, were used to determine fracture localization in the femoralshaft, degree of comminution and fracture type. For fracture localization the shaft was divided intoseven equal segments according to Kootstra [2] (Fig. 2.1).The following fracture types were distinguished (we made use of the fracture quotient Q=x/y;x:length of fracture; y:width of femoral shaft at place of fracture): transverse Q=1.0-1.1; shortoblique Q=1.2-1.3; oblique Q=1.4-1.7; longitudinal Q>1.7; spiral. The degree of comminution was

Table 2.1

Accident cause in isolated fractures

of the femoral shaft

Cause N %

Fall 18 11

Pedestrian 3 2

Driver/passenger of

Bicycle 16 9

Autobike 72 42

Motorcycle 9 5

Automobile 30 17

Hit by heavy object 7 4

Miscellaneous 17 10

Figure 2.1. Division of the femoral shaft intoseven equal segments.

Page 32: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism in patients with an isolated fracture of the femoral shaft

2

noted: none, slight comminution with two or more fragments of less than 7 mm, moderatecomminution with fragments from 7 mm to 2 cm, and serious comminution with multiple fragmentsgreater than 2 cm with or without fragments of smaller size. Open fractures were treated, preferablywith open reduction and internal fixation, on the day of the accident. In patients with a closedfracture, the fracture was reduced and immobilized in skeletal traction within 6 hours afteradmission. During the period 1967-1972 most closed fractures were treated conservatively; in theperiod 1972-1985 open reduction and internal fixation from day 10 onwards was usually the firstchoice of treatment. The diagnosis of fat embolism was made when at least two major symptoms asdescribed by Gurd [3] were observed: 1) petechial rash, 2) respiratory distress with bilateral clinicaland/or radiologic signs of pulmonary involvement, and 3) evidence of cerebral involvement unrelatedto head injuries (primarily excluded). Two main groups of patients were distinguished: those whohad a closed fracture and were initially (10 days) treated conservatively (non-decompressed group)and patients who had an open fracture, or a closed fracture but were treated initially (within 24 hours)operatively (decompression group).No corticosteroids, antipyretics, or antiphlogistic medicaments were given in the initial phase oftreatment. Student's t-test for unpaired values was used in the statistical evaluation of the results.

R

Owtip

2

ESULTS

f the 172 patients who satisfied the conditions of this study, 133 were males (77%) and 39 (23%)ere females. The accident causes are given in Table 2.1. The average delay between accident andme of admission was 45 ± 35 minutes. The distribution of ages is given in Fig. 2.2. None of theatients showed any sign of shock at admission or during the initial phase of treatment.

0

10

20

30

40

50

60

70

80

90

100

16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65

Years

Num

ber o

f pat

ient

s

Figure 2.2. Ages of the patients at the time of the accident.

Page 33: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 2

23

There were 21 open and 151 closed fractures. Twenty-two patients with closed fractures weretreated operatively on the day of the accident. Six patients (3.5%) were diagnosed as having fatembolism (Table 2.2), of whom four were given supplementary oxygen, and two patients had to betreated with mechanical ventilation. These patients all belonged to the primarily conservativelytreated group with a closed fracture (129 patients). Neither the patients with an open fracture nor thepatients with a closed fracture, initially treated operatively, developed two or more major signs ofclinical fat embolism. The fat embolism patients showed a fracture localization in segment two or three of the femoral shaft(mean 2.3). The average localization in the other patients was significantly more distal (mean 4.2;p<0.025). Fracture localization and degree of comminution are given in Table 2.3. Temperature recordings revealed significantly higher temperatures on the day of the accident and day1 in the fat embolism patients compared to the other patients in the non-decompressed group (p<0.01,day 0; p<0.005, day 1). Low initial temperatures were found in the decompressed group compared to

the non-decompressed group (p<0.01) (Fig. 2.3).In the fat embolism group the initial pulse rate was as low as in the remaining patients of the non-decompressed group, but increased to significantly higher values during the rest of the observationperiod (p<0.025). Pulse rate values in the decompressed group generally followed the temperaturepattern (Fig. 2.4). All patients showed comparable decreases in hemoglobin and hematocrit values in the first 24 hoursafter the accident. Significantly lower values were observed in the fat embolism group from day 2onwards (p<0.025) compared to the remaining patients in the non-decompressed group. Thisdecrease was comparable to those of the open and/or primarily operatively treated patients (p<0.001)(Fig. 2.5).

Table 2.2.Major fat embolism symptoms present in patients of non-decompressedand decompression group.

Non-decompressed Decompressed

FES (6) No FES (123) (43)

Petechial rash 4 1 0

Pulmonary dysfunction 5 4 0

Mental dysfunction 4 1 0

Page 34: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism in patients with an isolated fracture of the femoral shaft

24

All groups showed a temporary decrease in platelet count during the observation period. Plateletcounts were slightly lower in the fatembolism group (p<0.05, day 1) (Fig.2.6) and in the primarilydecompressed group.Arterial blood gas analysis revealedabout equal values directly after thetrauma in all groups. The fatembolism patients showed asignificant decrease in PaO2 andoxygen saturation levels on day 1 afterthe trauma (p<0.001) (Fig. 2.7). Thefat embolism group had a PaO2 of 8.0kPa and an oxygen saturation of 90%,compared to 11.2 kPa and 96% for thenon-decompressed group and 14.1kPa and 97% in the decompressedgroup. After (symptomatic) treatmentof the pulmonary dysfunction in thefat embolism patients, this groupreached similar PaO2 and oxygensaturation levels to the other groupsaround day 2 after the trauma. Noimportant differences between the groupand creatinine levels, plasma protein, anand left the hospital in good health.

DISCUSSION

Fat embolism is a syndrome which idisturbances, and petechiae. The onset indications known which invariably

Table 2.3.

Fracture localization and degree of comminution inpatients in the fat embolism group, non-decompressedgroup and decompression group.

Fat embolism Nondecompression

Decompression

Segment

None

Slight

Moderate

Serious

None

Slight

Moderate

Serious

None

Slight

Moderate

Serious

1 0 0 0 0 6 1 1 1 0 0 1 02 1 0 2 1 4 7 8 1 0 2 2 13 0 1 0 1 7 9 11 1 0 3 4 14 0 0 0 0 7 6 6 0 3 1 7 35 0 0 0 0 10 6 13 2 2 0 3 16 0 0 0 0 3 2 6 0 0 1 0 17 0 0 0 0 1 0 2 0 1 0 2 2

total 1 1 2 2 38 31 47 5 6 7 19 9

s were observed in leukocyte counts, electrolytes, or BUNd albumin, as well as coagulograms. All patients survived

ncludes the symptoms of respiratory failure, sensoriumof the syndrome is unpredictable and there are no hard

Page 35: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 2

Temperature

37.0

37.5

38.0

38.5

39.0

0 1 2 3 4 5

degr

ees

Cel

cius

Hemoglobin

80

100

120

140

160

0 1 2 3 4 5

g/l

5

6

7

8

9

mm

ol/l

Hemoglobin

80

100

120

140

160

0 1 2 3 4 5

g/l

5

6

7

8

9

mm

ol/l

Temperature

37.0

37.5

38.0

38.5

39.0

0 1 2 3 4 5

degr

ees

Cel

cius

Figure 2.3. Mean with standard error of the mean of temperature values in patients of the fat embolism group (opensquares), non-decompressed group (open circles) and decompressed group (filled circles) during the 6 day periodfollowing trauma. Statistically significant differences are marked with a star.

120

Figure 2.5. Mean with standard error of the mean of hemoglobin values in patients of the fat embolism group (opensquares), non-decompressed group (open circles) and decompressed group (filled circles) during the 6 day periodfollowing trauma. Statistically significant differences are marked with a star.

lead to its development. The incidence - relativelwith the number of fractures or injuries [4]. In this study six cases of clinical fat embolism (3.5to other authors [5], considering the fact that this

Heart Rate

70

80

90

100

110

0 1 2 3 4 5

/min

ute

20

Figure 2.4. Mean with standard error of the mean of heart rsquares), non-decompressed group (open circles) and decomfollowing trauma. Statistically significant differences are ma

Platelet count

100

150

200

250

300

0 1 2 3 4 5

10E9

/l

300

Figure 2.6. Mean with standard error of the mean of platesquares), non-decompressed group (open circles) and decofollowing trauma. Statistically significant differences are m

y

%s

atpr

lema

r

tu

e reke

t cpr

1

25

are in patients with single fractures - increases

) were observed. This percentage is comparabledy only included solitary fractures. Clinical fat

Heart Rate

70

80

90

100

110

0 1 2 3 4 5

/min

ute

values in patients of the fat embolism group (openssed group (filled circles) during the 6 day periodd with a star.

Platelet count

100

150

200

250

0 1 2 3 4 5

10E9

/l

ounts in patients of the fat embolism group (openressed group (filled circles) during the 6 day periodked with a star.

Page 36: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism in patients with an isolated fracture of the femoral shaft

2

ehofdcoecthNwestith(inwnIbsv

15 15

Fsf

6

mbolism developed in patients who had a closedours (non-decompressed group). In contrast, patiperated upon early (within 24 hours; decomprixation showed no clear evidence of pulmoniscrepancy between decompressed and non-decolosed fractures who were operated upon within 2nset of fat embolism occur in the first 24 hoursmbolization of marrow fat caused by fat globirculation after injury [5]. Although authors havee incidence of fat embolism, it is unlikely that one of the patients with an open fracture or wiithin 24 hours showed any evidence of fat

mbolization alone is unlikely to provoke fat emtresses in an injured patient to induce clinicallyssue ischemia by fracture hematoma may play ane fracture hematoma and reduces tissue pr

decompression) hypothesis is the investigation cidence of fat embolism in patients with open fhy cases of postoperative clinical fat embolismailing of the femoral shaft fractures [8].s the fat embolism syndrome related to fracture een paid to this criterion. All fat embolism paegment two or three of the femoral shaft, just cenous sinusoids within the marrow with large ca

PaO2

5

6

7

8

9

10

11

12

13

14

0 1 2 3 4 5

KPa

igure 2.7. Mean with standard error of the mean of arteriquares), non-decompressed group (open circles) and decoollowing trauma. Statistically significant differences are m

fracture and had not been operated on within 24ents with an open fracture or those who had beenession group) using open reduction and internalary or cerebral disturbances or petechiae. Thismpressed patients, especially in the patients with

4 hours, suggests that induction phenomena for the. In this regard a well known factor is pulmonaryules, which are invariably found in the venous tried to correlate the amount of fat globules withthe syndrome is caused by this mechanism only.th a closed fracture who had been operated uponembolism symptoms. Therefore, bone marrowbolism but it may compound the effect of other apparent pulmonary problems. Especially local important role. Opening of the fracture evacuates

essure around the fracture. In support of thisby Kallio in 1941 [6,7], who described a lowerractures. Concomitantly, this might be the reason were observed after acute 'closed' intramedullary

type or localization? Until now less attention hastients in this series had a fracture localization inranial of the isthmus. At this place there severalpillaries which do not have any wall musculature.

PaO2

5

6

7

8

9

10

11

12

13

14

0 1 2 3 4 5K

Pa

al pO2 values in patients of the fat embolism group (openmpressed group (filled circles) during the 6 day periodarked with a star.

Page 37: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 2

27

The walls of these veins are very fragile here and are only embedded in depots of marrow fat [4].This might explain why marrow fat just in this place might be released into the venous circulation.The fat embolism patients were shown to have a short oblique or oblique fracture with a moderatedegree of comminution (except the eldest patient with a spiral fracture) especially of fragments up to2 cm. These fracture characteristics are often related to a certain degree of energy impact and aredesignated as high-energy injuries. Consequently, this moderate degree of bone marrow destructionwill invariably be accompanied by fracture hematoma formation, soft-tissue injury, andconsequently, in closed fractures, by local tissue hypoxia.Interesting in this regard was that differences in blood loss reflected in hemoglobin and hematocritdecrease could not be found during the first 24 hours between the fat embolism group and the non-decompressed group. Significant differences were detected from day 2 onwards and seem thereforenot related to the initial amount of hematoma formation.The temperature pattern in fat embolism patients generally showed significantly elevated valueswithin 24 hours after the accident, before any sign of pulmonary insufficiency was noticed. Herethere was a significant difference to the main (non-decompressed) group with a highest mean of37.9°C and 37.6°C in the group with any form of decompression. This pyrexia is a well knownfeature in patients with clinically apparent fat embolism, and it is probably induced through therelease of interleukin-1 (endogenous pyrogen) by activated macrophages [9]. Fat globuli or toxicproducts released from the local ischemic tissues could serve as activators. The low initial pulse ratevalues (day of accident) in the fat embolism patients are of special interest because a rise intemperature is normally accompanied by an increase in pulse rate. During the entire 6-day observation period the decompressed group was the least compromisedconcerning respiratory dysfunction. As expected, arterial PaO2 and oxygen saturation were low at theonset of the fat embolism syndrome, although on admission differences between the groups could notbe detected. As only two patients had to be treated by mechanical ventilation, the degree ofrespiratory distress was limited. Following the assumption that the pulmonary distress is an integralpart of complement-induced PMN and platelet activation, the moderate degree of respiratory distressis in accordance with the only slight decrease in platelet numbers and the absence of severedisturbances in coagulograms.The form of fat embolism described in this article is considered the nonfulminant subacute orclassical form classified by Sevitt [11]. The fulminant fat embolism syndrome, which is oftenmisdiagnosed and develops suddenly within a few hours after the accident, with an often fataloutcome was not observed in our selective series. It is unlikely that there will be any predictable signfor this form of fat embolism.To our knowledge the patients described represent the first large series of patients investigated for theoccurence of fat embolism in a 'standardized' injury.In this series a temperature increase to levels of 38.5°C or higher within 24 hours combined with afracture of the femoral shaft just proximal to the isthmus seems to have some predictive value. If apatient shows these symptoms within 24 hours after the accident, one should be aware of the dangerof a fat embolism syndrome during the following few days.

Page 38: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism in patients with an isolated fracture of the femoral shaft

28

REFERENCES1. Peltier LF. Fat embolism: a current concept. Clin Orthop 1969;66:241-253.2. Kootstra G. Femoral shaft fractures in adults. A study of 329 consecutive cases with a statistical

analysis of different methods of treatment. Thesis, Assen, the Netherlands, van Gorcum, 1973.3. Gurd AR. Fat embolism: an aid to diagnosis. J Bone Jt Surg 1970;52-B;732-737.4. Gossling HR, Pelligrini VD Jr. Fat embolism syndrome: a review of the pathophysiology and

physiological basis of treatment. Clin Orthop 1982;165:68-82.5. Allardyce DB, Meek RN, Woodruff B, Cassim MM, Ellis D. Increasing our knowledge of the

pathogenesis of fat embolism:a prospective study of 43 patients with fractured femoral shafts. JTrauma 1974;14:955-962.

6. Editorial comment to (7) J Trauma 1982;22:894.7. Riska EB, Myllynen P. Fat embolism in patients with multiple injuries. J Trauma 1982;22:891-894.8. King KF, Rush J. Closed intramedullary nailing of femoral shaft fractures: a review of 112 cases

treated by Kuntscher technique. J Bone Jt Surg 1981;63-A:1319-1323.9. Dinarello CA. Interleukin-1. Rev Infect Dis 1984;6:51-95.10. Manning JB, Bach AW, Herman CM, Carrico CJ. Fat release after femur nailing in the dog.

J Trauma 1983;23:322-326.11. Sevitt S. Fat embolism. London, Butterworths, 1962.

Page 39: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 3

FAT EMBOLISM AND PATENT FORAMEN OVALE

M.W.N. Nijsten, J.P.M. Hamer, H.J. ten Duis, J.L. Posma

Part of this chapter was published as a Letter inthe Lancet 1989; i: 1271

Page 40: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism and patent foramen ovale

32

INTRODUCTIONFat is an integral constituent of the soft tissues and the bone marrow. After skeletal trauma, bonemarrow emboli can rapidly enter the circulation. Especially long bone fractures are known togenerate circulating, abnormally large fat globules that sometimes have diameters>10 µm, incontrast to the normal fat that circulates in the form of small lipoproteins [1].

Acute massive fat embolismAs forensic physicians know, a dead person with multiple fractures found three floors below anopen window should have his lungs examined. If fat emboli are present in his lung he was stillalive at the moment he hit the ground. However, if no fat emboli or marrow emboli are present hemay have been dead before he fell, and thus may have been murdered [1]. The sometimeshyperacute nature of this embolization process is illustrated by a large post-mortem study doneon aircraft accident victims. In 1979 a New Zealand jetliner crashed into Mount Erebus onAntarctica, killing all persons aboard. Detailed post-mortem analysis [2] in 205 of the 257 victimsshowed that despite an nearly instantaneous death, many had fat emboli (65%) and bone marrowemboli (29%) in their pulmonary capillary beds. In a different study of 56 patients who died within 24 hours of blunt injury (including long bonefractures) 68% had significant pulmonary fat emboli upon post-mortem examination [3]. Acutemassive fat embolism, as described above, should not be confused with classic fat embolismsyndrome (FES) as defined below.

Classic fat embolism syndrome (FES)Some trauma patients can develop the fat embolism syndrome (FES). The classical fat embolismsyndrome is generally defined by three major symptoms that occur with a delayed onset of hoursto days after the trauma [4]:• A petechial rash on the anterior chest, face, axillae, conjunctivae and other non-dependent

parts in general.• Respiratory distress with diffuse bilateral abnormalities on the chest X-ray.• Cerebral disturbances varying from mild to coma. In addition to these three major criteria aspecific minor signs are usually observed, the mostimportant being fever, tachycardia and thrombocytopenia.Whereas acute massive fat embolism is often diagnosed post-mortem in very severely injuredpatients who rapidly succumb, patients with FES will usually survive. Apart from severity, thecrucial difference between these two entities is the delayed onset that is seen in FES. In apparentlysimilar patients with long bone fractures, only a minority will actually develop FES after thesymptom-free period. Two central problems in FES-research are :• What is the precise origin and what are the effects of abnormal circulating fat that is observed in

most patients after trauma?

Page 41: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 3

33

• Why does only a minority develop FES?

Origin of circulating fatTwo main theories on the genesis of circulating fat emboli exist: the mechanical and the chemicaltheory [1,4]. The mechanical theory explains the strong association between long bone fractures andFES through the entrance of bone marrow fat into the circulation. The chemical theory [5,6] isinspired by observations that fat emboli can occur in patients who have no trauma, such as patientstreated by steroids or patients with sickle cell disease [7]. Also some biochemical changes can lead tothe agglutination of fat and fat emboli, as has been observed in the early days of total parenteralnutrition [8]. The acute phase protein CRP can agglutinate fat - a property that has even been used fora new rapid bedside CRP-assay [9].Although the mechanical theory is now the most popular, the mechanical and chemical models arenot mutually exclusive and may act in concert. The mechanical theory may not only involve fatembolization from the bones, but also from the soft tissues, as confirmed in a recent study [3].

Predisposing factors for FESIt has proven very difficult to predict who will develop FES. Many investigators have wondered whyFES only occurs in a minority of apparently similar patients. In a previous study we identifieddelayed stabilization of femoral fractures and early development of fever as a risk factors for thedevelopment of FES [10]. When venous fat particles reach the arterial circulation, the lungs musthave been (by)passed somehow. In addition to exchanging oxygen and carbon dioxide with thesurroundings, the lung has an important filtering function. The lungs continuously interact withand filter many particles that would otherwise enter the systemic arterial circulation. This includesthrombo-emboli, bone marrow emboli, air emboli and tumor emboli.The most prevalent occurring right-to-left shunt at the level of the heart is the patent foramen ovale.The presence of a patent foramen ovale (PFO) as detected by echocardiography is strongly increasedin young patients who sustained ischaemic stroke [11,12] and divers who developed decompressionsickness [13]. By analogy, it could be hypothesized that a patent foramen ovale might allow entry offat globules into the systemic circulation. Might patients with a history of post-traumatic fatembolism have a patent foramen ovale? We investigated the presence of PFO by echocardiographyin patients who developed FES after sustaining isolated skeletal trauma.

Page 42: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism and patent foramen ovale

34

Table 3.1.Patients who developed FES and were later investigated bytrans-esophageal echocardiography.

Male/Female 5/1Mean Age (+/-SD) 24 +/- 11Tibial fracture 1Isolated femoral fracture 4Bilateral femoral fracture 1

PATIENTS AND METHODSWe selected otherwise healthy patients with a history of fat embolism syndrome after isolated long-bone fractures. Of all trauma patients with isolated fractures of the long extremities that were seen atour hospital between 1968 and 1985, 12 met this condition. In a retrospective series of 172 patientswith an isolated femoral fracture admitted to our hospital between 6 developed FES [10]. Anadditional 6 patients with FES were identified after screening patients with tibial, combined tibial andfemoral or bilateral femoral fractures without significant additional injury [4]. To detect the presenceof PFO, transesophageal color-coded Doppler echocardiography with a Toshiba 65A 3.75 MHztransducer was performed as an outpatient procedure. The Valsalva maneuver was performed duringechocardiography to maximize the sensitivity for detecting a shunt across a patent foramen ovale.

RESULTSOf the total of 12 patients with diagnosed FES, 6 patients (Table 3.1) volunteered to undergoechocardiography. Echocardiography was performed without complications, and resulted inadequate imaging of the atria in all cases. In 5 patients no right-to-left shunt was present. 1 patienthad a minute right-to-left shunt during the Valsalva maneuver. This short-lasting and barelydetectable shunt was of no pathophysiological significance.

DISCUSSIONThe fact that no patients in our series had relevant right-to-left shunting as measured with a sensitivetechnique [14], contrasts with the reported frequencies of PFO in patients who sustained idiopathicischaemic stroke and serious air embolism (50% and 61% respectively) [11-13]. Whereas PFOobviously is of major importance in stroke and air embolism, the absence of a relevant foramen ovalein the patients we studied makes it unlikely that the presence PFO has a causal role in thedevelopment of FES. Although our series of FES patients is small, the observed incidence of 0 out of6 makes PFO very unlikely as a necessary factor for the development of FES. It is no surprise thatwe found no relevant PFO in patients who had well-defined FES. In fact an important role ofPFO in the pathogenesis of FES has long ago already been proposed and challenged [1]. In post-mortem investigations in seven patients who died with FES, Sevitt found no PFO in any of thepatients [15]. With the availability of trans-esophageal echocardiography, assessing PFO afterFES was a logical step. Our study is the first that confirms this observation in patients who arestill alive (long) after recovering from FES.

Page 43: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 3

35

Another argument against a causal role of PFO in FES is the presence of circulating fat globulesin the majority of trauma patients [16], while the population incidence of PFO is below 50% evenwhen measured according to the most sensitive criteria. Thus systemically circulating fat ispresent regardless of a PFO. The fat emboli somehow (repeatedly) pass the lungs. Possibly the fatemboli deform to pass the lungs' capillaries (Fig 3.1). In the majority of patients with skeletalinjury fat excretion in the urine can be shown according to some investigators [17], underscoringthe ease with which fat globules reach the systemic circulation, and pass the glomerular filter aswell.

Pell's case report in 1993The discussion of the relevance of PFO would probably have been closed, had not a paper on thissubject appeared in the New England Journal Medicine [18]. The editorial [19] accompanyingthis brief report was even optimistically titled "Unravelling the fat embolism syndrome". In theirpaper Pell and colleagues suggested a major role of PFO in the pathogenesis of FES on the basisof a report on a single patient who died shortly after intramedullary nailing of a femoral shaftfracture. Transesophageal echocardiography was performed during the operation. Showers ofechogenic masses were seen passing through the right heart. Even a lump of 7 cm was seen.Material was seen passing through a PFO. The fatal outcome the authors vividly describe is oneof acute cor pulmonale with secondary paradoxical embolism. The floating objects imaged bytransesophageal echocardiography, must have been large thrombi or bone marrow fragments.Finally, the patient described was not previously healthy, and he did not display the characteristicsymptom-free interval. The case report by Pell illustrates the devastating consequences of acute cor pulmonale in patientswho often are already in a compromised cardiopulmonary condition. Unfortunately, the patientdescribed in the case report in the New England Journal of Medicine did not have typical FES.Contrary to what might be inferred from the report's title, this patient did in all likelihood not diefrom FES or from PFO, and he certainly did not show the classic picture of FES. Whereas thegenesis of FES is not understood, acute right heart failure in patients where embolic material isdislodged during operative procedures is well-known and well-understood. In fact today morepatients may die from this form of acute pulmonary embolism than patients who die from theclassical FES.Pell later published a series of 24 patients who were monitored with TEE per-operatively [20]:small emboli were observed in 14 patients; in 6 patients moderately large emboli (up to 10 mm)and in 4 patients large emboli >10mm were observed. The dimensions of the observed emboliunderscore that 70mm is very large indeed for an embolic 'lump' as was described in the casereport [17].

Animal models of FESOne of the difficulties in investigating FES is finding an appropriate animal model. A correctanimal model should include a trauma or at least a simulation of the effects of trauma, asubsequent interval that is relatively free of signs that is later followed by organ manifestations offat emboli in affected organs. Despite the fact that many animal experiments designed to model

Page 44: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism and patent foramen ovale

36

fat embolism have been carried out, no model reproduces the events observed in humans. Eitherthe animals rapidly succumbed from traumatic shock [21], or immediate pulmonary edema wasinduced by fatty acid injury [22,23], or unrealistically massive neutral fat infusions resulted inobstructive shock [24]. None of the models reproduce the intriguing 'incubation period' that isobserved in classical FES.

Decreasing incidence of FESAlthough FES undoubtedly remains a real entity [25], the incidence of clinically manifest FEShas decreased to such extent that many clinicians have never seen a patient with the classical FES.Probably a combination of changes in modern management of trauma patients has contributed tothis decrease [26]. Except for the more rapid stabilization of fractures [27] is not clear whichother changes have been decisive in reducing the incidence of FES. Potential factors are: moreaggressive fluid infusions, early application of positive end-expiratory pressure (PEEP) or theroutine early administration of low-molecular weight heparins. Although this decreased incidenceof manifest FES may be good news, observational studies into the causes of FES are hampered bythe low incidence of the syndrome. Thus it may be wise to direct research efforts to detectsubclinical FES, for example with modern imaging tools.

Imaging in FESIn a further advance in real-time monitoring of embolic events, Edmonds recently reported on theuse of transcranial Doppler to detect fat or air emboli during total hip arthroplasty [28]. Embolicsignals in the middle cerebral artery were found in 8 of the 20 patients that were monitored. Thenumber of embolic signals in these patients varied from 1 to 200 - in all cases coinciding with theimpaction of a cemented component or after hip relocation. Another useful application of real-time imaging is the use of peroperative TEE to compare two different techniques of artificial hipfixation for their potential to generate emboli.Whereas conventional cementing generated emboli in 95% of the patients, a new 'bone vacuum'technique resulted in only 5% emboli [30]. Such studies have also shown that patients with pre-existing disease are especially prone for complications from iatrogenic emboli. CT-scans of thelungs have shown that the radiological picture of FES [31] differs from acute pulmonary edema orARDS. CT-scanning and magnetic resonance imaging of the brain has shown lesions caused bycerebral FES [32], indicating that although patients survive FES, it is may leave permanentdefects.

Page 45: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 3

CONCLUSIONThe pathogenesis of FES is still not understood, but patent foramen ovale is unlikely to berelevant in FES. Early inflammatory responses and especially timing of the operation are probablyimportant in FES, although the mechanisms are not clear. Investigating FES in its isolated form inpatients with solitary long bone injuries has become more difficult due to the decreased incidenceof FES.

Nowadayin patientsby the eftechnique

Figure 3.1 Illustration from Zenker [29] showing a fat droplet deformed intosausage shape at the upper right of the capillary bed.

37

s, it may be more relevant and fruitful to study subclinical fat embolism or fat embolism with multiple injuries where the classical manifestations of FES will be overshadowedfects of the injuries. Such an approach will probably benefit from new sensitives that can determine the behavior of fat emboli.

Page 46: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism and patent foramen ovale

38

REFERENCES

1. Sevitt S. Fat embolism. London, Butterworths, 1962. 2. Bierre AR, Koelmeyer TD . Pulmonary fat and bone marrow embolism in aircraft accident

victims. Pathology 1983;15:131-135.3. Mudd KL, Hunt A, Matherly RC, Goldsmith LJ, Campbell FR, Nichols GR 2nd, Rink RD.

Analysis of pulmonary fat embolism in blunt force fatalities. J Trauma 2000;48:711-715.4. ten Duis HJ. The fat embolism syndrome. Injury 1997;28:77-85. 5. Bergentz SE, Studies on the genesis of post-traumatic fat embolism. Acta Chir Scand 1962:282. 6. Hulman G. Fat macroglobule formation from chylomicrons and non-traumatic fat embolism. Clin

Chim Acta 1988;177:173-178. 7. Huang JC, Gay R, Khella SL. Sickling crisis, fat embolism, and coma after steroids. Lancet

1994;344:951-2.8. Panter-Brick M, Wagget J, Dale G. Intralipid and thrombocytopenia. Lancet 1975;i:857-858.9. Richter D, Rohricht AM, Nurnberger W, Wahn V, Schroten H. The fat emulsion agglutination

test: a reliable and cost effective alternative to the latex agglutination test for rapid bedside CRPmeasurement. Clin Chim Acta 1997; 261:141-148.

10. ten Duis HJ, Nijsten MWN, Klasen HJ, Binnendijk B. Fat embolism in patients with an isolatedfracture of the femoral shaft. J Trauma 1988;28:383-90.

11. Lechat Ph, Mas JL, Lascault G, Loron P, Theard M, Klimczac M, Drobinski G, Thomas D,Grosgogeat Y.Prevalence of patent foramen ovale in patients with stroke. N Eng J Med1988;318:1148-52.

12. Webster MWI, Chancellor AM, Smith HJ, Swift DL, Sharpe DN, Bass NM, Glasgow GL. Patentforamen ovale in young stroke patients. Lancet 1988;ii:11-12.

13. Moon RE, Camporesi EM, Kisslo JA. Patent foramen ovale in decompression sickness in divers.Lancet 1989;i:513-14.

14. Mügge A, Daniel WG, Wenzlaff P, Lichtlan PR. Patent foramen ovale or left atrial thrombi inunexplained arterial embolism. Lancet 1989;i:282-283.

15. Sevitt S. Pulmonary and systemic fat embolism. Proc Path Soc Gr Britain Ireland. 1956.16. Peltier LF. Fat embolism. Detection of fat droplets in the circulating blood. Surgery 1954;36:198.17. Musselman MM, Glas WW, Grekin TD. Fat embolism, a clinical investigation. Arch Surg

Chicago 1952;65:551.18. Pell AC, Hughes D, Keating J, Christie J, Busuttil A, Sutherland GR. Brief report: fulminating fat

embolism syndrome caused by paradoxical embolism through a patent foramen ovale. N Engl JMed 1993;329:926-929.

19. Fabian TC. Unravelling the fat embolism syndrome. N Engl J Med 1993;329:961-963.20. Pell AC, Christie J, Keating JF, Sutherland GR The detection of fat embolism by transesophageal

echocardiography during reamed intramedullary nailing. A study of 24 patients with femoral andtibial fractures. J Bone Joint Surg Br 1993;75:921-925.

21. Derks CM, Peters RM. The role of shock and fat embolus in leakage from pulmonary capillaries.Surg Gynecol Obstet 1973;37:945-948.

22. Scuderi CS. Fat embolism: a clinical and experimental study. Surg Gynec Obstet 1941;72:732.23. Tornabene VW, Fortune JB, Wgner PD, Halasz NA. Gas exchange after pulmonary fat embolism

in dogs. J Thorac Cardiovasc Surg 1979;78:589.

Page 47: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 3

39

24. Jones JG, Minty BD, Beeley JM, Royston D, Crow J, Grossman RF Pulmonary epithelialpermeability is immediately increased after embolization with oleic acid but not with neutral fat.Thorax 1982;37:169-174.

25. van den Brand JG, van der Hoeven JH, Olsman JG. Dyspnea or confusion after trauma? Considerfat embolism syndrome. Ned Tijdschr Geneeskd 2000;144:1513-1517.

26. Gossling HR, Pellegrini VD. Fat embolism syndrome: a review of the pathophysiology andphysiological basis of treatment. Clin Orthop 1982; 165:68-82.

27. Behrman SW, Fabian TC, Kudsk KA, Taylor JC.Improved outcome with femur fractures: earlyvs. delayed fixation. J Trauma 1990;30:792-797.

28. Edmonds CR, Barbut D, Hager D, Sharrock NE. Intraoperative cerebral arterial embolizationduring total hip arthroplasty. Anesthesiology 2000;93:315-318

29. Zenker FA. Beiträge zur Anatomie und Physiologie der Lunge. Braunsdorf 1861. p61.30. Pitto RP, Blunk J, Kossler M. Transesophageal echocardiography and clinical features of fat

embolism during cemented total hip arthroplasty. A randomized study in patients with a femoralneck fracture. Arch Orthop Trauma Surg 2000;120:53-58.

31. Arakawa H, Kurihara Y, Nakajima Y. Pulmonary fat embolism syndrome: CT findings in sixpatients. J Comput Assist Tomogr 2000;24:24-29.

32. Takahashi M, Suzuki R, Osakabe Y, Asai JI, Miyo T, Nagashima G, Fujimoto T, Takahashi Y.Magnetic resonance imaging findings in cerebral fat embolism: correlation with clinicalmanifestations. J Trauma 1999;46:324-327.

Page 48: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Fat embolism and patent foramen ovale

40

Page 49: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 4

INTERLEUKIN-6, FEVER AND ACUTE PHASE RESPONSES IN PATIENTS WITHBURNS

M.W.N. Nijsten, E.R. de Groot, H.J. ten Duis, H.J. Klasen, C.E. Hack, L.A. Aarden

Part of this chapter was published in as a letter inthe Lancet 1987;2: 921

Page 50: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Serum levels of interleukin-6 and acute phase responses

38

INTRODUCTIONFever has been defined as "a state of elevated core temperature, which is often, but notnecessarily, part of the defensive responses of multicellular organisms (host) to the invasion oflive (microorganisms) or inanimate matter recognized as pathogenic or alien by the host" [1]. Thisdefinition clearly recognizes that fever is often a response to foreign substances, called exogenouspyrogens. The bacterial cell-wall constituent lipopolysaccharide (endotoxin) is an importantexogenous pyrogen. However, many non-infectious diseases are accompanied by fever as well.Experiments with sterile inflammation models led to the hypothesis that the body itself producespyrogenic substances. The rabbit fever assay has long been a standard for measuring pyrogenicactivity of materials [2]. Upon injection of various pyrogens the rabbit develops fever in areproducible manner. Experiments showed that supernatants from in vitro endotoxin-activatedmonocytes induced fever upon injection, pointing to the existence of an endogenous pyrogen [3].It was further hypothesized that the induction of endogenous pyrogen might be a commonpathway in the generation of fever. The fact that prostaglandin synthetase inhibitors (e.g. aspirin)depress fever of diverse origins also suggests a common pathway of fever induction. Theendogenous pyrogen as produced by monocytes for example was apparently pyrogenic in minutequantities [3], but its precise characterization had been impossible until more sensitive andspecific technologies, such as recombinant DNA and monoclonal antibodies were available. Fever is a manifestation of a systemic inflammatory response that involves multiple organsystems. The coordinated reaction that follows trauma or infection is often called the acute phaseresponse. In addition to fever, its components are tachycardia, leukocytosis and changes incirculating protein levels. Acute phase proteins such as C-reactive protein (CRP) and α1-antitrypsin (α1AT) are increased, whereas albumin levels are decreased.On the basis of partially purified protein extracts the existence of the monokine interleukin-1 (IL-1), which is produced by activated monocytes was proposed. IL-1 preparations induced the acutephase response and mediated fever through the release of prostaglandin-E2 in the hypothalamus[4,5]. However, partly because of the difficulties in measuring IL-1 in biological fluids with theIL-1 bioassay, only scant evidence for a correlation between circulating IL-1 and the acute phaseresponse had been put forward [6]. In 1987, the cDNA was cloned of another monocyte product that seemed to be involved in theacute phase response. Interleukin-6 (IL-6) is a 184 amino acid protein with a molecular weight of20 to 30 kD (dependent on glycosylation) and was originally described as interferon-ß2, 26Kprotein, B-cell stimulating factor-2, and hybridoma growth factor. In human peripheral blood,monocytes are the main source of IL-6 [7]. IL-6 appeared to be involved in the acute phaseresponse since Gauldie et al. [8] found that IL-6 is also identical to hepatocyte-stimulating factor,a major inducer of acute phase protein production in cultured liver cells. We have observed thatrecombinant IL-6 (rIL-6) is active in the thymocyte co-stimulation assay, the classical assay forIL-1. Furthermore, rIL-6 was found to be strongly pyrogenic in rabbits [9].

Page 51: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 4

39

In this study we investigated the involvement of IL-6 in the acute phase response in humans. Inpatients with severe burns and in healthy controls we applied a sensitive IL-6 bio-assay to detectthe presence of increased circulating IL-6 levels. Patients with burns display an immediate severeinflammatory reaction, including early high fever without apparent infection, suggestive of highendogenous pyrogen production. Since the time of the injury is well defined, this allows optimalidentification of time-dependent changes of inflammatory parameters.

PATIENTS AND METHODSIn patients admitted to the Burns Unit of the Martini Hospital Groningen serum and plasmasamples were taken, that were frozen directly after centrifugation. For IL-6 a very sensitive bioassay, that allows detection of as little as 0.1 pmol/l IL-6 in biologicalfluids, was used [7,10]. The assay is based on the measurement of 3H-thymidine incorporation inan IL-6-dependent B-cell line. IL-6 levels were measured in serum with this B9 bioassay asdescribed previously [7]. In this assay an activity of 1 U/ml corresponds to 1 pg/ml or 5.10-14 mol/l ofIL-6. The samples were preincubated at 56o C for 30 minutes, and tested in triplicate and at multipledilutions. Specificity was checked by inhibition with polyclonal goat antibodies raised againstrecombinant human IL-6. CRP and α1-antitrypsin (α1-protease inhibitor) were detected in serum bylaser nephelometry (Behring Nephelometer Analyzer, Behringwerke AG, Marburg, Germany).Samples from healthy controls were run in parallel in all immunological assays. IL-6 values werelog-transformed to approximate a normal distribution. Pearson's regression analysis was used tocalculate coefficients of correlation between two parameters. To determine the significance ofcorrelations or differences between means, Student's t-test was used. A two-sided P-value of less than0.05 was considered to represent a significant difference. Means are expressed with standard errors.

RESULTS13 patients with a mean percentage of burned body surface (TBSA) of 31% were included.When we monitored circulating IL-6, CRP and α1-antitrypsin we found that within hours of theinjury, IL-6 had risen from 2 to 100 times the normal level, whereas CRP and α1-antitrypsin rose

Table 4.1Serum IL-6 levels and the development of the acute phase response in patientswith severe burns.Mean (SE) N IL-6 CRP α1AT temperature

(pmol/l) (mg/l) (IU/ml) (oC)On admission

68.6(5.4) 34(17) 81(6) 37.8(0.1)

One day post-burn 6 3.9(1.5) 96(21) 116(9) 38.8(0.1)2 months post-burn 9 1.3(0.4) 67(17) 220(9) 37.6(0.1)Controls 5 0.4(0.2) <19 80(10) 37.0(0.1)

Page 52: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Serum levels of interleukin-6 and acute phase responses

40

more gradually (Table 4.1). On the other hand, while the acute phase proteins were still raised,temperature and IL-6 were already decreasing. IL-6 was correlated with temperature (R=0.61,p<0.0001), and with CRP (R=0.44, p<0.001). Antiserum raised against rIL-6 inhibited the activityof serum in the IL-6 assay, which proves the assay’s specificity for IL-6.

DISCUSSIONIn this study it was shown that IL-6 is strongly elevated in burns patients, and that IL-6 levelswere correlated with manifestations of the acute phase response such as fever and increased CRP-levels. The time-lag between the peak in IL-6 and CRP can be explained by the fact that IL-6 isdirectly produced by stimulated monocytes and activated endothelial cells, whereas acute phaseproteins are synthesized in the liver after induction by mediators. The time-lag between the CRP-

peak and α1AT-peak is not unexpected since CRP is known to be a faster responding acute phaseprotein than α1AT [11]. Our study is obviously limited by the relatively small number of patientsthat were included. Although the overall correlation between IL-6 and fever was relatively strongwith an R of 0.61, the factor time was not taken into account, as the limited set of patients did notallow correlation of IL-6 with fever on fixed times for all patients. But even a correlation of IL-6with fever at a fixed time after admission as such is no proof of causality. A causal role of IL-6 infever in man could only be proved by administration of IL-6. At the time of the study no rhIL-6for administration to humans was available.

35

36

37

38

39

40

1.0E-13 1.0E-12 1.0E-11 1.0E-10

Interleukin-6 mol/l

Tem

pera

ture

Figure 4.1. Relation between body temperature and IL-6 levels in 13 patients withsevere burns. IL-6 was measured in triplicate at three different dilutions; temperaturesare daily means of four measurements. Normal IL-6 range is < 5 10-13 mol/l (10U/ml).

10-13 10-12 10-11 10-10

Page 53: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 4

41

Induction of acute phase response by IL-6 and other cytokinesSeveral purified recombinant cytokines have been shown to induce both fever and othercomponents of the acute phase response. Although IL-1 was the first substance to be identified asan endogenous pyrogen, IL-1, IL-6 and TNFα now all appear to be major endogenous pyrogens.Studies have shown that both in animals and humans injected with IL-1α [12], IL-1β [13], IL-6[14,15] and TNFα [16,17], fever is induced. In many models of systemic inflammation IL-6 isinduced by IL-1 and TNFα, with a marked synergistic effect of IL-1 and TNFα on IL-6production [2]. In 1994 [15] it was shown that administration of IL-6 is able to induce all aspectsof the acute phase response in man. Later it was also observed that IL-6 also inducesprocalcitonin, a protein with even faster and more pronounced kinetics than CRP [18]. Althoughthe pleiotropic clinical manifestations after IL-6 administration are clearly inflammatory, incomparison with the more potent inflammatory cytokines TNFα or IL-1 some authors havecharacterized IL-6 as an anti-inflammatory cytokine[19].

IL-6 as a quantitative marker of inflammationIn the field of plasma cytokine measurements, after more than 10 years of extensiveinvestigations, IL-6 has emerged as one of the most useful cytokines for quantifying acuteinflammatory responses. Several investigations have shown that higher, and also persistentlyhigher IL-6 levels are associated with poor outcome in sepsis [20,21,22]. In fact, levels of IL-6>1000 pg/ml, as assayed by a semi-quantitative point-of-care test, have been used as an inclusioncriterion in anti-TNF-trials [23]. In early triage for trial purposes it will be of interest to see howearly procalcitonin (PCT) levels will compare with IL-6 levels as a criterion for patient selectionwith sepsis trials. PCT is also induced by IL-6 and TNFα and rises much faster than CRP [18]. Inaddition some investigators have claimed [24,25,26] that PCT-levels can better discriminate septicfrom non-septic patients than CRP. Cytokine intervention studiesSince TNF and IL-1 appear to be more responsible for the adverse effects of the inflammatoryresponse, these cytokines have been intensively studied as target for therapeutic interventions.Many experimental models have shown benefit, with clinically the most impressive beneficialeffect of anti-TNF treatment in the Jarisch-Herxheimer syndrome [27]. It should be noted that inthis successful study anti-TNF was given immediately before the expected Jarisch-Herxheimerreaction. But in clinical sepsis trials significant effects on mortality have not materialized to date[28]. The prospective studies that did not show a beneficial effect on mortality in sepsis patientsincluded human recombinant IL-1 receptor antagonist [29,30], antiTNF antibodies [23,31,32] andTNF receptor fusion protein [33,34]. The disappointing clinical results with inhibition of a singlecytokines has forced investigators to reassess how elevated cytokine levels in sepsis should beinterpreted. Dozens of cytokines and cell types have been identified to have a role ininflammation [35], with an overwhelming number of potential interactions. It is now accepted thatcytokines should be viewed as part of a network were information is transmitted by parallel andsequential induction of cytokines and other mediators. Consequentially it is not surprising that theinhibition of only one mediator will have limited effects [28].

Page 54: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Serum levels of interleukin-6 and acute phase responses

42

ConclusionIL-6 is an important endogenous pyrogen and inducer of other aspects of the acute phaseresponse. IL-6 induces these effects directly and together with IL-1 or TNFα. The measurementof circulating IL-6 continues to provide one of the most valuable tools for studying inflammatoryresponses in patients.

REFERENCES1. IUPS Thermal commission. Glossary of terms for thermal physiology: second edition. Pflugers

Arch 1987;410:567-587.2. Dinarello CA. Cytokines as endogenous pyrogens. J Infect Dis 1999;179:S2:S294-304.3. Bodel P, Atkins E.Release of endogenous pyrogen by human monocytes. N Engl J Med

1967;276:1002-1008.4. Editorial. Interleukin-1 in defense of the host. Lancet 1985; ii: 536-537.5. Dinarello CA. Interleukin-1. Rev Infect Dis 1984;6:51-95.6. Bendtzen K, Baek L, Berild D, Hasselbach H, Dinarello CA, Wolff SM. Demonstration of

circulating leukocytic pyrogen/interleukin-1 during fever. N Eng J Med 1984; 310:596.7. Aarden LA, de Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth factor by

human monocytes. Eur J Immunol 1987;17:1411-1416.8. Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H. Interferon ß2/B-cell stimulatory factor

type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the majoracute phase response in liver cells. Proc Natl Acad Sci USA 1987;84:7251-7256.

9. Helle M, Brakenhoff JP, De Groot ER, Aarden LA, Interleukin 6 is involved in interleukin 1-induced activities. Eur J Immunol 1988;18:957-959

10. van Oers MHJ, van der Heyden AAPAM, Aarden LA, A novel interleukin in serum and urine ofrenal transplant recipients. Clin Exp Immunol 1988;71:314-319.

11. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N EnglJ Med 1999;340:448-454.

12. Smith JW, Urba WJ, Curti BD, et al. The toxic and hematologic effects of interleukin-1 alphaadministered in a phase I trial to patients with advanced malignancies. J Clin Oncol1992;10:1141-1152.

13. Crown J, Jacubowski A, Kemeny N, Elwood LJ, Steis RG, Janik JE, Sharfman WH, Miller LL,Fenton RG, Conlon KC, et al. A phase I trial of recombinant human interleukin 1β alone and incombination with myelosuppressive doses of 5-fluorouracil in patients with gastrointestinalcancer. Blood 1999;78:1420-1427.

14. Dinarello CA, Cannon JG, Mancilla J, Bishai I, Lees J, Coceani F. Interleukin-6 as anendogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral bloodmononuclear cells. Brain Res 1991;562:199-206.

15. van Gameren MM, Willemse PH, Mulder NH, Limburg PC, Groen HJ, Vellenga E, de Vries EG.Effects of recombinant human interleukin-6 in cancer patients: a phase I-II study. Blood1994;84:1434-1441.

Page 55: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 4

43

16. Dinarello CA, Cannon JG, Wolff SM, Bernheim HA, Beutler B, Cerami A, Figari IS, PalladinoMA Jr, O'Connor JV. Tumor necrosis factor (cachectin) is an endogenous pyrogen and inducesproduction of interleukin 1. J Exp Med 1986;163:1433-1450.

17. van der Poll T, Buller HR, ten Cate H, Wortel CH, Bauer KA, van Deventer SJ, Hack CE,Sauerwein HP, Rosenberg RD, ten Cate JW. Activation of coagulation after administration oftumor necrosis factor to normal subjects. N Engl J Med 1990;322:1622-1627.

18. Nijsten MWN, Olinga P, The TH, de Vries EG, Schraffordt Koops H, Groothuis GM, LimburgPC, ten Duis HJ, Moshage H, Hoekstra HJ, et al. Procalcitonin behaves as a fast responding acutephase protein in vivo and in vitro. Crit Care Med 2000 ;28:458-461.

19. van der Poll T, van Deventer SJH. Interleukin-6 in bacterial infection and sepsis: innocentbystander or essential mediator? In: Vincent JL (Ed) Yearbook of intensive care and emergencymedicine. Springer-Verlag Berlin Heidelberg 1999:43-53.

20. Waage A, Brandtzaeg P, Halstensen A, Kierulf P, Espevik T. The complex pattern of cytokinesin serum from patients with meningococcal septic shock. Association between interleukin 6,interleukin 1, and fatal outcome. J Exp Med 1989;169:333-338.

21. Lowry SF, Calvano SE, van der Poll T. Measurement of inflammatory mediators in clinicalsepsis. In: Sibbald WJ, Vincent JL (eds) Clinical trials for the treatment of sepsis. Springer-Verlag Heidelberg. 1995:86-105.

22. Hack CE, De Groot ER, Felt-Bersma RJ, Nuijens JH, Strack Van Schijndel RJ, Eerenberg-Belmer AJ, Thijs LG, Aarden LA. Increased plasma levels of interleukin-6 in sepsis. Blood1989;74:1704-1710.

23. Reinhart K, Wiegand-Lohnert C, Grimminger F, Kaul M, Withington S, Treacher D, Eckart J,Willatts S, Bouza C, Krausch D, Stockenhuber F, Eiselstein J, Daum L, Kempeni J. Assessmentof the safety and efficacy of the monoclonal anti-tumor necrosis factor antibody-fragment, MAK195F, in patients with sepsis and septic shock. A multicenter, randomized, placebo-controlled,dose-ranging study. Crit Care Med 1996;24:733-742.

24. Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C. High serum procalcitoninconcentrations in patients with sepsis and infection. Lancet 1993;341:515-518.

25. Oberhoffer M, Karzai W, Meier-Hellmann A, Bogel D, Fassbinder J, Reinhart K. Sensitivity andspecificity of various markers of inflammation for the prediction of tumor necrosis factor-alphaand interleukin-6 in patients with sepsis. Crit Care Med 1999;27:1814-1818.

26. Reinhart K, Karzai W, Meisner M. Procalcitonin as a marker of the systemic inflammatoryresponse to infection. Intensive Care Med 2000;26:1193-1200.

27. Fekade D, Knox K, Hussein K, Melka A, Lalloo DG, Coxon RE, Warrell DA. Prevention ofJarisch-Herxheimer reactions by treatment with antibodies against tumor necrosis factor alpha. NEngl J Med 1996;335:311-315.

28. Abraham E. Why immunomodulatory therapies have not worked in sepsis. Intensive Care Med1999;25:556-566.

29. Fisher CJ Jr, Dhainaut JF, Opal SM, Pribble JP, Balk RA, Slotman GJ, Iberti TJ, Rackow EC,Shapiro MJ, Greenman RL, et al. Recombinant human interleukin 1 receptor antagonist in thetreatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group. JAMA 1994;271:1836-1843.

Page 56: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Serum levels of interleukin-6 and acute phase responses

44

30. Opal SM, Fisher CJ Jr, Dhainaut JF, Vincent JL, Brase R, Lowry SF, Sadoff JC, Slotman GJ,Levy H, Balk RA, Shelly MP, Pribble JP, LaBrecque JF, Lookabaugh J, Donovan H, Dubin H,Baughman R, Norman J, DeMaria E, Matzel K, Abraham E, Seneff M.Confirmatory interleukin-1 receptor antagonist trial in severe sepsis: a phase III, randomized, double-blind, placebo-controlled, multicenter trial. The Interleukin-1 Receptor Antagonist Sepsis Investigator Group.Crit Care Med 1997;25:1115-1124.

31. Abraham E, Anzueto A, Gutierrez G, Tessler S, San Pedro G, Wunderink R, Dal Nogare A,Nasraway S, Berman S, Cooney R, Levy H, Baughman R, Rumbak M, Light RB, Poole L, AllredR, Constant J, Pennington J, Porter S. Double-blind randomised controlled trial of monoclonalantibody to human tumor necrosis factor in treatment of septic shock. NORASEPT II StudyGroup. Lancet 1998;351:929-933.

32. Cohen J, Carlet J. INTERSEPT: an international, multicenter, placebo-controlled trial ofmonoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. InternationalSepsis Trial Study Group. Crit Care Med 1996;24:1431-1140.

33. Fisher CJ Jr, Agosti JM, Opal SM, Lowry SF, Balk RA, Sadoff JC, Abraham E, Schein RM,Benjamin E. Treatment of septic shock with the tumor necrosis factor receptor:Fc fusion protein.The Soluble TNF Receptor Sepsis Study Group. N Engl J Med 1996;334:1697-1702.

34. Abraham E, Glauser MP, Butler T, Garbino J, Gelmont D, Laterre PF, Kudsk K, Bruining HA,Otto C, Tobin E, Zwingelstein C, Lesslauer W, Leighton A. p55 Tumor necrosis factor receptorfusion protein in the treatment of patients with severe sepsis and septic shock. A randomizedcontrolled multicenter trial. Ro 45-2081 Study Group. JAMA 1997;277:1531-1538.

35. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997;66:101-195.

Page 57: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 5

INTERLEUKIN 6 AND ITS RELATION TO THE HUMORAL IMMUNERESPONSE AND CLINICAL PARAMETERS IN BURNED PATIENTS

M.W.N. Nijsten, C.E. Hack, M. Helle, H.J. ten Duis, H.J Klasen L.A. Aarden

Surgery 1991; 109: 761-767

Page 58: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

50

ABSTRACTThe cytokine interleukin-6 (IL-6), which has been shown to be increased in burn patients, isproduced by activated monocytes and endothelial cells and has many in vitro activities, includingstimulation of acute phase protein synthesis in hepatocytes, immunoglobulin-synthesis in B-lymphocytes and stimulation of growth of megakaryocytes. In 13 patients with a mean of 31% fullthickness burns, we studied the relation of serum IL-6 to clinical parameters and parameters of theacute phase response and immunoglobulin production. IL-6 was already elevated within hours aftersustaining the injury, and it remained elevated for several weeks. All components of the acute phaseresponse were observed: fever, tachycardia, leukocytosis with an associated left shift, elevation of C-reactive protein and α1-antitrypsin, and a decrease in albumin levels. In the second week after burninjury immunoglobulin M-levels peaked, followed by a prolonged elevation of immunoglobulin G-levels. Platelet counts initially decreased and rebounded to supranormal levels after two weeks. IL-6levels were positively correlated with acute phase responses. We believe that the production of IL-6 induces the synthesis of acute phase proteins. High IL-6 levelsmay also be an etiologic factor in the marked immunoglobulin response observed. Likewise, therelation between the megakaryocyte promoting activity of IL-6 and the rebound thrombocytosis,requires further investigation.

INTRODUCTIONInterleukin-6 (IL-6) is a hormone-like protein that plays a central role in inflammation. It is producedby various cells, such as monocytes, endothelial cells, and fibroblasts. The multifunctional propertiesof IL-6 are illustrated by the fact that after DNA analysis, several proteins known for different activi-ties turned out to be identical to IL-6: hepatocyte stimulatory factor (HSF), B-cell stimulatory factor-2 (BSF-2), interferon-beta-2 (IFN-ß2), hybridoma growth factor (HGF), and human myeloiddifferentiation-inducing protein (MGI-2) [1]. IL-6 causes fever upon injection [2], and it can induce production of acute phase proteins like C-reactive protein (CRP), fibrinogen and α1-antitrypsin (α1-proteinase inhibitor) in hepatocytes [3].Severely burned patients display a strong acute phase response in the first hours to days after burninjury. They often have a high fever within 24 hours and produce acute phase proteins such as CRPand α1-antitrypsin. As we have shown before, these patients have markedly increased serum IL-6levels, which correlate with body temperature [4].As a B-cell stimulatory factor IL-6 induces immunoglobulin production [5]. Burned patients displayelevated immunoglobulin levels in the weeks following the burn [6]. Immunoglobulin M (IgM) risesto supranormal levels after 1 week, whereas IgG shows a slower increase to levels above the normalrange after several weeks. Lymphocytes isolated from burn patients have an elevated spontaneous,polyclonal immunoglobulin production [7].

Page 59: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

51

The genesis of this immunoglobulin response in burn patients is unclear and has been related to stresshormone levels, the presence of infection, T-cell alloreactivity and antigens from traumatized tissue[8,9,10].Recently it has become apparent that IL-6 is also a direct-acting growth factor for megakaryocytes,and as such plays a key role in thrombopoiesis [11]. Platelet counts have long been known to bedramatically increased in burn patients, but this phenomenon has received little attention. It issupposed to be a 'rebound' after initial sequestration of platelets in the burn wound [12], comparableto other conditions characterized by a temporary depletion of platelets. The goal of this study was to assess the clinical and laboratory characteristics of inflammation inburn patients, focusing on IL-6 production. We specifically looked at the time course of IL-6, acutephase responses, immunoglobulin synthesis and platelet counts.

PATIENTS AND METHODS

Patients Eight females and five males (mean age of 33 years), were selected for this study (Table 5.1). Themean percentage of total body surface area covered with full thickness burns was 31%. None of thepatients had physical disorders before the accident. This patient group is identical with the one brieflydescribed by us before [4]. On admission patients were vaccinated with tetanus toxoid. Surgicaltreatment was performed in phases. Each week approximately 10% of the full thickness burn wasexcised and covered by an autologous skin graft. In addition, an essential component of our treatmentwas selective decontamination of the intestinal tract and early oral feeding to prevent colonization bypotential pathogens [13]. This decontamination regimen consisted of daily doses of oral colistine,polymyxin B and co-trimoxazole. Blood cultures were taken when bacteremia was suspected.Systemic antibiotics were only administered in case of proven bacteraemia. Low-dose diclofenac(VoltarenR, 5 or 10mg), was administered in case of fever greater than 40o C.

Routine parametersRecords of temperature, heart rate and blood pressure were taken, as well as of fluids and medicationadministered. The core temperature and the heart rate values used were the daily means of at leastfour measurements. Routine daily examinations included total protein, albumin, hemoglobin, plate-lets, white cell numbers and white cell differentiation count, blood urea nitrogen (BUN), creatinineand creatinine clearance.

Immunological assays Serum and EDTA-plasma samples were taken on admission and during the five subsequent days;thereafter samples were taken weekly until discharge from the burn unit. Samples were stored inthree aliquots at -70o C and were analyzed directly after thawing. IL-6 levels were measured in serumwith the B9 bioassay as described previously [14]. In this assay an activity of 1 U/ml corresponds to 1pg/ml or 5.10-14 mol/l of IL-6. The samples were preincubated at 56o C for 30 minutes, and tested in

Page 60: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

52

triplicate and at multiple dilutions [15]. Specificity was checked by inhibition with polyclonal goatantibodies raised against recombinant human IL-6. To assess complement activation, C3a, the activa-tion product of the third component of complement, was measured by radioimmunoassay in plasma[16] and C3dg was measured with a similar radioimmunoassay. C-reactive protein, α1-antitrypsin,IgG, IgM, complement factor C4 and α2-macroglobulin were measured in serum by lasernephelometry (Behring Nephelometer Analyzer, Behringwerke AG, Marburg, Germany) usingappropriate antisera. Samples from healthy controls were run in parallel in all immunological assays.

Analysis of data IL-6 and C3a values were log-transformed to approximate a normal distribution. Pearson's regressionanalysis was used when appropriate to calculate coefficients of correlation between two parameters.To determine the significance of correlations or differences between means, Student's t-test was used.A two-sided P-value of less than 0.05 was considered to represent a significant difference. Unlessindicated otherwise, means are expressed with associated standard errors.

RESULTS

Patients Table 5.1 shows the general characteristics of the 13 patients studied. All patients survived, and onlyone episode of proven bacteremia was encountered in this group (in the 6th week post-burn). Onepatient suffered from inhalation injury, requiring mechanical ventilation. No patients were inhypotensive shock during any phase of the study. We decided to analyze the data from all patientstogether. Figs. 5.1, 5.2 and 5.3 display means of measurements for 15 parameters.

Interleukin 6IL-6 levels were already elevated at the time of admission and continued to be elevated afterward(Fig. 5.1). Although the levels varied considerably, in general they were 10- to 100-fold elevatedabove normal levels. (controls: <5.10-13 Mol/l ). There was no relation between the extent of theburns and IL-6 levels, but patients with more severe burns had elevated IL-6 for longer periods.

Table 5.1 General clinical data Numbers, meansSex 8 females, 5 malesAge 33 (range 16-33) yearFull thickness TBSA 31 (range 10-56) percentPartial and full thickness TBSA 38 (range 17-56) percentOperations 4.0 (range 2-6) operations/patientStay at burn unit 77 (range 26-187) daysDeaths 0TBSA:Total body surface area

Page 61: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

53

Acute phase proteinsCRP-levels were elevated from the first day post-burn onward, rose a 100-fold and were down tocontrol values after two months. The levels of α1-antitrypsin displayed a less pronounced and slowerrise, but remained at twice control levels after two months.

Immunoglobulins The levels of IgM and IgG initially dropped and later rose to supranormal levels. IgM levels showeda very significant rise in the second week after burn injury. IgG rose later and remained elevated attwice normal levels for longer periods.

Complement activation Increased levels of C3a and C3dg indicated activation of complement factor C3. Levels variedwidely and could not be related to the extent of the burns. Mean levels of C3a, as well as C3dg, werehighest at the end of the second week (Fig. 5.2). C4 levels did not significantly change during thefirst week and increased steadily during subsequent weeks.

Routine parametersTemperature was elevated within hours after admission and remained above 38o C for more than 1month. One patient reached a body temperature of 41o C within 12 hours after the injury. The heartrate followed the temperature response and was well over 120/min in the first week, and averagedover 100/min during the entire stay in the burn unit. Leukocyte counts were increased sharply to28·109 /l on admission, and remained elevated around 12·109/l for several weeks (Fig. 5.3). In theleukocyte differentiation a sharp elevation of the percentage of rods (young neutrophils) was noted(rods in controls <3%). Because of the presence of abnormal leukocytes like myelocytes andmetamyelocytes in the differentiation, the stacked bars in Fig. 5.1 do not always add up to 100%.High hemoglobin during the first 48 hours reflected hemoconcentration as a result of extensive lossof fluid. Platelet counts were low during the first week and then rapidly rebounded to supranormallevels, not returning to normal levels until the second month. Total protein remained below normallevels for many weeks.

CorrelationsCorrelations between IL-6 and parameters of the acute phase response are shown in Table 5.2. Allmeasurements in all patients were used for this analysis. Values of concentrations measured wereonly correlated when determined in the same sample. IL-6 appeared to be most strongly correlatedwith body temperature. IL-6 was negatively correlated with total protein and platelet counts. Wefound no correlation between complement levels and IL-6.

Page 62: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

200

Temperature and Heart rate

36.0

36.5

37.0

37.5

38.0

38.5

39.0

39.5

0 1 2 3 4 9 14 21 30 45 65

deg

rees

Cel

cius

80

90

100

110

120

130

140

/min

��������Rods

������ Mature neutrophils

������ Lymphocytes

������Monocytes

Interleukin-6

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

2.00

0 1 2 3 4 9 14 21 30 45 65

log

units

/ m

l

54

C-reactive protein

0

50

100

150

0 1 2 3 4 9 14 21 30 45 65

mg

/ l

Figure 5.1. Time course of means of IL-6, temperature (fildifferentiation for the entire patient group. Note that the time sneither linear nor logarithmic. IL-6 levels are shown on a differentiation do not always add up to 100%, due to the presenc

��������� ���������� ��������� ��������� �������������������� ��������� ��������� ����������������������������� ���������� ��������� ��������� �������������������� ��������� ��������� ��������������������������������������

��������������������

������������������������������

������������������

������������������������������������

������������������������������

����������������������������������������

������������������������������������

������������������������������������

��������������������

��������������������

������������������������������������������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������������������������������������

��������������������������������������������������������������������������������������������������������������

���������������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������

������������������������������������������������������������������������������������������

������������������������������������������������������������������������

��������������������������������������������������������������������������������������������������������������

����������������������������������������������������������������������������������������������������

������������������

����������������������������������������

����������������������������������������

������������������������������������

���������������������������

����������������������������������������

����������������������������������������

������������������������������������

���������������������������������������������

��������������������������������������������������

��������������������������������������������������

������������������

��������������������

�������������������� ���������

���������������������������

��������������������

��������������������

��������� ������������������

������������������������������

0

20

40

60

80

100

0 1 2 3 4 9 14 21 30 45 65

perc

enta

ge

led circles), heart rate (squares), CRP and leukocytecale is chosen to accentuate the important phases; it islogarithmic scale. The percentages in the leukocytee of non-standard types of leukocytes.

Page 63: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

200

IgG en IgM

0

50

100

150

200

250

300

0 1 2 3 4 9 14 21 30 45 65

IU /

ml

100 100

alpha-1-Antitrypsin

0

100

200

300

0 1 2 3 4 9 14 21 30 45 65

IU /

ml

Complement factor C4

0

50

100

150

0 1 2 3 4 9 14 21 30 45 65

IE /

ml

Figure 5.2. Time course of means of α1antitrypsin, IgM (filled circlecircles) and C3dg (open circles) for the entire patient group. Note thavalues are shown on a logarithmic scale.

55

C3a and C3dg

1

10

0 1 2 3 4 9 14 21 30 45 65

C3a

nm

ol/m

l

0

20

40

60

80

C3d

g nm

ol/l

s), IgG (squares), complement factors C4, C3a (filledt the time scale is neither linear nor logarithmic. C3a

Page 64: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

200

Total Protein

0

10

20

30

40

50

60

70

80

0 1 2 3 4 9 14 21 30 45 65

g / l

600

Leukocytes

0

10

20

30

40

0 1 2 3 4 9 14 21 30 45 65

10E9

/ l

56

Hemoglobin

0

40

80

120

160

0 1 2 3 4 9 14 21 30 45 65

g / l

Figure 5.3. Time course of means of leukocyte count, total protein,group. Note that the time scale is neither linear nor logarithmic.

h

Platelet Count

0

100

200

300

400

500

0 1 2 3 4 9 14 21 30 45 65

10E9

/ l

emoglobin and platelet count for the entire patient

Page 65: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

57

DISCUSSIONThis study examined the relation of IL-6 to acute phase responses and humoral and hematologicalparameters in severely but not critically burned patients. Such patients tend to display maximalpathophysiological responses, whereas in critically burned patients acute organ failure often stronglyaffects many responses. The in vivo responses of several of the parameters shown in Figs. 1,2 and 3can be interpreted in terms of in vitro activities of IL-6 (Fig. 5.4). The results support the hypothesisthat IL-6 has a causal role in the acute phase reaction. Moreover, the results indicate a possible effectof IL-6 on changes of immunoglobulin levels and thrombocyte synthesis. The sustained elevation of IL-6 indicates continuous production by monocytes, endothelial cells orfibroblasts. Because IL-6 probably has a half-life of less than one hour [17], it must be produced forprolonged periods. In theory, levels of a low molecular weight protein such as IL-6 (MW 20 to 30kD) can also increase through decreased glomerular filtration, but the patients displayed onlymarginal renal disturbances as reflected by creatinine, creatinine clearance and BUN. These renalparameters were not correlated with any of the measured proteins, also making a renal effect onprotein levels unlikely. Monocytes are potent producers of IL-6 [14], and they are known to beactivated in burn patients [18]. Endotoxin or complement can induce the production of severalcytokines by monocytes. The absence of correlation between the complement factors we measured(C3a, C3d and C4) and IL-6, does not rule out a causal relation, since the levels of such short-livedagents are always difficult to correlate. Endotoxemia can occur in burns patients without a focus ofinfection being present. Guo et al. recently showed that in burn patients levels of circulatingendotoxin and IL-6 can be reduced by polymyxin B therapy [19]. IL-6 is only one of many cytokines involved in inflammation, and in the context of acute phaseresponses tumor necrosis factor (TNF) and interleukin-1 (IL-1) are important mediators [20]. Wewere unable to detect IL-1 with the standard assay, and we did not attempt to measure TNF. Yet it

Table 5.2Correlation (R) of interleukin-6 with parameters of the acute phaseresponse. (n=88 to 91)Parameter Parameter R PIL-6 Temperature +0.61 < 0.0001

Heart rate +0.55 < 0.0001Rods +0.48 < 0.0001CRP +0.44 < 0.0001Leukocytes +0.36 < 0.0005IgM 0.00 N.S.α1-antitrypsin -0.06 N.S.Trombocytes -0.44 < 0.0001Total protein -0.48 < 0.0001

Page 66: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

58

seems logical that both TNF and IL-1 were also produced. So it would be interesting to measure IL-1and TNF in such patients with sufficiently sensitive assays.The course of temperature, heart rate, CRP, α1-antitrypsin, leukocytes and the 'left shift' in theleukocyte differentiation indicate a huge acute phase response. The rapidly developing fever occursbefore an established infection can be present. The correlations between IL-6 and temperature, heartrate, CRP, leukocytes and percentage of rods are in accordance with a central role of IL-6 in the acutephase response [21]. Table 5.2 shows that the heart rate is correlated mainly with body temperature(R=0.67), which suggests that the tachycardia was induced by fever, and indirectly by IL-6. Thistachycardia was more pronounced than that in patients with fever following an uncomplicated viralinfection [22]. The hypermetabolic state and the lowered hemoglobin levels of the burn patients

probably explain this 'additional' tachycardia. The rise in CRP within one day and the later rise of α1-antitrypsin are in accordance with the well-known property of CRP as a fast and strongly respondingacute phase protein. The sharply dropped albumin levels (closely following total protein levels, datanot shown) are in accordance with the down-regulation of albumin synthesis as part of the acutephase response but are primarily caused by capillary leakage and the massive loss of protein throughthe burned skin.The typical pattern of IgM and IgG (Fig. 5.2) has been observed by other authors [6,9,10]. Onecould argue that the increase of the high molecular weight protein IgM (MW 900 kD) was caused by

Figure 5.4. Diagram of the processes possibly related to interleukin-6 (IL-6) production.

Page 67: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

59

the infusion of large amounts of protein coupled with the selective loss of other proteins throughtissue or wound leakage. This is unlikely because of the distinct pattern in time of the IgM-peak andits presence in virtually all patients. Also this pattern was not observed for α2-macroglobulin, anotherhigh molecular weight protein (data not shown). Moreover the protein spectrum in blister fluid isonly marginally determined by molecular weight [23]. C3a en C3dg levels, both indicative of activation of complement factor C3, were maximal in thesecond week post-burn and the C4 pattern indicates consumption of C4. The rapid decrease of IgMin the second week coincides with increases in C3a, which resemble the changes that occur in serumsickness. Yet, we saw no clinical signs of immune complex deposition and we could not detectimmune-complexes with the C1q binding assay (data not shown).The patterns of IgG and IgM levels bear a strong resemblance to the textbook primary immuneresponse when the immune system is challenged by a new antigen. An autoimmune origin of thisresponse is unlikely, since screening of 15 serum samples from the second week postburn withindirect immunofluorescence for the presence of autoantibodies was negative. The polyclonal IgGand IgM response in patients after uncomplicated surgery has been shown to be directed against'recall' antigens [24]. Moreover, IL-6 has been shown to augment the production of immunoglobulinsby B- cells in vitro and in vivo [5,25].The sharp rebound in platelet numbers, preceding the slower recovery of hemoglobin levels reflects astrong stimulation of bone marrow. This rise of platelets is a familiar phenomenon following traumaand burns [12]. When rats are selectively depleted of platelets by exchange transfusion, they alsodisplay a rebound thrombocytosis [26]. In fact, some authors consider thrombocytosis an integral partof the acute phase response [27,28]. Sustained thrombocytosis is also often seen in chronicinflammatory disease states like rheumatoid arthritis [29]. In this disease IL-6 levels in serum and inparticular in synovial fluids, are increased markedly [30]. Recently IL-6 has been demonstrated to bea crucial factor for thrombopoiesis in vitro and in vivo [11,31]. Although platelet production dependson the interaction of several cytokines [32], IL-6 may be instrumental in the thrombocytosis in burnpatients. The negative correlation between IL-6 and platelet counts can be explained by the time-lagbetween stimulation of precursor cells in the bone marrow and the appearance of platelets in theperipheral blood.

In summary, this investigation focused on the role of one important cytokine (IL-6) in burn patients.The high levels of IL-6 we found may be related to three distinct responses: the acute phase response,immunoglobulin production and thrombocytosis. Although each of these responses is already knownto be influenced by several other factors, IL-6 may play an essential role in each of them. Currentinvestigations on the effect of IL-6 administered in vivo will help to answer this question.

ACKNOWLEDGEMENTThe authors wish to thank Douwe Buiter for drawing Figure 5.4 and Mereke Schaub for technicalassistance.

Page 68: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

60

REFERENCES1. Kishimoto T. The biology of interleukin-6. Blood 1989;74:1-10.2. Helle M, Brakenhoff JPP, de Groot ER, Aarden LA. Interleukin 6 is involved in interleukin 1-

induced activities. Eur J Immunol 1988;18:957-959.3. Gauldie J, Richards C, Harnish D, Lansdorp P, Bauman H. Interferon ß2/ B-cell stimulatory factor

type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the majoracute phase protein response in liver cells. Proc Nat Acad Sci USA 1987;84:7251-7255.

4. Nijsten MWN, de Groot ER, ten Duis HJ, Klasen HJ, Hack CE, Aarden LA. Serum levels ofinterleukin-6 and acute phase responses. Lancet 1987;ii:921.

5. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K,Koyama K, Iwamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Takahara Y, Taniguchi T, KishimotoT. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes toproduce immunoglobulin. Nature 1986;324:73-76.

6. Arturson G, Johansson SGO, Högman CF, Killander J. Changes in immunoglobulin levels inseverely burned patients. Lancet 1969;i:546-548.

7. Teodorczyk-Injeyan JA, Sparkes BG, Falk RE, Peters WJ. Polyclonal immunoglobulin production inburned patients: kinetics and correlations with T-cell activity. J Trauma 1986;26:834-839.

8. Shorr RM, Ershler WB, Gamelli RL. Immunoglobulin production in burned patients. J Trauma1984;24:319-322.

9. Hershman MJ, Cheadle WG, George CD, Cost KM, Appel SH, Davidson PF, Polk HC Jr. Theresponse of immunoglobulins to infection after thermal and non-thermal injury. Am Surg1988;54:408-411.

10. Kagan RJ, Bratescu A, Jonasson O, Matsuda T, Teodorescu M. The relationship between thepercentage of circulating B-cells, corticosteroid levels and other immunologic parameters inthermally injured patients. J Trauma 1989;29:208-213.

11. Lotem J, Shabo Y, Sachs L. Regulation of megakaryocyte development by interleukin-6. Blood1989;74:1545-1551.

12. Eurenius K, Mortensen RF, Meserol PM, Curreri PW. Platelet and megakaryocyte kinetics followingthermal injury. J Lab Clin Med 1972;79:247-257.

13. Manson WL, Westerveld AW, Klasen HJ, Sauër EW. Selective decontamination of the digestivetract for infection propylaxis in severely burned patients. Scand J Plast Reconst Surg 1987;21:269-272.

14. Aarden LA, de Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth factor byhuman monocytes. Eur J Immunol 1987;17:1411-1416.

15. van Oers MHJ, van der Heyden AAPAM, Aarden LA. Interleukin 6 (IL-6) in serum and urine ofrenal transplant recipients. Clin Exp Immunol 1988;71:314-319.

16. Hack CE, Paardekooper J, Eerenberg AJM, Navis GO, Nijsten MWN, Thijs LG, Nuijens JH. Amodified competitive inhibition radioimmunoassay for the detection of C3a. J Immunol Methods1988;108:77-84.

Page 69: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 5

61

17. van Deventer SJH, Büller HR, ten Cate JW, Aarden LA, Hack CE, Sturk A. Endotoxin-inducedbiological effects: the role of cytokines. In: Endotoxins in the pathogenesis of gram-negativesepticemia, Thesis. Amsterdam, 1988.

18. Moore FD, Davis CF. Monocyte activation after burns and endotoxemia. J Surg Res 1989;46:350-354.

19. Guo Y, Dickerson C, Chrest FJ, Adler WH, Munster AM, Winchurch RA. Increased levels ofcirculating interleukin 6 in burn patients. Clin Immunol Immunopathol 1990;54:361-371.

20. Dinarello CA, Interleukin 1 and its biologically related cytokines. Adv Immunol 1989;44:153-205.21. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J

1990;265:621-636.22. Karjalainen J, Viitasalo M. Fever and cardiac rhythm. Arch Intern Med 1986;146:1169-1171.23. Deitch EA, Emmet M. Early protein alteration in blister fluid and serum associated with burn injury.

J Trauma 1986;26:34-39.24. Di Padova F, Durig M, Di Padova C, Pozzoli M, Tritapepe R. Spontaneous and polyclonal Ig

secretion by circulating B cells after surgery. Surgery 1988;103:547-552.25. Nawata Y, Eugui EM, Lee SW, Allison AC. IL-6 is the principal factor produced by synovia of

patients with rheumatoid arthritis that induces B-lymphocytes to secrete immunoglobulins. Ann N YAcad Sci 1989;557:230-238.

26. Mimura H, Segal GM, Lee MY, Adamson JW. Megakaryocytosis in the rat: response totrombocytopenia induced by exchange tranfusion. Exp Hematol 1985;13:1048-1054.

27. Parry MF, Jacobs B, Scully, Neu HC. Thrombocytosis: an acute-phase reactant, not an adversereaction to the new beta-lactam antibiotics. Diagn Microbiol Infect Dis 1984;2:229-231.

28. Williams JE, Cypher JJ, Mosesson MW. Evidence that production of platelet fibrinogen issynchronous with platelet production in the turpentine-induced acute phase response. J Lab ClinMed 1985:106;343-348.

29. Farr M, Scott DL, Constable TJ, Hawker RJ, Hawker CF, Stuart J. Thrombocytosis of active disease.Ann Rheum Dis 1983;42:545-549.

30. Waage A, Kaufmann C, Espevik T, Husby G. Interleukin 6 in synovial fluid from patients witharthritis. Clin Immunol Immunopathol 1989;50:394-398.

31. Ishibashi T, Kimura H, Shikama Y, Uchida T, Karyone S, Hirano T, Kishimoto T, Takatsuki F,Akiyama Y. Interleukin 6 is a potent thrombopoeitic factor in vivo in mice. Blood 1989;74:1241-1244.

32. Bruno E, Hofman R. Effect of interleukin 6 on in vitro human megakaryocytopoiesis: its interactionwith other cytokines. Exp Hematol 1989;17:1038-1043.

Page 70: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Interleukin-6 and humoral and clinical responses in burned patients

62

Page 71: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 6

PROCALCITONIN BEHAVES AS A FAST RESPONDING ACUTE PHASEPROTEIN IN VIVO AND IN VITRO

M.W.N. Nijsten, P. Olinga, T.H. The, E.G.E. de Vries, H.Schraffordt Koops, G.M.M. Groothuis, P.C. Limburg, H.J. ten Duis,

H. Moshage, H.J. Hoekstra, J. Bijzet, J.H. Zwaveling

Crit Care Med 2000;28:458-461.

Page 72: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Procalcitonin in vivo and in vitro

64

ABSTRACT Objectives: Procalcitonin (PCT) is a 13 kD protein of which plasma levels are strongly increasedin inflammatory states. PCT-levels are claimed to have a more powerful discriminatory value forbacterial infection than the acute phase proteins serum amyloid A (SAA) or C-reactive protein(CRP). The source of production and its mechanism of induction are unknown. We investigatedthe inducibility of PCT both in vivo and in vitro, and compared PCT’s behavior with SAA andCRP.Design: A prospective descriptive patient sample study and a controlled liver tissue culture study.Setting: A university hospital.Patients: Cancer patients who were treated with human tumor necrosis factor α (rhTNFα; 5patients) or interleukin-6 (rhIL-6; 7 patients).Measurements and Main results: Serial serum samples were collected for analysis of levels ofPCT, SAA and CRP. In the TNFα group frequent sampling was performed on the first day toallow analysis of initial responses. In a human liver slice model the release of PCT, SAA and CRPwas measured upon induction with rhTNFα and rhIL-6 for 24 hours. We found that in vivo, afteradministration of both rhTNFα and rhIL-6, PCT displayed acute phase reactant behavior. AfterrhTNFα-administration PCT reached half-maximal levels within 8 hours, 12 hours earlier thaneither SAA or CRP. In vitro PCT, SAA and CRP were produced in detectable quantities by livertissue. PCT production by liver slices was enhanced after stimulation with rhTNFα or rhIL-6;SAA and CRP levels were elevated after stimulation with rhTNFα. Conclusions: We found that PCT and acute phase proteins such as CRP are induced by similarpathways. The liver appears to be a major source of PCT-production. Thus PCT may beconsidered an acute phase protein. PCT’s different kinetics, and not a fundamentally differentafferent pathway may explain its putative diagnostic potential to discriminate bacterial infectionfrom other causes of inflammation.

INTRODUCTIONAny significant inflammatory process is associated with an acute phase response including theproduction of a broad range of effector proteins such as coagulation factors and proteaseinhibitors. Acute phase proteins (APP) such as the archetypal C-reactive protein (CRP) areelevated in a manner proportional to the extent of the inflammatory process. The cytokines tumornecrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1 have been identified as pivotal inthe induction of APPs. Although the theoretical benefits of measuring plasma cytokine levels areobvious, in clinical practice only APPs such as CRP serve to “quantify” inflammation. The shorthalf-life (minutes to hours) of cytokines makes relatively infrequent measurements useless -certainly if elaborate assay procedures are involved. It has been shown that cytokine levels arehigher in bacteraemia, but clinically it has proven difficult to discriminate causes of inflammationon the basis of patterns of cytokine or APP responses. In many patients with fever, it is ofparamount importance to discriminate bacterial causes from other causes of inflammation.

Page 73: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 6

65

It has been claimed that the serum level of the protein procalcitonin (PCT; 116 amino acids; 13kD) is superior to APPs in differentiating infectious from non-infectious causes of inflammation[1]. PCT levels are proportional to the inflammatory stimulus and elevated levels are detectablewithin 6 hours after the insult and subsequently decrease with a half-life of about one day [2]. Thedynamic range of PCT is very large: in a healthy person PCT-levels are lower than 0.5 µg/L but inseptic patients PCT-levels of up to 1000 µg/L have been measured. In the differentiation ofinfected from non-infected pancreatitis PCT was better than CRP [3]. Likewise in one study PCTappeared better than CRP in detecting bacterial infection in patients with autoimmune disease [4].On the basis of these results it has been suggested that, in contrast to APPs, PCT is induceddirectly by endotoxin without intervening cytokines [2]. Induction of PCT after injection ofendotoxin has been demonstrated in human volunteers [5], but the role cytokines play is unknown.Many basic questions about PCT are still unanswered. The question which tissue or organproduces PCT has not been addressed. Mainly based on the fact that PCT is a precursor ofcalcitonin, a neuro-endocrine origin has been assumed – but not found. Likewise the mechanismof induction is unknown. We hypothesized that, analogous to acute phase proteins, PCT isproduced by the liver and induced by TNFα and IL-6 in the absence of endotoxin

In two in vivo studies we investigated whether rhTNFα and rhIL-6 administered to patients candirectly induce PCT. In vitro, the production of PCT by human liver tissue, and its inducibility byrhTNFα and rhIL-6 were assessed. In vivo as well as in vitro we compared PCT with serumamyloid A (SAA) and CRP, since SAA and CRP are the fastest responding APPs. The first study concerned patients treated with hyperthermic isolated limb perfusion with rhTNFα[6]. Due to leakage of rhTNFα to the systemic circulation these patients display an intense short-lived systemic inflammatory response [7].The second study concerned cancer patients who received rhIL-6 as a hematopoietic growth factor[8].In the in vitro study we used precision-cut liver slices, which are extensively applied inpharmacology and toxicology [9]. Luster et al. [10] have shown that endotoxin in human liverslices induced TNFα and IL-6 release. The inflammatory response of the liver is a multi-cellularphenomenon, involving Kupffer cells, endothelial cells and hepatocytes. In the liver slice all celltypes of the liver are retained. Therefore, liver slices are an attractive model to study integratedinflammatory responses.

PATIENTS AND METHODS In all studies pure E.coli-derived rhTNFα (Boehringer, Ingelheim, Germany) or rhIL-6 (Novartis,Basel, Switzerland) were used. PCT was measured with an immunoluminometric sandwich assay(LUMItest PCT, Brahms Gmbh, Berlin, Germany; certified normal levels <0.5 µg/L; certifieddetection range >0.08 µg/L [2]). SAA and CRP were measured with enzyme linkedimmunosorbent assays [11]. (normal SAA < 2.7 mg/L; normal CRP<2.3 mg/L; detection limit forboth assays is 0.001 mg/L).

Page 74: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Procalcitonin in vivo and in vitro

66

Patients treated with TNFα or IL-6.Five patients with nonresectable soft tissue sarcomas of the lower extremity received 90 min of

hyperthermic isolated limb perfusion with 4 mg of rhTNFα and melphalan (10 mg/L limbvolume) [6]. Systemic serum samples were analyzed for PCT, SAA and CRP at baseline, at 30and 90 min after start of rhTNFα-perfusion, as well as at 2, 6, 12, 24, 48 and 72 h aftertermination of perfusion [7]. In a study to define toxicity and effects on hematopoiesis and biochemical parameters, patientswith stage III-IV breast cancer or non-small cell lung cancer received rhIL-6 in a constant dailydosage of 0.5 to 20 µg/kg for 7 successive days (first day intravenously; subsequent 6 dayssubcutaneously) [8]. Of a total of 20 patients, 7 patients with the lowest initial SAA and CRPvalues were further studied, in order to exclude patients with substantial inflammation beforeadministration of rhIL-6. These patients received rhIL-6 doses of 0.5, 1, 2.5, 2.5, 5, 5 and 20µg/kg/day respectively. At the start of rhIL-6 administration patients had a good performancestatus. PCT, SAA and CRP were determined at baseline and after 1, 2, 7 and 14 days respectively.PCT was determined in serum samples that were stored for up to 7 years at -20oC. SAA and CRPvalues have been published earlier [8] but a subset of these data are re-used here to facilitatecomparison with PCT.

Liver slices incubated with TNFα or IL-6.

0.01

0.1

1

10

100

0 20 40 60 80Hour

PCT

(ug/

L)

0.1

1

10

100

1000

SAA

and

CR

P (m

g/L)

������

Figure 6.1. Mean levels (± SEM) of procalcitonin (PCT; filled circles; normal < 0.08 µg/L), serum amyloidA (SAA; squares; normal < 2.7 mg/L) and C-reactive protein (CRP; diamonds; normal < 2.3 mg/L) in 5patients in the first 72 hours after the start of perfusion with tumor necrosis factor alpha. Note the twoseparate logarithmic scales.

Page 75: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 6

67

Human liver tissue was obtained from redundant material after bipartitioning of livers procuredfrom multi-organ donors. The human livers were handled as described before [12]. From thesepieces of liver tissue, cores (diameter 8 mm) were made as previously described, and stored in ice-cold University of Wisconsin organ preservation solution until slicing. Liver slices (200-300 µmthickness; wet weight 10-14 mg) were prepared with the Krumdieck slicer. The slices wereincubated 24 h in 3.2 mL Williams’ medium E supplemented to 25 mM glucose and 50 µg/mLgentamicin. The 6-well tissue culture plates were continuously rocked back and forth (90/min) at37 oC under 95% O2 and 5% CO2. In addition to unstimulated control slices, slices werestimulated with rhTNFα or rhIL-6 (final concentration 15.6 ng/mL) that was added at the start ofslice incubation. The liver slices were incubated in triplicate, and both induction studies andcontrols were performed in slices from three donor livers. PCT, SAA and CRP were measured inthe culture medium after 24 h.

All studies were approved by the medical ethical commission of our hospital. Patients treated withrhTNFα or rhL-6 gave written informed consent. Consent from the legal authorities and from thefamilies concerned was solicited for the explantation of organs for transplantation purposes.To examine the statistical significance of induction of PCT, SAA and CRP in the two patient-groups, areas under the curve were calculated for each patient after log-transformation. Likewise,in vitro PCT, SAA and CRP results were also log-transformed for statistical analysis. Student’s t-

0.01

0.1

1

10

100

0 7 14Day

PCT

(ug/

L)

0.1

1

10

100

1000

SAA

and

CR

P (m

g/L)

������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������

Figure 6.2. Mean levels (±SEM) of procalcitonin (PCT; filled circles), serum amyloid A (SAA;squares) and C-reactive protein (CRP; diamonds) in 7 patients in the two weeks following the start ofinterleukin-6 administration. The period of interleukin-6 administration is indicated by the hatched bar.

Page 76: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Procalcitonin in vivo and in vitro

68

test was used to assess the significance of differences. A p-value <0.05 was consideredsignificant.

RESULTSPatients treated with TNFα or IL-6.As early as 3.5 h after the start of the rhTNFα-perfusion patients had already mean PCT-levels of1.3 µg/L compared to baseline levels of less than 0.1 µg/L. PCT rose to a maximal level of 28µg/L. Maximal SAA and CRP-levels were 392 and 196 mg/L respectively, compared to baselinelevels of 1.9 and 0.9 mg/L respectively (Fig. 6.1). PCT reached a half-maximal level within 8 h,whereas SAA and CRP reached half-maximal values after 20 h. Stimulation of PCT, SAA andCRP by rhTNFα as assessed by the areas under the curve was significant with a p-value <0.0001. The patients that received rhIL-6 had maximal levels of PCT, SAA and CRP of 7.8 µg/L, 634mg/L and 196 mg/L respectively, compared to baseline levels of 0.1 µg/L, 6 mg/L and 7 mg/Lrespectively (Fig. 6.2). Stimulation of PCT, SAA and CRP by rhIL-6 as assessed by the areasunder the curve was significant with a p-values of 0.02, 0.0004 and 0.0002 respectively. Ascomparison of Fig. 6.1 and Fig. 6.2 shows, increased PCT levels after 1 and 2 days were 9 timeshigher in the TNFα group than in the IL-6 group.

Liver slices incubated with TNFα or IL-6.After incubation of the liver slices unstimulated mean (±SEM) production of PCT (0.4±0.2 µg/L),SAA (0.27±0.06 mg/L) and CRP (0.52±0.09 mg/L) could be detected. Synthesis of PCT wasincreased by stimulation with rhTNFα and rhIL-6 to 274 (p=0.008) and 223 percent (p=0.001) ofcontrol levels respectively. rhTNFα induced significant increases in SAA and CRP production to194 percent (p=0.02) and 147 percent (p=0.03) (Fig. 6.3); increases of SAA and CRP after rhIL-6were not significant (p=0.13 and p=0.24 respectively).

DISCUSSIONTo our knowledge this is the first study to demonstrate PCT induction in vivo after administrationof TNFα or IL-6. In addition this is the first study to show that PCT was produced by liver tissuein vitro after stimulation with TNFα or IL-6. In vivo PCT has a unique time response. The early rise of PCT we observed after theadministration of TNFα was as rapid as observed by Dandona et al. after endotoxin administration[5]. When compared to SAA and CRP the rise of PCT occurred 12 h earlier, with a subsequentfall in circulating levels that is comparable to the fall of SAA or CRP levels. In vitro, after stimulation with TNFα the levels of PCT, SAA and CRP were all increased abovecontrol values. The fact that no significant increase in SAA and CRP was found after stimulationwith IL-6, is in accordance with the concept of cytokine-specific APP-activation: in vitro so-called type I APPs such as SAA and CRP are preferentially induced by TNFα or IL-1, whereastype II APPs such as fibrinogen are preferentially induced by IL-6 [13]. However, for PCT-induction, under the culture conditions used, no such selectivity was observed. When comparingresponses it is important to realise that differences found in PCT release in vivo and in vitro maystill be related to a dose dependency of the induction by TNFα and IL-6.

Page 77: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 6

69

PCT offers interesting opportunities for further study in the liver slice model, since in vitro PCT-levels were far above the detection threshold of the commercial PCT-assay we used. Sinceendotoxin can induce cytokines in liver slices [10], it needs to be verified if endotoxin can inducePCT in liver slices, possibly by signalling between the different cell types present in the liverslice. Such an integrated in vitro model may offer possibilities to study intervention in theinflammatory cascade as it occurs in the human liver. It is not necessary to postulate that PCT is principally induced by endotoxin. In fact, a recentstudy in trauma patients showed that PCT and CRP were related to inflammation caused by injuryand were not related to infection [14]. Early sampling after the onset of inflammation and theinitially discordant responses of PCT and CRP (or SAA) may explain why some studies revealeda better association of infection with PCT than CRP, whereas a recent study showed that PCT was

not a better predictor than CRP for infection [15]. In estimating the sensitivity and specificity ofelevated PCT in regard to serious inflammation it is also important to carefully define normalvalues. PCT’s normal upper limit may well be much lower than the 0.5 µg/L value currently used[2], since others report a PCT-level of only 0.003 µg/L in pooled serum from 13 healthyindividuals [16]. Although neither “acute phase protein” nor “acute phase response” has been uniformly defined[13], we think PCT should seriously be considered an APP since it increases sharply afterinflammation, it is produced by liver tissue and it can be induced by TNFα or IL-6. The half-lifeof elevated PCT-levels of about one day is also characteristic for APPs. As nearly all APPs aresynthesized in hepatocytes, this still needs to be verified for PCT, since we only measured PCT in

100

200

300

400

PCT SAA CRP PCT SAA CRP

% s

timul

atio

n

Figure 6.3: Stimulation of production procalcitonin (PCT), serum amyloid A (SAA) and C-reactive protein (CRP)by liver slices by tumor necrosis factor α (closed bars) and interleukin-6 (open bars). Mean productionlevels after 24 h (±SEM) in 9 stimulated slices are expressed as percentage of mean unstimulatedcontrol levels in 9 slices. Significant increases are denoted by a star.

Page 78: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Procalcitonin in vivo and in vitro

70

a system containing multiple cell types (i.e. hepatocytes, endothelial cells and Kupffer cells).Whether PCT qualifies as an APP will ultimately depend on the clarification of its function.Recent experiments by Nylen and colleagues with a peritonitis model in Syrian hamsters showedexcess mortality after administration of PCT [17]. However, this study is hampered by the use ofhuman PCT in animals, the use of non-specific antibodies and mortality differences betweencontrol groups [18]. If PCT turns out to have a paracrine or endocrine function it might be acytokine, if PCT has effector functions (e.g. protease inhibition) it belongs to the APP-family. In conclusion, our results as well as those published by others strongly suggest that PCT is anAPP. Irrespective of PCT’s unidentified function, its unique kinetics compared with both acutephase proteins and with cytokines may establish PCT as a particularly useful parameter ofinflammation in critical care areas.

ACKNOWLEDGEMENTSParts of these studies were supported by grants from Organon International NV and SolvayPharmaceuticals BV.

REFERENCES

1 Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C. High serum procalcitoninconcentrations in patients with sepsis and infection. Lancet 1993; 341:515-518.

2 Meisner M. Procalcitonin. A new innovative infection parameter. Biochemical and clinical aspects.Meisner M. Berlin: Brahms Diagnostica 1996. ISBN 3-00-000803-9.

3 Rau B, Steinbach G, Gansauge F, et al. The potential role of procalcitonin and interleukin 8 in theprediction of infected necrosis in acute pancreatitis. Gut 1997; 41:832-840.

4 Eberhard OK, Haubitz M, Brunkhorst FM, Kliem V, Koch KM, Brunkhorst R. Usefulness ofprocalcitonin for differentiation between activity of systemic autoimmune disease (systemic lupuserythematosus/systemic antineutrophil cytoplasmic antibody-associated vasculitis) and invasivebacterial infection. Arthritis Rheum 1997; 40:1250-1256.

5 Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, Bohuon C. Procalcitonin increaseafter endotoxin injection in normal subjects. J Clin Endocrinol Metab 1994; 79:1605-1608.

6 Eggermont AM, Schraffordt Koops H, Lienard D, Kroon BB, van Geel AN, Hoekstra HJ, LejeuneFJ. Isolated limb perfusion with high dose tumor necrosis factor α in combination with interferon γand melphalan for irresectable extremity soft tissue sarcomas: multicenter trial. J Clin Oncol 1996;14:2653-2665.

7 Zwaveling JH, Maring JK, Clarke FL, van Ginkel RJ, Limburg PC, Hoekstra HJ, Koops HS,Girbes AR. High plasma tumor necrosis factor (TNF)-alpha concentrations and a sepsis-likesyndrome in patients undergoing hyperthermic isolated limb perfusion with recombinantTNF-alpha, interferon-gamma, and melphalan. Crit Care Med 1996; 24:765-770.

Page 79: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 6

71

8 van Gameren MM, Willemse PHB, Mulder NH, Limburg PC, Groen HJ, Vellenga E, de Vries EG.Effects of recombinant human interleukin-6 in cancer patients: a phase I-II study. Blood 1994;84:1434-1441.

9 Olinga P, Meijer DKF, Slooff MJH, et al. Liver slices in in vitro pharmacotoxicology with specialreference to the use of human liver tissue. Toxicol in Vitro 1998; 12:77-100.

10 Luster MI, Germolec DR, Yoshida T, et al. Endotoxin-induced cytokine gene expression andexcretion in the liver. Hepatology 1994; 19:480-488.

11 Hazenberg BPC, Limburg PC, Bijzet J, et al.: Monoclonal antibody base ELISA for human SAA.In: Amyloid and Amyloidosis 1990. Eds Natvig JB, Forre O, Husby G, et al. Kluwer AcademicPublishers, Dordrecht/ Boston/ London 1991:898-901.

12 Olinga P, Merema MT, Hof IH, de Jong KP, Slooff MJ, Meijer DK, Groothuis GM. Effect ofhuman liver source on the functionality of isolated hepatocytes and liver slices. Drug MetabDispos 1998; 26:5-11.

13 Moshage H: Cytokines and the hepatic acute phase response. J Pathol 1997; 181:257-266.14 Mimoz O, Benoist JF, Edouard AR, Assicot M, Bohuon C, Samii K. Procalcitonin and C-reactive

protein during the early posttraumatic systemic inflammatory response syndrome. Intensive CareMed 1998; 24:185-188.

15 Ugarte H, Silva E, Mercan D, de Mendonca A, Vincent JL. Procalcitonin used as a marker ofinfection in the intensive care unit. Crit Care Med 1999; 27:498-504.

16 Snider RH Jr, Nylen ES, Becker KL. Procalcitonin and its component peptides in systemicinflammation: immunochemical characterisation. J Investig Med 1997; 45:552-560.

17 Nylen ES, Whang KT, Snider RH, Steinwald PM, White JC, Becker KL. Mortality is increased byprocalcitonin and decreased by an antiserum reactive to procalcitonin in experimental sepsis. CritCare Med 1998;26:1001-1006.

18 Braithwaite SS. Procalcitonin – marker or mediator? Crit Care Med 1998; 26:977-978.

Page 80: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 7

EARLY POST-TRAUMATIC THROMBOCYTOPENIA IS NOT AFFECTEDBY HIGH-DOSE METHYL-PREDNISOLONE

M.W.N. Nijsten, K. Bartelet, M. Bos, R.J. Porte, T.H. The, H.J. ten Duis

Submitted

Page 81: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Early post-traumatic thrombocytopenia and methylprednisolone

74

ABSTRACTBackground: After trauma and sepsis platelet counts (PC) decrease during the first two days.Except in massive bleeding,

this is primarily due to inflammation-induced platelet sequestration. Changes in PC are stronglyrelated with outcome. In contrast to animal experiments, the effect of early steroids on PC incritically ill patients has not been studied. Spinal cord injury is currently the only widely acceptedindication for the administration of high-dose steroids. We investigated if earlymethylprednisolone (MPS) affected platelet sequestration in patients with spinal injury.Methods: In the MPS-group, patients with vertebral and spinal cord injury received 30 mg/kg ofMPS followed by an infusion of 5.4 mg/kg/hr for the next 23 hrs. Controls were patients withvertebral fractures who received no steroids. Patients who received any platelet transfusion orexcessive red cell transfusion were excluded. PC and hemoglobin were determined on day 0, 1and 2.Results: In 24 patients MPS was administered on average 3.8 hrs after the injury. 41 patients wereincluded in the control group. Both groups were comparable in age, Injury Severity Score andblood loss. Between day 0 and day 2 the PC decreased by 41% and 37% in the MPS and controlgroup respectively. In both groups hemoglobin decreased by 14%.Conclusion: High-dose MPS started within 4 hours does not inhibit platelet sequestration duringthe first 2 days after trauma. This inability of MPS to prevent platelet sequestration may berelated to the failure of MPS to affect mortality in clinical trials.

INTRODUCTIONIn major trauma and sepsis, platelet sequestration is intimately linked with systemic inflammationand possibly with disseminated intravascular coagulation (DIC) [1].As a result of the trauma theplatelet count (PC) decreases during the first days. In severely injured patients the extent of earlythrombocytopenia is strongly associated with the subsequent occurrence of multiple organdysfunction syndrome [2]. In a recent study on 1415 patients admitted to the surgical intensivecare unit (ICU) we found that the rate of change in platelet count was associated with mortality[3]. This phenomenon was observed in all subgroups: abdominal surgery, vascular surgery, livertransplantation and trauma. In medical ICU patients it has also been observed that the magnitudeof decreases in PC constitutes an important, independent marker for mortality [4].Experimental trauma and sepsis studies have shown that very early (i.e. before or <60 minutes ofthe insult) administration of corticosteroids has a positive effect on mortality in parallel with avery marked reduction of platelet sequestration [5,6]. On the other hand, well-conducted clinicalintervention studies in critically ill patients have all consistingly failed to improve survival. Inaddition to high-dose corticosteroids [7,8,9] these interventions included anti-endotoxinantibodies, several cytokine inhibitors, platelet activating factor (PAF) antagonist and bradykininantagonist [10]. These studies did not report on the effect of the intervention on quantitativechanges in platelet counts.

Page 82: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 7

75

To our knowledge, acute spinal cord injury is the only widely accepted indication for theadministration of high-dose corticosteroids after acute injury in particular and acute critical illnessin general. The second and third National Acute Spinal Cord Injury Studies [11,12] (NASCIS-2and -3) reported a significantly better neurologic outcome for patients treated withmethylprednisolone (MPS) within 8 hours of the injury (30 mg/kg bolus followed by 5.4 mg/kg/hrfor 23 hrs or 48 hrs if started more than 3 hrs after the injury). Although the design andinterpretation of the NASCIS studies have been questioned [13], the NASCIS-protocol is widelyapplied, not in the least because of the devastating nature of this trauma. The effect of MPS onplatelet counts was not studied by the NASCIS investigators. In the current study we investigatedpatients with spinal cord injury who received MPS according to NASCIS guidelines. Patients withvertebral fractures and otherwise comparable injuries, but without spinal cord injury were used ascontrols. The aim of the study was to assess if high-dose corticosteroids inhibit early plateletsequestration.

PATIENTS AND METHODSPatient data were gathered from the hospital data base, from a dedicated trauma database that usesInternational Classification of Diseases (ICD-9) codes and from patient files. Patients with ICD-9code 806 (vertebral injury with spinal cord lesion) who received MPS (methylprednisolonesodium succinate, Pharmacia-Upjohn, Peapack , New Jersey, USA)according to the NASCIS protocol were included in the MPS group. Patients with ICD-9 code 805who received no steroids since they had no spinal injury, were included in the control group. Tominimize confounding effects of excessive injury and bleeding necessitating red cell or platelettransfusion the following patients were excluded: patients who died within one week, patientswho received any platelet transfusion and patients who received more than 4 units of red cells perday during the first three days. In all patients the injury severity score (ISS) was determined [14].Platelet count and hemoglobin were determined on day 0, 1 and 2. Statistical differences betweenmean values were assessed with the Student t-test.

Table 7.1Patient characteristics

MPS group Control groupNumber of patients 24 41Age (+/-SD) 38+/- 15 39+/-19Male/Female 18/6 35/6Died 2 1

Vertebral fractures Cervical 6 9 Thoracic 13 15 Lumbal 4 19 Sacral 2 4

Mean ISS ±SD (range) 24±11 (9-41) 21±8 (4-34)Patients operated on day 0,1, 2 8 16

MPS: methylprednisolone; ISS: injury severity score.

Page 83: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Early post-traumatic thrombocytopenia and methylprednisolone

76

RESULTSThe MPS group and control group included 24 and 41 patients respectively. Groups werecomparable in age and ISS (Table 7.1). The mean delay between injury and MPS-treatment was3.8 hrs (range 0.5-7 hrs). Two MPS patients died and one control patient died during hospital stay(after 9, 13 and 29 days respectively). In the MPS group 9 patients received a total of 23 red cellunits on day 0, 1 and 2; in the control group 13 patients received 38 units over the same period.For the two groups taken together, the mean±SD platelet count dropped from 215±56 on day 0 to133±41 ·103/mm3 on day 2 (a 38% decrease; p<0.001). Over the same period overall hemoglobindropped from 11.0±2.6 to 9.4±1.9 g/L (a 14% decrease; p<0.001). The difference between therelative decrease in PC and the relative decrease in hemoglobin was more than a factor 2 andsignificant (p<0.001).Fig. 7.1 displays the changes of platelet count and hemoglobin for the MPS and the controlgroups. No differences between the two groups were observed in the decrease of platelet count

and hemoglobin. Also within both groups the relative or fractional drop in platelet count was againsignificantly higher than the relative drop in hemoglobin (p<0.001 and p<0.001 respectively). To

0

50

100

150

200

250

0 1 2

Days post Trauma

Plat

elet

cou

nt 1

03 /mm

3

0

2

4

6

8

10

12

Hem

oglo

bin

g/dLPlatelet count

Hemoglobin

Methylprednisolone

Figure 7.1. Time course (means+/- SE) of platelet counts (filled symbols) and hemoglobin (open symbols) inpatients with vertebral fractures treated with high-dose methylprednisolone (MPS group; circles) and patients(control group; squares) that were not treated with MPS. No significant differences were observed between theMPS-group and the control group for the decrease in PC (41 % and 37 % respectively) and the decrease inhemoglobin (14 % for both groups). Thus early high-dose MPS does not inhibit platelet sequestration. Theperiod during which the patients received MPS is shown on the time axis.

Page 84: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 7

77

further minimize confounding effects of transfusion or operation we also analyzed patients whoreceived no red cell transfusion and who were not operated (11 MPS patients and 22 controlpatients). Again, no significant differences between the MPS and the control group were seen; therelative drop in PC of 34% was significantly higher than the 15% drop in hemoglobin (p<0.001).

DISCUSSIONPlatelet counts significantly decreased after admission in the patients studied. The observeddecrease in platelet count is much greater than can be explained on the basis of blood loss alone.The 38% drop in platelet count in all patients is more than double the fractional decrease inhemoglobin. When the effect of the spleen as an exchangable pool of platelets is taken intoaccount, even more platelets must have disappeared from the circulation compared to red cells.Since in comparable groups the same decrease in platelet count was observed with or withoutMPS, MPS clearly was not effective in preventing platelet sequestration.Both the unparalleled succes of steroids and the difficulties in replacing corticosteroids with moremodern treatments are related to the very broad spectrum of their anti-inflammatory actions. Theseeffects include inhibition of many cytokines and mediators, reduction in vascular permeabilityand inhibition of several leukocyte types [15]. In the patients studied, even if additional or‘secondary’ inflammatory responses are suppressed by high-dose MPS the initial eventsapparently irreversibly induce sustained platelet sequestration. Whatever the nature of anypharmacologic intervention after trauma, administering it much earlier than 3.8 hours post-traumaappears will be difficult in the majority of cases. In the NASCIS-2 and -3 studies, where theparticipants were obviously aware of the importance of rapid treatment, the delay to MPS-administration was 8.7 and 3 hrs respectively [11,12]. We prefer to use the term platelet sequestration since it includes both adhesion of platelets to thevascular wall, and aggregation of platelets in clots. Although DIC is not uniformly defined [16],demonstrating the consumption of platelets as well as coagulation factors is essential for thediagnosis. To some extent DIC is present in many patients after major trauma [2], but we did notmeasure coagulation parameters to assess the extent of possible DIC in our patients. But DIC isnot a prerequisite for platelet sequestration, since thrombocytopenia often occurs in the absence ofDIC [17]. The 'target' organs of platelet sequestration after trauma or sepsis are many: 111Indium-labelled platelets have been located in the gut, lung, liver and spleen, especially in patients withpoor outcomes [18]. Regardless of the target organ, the endothelial cell is (by definition) the maincell with which platelets interact. Various inflammatory stimuli can induce endothelial cellactivation [19] that can trigger coagulation and platelet adhesion, e.g. by inducing release oftissue factor [20]. Correlating the prolonged platelet sequestration observed in vivo with in vitrostudies is not trivial since it is very difficult to realistically reproduce the interaction betweenendothelial cells and platelets. In an interesting in vivo experiment [21] endothelial dysfunctiondeveloped after a 1-hr perfusion with endotoxin of the forearm vessels in healthy persons. Thisendothelial "stunning" lasted for more than 48 hours. Thus activation of platelets and activation ofendothelial cells may be present in the absence of significant DIC.As indicated above, in many trauma and sepsis experiments but not in clinical studies, limiting thedecreases in platelet count has been used as a surrogate goal. In observational studies low early

Page 85: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Early post-traumatic thrombocytopenia and methylprednisolone

78

platelet counts were predictive of poor outcome in patients with sepsis or patients with a rupturedabdominal aortic aneurysm [22,23]. In prognostic models in critical care, the platelet count hasemerged as an important component. For example the multi-organ dysfunction [24] and thesequential organ failure assessment scores [25] use the platelet count and not the leukocyte countas one of their components parameters, which in contrast to the older Acute Physiology andHealth Evaluation (APACHE) score [26]. Recent studies have shown that the rate of change inthe platelet count is even a better marker of outcome. Failure of the platelet count to recoversufficiently after reaching nadir values was associated with poor outcome in medical [4] andsurgical [3] intensive care patients. In surgical ICU patients we found that the rate of change inplatelet count had the the same predictive power for mortality as APACHE-II scores [3].However, in the immunomodulatory clinical intervention trials [10] platelet count has not been usedas a major parameter. No intervention effects on the platelet count have been reported - although theplatelet count was measured in many trials as part of semiquantitative hematological or coagulationscores. It would be of interest to re-analyse existing clinical trial databases and compare quantitativechanges in platelet count with the intervention and with outcome. Thus many lines of evidence point to a relation of platelet count with outcome in a variety ofseriously ill patients. The pathophysiologic importance of platelet sequestration in regard tooutcome can only be proven by selectively blocking this process. Promising interventions'downstream' the inflammatory cascade may be possible at clinically feasible times. Inhibitors oftissue-factor activity may be very effective in limiting DIC and platelet sequestration [16].Antithrombin-III and especially glycoprotein IIb/IIIa (GP IIb/IIIa) -inhibitors have both shown anti-inflammatory effects. The GP IIb/IIIa-inhibitors that selectively inhibit platelet adhesion haveshown dramatic effects on coronary platelet sequestration and mortality in several clinical trials [27].

In conclusion we have shown that high dose steroids started within hours after major injury do notinfluence subsequent systemic platetet sequestration. We believe that limiting the decrease inplatelet count as a goal in intervention studies remains useful. How instrumental the role of plaletetsequestration is in systemic inflammation, needs to be addressed by specific intervention studies.

Page 86: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 7

79

REFERENCES1. Gando S, Kameue T, Nanzaki S, Nakanishi Y. Disseminated intravascular coagulation is a

frequent complication of systemic inflammatory response syndrome. Thromb Haemost1996;75:224-228.

2. Gando S, Nanzaki S, Kemmotsu O. Disseminated intravascular coagulation and sustainedsystemic inflammatory response syndrome predict organ dysfunctions after trauma: application ofclinical decision analysis. Ann Surg 1999;229:121-127.

3. Nijsten MWN, ten Duis HJ, Zijlstra JG, Porte RJ, Zwaveling JH, Paling JC, The TH. Blunted risein platelet count in critically ill patients is associated with worse outcome. Crit Care Med2000;28:3843-3846.

4. Vanderschueren S, de Weerdt A, Malbrain M, Vankerschaever D, Frans E, Wilmer A, BobbaersH. Thrombocytopenia and prognosis in intensive care. Crit Care Med 2000;28:1871-1876.

5. Hardaway RM, Williams CH, Dozier SE. Influence of steroids on hemorrhagic and traumaticshock. J Trauma 1987;27:667-670.

6. Yoshikawa T, Murakami M, Furukawa Y, Takemura S, Kondo M. Prevention bymethylprednisolone of disseminated intravascular coagulation induced by sustained infusion ofendotoxin in rats. Haemostasis 1983; 13:268-273.

7. Sprung CL, Caralis PV, Marcial EH, Pierce M, Gelbard MA, Long WM, Duncan RC, TendlerMD, Karpf M. The effects of high-dose corticosteroids in patients with septic shock. Aprospective, controlled study. N Engl J Med 1984;311:1137-1143.

8. Bone RC, Fisher CJ Jr, Clemmer TP, Slotman GJ, Metz CA, Balk RA. A controlled clinical trialof high-dose methylprednisolone in the treatment of severe sepsis and septic shock. N Engl J Med1987;317:653-658

9. The Veterans Administration Systemic Sepsis Cooperative Study Group. Effect of high-doseglucocorticoid therapy on mortality in patients with clinical signs of systemic sepsis. N Engl JMed 1987;317:659-665.

10. Abraham E. Why immunomodulatory therapies have not worked in sepsis.Intensive Care Med 1999;25:556-566

11. Bracken MB, Shepard MJ, Collins WF, et al. A randomized, controlled trial ofmethylprednisolone or naloxone in the treatment of acute spinal cord injury: results of the secondNational Acute Spinal Cord Injury Study. N Eng J Med 1990;322:1405-1411.

12. Bracken MB, Shepard MJ, Holford TR, et al. Administration of methylprednisolone for 24 or 48hours or tirilazad mesylate for 48 hours after acute spinal cord injury: results of the third NationalAcute Spinal Cord Injury Randomized Controlled Trial. JAMA 1997;277:1597-1604.

13. Nesathurai S. Steroids and spinal cord injury: revisiting the NASCIS 2 and 3 trials. J Trauma1998. 45:1088-1093.

14. Baker SP, O'Neill B. The injury severity score: un update. J Trauma 1976;16:882-885.15. Schleimer RP. An overview of glucocortocoid anti-inflammatory actions. Eur J Clin Pharmacol

1993;45 Suppl 1;S3-7.16. Levi M, ten Cate H. Disseminated Intravascular coagulation. N Eng J Med 1999;341:586-592.

Page 87: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Early post-traumatic thrombocytopenia and methylprednisolone

80

17. Neame PB, Kelton JG, Walker IR, Stewart IO, Nossel HL, Hirsch J. Thrombocytopenia insepticemia:the role of disseminated intravascular coagulation. Blood 1980;56:88-92.

18. Sigurdsson GH, Christenson JT, el-Rakshy MB, Sadek S. Intestinal platelet trapping after traumaticand septic shock. An early sign of sepsis and multiorgan failure in critically ill patients? Crit CareMed 1992; 20:458-467.

19. Hunt BJ, Jurd KM. Endothelial cell activation. A central pathophysiological process. BMJ1998;316:1328-1329.

20. Gando S, Nanzaki S, Sasaki S, Kemmotsu O. Significant correlations between tissue factor andthrombin markers in trauma and septic patients with disseminated intravascular coagulation.Thromb Haemost 1998;79:1111-1115.

21. Bhagat K, Moss R, Collier J, Vallance P. Endothelial stunning following brief exposure toendotoxin: a mechanism to link infection to infarction? Cardiovasc Res 1996;32:822-829.

22. Bonfiglio MF, Traeger SM, Kier KL, Martin BR, Hulisz DT, Verbeck SR. Thrombocytopenia inintensive care patients: a comprehensive analysis of risk factors in 314 patients. Ann Pharmacother1995;29:835-842.

23. Bradbury AW, Bachoo P, Milne AA, Duncan JL. Platelet count and the outcome of operation forruptured abdominal aortic aneurysm. J Vasc Surg 1995;21:484-491.

24. Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. Multiple organdysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med1995;23:1638-1652.

25. Vincent JL, de Mendonca A, Cantraine F, et al. Use of the SOFA score to assess the incidence oforgan dysfunction/failure in intensive care units: results of a multicenter prospective study. CritCare Med 1998; 26:1793-1800.

26. Knaus WA, Draper EA, Wanger DP, Zimmerman JE. APACHE-II: a severity of diseaseclassification system. Crit Care Med 1985;13:818-829.

27. Cannon CP. Incorporating platelet glycoprotein IIb/IIIa inhibition in critical pathways: unstableangina/non-ST-segment elevation myocardial infarction. Clin Cardiol 1999;22(Suppl):IV30-36.

Page 88: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 8

BLUNTED RISE IN PLATELET COUNT IN CRITICALLY ILL PATIENTSIS ASSOCIATED WITH WORSE OUTCOME

M.W.N. Nijsten, H.J. ten Duis, J.G. Zijlstra, R.J. Porte, J.H. Zwaveling, J.C. Paling, T.H. The

Crit Care Med 2000;28:3843-3846.

Page 89: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

82

ABSTRACT Objective: Low platelet counts (PC) are known to be associated with disease severity in critically illpatients, but the relevance of time-dependent changes of PC has not been investigated. We tested thehypothesis that a low rate of change of platelet counts (PC) after admission to the intensive care unit(ICU) is associated with mortality.Design: Retrospective study.Setting: A 12-bed surgical ICU of a university hospital.Patients: All adult patients admitted for at least 4 days to the ICU over a 7-year period.Methods On admission Acute Physiology and Chronic Health Evaluation (APACHE-II)-scoreswere calculated. PC and leukocyte counts were analyzed from admission to day 10. The daily rise ofthe platelet count (∆PC/∆t) from day 2 to day 10 was calculated. 30-day mortality as well as hospitalmortality were determined. Results: 1415 admissions were studied. Median PC (interquartile range) initially decreased andsubsequently increased, with a higher PC in 1203 survivors than in 212 non-survivors from day 2onward (302 (181- 438) vs. 129 (62- 228) ⋅109/L at day 10; p<0.001). After stratification of patientsper type of surgery, within each group PC was also higher in survivors. Mean ∆PC/∆t was more than5 times higher in survivors compared to non-survivors: 30±46 vs. 6±28 ⋅109/L/day (p<0.001). Thearea under the receiving-operating-characteristic curve of ∆PC/∆t for 30-day survival was 0.743,compared to 0.728 for APACHE-II. Leukocyte counts showed marginal differences between non-survivors and survivors.Conclusion: A blunted or absent rise in PC in critically ill patients is associated with increasedmortality. ∆PC/∆t is a readily available and cheap parameter to improve assessment of critically illpatients.

INTRODUCTIONIncreased platelet production is a normal response after inflammatory insults such as trauma orinfection. When patients recover after a significant inflammatory event, thrombocytosis is typicallyobserved after approximately one week, with platelet counts leveling off to normal levels afterwards.This phenomenon can be seen as a late part of the acute phase response and has been shown to bemediated by cytokines, especially interleukin-6 [1,2]. In fact, in patients with chronic diseases suchas rheumatoid arthritis or inflammatory bowel disease, thrombocytosis is a marker of persistentinflammation.On the high end of the spectrum of disease severity - e.g. sepsis or major bleeding - low plateletcounts are correlated with disease severity. Significant initial thrombocytopenia (platelet count<50⋅109/L) is known to be predictive of poor outcome in patients with sepsis or with a rupturedabdominal aortic aneurysm [3,4]. Once such patients have survived the first days following the acuteevent a complex interplay of factors will determine the ultimate outcome. Many parametersconsidered either pro-inflammatory or anti-inflammatory have been studied in the quest to predictand to influence outcome. Clinically useful measurement of parameters such as cytokines is difficultand associated with considerable costs. Although many studies have recognized the value of baseline platelet counts in predicting outcome,

Page 90: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 8

the meaning of subsequent time-dependent changes in platelet counts that do not excessivelydeviate from so-called normal values has not been studied. We have often noted that critically illpatients do not display thrombocytosis, although such a response might be expected because of thestrong inflammatory stimuli that are present. To assess the clinical relevance of this phenomenon wesystematically looked at platelet counts after admission to the intensive care unit (ICU), In additionleukocyte counts were also studied since leukocyte counts are frequently measured as an indicator ofinflammation. We hypothesized that a lower rise in platelet counts in the days following ICU-admission would beassociated with adverse outcome. Since the objective was to observe sequential changes in theplatelet count in patients that were especially at risk for a complicated course, we only studiedpatients that stayed at least 4 days in the unit.

PATAll tertithusfolloand (noradm

Survivors Non-survivors

N 1203 212

First admissions 1109 202

Readmissions 94 10

Male/Female 777 / 426 133 / 79

Age 55±19 57±19

APACHE-II 18.0±6.7 23.6±6.9

Groups

Trauma 259 27

Vascular surgery 137 32

Abdominal surgery 185 38

Liver transplantation 186 21

Miscelleneous 436 94

Table 8.1 Patients studiedNote that patients are counted on admission basis. APACHE-II scores are significantlydifferent between the two groups (p<0.001)

83

IENTS AND METHODSpatients 15 years and older who were admitted between 1992 and 1998 to the surgical ICU of aary teaching hospital for 4 days or more were retrospectively studied. Patients had to stay (and survive) at least 4 days in the unit in order to be included. Based on type of surgery, thewing groups were defined: trauma, vascular surgery, abdominal surgery, liver transplantation miscellaneous. All platelet count (normal range 150 to 350 ⋅109/L) and leukocyte countmal range 4 to 10 ⋅109/L) measurements performed from ICU-admission to 10 days after ICUission were analyzed. During ICU-stay, these measurements were performed daily by Coulter

Page 91: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

8

CHa

SRprdvpp(

FTs

vivors (openon-survivors-

0

100

200

300

400

500

0 2 4 6 8 10

Days after ICU-admission

Plat

elet

cou

nt

Figure 8.1. Overall median platelet counts (109/L ; interquartile range) after ICU-admission for surcircles) and patients that died within 30 days of ICU-admission (non-survivors; closed circles). N

0

100

200

300

400

500

0 2 4 6 8 10

Days after ICU-admission

Plat

elet

cou

nt

Figure 8.2a. Median platelet counts (109/L ; interquartile range) for survivors (open circles) and nonsurvivors (closed circles) in trauma patients. The difference is significant (p<0.001).

4

ounter (Beckman Coulter, Fullerton, California, United States). Acute Physiology and Chronicealth Evaluation (APACHE-II; [5]) scores were calculated on admission. Survival at 30 days after

dmission to the ICU and hospital mortality were determined.

tatisticseadmissions to the ICU were also included, provided those occurred more than 30 days after arevious ICU-admission. Thus a patient who was admitted previously once and died during a latereadmission was counted once as a survivor, and once as a non-survivor. When multipleeterminations were made for a single patient on the same day, these values were averaged to onealue for that day before daily group means were calculated. To quantify the daily increase of thelatelet count linear regression estimates were calculated for each individual patient, as well as foratient groups. On the basis of the known bimodal change of platelet counts, the platelet countPC) was assumed to change in a linear fashion from day 2 to day 10, as described by

PC=a⋅t + b.

or each patient the constant a (slope) was calculated, denoted by ∆PC/∆t hereafter.o assess the relation between the APACHE-II score and ∆PC/∆t with 30-day survival and hospitalurvival respectively, receiving-operating-characteristic (ROC) curves were constructed,

do not show the rise in platelet counts observed in survivors (p<0.001).

Page 92: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 8

85

with the area under the curve as a measure of discriminatory ability [6]. Data are expressed as medians with interquartile ranges or means±SD. Significances of differences were assessed with aStudent’s t-test, and corrected according to Bonferroni in case of multiple comparisons.

RESULTSDuring the 7-year study period a total of 3286 different patients aged 15-96 years, had 3940admissions to our surgical ICU. Of these patients 383 died within 30 days. When only patientswho were admitted for more than 4 days were included, 1311 patients (1415 admissions) remainedfor further analysis. In this group 212 (15% of admissions) patients died within 30 days after ICU-admission (Table 8.1). In 277 admissions the patients died during hospital stay. A total of 17364platelet counts and 13695 leukocyte counts were analyzed. The median platelet count of theentire patient group initially dropped to a nadir of 113 (64- 192) ⋅109/L on the second day afteradmission, and subsequently increased to 277 (157- 424) ⋅109/L after 10 days.When survivors and non-survivors were considered separately (Fig. 8.1) the platelet count onadmission showed no difference. From day 2 to day 10 the platelet count in survivors wassignificantly higher than in non-survivors resulting in a platelet count at day 10 of 302 (181-428) ⋅109/L and 129 (62- 228) ⋅109/L respectively. Although the mean platelet count differedmarkedly between patients groups, within all groups (i.e. trauma, vascular surgery, abdominalsurgery, liver transplantation and miscellaneous) non-survivors consistently had lower rise in

platelet counts as measured by mean ∆PC/∆t (Fig. 8.2a-d). These differences were highly

0

100

200

300

400

500

0 2 4 6 8 10

Days after ICU-admission

Plat

elet

cou

nt

Figure 8.2b. Median platelet counts (109/L ; interquartile range) for survivors (open circles) and non-survivors (closed circles) in vascular surgery patients. The difference is significant (p<0.001).

Page 93: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

0

100

200

300

400

500

0 2 4 6 8 10

Days after ICU-admission

Plat

elet

cou

nt

Figure 8.2c. Median platelet counts (109/L ; interquartile range) for survivors (open circles) andnon-survivors (closed circles) in abdominal surgery patients. This difference is significant(p<0.001).

86

significant (p<0.001) for all groups with the exception of the liver transplantation group. Forreadmitted patients the time-dependent changes of the platelet count did not differ from patientswho were first admitted.As opposed to the platelet count, overall and subgroup leukocyte counts in survivors were slightlylower compared to non-survivors, with values of 13.4 (10.2- 18.0) ⋅109/L and 15.5 (11.4-21.2) ⋅109/L at day 10 respectively (p=0.03). Patients with low APACHE-II-scores (<18) showed asomewhat higher platelet count than patients with high APACHE-II scores (>=18) with values of308 (126-446) and 260 (153- 416) ⋅109/L at day 10 (p=0.03).In 1290 admissions the daily change in platelet counts (∆PC/∆t) between day 2 and 10 could becalculated: 30-day survivors had a value of 30±46 ⋅109/L/day whereas non-survivors had a valueof 6±28 ⋅109/L/day (p<0.001). With respect to 30-day survival the area under the ROC was 0.743 for ∆PC/∆t (Fig. 8.3) and 0.728 for APACHE-II. The areas under the ROC for ∆PC/∆t andAPACHE-II when calculated in respect to hospital survival where 0.736 and 0.708 respectively.

Page 94: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 8

87

DISCUSSIONThe aim of this study was to analyze the time-response of the platelet count in relation to outcomein critically ill patients. In patients who uneventfully recover from an inflammatory insult theplatelet count displays a bimodal response with an initial decrease below baseline values for thefirst days, followed by an increase above the normal range after one week. In our study, thesurviving trauma patients clearly showed such a response (Fig. 8.2a). Trauma patients sustainedtheir injury and subsequent definitive surgery in most cases within 24 hours before ICU admission.Combined with the fact that nearly all of these patients were previously healthy and relativelyyoung - as opposed to most patients in the other patient groups – this may explain why the bimodaltime course of platelet counts was most marked in trauma patients. In the other patient groups lower overall platelet counts were observed, especially in livertransplants (Fig. 8.2d). In this latter group, splenomegaly is a major determinant of low plateletcounts. Nevertheless, in all patients groups we saw the same diverging pattern between those whoeventually died and those who survived. Platelet counts in survivors were twice the value of non-survivors at day 10. When the rate of change ∆PC/∆t from day 2 onward was examined, this valuewas 5-fold higher in the survivors compared to non-survivors. If the change in platelet counts isconsidered from day 0 onward instead of from day 2 onward, the difference between survivors andnon-survivors would be even more even more striking. But the reason for calculating ∆PC/∆t fromday 2 onward, and not from day 0 onward, was to appropriately fit the bimodal time course ofplatelet counts.

The constancy and reproducibility of the plaletet count in normal individuals has already been

0

100

200

300

400

500

0 2 4 6 8 10

Days after ICU-admission

Plat

elet

cou

nt

Figure 8.2d. Median platelet counts (109/L ; interquartile range) for survivors (open circles) andnon-survivors (closed circles) in liver transplantation patients.

Page 95: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

88

established by Brecher [7] in 1953 and later by Ross [8] who showed a remarkable intra-individual stability of the platelet count over a 9-month period. Although for a normal populationthe platelet count may vary by 200 ⋅109/L, from 150 to 350 ⋅109/L, in any normal individual therange is only approximately 60 ⋅109/L. The use of ∆PC/∆t removes baseline inter-individualdifferences. That ∆PC/∆t shows greater differences between survivors and non-survivors than theplatelet count itself, underscores that Brecher’s observation on normal individuals is very relevant in patients as well. The relation between a blunted rise in platelet counts and outcome is not restricted to surgical ICUpatients, as shown for 206 patients at the medical ICU [9]. In this study which excluded patients withentities known to directly effect platelet counts (i.e. AIDS, leukemia, chemotherapy, immunethrombocytopenia and hemolytic uremic syndrome) survivors also showed significantly higherplatelet counts from ICU-day 3 onward. In a recent study on the international EURICUS-II databasecompiling data from 1636 patients from 61 centers with both medical and surgical ICU patients, wealso found that ∆PC/∆t discriminates survivors from non-survivors [10]. That the platelet count can be a good parameter to follow-up critically ill patients is also reflectedin the new emphasis on this parameter in recently introduced scoring systems for critically illpatients. The sequential organ failure assessment (SOFA) score [11] uses the platelet count andnot the leukocyte count as one of its 6 components. Likewise, the multi-organ dysfunction score[12] uses the platelet count and not the leukocyte count as one of 7 components. The older, andstill widely used APACHE-II uses the leukocyte count, but not the platelet count. It is obviouslynot our intention to promote ∆PC/∆t as yet another prognostic scoring system, especially since asingle parameter can never be the foundation of a prognostic score for such complex patients asthere are at the ICU. But the fact that ∆PC/∆t is as good as APACHE-II in predicting mortality isremarkable and makes it a candidate component of scoring systems. It should not be difficult toverify our findings on the existing databases that are the foundation of the above mentionedscores [11,12]. Since these scores already use platelet count as a component, they could bereanalyzed with the inclusion of ∆PC/∆t.

Page 96: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 8

89

The blunted rise of the platelet counts in the non-survivors must be the result of consumption that isrelatively higher than production. Although it is difficult to discriminate between decreased plateletsynthesis and increased consumption, it is probable that especially increased consumption ofplatelets is present. If bone marrow insufficiency or regulatory abnormalities are assumed to causedecreased platelet synthesis, this phenomenon is not reflected by decreased leukocyte counts in

our patient set, since non-survivors even had higher leukocyte counts than survivors. Consumption of platelets can be caused by disseminated intravascular coagulation (DIC) andsequestration in organs. DIC is frequently present in critically ill patients: it was found in 29 of 35ICU-patients with systemic inflammatory response syndrome [13]. In the acute respiratory distresssyndrome, the trapping of platelets in the lung has long been known [14]. Sigurdson et al.investigated platelet sequestration in ICU patients with 111Indium-labelled platelets at the bedside[15]. Patients with poor outcomes showed trapping of platelets in the gut as well as in the lung, liverand spleen. This phenomenon was observed 1 to 4 days before clinical sepsis or multiorgan failure.After liver transplantation, about 50% of the circulating platelets are sequestrated in the transplantedliver immediately after reperfusion [16]. It has been shown that platelet sequestration in the liver

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

1 - Specificity

Sens

itivi

ty

0

30

Figure 8.3. Receiver-operating characteristic curve that describes the relation between of various cut-off levels of the daily change in the platelet count (∆PC/∆t) and the sensitivity (true positive fraction) and 1-specificity (false positive fraction) in predicting death within 30 days. The area under the ROC-curve is 0.743.The straight diagonal indicates the ROC of a parameter that has no predictive value at all. The upper arrowindicates that values of ∆PC/∆t ≤30 ⋅109/L/day have a sensitivity of 0.89 for predicting mortality, with aspecificity of 0.42. A cut-off of ∆PC/∆t ≤0 corresponds to a sensitivity of 0.38 and a specificity of 0.90.

Page 97: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

90

graft is associated with increased reperfusion damage. Persistent thrombocytopenia aftertransplantation was found to be associated with decreased survival [17], which is in agreement withthe observations in the current study.Naturally the question arises if interventions aimed at preventing platelet consumption wouldaffect outcome. Again, reanalysis of existing data sets of intervention studies with regard to∆PC/∆t would be of great interest. Such analyses could answer: a) if ∆PC/∆t is related to outcomeand b) if the intervention affected ∆PC/∆t. Thus, in patients that are not critically ill increasedplatelet counts often indicate ongoing inflammation and disease. But in critically ill patients theopposite is the case since a blunted rise in platelet counts - i.e. a low ∆PC/∆t - has an unfavorableprognosis. The presence of a ‘normal’ platelet count after ICU-admission should not beautomatically interpreted as desirable. Therefore the platelet count, routinely determined at lowcost, should not only be used to detect thrombocytopenia or thrombocytosis. It should also beused to actively follow its time-dependent changes.

REFERENCES 1. Lotem J, Shabo Y, Sachs L. Regulation of megakaryocyte development by interleukin-6. Blood

1989;74:1545-1551.2. Nijsten MWN, Hack CE, Helle M, et al. Interleukin-6 and its relation to the humoral immune

response and clinical parameters in burned patients. Surgery 1991;109:761-767.3. Bonfiglio MF, Traeger SM, Kier KL, et al. Thrombocytopenia in intensive care patients: a

comprehensive analysis of risk factors in 314 patients. Ann Pharmacother 1995;29:835-842.4. Bradbury AW, Bachoo P, Milne AA, et al. Platelet count and the outcome of operation for ruptured

abdominal aortic aneurysm. J Vasc Surg 1995;21:484-491.5. Knaus WA, Draper EA, Wanger DP, et al. APACHE-II: a severity of disease classification system.

Crit Care Med 1985;13:818-829. 6. Zweig MH, Campbell G. Receiver-operating characteristic (ROC) plots: a fundamental evaluation

tool in clinical medicine. Clin Chem 1993;39:561-577.7. Brecher GB, Schneiderman M, Cronkite EP. The reproducibility and constancy of the platelet count.

Am J Clin Pathol 1953;23:15-26.8. Ross DW, Ayscue LH, Watson J, et al. Stability of hematologic parameters in healthy subjects.

Intraindividual versus interindividual variation. Am J Clin Pathol 1988;90:262-267.9. Nijsten MWN, Tulleken JE, vd Werf TS, et al. Absence of secondary thrombocytosis is associated

with prolonged ICU-stay and decreased survival [abstract]. Intensive Care Med 1997;23 Suppl1:59.

10. Nijsten MWN, Nap R, Reis Miranda D. Rate of change in platelet count is associated withmortality in EURICUS-II dataset [abstract]. Intensive Care Med 1999;25 Suppl 1:26.

11. Vincent JL, de Mendonca A, Cantraine F, et al. Use of the SOFA score to assess the incidence oforgan dysfunction/failure in intensive care units: results of a multicenter prospective study. CritCare Med 1998; 26:1793-1800.

12. Marshall JC, Cook DJ, Christou NV, et al. Multiple organ dysfunction score: a reliable descriptor ofa complex clinical outcome. Crit Care Med 1995;23:1638-1652.

Page 98: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 8

91

13. Gando S, Kameue T, Nanzaki S, et al. Disseminated intravascular coagulation is a frequentcomplication of systemic inflammatory response syndrome. Thromb Haemost 1996;75:224-228.

14. Heffner JE, Sahn SA, Repine JE. The role of platelets in the adult respiratory distress syndrome.Culprits or bystanders? Am Rev Respir Dis 1987;135:482-492.

15. Sigurdsson GH, Christenson JT, el-Rakshy MB, et al. Intestinal platelet rapping after traumaticand septic shock. An early sign of sepsis and multiorgan failure in critically ill patients? Crit CareMed 1992;20:458-467.

16. Porte RJ, Blauw E, Knot EAR, et al. Role of the donor liver in the origin of platelet disorders andhyperfibrinolysis in liver transplantation. J Hepatol 1994;21:592-600.

17. McCaughan GW, Herkes R, Powers B, et al. Thrombocytopenia post liver transplantation.Correlations with pre-operative platelet count, blood transfusion requirements, allograft functionand outcome. J Hepatol 1992;16:16-22.

Page 99: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Blunted rise in platelet counts in SICU patients

92

Page 100: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 9

PRIMARY AND SECONDARY CHANGES IN PLATELET COUNT AND OUTCOME IN

MEDICAL AND SURGICAL ICU PATIENTS

M.W.N. Nijsten, R.E. Nap , H.J. ten Duis, R.J. Porte, D. Reis Miranda

Submitted

Page 101: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

94

ABSTRACT

Objective: Thrombocytopenia is related to adverse outcome in critically ill patients. In manypatients admitted to the intensive care (ICU), the platelet count (PC) shows an initial decreasefollowed by a rebound of varying amplitude. Opposed to primary changes, the relation ofsecondary changes of PC with outcome has not been systematically studied. In this study we haveanalyzed changes in a large, heterogeneous set of ICU patients. Based on the changes of PC withtime we established previously, PC-changes were evaluated with a simple model that incorporatesthe biphasic changes in PC.Design: Analysis of the prospectively collected EURICUS-II database. Setting: 53 ICUs in 11 European countries.Patients: All patients for whom PC were available. Measurements and results: 5206 Patients were classified as medical (55%), unscheduled surgery(24%) or scheduled surgery (21%). During ICU-stay daily PC were recorded. The rate of changeof PC between day 0 and 2 was expressed as ∆PC/∆t 0→2, PC on day 2 as PC2 and the rate ofchange in PC between day 2 and 10 as ∆PC/∆t 2→10. In non-survivors ∆PC/∆t2→10 was only 1±26versus 13±31 ·109/l/d for survivors (p<0.001) Differences in early PC as reflected by PC2

(167±112 vs. 188±102 ·109/l; p<0.001) and ∆PC/∆t0→2 (-16±54 vs. -13±41 ·109/l/d; NS) were lesspronounced. Conclusions:A low increase or even a decrease in PC after day 2 is associated with poor outcome both inmedical and in surgical ICU patients. In individual patients, clinicians should bear in mind thetypical time course of the PC after ICU admission in order to recognize abnormal patterns. Inpatient groups, taking ∆PC/∆t2→10 into account may aid in improving outcome assessment.

INTRODUCTION

Both production and consumption of platelets are increased in diseases that involve systemicinflammation. Initial changes in PC in patients admitted to the ICU mostly concern decreases inPC. The magnitude of thrombocytopenia after ICU-admission has proven to be of importantnegative prognostic value in trauma [1], ruptured aortic aneurysm [2], malaria [3], meningitis [4],and sepsis patients [5], as well as in patients in general that were admitted to the ICU [6,7,8,9].The studies that investigated PC and outcome all focused on changes in PC to subnormal values,and mostly early after ICU-admission. Whereas systemic inflammation is initially accompanied by a drop in PC, in uncomplicatedpatients a rebound increase in PC is observed. For example in a trauma patient who does not needprolonged ICU-support, the PC will usually start to rise 2 to 3 days after the injury and reach

Page 102: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

95

supranormal levels after two weeks [10]. Increased production of platelets induced by interleukin-6, thrombopoietin and other cytokines [11] is responsible for this secondary thrombocytosis. We have noted that in critically ill patients who have an ongoing inflammatory response thisrebound thrombocytosis is absent in many cases. This observation led us to the hypothesis thatmore severe inflammation and thus adverse outcome, may be related to an absent or bluntedthrombocytotic response. In a recent study in patients admitted to a surgical ICU we found thatnon-survivors have a lower rate of change in PC after day 2 [10]. In the present study we attempted to systematically analyze both medical and surgical patients,early and late changes in PC, and their relation with outcome. A large multicenter database thathad been prospectively collected, was used for this purpose. We hypothesized that theinformation provided in the secondary changes of the platelet count would be distinct from theinformation carried by initial changes in the platelet count. A simple mathematical model basedon the time course of PC as identified earlier [10] was applied. This model (Fig. 9.1) assumes thatPC decreases linearly during the first two days after ICU-admission, reaches a nadir value aftertwo days, and changes linearly between day 2 and day 10.

ICU day

PC

0

500

0 102

Figure 9.1. Three-parameter model for time dependent changes in platelet count (PC) that was used in thisstudy. For each patient, the rate of change in the platelet count, ∆PC/∆t was calculated by linear regressionbetween day 0 and day 2 (∆PC/∆t0→2) and between day 2 and day 10 (∆PC/∆t2→10). Nadir PC was assumed tooccur at day 2 (PC2). In this example ∆PC/∆t0→2 equals the slope of the descending line on the left (-110·109/l/d), PC2 is the PC at day 2 (135 ·109/l) and ∆PC/∆t2→10 equals the slope of the ascending line (+34·109/l/d).

Page 103: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

96

PATIENTS AND METHODS

EURICUS-II DatabaseThe EURICUS-II dataset contains data of 53 ICUs (medical and surgical) in 11 Europeancountries [12]. Patient data were recorded from admission to the ICU until discharge from the

ICU. On admission patients were divided into one of three categories: unscheduled surgery,scheduled surgery or medical as has been described before [13]. In addition, age, the simplifiedacute physiology score (SAPS-II; [13]) on admission, daily PC (normal range 150-350·109/l)between day 0 (admission to the ICU) and day 10, were collected. Patients were classified intonon-survivors and survivors on the basis of hospital mortality.

Model of time dependent changes in PCThe platelet count was assumed to change linearly until day 2 (mostly a decrease), and to changelinearly (increase or decrease) afterwards. For each individual patient three descriptors of serialchanges in platelet count were extracted with a special program that calculated individualregression coefficients (Fig. 9.1):

∆PC/∆t0→2 109/l/d the early rate of change of the PC, between day 0 and day 2PC2 109/l platelet count on day 2∆PC/∆t 2→10 109/l/d the late rate of change of the PC, between day 2 and 10

Thus for each patient the course of the PC was approximated by two linear regressioncoefficients reflecting the rates of change (∆PC/∆t0→2 and ∆PC/∆t2→10) and the PC on day 2 (PC2)which was assumed to approximate nadir PC at any time. To verify the assumption that PC2

estimates the nadir PC, PC2 was compared with nadir PC.

Table 9.1

Patient characteristics

Survivors Non-survivors P

N 4034 1172

Age Years 59±21 66±18 <0.001

SAPS-II 31±15 49±20 <0.001

∆PC/∆t 0→2 109/l/d -13± 41 -16±54 0.086

PC2 109/l 188±102 168±112 <0.001

∆PC/∆t 2→10 109/l/d 13±31 1±26 <0.001

Values ± SD

Page 104: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

97

Statistical analysis.Means of all (derived) parameters were compared between non-survivors and survivors for theoverall patient group, as well as for the three admission groups. For the various analyses whichwere performed, our approach was to use the largest available subsets of patients possible for eachspecific analysis. Thus the total number of patients used varies in the different analyses. Thisvariation is mainly dependent upon length of stay, since the EURICUS-II study only recordeddata during ICU-stay. Unless indicated otherwise standard deviations are used. The two-sidedStudent's t-test was used for comparison of continuous parameters, without assuming equalvariances. The Bonferroni-correction was used in case of multiple testing.

RESULTS

Overall patient characteristics20508 PC values collected between day 0 and day 10 were available for 5206 patient admissions.Of these admissions, 55% were medical, 24% unscheduled surgery and 21% scheduled surgery.Mean ICU-length of stay was 5.9±10.5 days with a median (interquartile range) value of 2 (1-6)days. The overall mortality rate was 23% (Table 9.1), with group mortalities of 36% for medical,35% for unscheduled surgery and 11% for scheduled surgery respectively.

Platelet CountsOn admission, mean PC in the entire group was 217±115·109/l. At this time only 4% had aPC<50·109. Fig. 9.2 shows the diverging time course of the PC between non-survivors andsurvivors. On day 0 the non-surviving patients had a mean PC of 203±122 that dropped to167±112 on day 2 and rose to 222±137·109/l on day 10. In the survivors this value at day 10 was317±166, which is 95·109/l higher than in non-survivors (p<0.001). This pattern of an initialdecrease with a subsequent increase was also observed in the three major subgroups: medical,unscheduled surgery and scheduled surgery, with mean PC reaching a minimal value on day 2. Insupport of the model’s assumption, the nadir PC showed a strong correlation with the PC2, with aSpearman correlation coefficient of 0.89 (p<0.001).In non-survivors ∆PC/∆t 2→10 was only 1±26 compared to 13±31·109/l/d for survivors (p<0.001).The difference in PC2 (167±112 vs. 188±102 ·109/l; p<0.001) was less pronounced. ∆PC/∆t0→2

showed no difference (-16±54 vs. -13±41 ·109/l/d; NS).

Page 105: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

98

A value of ∆PC/∆t2→10 below -30·109/l/d was associated with a 40% mortality, whereas a rise ofmore than +60·109/l/d was associated with a 3% mortality. Figure 9.3 shows the distributions andassociated mortalities of the three parameters ∆PC/∆t0→2, PC2 and ∆PC/∆t2→10 for the threeadmission groups. Fig. 9.3 illustrates that in the majority of patients PC indeed decreases betweenday 0 and day 2 in most patients, but drops to levels still over 150·109/l in most patients. Only fewpatients have severe thrombocytopenia on day 2 and after day 2 PC rises in most patients. Fig 9.3

also indicates that ∆PC/∆t2→10 has the clearest association with mortality. Since PC2 and ∆PC/∆t2→10 had the strongest relation with mortality, the interaction of these twoparameters (Table 9.2) and their combined relation with mortality (Fig. 9.4) were analyzed. Heresubranges were chosen to approximate interquartile ranges for the two respective parameters. Theordinal by ordinal Spearman correlation coefficient was -0.16, revealing that PC2 and ∆PC/∆t2→10

are nearly independent. Fig. 9.4 shows that the relations of low PC2 and a low ∆PC/∆t2→10 withmortality are additive. For example the 108 patients with PC2<100·109/l and ∆PC/∆t2→10 < 0 have60% mortality rate in contrast to an 8% mortality rate in the 113 patients with PC2>200·109/l and∆PC/∆t2→10 > 30·109/l/d. ChildrenIn the small subgroup of children (age<=15 year; 13 nonsurvivors; 39 survivors) only ∆PC/∆t2→10

showed a difference with -9±40 versus 24±40 ·109/l/d (p=0.016) in non-survivors and survivorsrespectively.

Figure 2

0

100

200

300

400

500

0 2 4 6 8 10ICU day

Plat

elet

cou

nt

0.00

0.20

0.40

0.60

0.80

1.00

Frac

tion

of P

atie

nts

Figure 9.2. Mean PC +/- SD in non-survivors (ICU and hospital mortality; filled circles) and survivors(circles). The mean PC values are significantly different between survivors and non-nonsurvivors. The thickdescending line indicates the fraction of patients in which PC was measured; the 100% at day 0 corresponds to4862 patients.

Page 106: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

99

Late mortality and early changes in PCSince changes in PC only shortly before death might disproportionately affect PC2 and∆PC/∆t2→10, we performed an additional analysis on the predictive value of PC2 and ∆PC/∆t for alonger period ahead. In the subgroup of patients that had a stay of at least 10 days at the ICU, PC2

and the rate of change between day 2 and day 5 (i.e. ∆PC/∆t2→5) were compared. PC2 was184±124 and 186±100·109/l respectively (NS) and ∆PC/∆t2→5 was 0±30 and 10±32·109/l/drespectively (p<0.001).

DISCUSSION

The relationship between PC and mortality in critically ill patients was analyzed in this study. In alarge group of medical and surgical ICU patients the prognostic importance of PC extends wellbeyond initial changes. With regard to PC, day 2 after ICU admission can be viewed as a turningpoint (Fig 9.1). The extent of the decrease in PC during the first two days was not related withmortality. Many investigators observed a relation with early nadir PC and mortality. In this studywe also found that the PC at day 2, which adequately reflected nadir PC, is related with mortality.But the daily rate of change in PC after day 2 showed the strongest relation with mortality. Thisphenomenon was especially marked in unscheduled surgery patients (Fig. 9.3), where thesemortalities were 53% and 0% for low (i.e. <0) and high (>30·109/l/d) values of ∆PC/∆t 2→10

respectively. Vanderschueren [6] reported that in 329 predominantly medical ICU patients that nadir PC wasassociated with mortality. Unfortunately, the authors do no report the time interval betweenmeasuring a nadir PC and the patients' death. Stephan observed in 147 surgical ICU patients [7]that those patients who had a PC below 100 at any time had a mortality rate of 38%, compared

Table 9.2

Number of patients (percentage) according to PC2 and ∆PC/∆t 2→10

∆PC/∆t 2→10 (109/l/d)

PC2 (109/l) <0 0 ~ 10 10 ~ 30 >30 Totals

<=100 106 (5) 100 (5) 134 (7) 98 (5) 438 (23)

100 ~ 150 107 (6) 99 (5) 127 (7) 87 (5) 420 (22)

151 ~ 200 136 (7) 91 (5) 117 (6) 38 (2) 382 (20)

> 200 299 (16) 123 (6) 153 (8) 113 (6) 688 (36)

Totals 648 (34) 413(21) 531(28) 336 (17) 1928 (100)

Page 107: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

100

with a 20% mortality rate for PC >100·109/l. In the latter study thrombocytopenia occurred1.8±0.5 days after admission.In a previous study of 1415 admissions to a surgical ICU [10] we also observed an associationwith mortality of a low rate of change in PC after day 2. The current study generalizes theseresults to a larger, more heterogeneous patient group that includes medical patients as well aschildren. That changes in PC between day 2 and day 5 were also associated with the mortality thatoccurred after day 10, underscores that the phenomenon we observed is not solely the result ofchanges occurring directly before death.

Limitations of this studyThe primary purpose of EURICUS-II was to investigate the role of collaborative practice at theICU, and to find parameters to measure improvements in nurse-doctor interaction [12]. Thus weretrospectively analyzed data that were prospectively collected for other purposes. The databasecontains very heterogeneous patients without detailed information on their condition before ICU-admission, making analysis of PC behavior in specific syndromes or diseases impossible.Especially in the medical patients, PC before ICU-admission would have been useful. Pre-admission PC would obviously allow a better estimation of ∆PC since in many cases PC will havedecreased already before ICU-admission. Many relevant physiological parameters were notrecorded, such as leukocyte counts or quantitative indicators of systemic inflammation (e.g. C-reactive protein). We did not study the mechanisms that lead to decreased PC. Nevertheless, aspointed out below, we think other studies provide compelling arguments to assume thatinflammation-induced platelet sequestration plays a major role. Pathophysiology of decreasing platelet countsDecreases in PC are by definition the result of platelet consumption that is higher than plateletproduction. We assume that increased platelet consumption is the main contributor to (relatively)low PC in ICU patients both during the primary and secondary phases of ICU-stay. Acute PC-decreases must result from increased consumption of platelets since platelets have a normal life-span of 8 to 11 days [14]. The fact that only 4% had a PC below 50·109/l excludes a significantimpact of patients with acquired bone marrow failure on our results, since such patients usuallyhave a PC well below 50·109/l. In a study on thrombocytopenia in the surgical ICU 8 out of the 9patients with sepsis and thrombocytopenia who underwent bone marrow examination during laterphases of their ICU-stay, displayed a normocellular or hypercellular marrow with a normalnumber of megakaryocytes [7]. The same observation was made in 15 children withthrombocytopenia after sepsis without signs of disseminated intravascular coagulation (DIC) [15].In our previous study in surgical ICU patients [10], non-survivors had higher leukocyte countsthan survivors, which also argues against marrow failure. The most direct observation ofincreased platelet sequestration in trauma and sepsis patients in the ICU was seen afteradministration of 111Indium-labelled platelets. Increased sequestration was found in several organsincluding the lung, liver and gut, especially in patients with adverse outcomes [16].

Page 108: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

0

500

1000

1500

2000

<-30 -30~0 0~30 30~60 >60

Num

ber o

f pat

ient

s

0~20 21~50 51~100 101~150 >150

<-30 -30~0 0~30 30~60 >60

100%

PC2 ∆PC/∆t 2→10

0%

20%

40%

60%

80%

<-30 -3

Mor

talit

y

Figure 9.3. Di(left panels; 38patients). Bar black bars and The upper left PC. The upperpatients (1%) hhave an increasThe lower midassociated withpatients had 53

<0

10 ~ 30

Figure 9.4. An∆PC/∆t2→10 areTable 9.2 for dacross axis shodimensional frthan PC2.

S

∆PC/∆t 0→2

101

0~0 0~30 30~60 >60 0~20 21~50 51~100 101~150 >150 <-30 -30~0 0~30 30~60 >60

stribution of patients (upper panels) and mortality (lower panels) according to ∆PC/∆t0→2

43 patients) , PC2 (Middle panels ; 2645 patients) and ∆PC/∆t2→10 (Right panels; 2049shades denote admission categories: scheduled surgery: gray bars; unscheduled surgery:medical: white bars.panel shows that ∆PC/∆t0→2 is negative in most patients, indicating an initial decrease in middle panel shows that a day 2, most patients still have a PC>150 ·109/l, and only 35ave a PC< 20 ·109/l. The right upper panel indicates that after day 2, most patients displaye in PC.dle and lower right panels indicate that decreased values of PC2 and ∆PC/∆t2→10 are increased mortality. In the unscheduled surgery group with ∆PC/∆t2→10 <-30·109/l/d 19% mortality; while 37 patients with . ∆PC/∆t2→10 > 60·109/l/d had zero mortality.

0%

20%

40%

60%

Mor

talit

y

<100 100-150 150-200 >200

alysis of combined relations of PC2 and ∆PC/∆t2→10 with mortality. Subranges for PC2 and different from Fig. 3., as they were chosen to best correspond to interquartile ranges (seeistribution of patient numbers). Mortality is displayed on the vertical axis; the horizontalws four PC2-categories; the depth axis shows four ∆PC/∆t2→10-categories. This two-

equency distribution illustrates that ∆PC/∆t2→10 has a stronger association with mortality

cheduled Surgery Unscheduled surgery Medical

Page 109: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

102

The term platelet sequestration is broad enough to include the distinct process of adhesion toendothelium and aggregation in clots. Apart from the effect of systemic inflammation that ispresent in the majority of ICU-patients [17], blood loss [18] and intravascular coagulation [19]are frequent causes of acute decreases PC. Although sometimes acting in concert [20], theseprocesses are separate and in most sepsis patients with thrombocytopenia significant diffuseintravascular coagulation (DIC) is not present [21]. Endothelial cell activation as part of systemicinflammation can trigger platelet adhesion by itself through the expression of a variety ofadhesion molecules [22]. Platelet sequestration is also observed without DIC in several importantmodels of systemic inflammation. In experimental models of TNF-administration [23] or malaria[24] mortality was related to the extent of thrombocytopenia, and not DIC. Experimentally and clinically it is difficult to measure the contribution of endothelial activation toplatelet consumption. It is also not known if sequestration of platelets is directly causal tomortality, but the inflammatory potential of adherent or aggregating platelets is well known. Inthe adult respiratory distress syndrome [25] or ischemia-reperfusion syndromes [26] such asobserved after liver transplantation [27] platelets are powerful and important mediators of tissueinjury. The pervasiveness of platelet sequestration in the critically ill hints that platelet sequestration maybe a pathogenic process many organs.

Theoretical advantages of ∆PC/∆t2→10

One reason that ∆PC/∆t2→10 may discriminate better than absolute PC is that inter-individualvariation is reduced. Intra-individual variation in PC amounts is only 30% of the inter-individualvariation in healthy persons [28,29]. Major methodological criteria which a physiologic measure used in a scoring system should fulfillare reproducibility, responsiveness and validity [30]. Responsiveness means that a measure detects clinically meaningful changes in the process ofinterest, and that changes in the measure correspond to clinically significant changes. With regardto this criterion it is contradictory to transform a responsive, continuous parameter into adiscontinuous parameter with only a limited number of values. Moreover scoring systems such asSOFA (sequential organ failure score; [31]) or MODS (multiple organ dysfunction score; [30])only start to score the PC as abnormal if it is subnormal. For example a PC>150 has 0 SOFApoints, and will still have 0 points when the PC drops 250 on day 2 to 160 on day 10, a drop thatwe find is associated with increased mortality. Although the practical advantage is obvious,namely calculating a total score without a computer, important information is lost in this process.Yet, the SOFA-authors [31], like the MODS-authors [30] have subsequently observed thatchanges in their organ scores (i.e. ∆SOFA or ∆MODS) are also powerful predictors of outcome inaddition to the initial scores themselves. The added value of ∆SOFA or ∆MODS is intuitivelylogical since these incremental increases in organ dysfunction portend by definition additional,more recent information. The special relevance of these incremental scores has been interpreted asreflecting de novo events that arise at the ICU, and may thus be amenable to therapeuticintervention. We believe that the same phenomenon is apparently true for ∆PC/∆t2→10.

Page 110: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

103

Validity is the extent to which a parameter is a meaningful representation of the entire spectrum ofthe process of interest. Although the authors who incorporated PC into their scoring systemsconsidered PC a useful and valid parameter, it is in fact unclear for which pathophysiologicalprocess it is valid. The SOFA authors call PC a "coagulation" parameter while MODS authorscall it an "hematological" parameter. Coagulation and hematology are two rather broad anddifferent processes. In fact it may turn out that PC and especially ∆PC/∆t reflect endothelialactivation in many instances, as argued above. Thus we think that although PC and ∆PC/∆t areuseful parameters, the process they reflected is not established. Although it is obviously futile for application as a solitary parameter, we think ∆PC/∆t2→10 couldbe considered for inclusion in future scoring systems.

Clinical and research implicationsIn clinical practice, PC2 and ∆PC/∆t2→10 are not difficult to assess, even without making a graphor performing calculations. Simple observation of the PC over a number of days will easily showwhether it is decreasing, increasing or does not change after day 2. Our results illustrate thatstable platelet counts, even in the so-called normal range (150-350 ·109/l) are not ideal in manycircumstances. In a patient who is recovering from severe injury the PC should be high-normal orsupranormal after 1 week [10]. If the PC fails to rise in the first week, even if it is 200·109/l, thisshould alert the clinician to potential complications. If platelet sequestration is a pathogenic process and ∆PC/∆t2→10 reflects platelet sequestration, thisraises the logical question if inhibition of platelet sequestration will improve outcome. Analysis ofintervention trial data with respect to ∆PC/∆t2→10 may be useful for addressing this question. Likeplatelets, the agents protein C [32], antithrombin-III [33], glycoprotein IIb/IIIa (GPIIb/IIIa; [34])and P-selectin [35] are all involved at intersection of the coagulatory and inflammatory processes.Thus these agents (i.e. activated protein C, antithrombin-III) or their inhibitors (i.e. GPIIb/IIIa-inhibitors or P-selectin inhibitors) are prime candidates for an examination of their impact on∆PC/∆t2→10.

In conclusion, serial platelet counts carry important information in critically ill patients. Takingthe biphasic changes of PC into account helps to assess what is normal and what is abnormal.Scoring systems might be improved by accommodating the information contained in changes inplatelet counts. Although some may take the process of platelet sequestration for granted, it isobvious that a fundamental process must be involved as it occurs in the large variety of criticallyill patients. Intervention studies may show whether platelet sequestration is a process that iscausal to mortality or whether it is only another of the many markers that have been associatedwith poor outcome in the ICU.

ACKNOWLEDGEMENTThis study was supported by a grant from the European Commission (BMH4-CT96-0817).

Page 111: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

104

REFERENCES 1. Sauaia A, Moore FA, Moore EE, Norris JM, Lezotte DC, Hamman RF. Multiple organ failure can

be predicted as early as 12 hours after injury. J Trauma 1998;45:291-301.2. Bradbury AW, Bachoo P, Milne AA, Duncan JL. Platelet count and the outcome of operation for

ruptured abdominal aortic aneurysm. J Vasc Surg 1995;21:484-491.3. Horstmann RD, Dietrich M. Haemostatic alterations in malaria correlate to parasitaemia. Blut

1985;51:329-335.4. van Deuren M, Neeleman C, van 't Hek LGFM, van der Meer JWM. A normal platelet count at

admission in acute meningococcal disease does not exclude a fulminant course. Intensive Care Med1998;24:157-161.

5. Peduzzi P, Shatney C, Sheagren J, Sprung C. Predictors of bacteremia and gram-negativebacteremia in patients with sepsis. The Veterans Affairs Systemic Sepsis Cooperative StudyGroup. Arch Intern Med 1992;152:529-535.

6. Vanderschueren S, de Weerdt A, Malbrain M, Vankersschaever D, Frans E, Wilmer A, BobbaersH.Thrombocytopenia and the prognosis in intensive care. Crit Care Med 2000; 28:1871-1876.

7. Stephan F, Hollande J, Richard O, Cheffi A, Maier-Redelsperger M, Flahault A.Thrombocytopenia in a surgical ICU. Chest 1999;115:1363-1370.

8. Baughman RP, Lower EE, Flessa HC, Tollerud DJ. Thrombocytopenia in the intensive careunit.Chest 1993;104:1243-1247.

9. Lee KH, Hui KP, Tan WC. Thrombocytopenia in sepsis: a predictor of mortality in the intensivecare unit. Singapore Med J 1993;34:245-246.

10. Nijsten MWN, ten Duis HJ, Zijlstra JG, Porte RJ, Zwaveling JH, Paling JC, The TH. Blunted rise inplatelet count in critically ill patients is associated with worse outcome. Crit Care Med 2000;28:3843-3846.

11. Uppenkamp M, Makarova E, Petrasch S, Brittinger G. Thrombopoietin serum concentration inpatients with reactive and myeloproliferative thrombocytosis. Ann Hematol 1998;77:217-223.

12. The effect of harmonising and standardising the nursing tasks on the Intensive Care Units of theEuropean Community – EURICUS II. Biomedical and health research programme 1994-1998.Summaries for research projects supported under BIOMED 2, volume II;494-495.

13. Le Gall Jr, Lemeshow S, Saulnier F . A new Simplified Acute Physiology Score (SAPS-II) based on aEuropean/ North American multicenter study. JAMA 1993;270:2957-2963.

14. Heyns AD, Badenhorst PN, Lotter MG, Pieters H, Wessels P . Kinetics and mobilization from thespleen of indium-111-labeled platelets during platelet apheresis. Transfusion 1985;25:215-218

15. Mitterstieler G, Kurz R, Haas H, Desoye G. Thrombopoiesis and blood coagulation in pediatricpatients with septicemia. Padiatr Padol 1981;16:257-265.

16. Sigurdsson GH, Christenson JT, el-Rakshy MB, Sadek S. Intestinal platelet trapping after traumaticand septic shock. An early sign of sepsis and multiorgan failure in critically ill patients? Crit CareMed 1992;20:458-467.

17. Pittet D, Rangel-Frausto S, Li N, Tarara D, Costigan M, Rempe L, Jebson P, Wenzel RP .Systemic inflammatory response syndrome, sepsis, severe sepsis and septic shock: incidence,morbidities and outcomes in surgical ICU patients. Intensive Care Med 1995;21:302-329.

Page 112: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 9

105

18. Harvey MP, Greenfield TP, Sugrue ME, Rosenfeld D . Massive blood transfusion in a tertiaryreferral hospital. Clinical outcomes and haemostatic complications. Med J Aust 1995;163:356-359.

19. Levi M, ten Cate H . Disseminated Intravascular coagulation. N Eng J Med 1999;341: 586-592. 20. Gando S, Kameue T, Nanzaki S, Nakanishi Y. Disseminated intravascular coagulation is a frequent

complication of systemic inflammatory response syndrome. Thromb Haemost 1996;75:224-228.21. Neame PB, Kelton JG, Walker. Stewart IO, Nossel HL, Hirsch J. Thrombocytopenia in septicemia:

the role of intravascular coagulation. Blood 1980;56:88-92.22. Hunt BJ, Jurd KM. Endothelial cell activation. A central pathophysiological process. BMJ

1998;316:1328-1329.23. Senaldi G, Piguet PF. Mortality and platelet depletion occur independently of fibrinogen

consumption in murine models of tumor necrosis factor-mediated systemic inflammatoryresponses. Cytokine 1998;10:382-389.

24. Piguet PF, Da Laperrousaz C, Vesin C, Tacchini-Cottier F, Senaldi G, Grau GE. Delayed mortalityand attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinasereceptor-deficient mice. Infect Immun 2000;68:3822-3829.

25. Heffner JE, Sahn SA, Repine JE. The role of platelets in the adult respiratory distress syndrome.Culprits or bystanders? Am Rev Respir Dis 1987;135:482-492.

26. Massberg S, Enders G, Leiderer R, Eisenmenger S, Vestweber D, Krombach F, Messmer K.Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood1998;92:507-515.

27. Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Platelets induce sinusoidalendothelial cell apoptosis upon reperfusion of the cold ischemic rat liver. Gastroenterology2000;118:183-191.

28. Buckley MF, James JW, Brown DE, Whyte GS, Dean MG, Chesterman CN, Donald JA. A novelapproach to the assessment of variations in the human platelet count. Thromb Haemost2000;83:480-484.

29. Ross DW, Ayscue LH, Watson J, et al: Stability of hematologic parameters in healthy subjects.Intraindividual versus interindividual variation. Am J Clin Pathol 1988;90:262-267.

30. Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. Multiple organdysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med1995;23:1638-1652.

31. Vincent JL, de Mendonca A, Cantraine F, et al. Use of the SOFA score to assess the incidence oforgan dysfunction/failure in intensive care units: results of a multicenter prospective study. Crit CareMed 1998; 26:1793-1800.

32. Esmon CT, Fukudome K, Mather T, Bode W, Regan LM, Stearns-Kurosawa DJ, Kurosawa S.Inflammation, sepsis, and coagulation. Haematologica 1999;84:254-259.

33. Fourrier F, Jourdain M, Tournoys A. Clinical trial results with antithrombin III in sepsis Crit CareMed 2000;28(9 Suppl):S38-43

34. Cannon CP. Incorporating platelet glycoprotein IIb/IIIa inhibition in critical pathways: unstableangina/non-ST-segment elevation myocardial infarction. Clin Cardiol 1999;22(Suppl):IV30-36

35. Frenette PS, Moyna C, Hartwell DW, Lowe JB, Hynes RO, Wagner DD. Platelet-endothelialinteractions in inflamed mesenteric venules. Blood 1998;91:1318-1324.

Page 113: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Primary and secondary changes in platelet count and outcome in ICU patients

106

Page 114: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

CHAPTER 10

SUMMARY, DISCUSSION AND LINES OF FURTHERINVESTIGATION

Page 115: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

108

SUMMARYPhysiological parameters and circulating markers in humans who display systemicinflammation were the main focus of this thesis. The patients investigated in this thesis variedfrom trauma patients with or without fat embolism (FES), patients with burns, patientsreceiving cytokine treatment to critically ill patients.

Fat embolism syndromeIn Chapter 2 we hypothesized that the nature of a femoral fracture, timing of the operationand early inflammatory responses would affect the risk for subsequent development of FES.We studied the incidence of FES in patients with an isolated fracture of the femoral shaftadmitted to our hospital in the period 1968-1985. In a detailed analysis of the factorsassociated with the occurence of FES an elevated temperature was the only factor thatdiscriminated FES patients from other patients on day 0, suggesting that early acute phaseresponses are intimately connected with the onset of FES. Chapter 3 addressed the hypothesis that an open foramen ovale is necessary for large fatglobules gain entrance the systemic arterial circulation. We demonstrated that the presence ofa patent foramen ovale with a right-to-left shunt is not relevant in FES, thus indicating that thefat globules are able to reach the systemic circulation through the lungs.

Interleukin-6 and acute phase responsesIn chapter 4, it was hypothesized that IL-6 is an endogenous pyrogen as well as an inducer ofacute phase responses. Thanks to the availability at that time of the sensitive and specific B9.9assay for IL-6, we were the first to show that circulating IL-6 is increased after inflammationin humans. IL-6 was already sharply increased when burns patients presented at the hospital.IL-6 was correlated with fever, and the IL-6 peak preceded increases in acute phase proteins.In chapter 5, all major subsequent parts of the acute phase response were looked for andactually observed in patients with severe burns: fever, tachycardia, leukocytosis, left shift inthe leukocyte differentiation, increased CRP and increased α1-proteinase inhibitor. As lateparts of the inflammatory response thrombocytosis, a distinct and short-lasted IgM-peak andfinally increased IgG levels were observed. There was no relation between the extent of theburns and IL-6 levels, but patients with more severe burns had elevated IL-6 for longer periods.The early parts of the acute phase response were postively correlated with increased IL-6levels, compatible with a causal role of IL-6 in the induction of these responses.

Page 116: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

109

Procalcitonin Chapter 6 addressed that hypothesis that endotoxin is not a sine qua non for the induction ofPCT. We found that in vitro, PCT is produced by human liver slices in significant amountsand that PCT-production could be stimulated with IL-6 or TNFα. In vivo, it was also foundthat IL-6 and TNFα induce PCT. Thus was concluded that cytokines without the presence ofendotoxin can induce PCT. In the patients treated with TNFα, the very pronounced acutephase response that was induced also allowed the comparison of response times of PCT andCRP. PCT reached half-maximal levels within 8 hours, compared to 20 hours for CRP.

Primary changes in platelet count - thrombocytopeniaEarly decreases in platelet count occur in nearly all trauma patients and in other critically illpatients. It is also known that the magnitude of the early drop in platelet count is related withoutcome. Chapter 7 investigated early platelet sequestration after major trauma, and the effect of high-steroids on platelet counts. Changes of the platelet count and hemoglobin in the first two daysafter moderate injury were measured retrospectively. In two groups of matched patients, onegroup received a very high dose of methylprednisolone starting 4 hours after the injury. Over48 hours, the platelet count dropped by 39% whereas hemoglobin only dropped by 14%. Bytaking transfusions and changes in hemoglobin into account, it was concluded that the post-traumatic thrombocytopenia is mainly caused by sequestration of platelets, not blood loss.Methylprednisolone had no effect on platelet-sequestration. Methylprednisolone is a verypowerful and pleiotropic inhibitor of inflammation that can prevent platelet sequestration ifadministered before an inflammatory stimulus. However, inflammation-induced plateletsequestration apparently becomes refractory to steroid treatment within hours.

Secondary changes in platelet count - (blunted) thrombocytosis Based on extensive evidence that links systemic inflammation with the sequestration ofplatelets we hypothesized that secondary changes in platelet count would be also be relatedwith outcome.In chapter 8 this conjecture was verified in patients in our own ICU. Surgical ICU patientswere analyzed for temporal changes in the platelet count as well as the leukocyte count. Bothin the large (N=1415) overall group and in all subgroups (trauma, abdominal surgery,vascular surgery and liver transplantation) the same phenomena were observed: Plateletcounts decrease to nadir values at 2 to 3 days after ICU admission and failure of the plateletcount to recover to normal or supranormal levels is associated with mortality. Leukocytecounts were not associated with mortality. The parameter ∆PC/∆t between day 2 and day 10was a better predictor of mortality then the APACHE-II score at ICU-admission.In chapter 9 we tested the assumption that the observations for surgical ICU patients inchapter 8 are also true for ICU patients in general. In addition we verified if secondarychanges in PC (i.e. after ICU day 2) have a stronger correlation with outcome than initial

Page 117: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

110

changes in PC. Thus the relation between temporal changes in PC and mortality were verifiedin a larger mixed patient set from the second European ICU Study (EURICUS-II; N=5206).In this population as well, low ∆PC/∆t values were associated with mortality. Changes afterday 2 indeed had a stronger relation with outcome than initial changes in platelet counts.

DISCUSSION AND FUTURE DEVELOPMENTS

Although we formally only studied patients and not healthy persons, trauma patients can beeffectively considered as previously healthy persons who subsequently sustained an injury.This is the justification for the title of this thesis "Acute systemic inflammation in health anddisease".Except for chapters 8 and 9 where a substantial number of patients with underlying chronicdisease were also included, the other chapters concern patients who were basically previouslyhealthy.

The main purpose ometers and thus hinflammatory sequTable 10.1 summawere observed. Thean ‘ideal’ inflammdisease severity. Inof inflammation an

Inflammation in he

Table 10.1. (See cover)Sequence of inflammatory parameter changes studied in this thesis

Time afterinflammatory

stimulus

Parameter

Hour TNFα , IL-6

Hours

fever, tachycardiaplatelet count

leukocyte count PCT

Day CRP , SAA

Days α1-antiproteinasealbumin

Week platelet count /=IgM

Weeks IgG

f this thesis was to define the time course of systemic inflammatory para-elp address hypotheses concerning the role of these parameters in theence. The implications of the findings are discussed in more detail below.rizes the time-scale and the associated order in which parameter changes final paragraph of this chapter discusses what should be the properties ofatory marker and why this is not necessarily the same as a marker of this context, procalcitonin and the platelet count are considered as markersd severity respectively.

alth and disease - Physiological and pathophysiological response

Page 118: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

111

Classifying responses as either physiological or pathophysiological depends to a certaindegree on an arbitrary cut-off. Nobody would probably call wound healing an unhealthyresponse. On the other hand, septic shock due to an exaggerated release of cytokines afterendotoxin stimulation is definitely not a beneficial response. Thus calling a responsephysiological depends on the extent of the primary stimulus and the induced responses andwhether the host is overwhelmed or not. We consider the trauma patients previously healthyand assume that their primary responses are physiological. The severity of illness varied in thedifferent studies: the patients studied in chapters 2, 3, 6 and 7 were mainly admitted to theward, patients in chapters 4 and 5 to the burn unit, while patients studied in chapters 8 and 9were admitted to the ICU. The systemic inflammatory responses in the moderately injuredpatients who stayed at the ward could still reasonably be called physiological, since theseresponses still are beneficial and do not appear to worsen the patient's condition. In intensivecare patients on the other hand, frequently disproportional host responses are observed thatcan be interpreted as pathophysiological.

The fat embolism syndrome: a peculiar case of inflammationThe first two chapters concerned the fat embolism syndrome (FES). A femoral shaft fractureis the typical trauma associated with FES. FES is the secondary systemic result (generalizedpetechiae, cerebral and respiratory disturbances) of a primary local trauma. Since FESdevelops in far fewer patients than the number of patients in which release of bone marrow fatis observed, additional pathogenic mechanisms must be present. In chapter 2 we found thatlocal factors, in particular a closed femoral fracture coupled with late operative stabilizationof this fracture, was associated FES. Thus we interpreted that femoral fractures where thesurrounding soft tissues (skin and muscle) were not decompressed, led to FES. A conceivablemechanical explanation is that increased pressure around the fracture enhances the entrance offat into the systemic circulation. The other major observation in chapter 2 is the association ofFES with an early onset of fever. Nowadays with modern early fracture stabilization and supportive volume therapy, FES israrely seen as an isolated syndrome [1]. But regardless of the decreasing incidence, FESremains a specific entity with an unknown pathogenesis. Causal treatment of FES is still notpossible, while positive end-expiratory pressure ventilation is the mainstay of supportivetherapy in cases of pulmonary insufficiency. Many prophylactic schemes have been tested,but none have shown real benefit. As we and others have observed, early operativestabilization of fractures lowers the incidence of FES. The higher incidence of FES [2] whenmultiple femoral fractures are present [3] implies that in multiple injured patients FES is oftenpresent, but can sometimes not be recognized due to the concomitant injuries to the brain orlungs. Several years after the our publication that dismissed patent foramen ovale as relevant in FES(chapter 3), a report based on one patient with presumed FES and a patent foramen ovaleappeared in the New England Journal of Medicine [4] The authors claimed that patentforamen ovale is important in the pathogenesis of FES. As pointed out in chapter 3 we

Page 119: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

112

disagree since the single patient described in this report did not conform to importantdiagnostic criteria of classical FES. Later, Forteza [5] and colleagues showed the presence ofcerebral fat emboli with transcranial doppler in 5 patients with long bone fractures. 4 of these5 patients did not have a patent foramen ovale. It should be noted that, in contrast to FES, thepresence of persisting foramen ovale is an important risk factor in ‘macro-embolic’ entitiessuch as cerebral thrombo-embolism or air-embolism.[6,7].The association of fat embolism with early fever, and thus with early systemic inflammation,that we found has also been observed by others [8]. Elevated levels of CRP have beenimplicated as a cause, since CRP has fat agglutinating potential under certain circumstances[9].

Some remaining questions regarding FES:⋅ Fat and marrow embolization can be imaged by TEE nearly always during orthopedic

surgery [10]. Why does it not harm the vast majority of patients? ⋅ Neutral fat which is relatively non-toxic can be converted to much more reactive fatty

acids by lipase or lipoprotein lipase. These fatty acids can be toxic to the pulmonaryvasculature [11]. But it is still not clear if this biochemical process plays a significant rolein FES.

⋅ How do large (up to 100 µm) fat globules manage to pass the glomerulus [16], a filter thatotherwise prevents the leakage of much smaller macro-molecules or lipoproteins?

After decades of experiments, a suitable animal model still needs to be developed. The so-called models of FES typically involved rather extreme stimuli, rapidly resultant in theanimals death. Typical models executed were: severe experimental shock [13], acute rightheart failure [14] after massive fat infusion or acute oleic acid pulmonary injury models [15].Rapidly developing shock is part of these models, but shock is not part of the classic FES.Also, none of these models reproduces the intriguing interval free of symptoms before theFES becomes manifest. Full-blown FES with petechiae and respiratory distress and cerebral disturbances has becomeless frequent, but subclinical manifestations with permanent damage may frequently occur.When a successful animal model of fat embolism is developed the mechanisms by which fatemboli are pathogenic may be understood more clearly. Such models could lead to directedtherapy that may limit subclinical damage by fat emboli that may be more pervasive andirreversible [17] than the acute clinical condition suggests.

Page 120: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

113

IL-6Since our first studies on IL-6, an extraordinary amount of research has been performed onIL-6. Together with many other cytokines, such as TNFα and IL-1, IL-6 is produced shortlyafter the local tissue damage [18] or systemic endotoxin stimulation occurs. Amongst others,monocytes and endothelial cells can produce large amounts of IL-6 [19,20]. After thediscovery of many other cytokines that have many functions, IL-6 still stands out for its verybroad range of actions. Platelet production is stimulated by IL-6 [21,22] and thrombopoietin[23]. The sharp IgM-peak during the second week and later the more sustained elevation inIgG, reflect immunoglobulin stimulation by the cytokines, especially by IL-6 [24]. IL-6appears to be involved in the pathogenesis of multiple myeloma [25] and Castleman's disease[26]. In fact, sustained inflammation is in general associated with hypergammaglobulinemiaand under these conditions high IgG levels contribute to the elevated ESR [27] as is shown bydetermining the ‘defibrinated’ ESR. Although obsolete now, measuring the ESR after fibrinremoval was an elegant way of quantifying chronic inflammation [28].Unlike in patients with mechanical injury, in sepsis cytokines often do more harm than good.It has now been extensively shown that shock in sepsis is mediated by sometimes extremelyhigh levels of cytokines such as TNFα, IL-6 and IL-1 [29], due to massive activation byendotoxin. Thus after limited trauma the inflammatory often is in a compensated state, but insepsis it is overwhelmed, resulting in a decompensated state. At that point cytokines have losttheir beneficial function. It is only logical to assume that the inflammatory system was'designed' by evolution for local purposes, i.e. containment of infection and restoration aftertrauma. The exquisite sensitivity of some myeloid cell types to endotoxin is beneficial underlocal circumstances. But when local defenses are overwhelmed, the massive induction ofcytokines serves no purpose, and will be lethal unless rapid medical intervention isperformed. Depending on the sort of disease, on the stage of the disease and on theinvestigator, some cytokines have been classified as inflammatory or anti-inflammatory.Although IL-6 levels are especially increased in sepsis, and higher levels associated with pooroutcome, some have nevertheless called IL-6 anti-inflammatory [30]. The observation thatelevated cytokine levels are correlated with increased mortality has generated an array ofsepsis intervention trials. In addition to high-dose corticosteroids, agents aimed at blocking orinhibiting the effects of endotoxin, TNFα, IL-1-receptor, platelet-activating factor receptor,bradykinin, prostacyclin and thromboxane-A2 were tested in randomized controlled trials[31]. Although it may appear conceptually helpful to divide cytokines into pro-inflammatoryand anti-inflammatory, such a division is artificial, because many cytokines have multiple“functions” and because many functions are performed by multiple cytokines. Thus it appearsmore appropriate to view cytokines not as sharply defined hormones, but as connections in asignalling network in which considerable overlap exists. Overlap and parallel pathways maypartly explain the failure of nearly all sepsis intervention trials to affect mortality. These trialshave been aimed at blocking a single pathway [31]. At the effector level it possibly easier todivide proteins into pro-inflammatory and anti-inflammatory. For example, the enzymeelastase that is released by neutrophils could be considered pro-inflammatory. Many acute

Page 121: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

114

phase reactants appear to be anti-inflammatory, like for example α1-proteinase inhibitor, aserine protease inhibitor that inactivates elastase by binding it. The high molar circulatingconcentration of α1-proteinase inhibitor compared to elastase keeps the proteolytic activity ofelastase restricted to a limited space in the direct neighborhood of the activated neutrophils[32].

ProcalcitoninTwo recent studies [32,33] found that in septic patients (35 and 24 patients respectively) PCTwas superior to IL-6, CRP, soluble CD14 and TNFα in early detection of non-survivors (13and 8 patients respectively). PCT levels may be an attractive tool for clinical monitoring, but the origin of increased PCT-synthesis may be even more important pathophysiologically. Endocrine cells in the lung havebeen initially proposed as a source of increased PCT synthesis after pulmonary injury likeinhalation burns [35] or pneumonitis [36] although others found no relation of PCT elevationwith the presence or absence burn injury with or without inhalation [37]. Despite theintensive search for a source of PCT, at the time of this writing no significant in vitroproduction equaling or surpassing the release of PCT that we found in liver slices [38], hasbeen reported, regardless of cell type or tissue used. In support of our finding that the liverproduces PCT, hepatic vein levels of PCT were higher than central venous levels inextracorporeal bypass patients [39].The in vitro PCT-concentrations that were found in the supernatant after stimulation (≈1µg/L) were considerable, both when compared to in vivo PCT concentrations, and whencompared to in vitro CRP and SAA concentrations (<1 mg/L). The measured liver synthesisrate of PCT could account for observed levels to in vivo. We could not find reports of PCT-measurements of supernatants of hepatocytes cultured with various cytokines, and cytokine-

stimulated HepG2 cells produced no PCT (unpublished data). Such measurements may

Table 10.2.Comparison of PCT with cytokines and acute phase proteins

Cytokines Procalcitonin Acute Phase Proteins

Function Signalling ?Effector. (Innate

bacteriostatic,coagulation,protease inhibition)

Source Leukocytes, endothelialcells

Liver ?Cell type ? Hepatocytes

Molecular weight <50 000 13 000 > 50 000Mass concentration 0.1-1 ng/l 1-100 ug/l 10 mg/L – 10 g/lInduction time to halfmaximal levels minutes- hours 8 hrs >20 hrs

Half life minutes - hours day day - days

Page 122: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

115

clarify the role of the liver and which cell type is involved in PCT synthesis, which isparticularly important as this organ is the sole producer of most acute phase proteins.

PCT - cytokine or acute phase proteinWhen Kushner’s broad definition of an acute phase protein is applied [40], PCT fits thedefinition since it is increased by more than 25% in inflammation. But the more importantquestion is whether PCT behaves as other ‘established’ acute phase proteins like CRP,fibrinogen or α1-proteinase inhibitor. Whereas CRP has been established as important inaspecific antibacterial defenses, the potential function of PCT is still unknown. Although, asthe name implies, PCT is a precursor of calcitonin, circulating PCT levels appear to be largelyunrelated to calcitonin levels. This is impressively illustrated by the fact that PCT levels canincrease orders of a magnitude, with unchanged calcitonin levels. The relatively largequantities of PCT that were produced in vitro by human liver slices, when extrapolated to thein vivo situation, could account for most PCT-levels observed in septic patients. Since thetime of publication, no other convincing source of PCT has been reported. But the questionremains which cell type within the liver (e.g. hepatocyte, endothelial cell or Kupffer cell) isthe source of PCT. The characteristics of PCT as indicated in the table are intermediatebetween cytokines and acute phase proteins.

In experiments in Syrian hamsters, Nylen [41] found that PCT exacerbates mortality inexperimental sepsis. The methodology of this study has been criticized [42] on several points.The investigators used non-recombinant human PCT in hamsters, and it was inhibited by goatanti-calcitonin antibodies. Thus the effects observed may not necessarily be due to PCT.Analogous to the debate if some cytokines are pro-inflammatory or anti-inflammatory, thefact that very high levels of PCT are associated with poor outcome does of course notautomatically imply that PCT is 'bad' or pro-inflammatory. Related to calcitonin's actions, ithas been suggested that PCT could play a role in osteoclast activity [43]. Maybe PCT isneither a cytokine, nor an acute phase protein. PCT could also simply have no function, or itmay be a useless by-product.

Clinical value of determining cytokines, acute phase proteins and PCTIn clinical practice CRP has generally substituted the ESR as a marker of inflammation,although for some acute diagnostic problems and especially chronic diseases clinicians stilluse the ESR [44]. The direct measurement of cytokines, such as IL-6 has limited practicalapplicability. Due to the short half-life of cytokines (minutes to hours) relatively infrequentpoint measurements are only of limited value in assessing the area under the curve.Nevertheless it has been shown that high IL-6 levels are related to mortality in sepsis patients,and IL-6-levels >1000 pg/ml have been used as a criterion to enroll patients in the anti-TNFintervention trials.

Page 123: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

116

The published evidence indicates that the overall response of parameters of the acute phaseresponse appears to be one-dimensional in the sense that the extent of the overallinflammatory stimulus determines the height of both cytokine levels and APP’s. A localinfection will hardly result in practically measurable cytokine levels and may give a discreteelevation of CRP. A major burn will result in elevated cytokine levels and a large increase inCRP and other acute phase proteins. Sepsis can result in extreme increases of cytokines andmaximal levels of acute phase proteins. According to this view daily CRP-determinations willinform the clinician on the extent of ongoing inflammation. As reported in chapter 6, PCTachieves half-maximal levels 12 hours earlier than CRP. This can be a very important clinicaladvantage of PCT since in acute situations the clinician is informed 12 hours earlier on theextent of the systemic inflammation present.Whatever may be the ultimate role of PCT in addition or even instead of CRP, it appears oflittle interest to pursue a role for SAA as an inflammatory marker [45]. SAA dynamics maybe somewhat more pronounced than CRP-dynamics, but both SAA and CRP display a time tohalf-maximal levels of 20 hours, as opposed to only 8 hours for PCT [38]. Thus measuringSAA levels appears to present little additional information when PCT and CRP levels areknown.

Open questions on the role of PCT ⋅ PCT shows an extraordinary rise after severe inflammation. This rise may be more

pronounced than that of any known acute phase protein. Baseline levels of PCT appear tobe below 0.01 µg/L [46,47]. To fully define its dynamic range, reliable baseline levels ofPCT in healthy persons should be determined.

⋅ Does most PCT originate in the liver? Is the endothelial cell the source of PCT, becauseinducing PCT in hepatocytes and myeloid cells has not succeeded?

⋅ Can endotoxin reproducibly induce PCT in liver slices? If so, this might represent anunique in vitro inflammatory model. If cytokine-induction is a prerequisite for PCT-synthesis, this would be a two stage system (stage 1: endotoxin induces cytokines; stage 2:cytokines induce PCT).

Platelet countsCauses of secondary (relative) thrombocytopeniaWe found that patients who do not survive after admission to a surgical ICU have a blunted orabsent secondary increase in platelet counts [48]. Data on the development of platelet countsin medical ICU patients in our hospital indicated that these patients also show a relationbetween platelet count and outcome [49]. Bleeding had only a minimal overall contribution tothis phenomenon in both groups. Bone marrow synthesis in these patients appears to be morethan adequate [50]. Probably very few of the surgical ICU patients (chapter 8) and few of the

Page 124: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

117

EURICUS-patients (chapter 9) had a compromised bone marrow before ICU-admission.Besides, leukocyte counts in the non-survivors were at least as high as in survivors [48], alsonot indicative of marrow failure. Thus drops in platelet count, or inappropriately low plateletcounts, very probably result from sequestration. We interpret this sequestration of platelets asa reflection of ongoing disease activity, i.e. SIRS or infection. The prognostic importance ofplatelet counts led to its inclusion (with the exclusion of the leukocyte count in some cases) inICU severity scores such as the SOFA-score [51].Two main causes of platelet sequestration can be distinguished: aggregation and adhesion.Aggregation occurs in the process of (disseminated) coagulation. The prime target of plateletadhesion is activated endothelium or subendothelium, a process that can occur independent ofcoagulation. Although we believe strong arguments exist to assume that platelets adhere toactivated endothelium that is associated with systemic inflammation, as also expanded onelsewhere in this thesis, some alternative hypotheses exist.

Alternative explanations for the disappearance of platelets Gando [52,53] contends in an number of studies that DIC with platelet consumption is nearlyalways present in several critically ill patient groups. But others found only in 40% of thethrombocytopenic surgical ICU patients proof of coexisting DIC. In a liver transplant modelextensive adherence of solitary platelets to apoptotic sinusoidal endothelial cells in theabsence of (occlusive) clots was detected and even visualized [54]. Tissue factor (TF), thegenerally accepted starting point of the (extrinsic) coagulatory cascade has been detected inelevated levels in trauma and sepsis [55] as further evidence of disseminated activation ofcoagulation. But TF-levels as well as changes in other coagulation parameters are relativelymodest in many patients compared to the extensive disappearance of platelets from thecirculation. For example platelet consumption often occurs in the absence of fibrinogendepletion [56]. Others have observed so-called hemophagocytosis in bone marrow in verysmall, strongly selected patients sets [57], while another study has implicated thatautoimmune phenomena such as platelet associated immunoglobulins have been detected incritically ill patients [58]. The importance of the qualitative observation of hemophagocytosisis not established, and quantifying platelet immunoglobulins is methodologically difficult.Patients who underwent a liver transplantation present a special case, since in these patientsthrombocytopenia is not only strongly coupled to graft function [59] but also to an increasedsplenic volume [60].

Early changes in PC and methylprednisoloneSteroids, including methylprednisolone, have been shown in many experimental models tostrongly inhibit endotoxin- or trauma-induced inflammation and limit or prevent thethrombocytopenia that is seen in these models. In healthy persons high-dose steroids do notaffect the platelet count [61]. But evidently it fails to affect the early deposition of platelets intrauma patients when given after within hours the trauma. Steroids have failed to affectoutcome in sepsis intervention trials. Unfortunately other sepsis-intervention studies have also

Page 125: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

118

not resulted in decisive benefits for other immunomodulatory agents. The fact that thesestudies also showed no impact on platelet counts or subscores based on the platelet countunderscores the apparently important relation of platelet counts and survival.

∆PC/∆t as a markerRecently a study was published that related the incidence of thrombocytopenia in two medicalICUs with ICU mortality [62]. After admission, nadir platelet counts below 150 109/L or adecrease of more than 50% was associated with higher death rates with an odds ratio of 6.0(CI 3.0-12). The occurrence of thrombocytopenia had more predictive power than eitherAPACHE-II, SAPS II and MODS scores. The study did not address the relevance of a bluntedrise in platelet counts. In chapter 9 we comprehensively studied time-dependent changes inPC. In was found that ∆PC/∆t after day 2 had a stronger relation with outcome than earlychanges in PC or nadir PC, thus pointing to the importance of day 2 as a ‘turning point’ withregard to platelet counts

Open questions on time-dependent changes in platelet counts.⋅ Based on periodic recordings of the ESR (or CRP) and platelet count [63], one could try

to deduce the ‘true individual normal’ platelet count in patients with chronic inflammatorydiseases. The platelet count that is measured at times when ESR and CRP are low couldbe assumed to be normal for that particular individual. The individual ∆ platelet countcould subsequently be analyzed in relation with elevated ESR and CRP levels.

⋅ Where do the platelets go to, when platelet consumption is observed during SIRS orsepsis? In one study that imaged labeled platelets in critically ill patients [68], theintestinal organs showed the highest platelet activity. It would be interesting to know ifthe homing behavior of the platelets is associated with clinical organ damage, for exampleacute renal failure or ARDS.

⋅ What are the clinically relevant receptors and ligands in platelet - endothelial cellinteraction in critically ill patients (in the absence of DIC)? In cardiovascular medicine ithas recently been proven that systemic inflammation is an important independent riskfactor for ischemic events [69,70], underscoring the clinical relevance of the relationbetween inflammation and coagulation. The great success of glycoprotein IIb/IIIa receptor(GP-IIb/IIIa) inhibitors in the CCU is [71] might in turn also be related with the anti-inflammatory effects of inhibiting GPIIb/IIIa. The P-selectin receptor, that is expressedby activated endothelial cells, has an important role both in leukocyte adhesion and infibrinogen-independent platelet-adhesion [69]. Sindram has suggested that P-selectin isimportant in mediating reperfusion injury in transplanted livers [54].

⋅ Is the platelet endothelial interaction (adhesion) always irreversible or is it under someconditions still reversible? Maybe marginated platelets, analogous to leukocytes, canreenter the circulation. Both in in vitro and in vivo [73] experiments this phenomenon hasbeen observed. The spleen is an example of a microenvironment where platelets arereversibly segregated from the circulation. If platelet re-entry is relevant, part of the

Page 126: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

119

effects of potential agents that selectively block platelet- endothelial interaction might bereflected by increases in platelet counts.

The ideal inflammatory markerAfter discussion of the merits of the several parameters that were studied as markers ofinflammation and disease severity, it is useful to define the "ideal” inflammatory marker as athought experiment. What should be its properties? It must have very low values in healthy persons. It increases rapidly once an inflammatorystate develops. Its increase is proportional to the extent of the underlying inflammatoryprocess - for example proportional to the total amount of damaged tissue or the total quantity

of cytokines produced. After the marker has rapidly increased to this proportional level, itpredictably decreases. The half-time should be around 1 day - a convenient unit of time inclinical practice. The marker can be determined for little cost in serum or plasma and is notsensitive to in vitro changes during procurement or storage.

1 10 100Hours

Arb

itrar

y un

its

Figure 10.1. Comparison of levels of ideal inflammatory marker (solid line) withlevels of cytokine (dashed line) after an acute inflammatory event. An idealmarker rises rapidly after inflammation in a manner proportional to theinflammatory stimulus. When the inflammation disappears, the marker decreaseswith a half-life of one day. The cytokine rises rapidly but also decreases rapidly.

Page 127: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

120

Indicators of inflammation and severity - Procalcitonin and the platelet count.On the basis of the formulated profile of the ideal inflammatory marker, procalcitoninprobably comes closest to these requirements. Disadvantages of PCT as an inflammatorymarker may be blunted responses in neutropenia [74] and interindividual differences that arelarger than for CRP [75]. The combination of CRP and PCT may turn out to be a particularlyeffective indicator of the inflammatory state and the possibility of bacterial infection.It is important to note that a marker of inflammation is not the same as a marker of severity. Agood marker of severity should be associated with outcome (i.e. mortality, hospital stay),whereas a marker of inflammation reflects the amplitude of the inflammatory response assuch, regardless of the impact this inflammatory response has on ultimate outcome. Well-known examples of markers of severity are the severity scores: Acute physiological andchronic health evaluation (APACHE-II) [76], injury severity score (ISS) [77] or multipleorgan dysfunction score (MODS) [78]. Changes in platelet counts (nadir platelet count and ∆PC/∆t) are also strongly related tooutcome. The fact that ∆PC/∆t after ICU-day 2 is as good a predictor of outcome as theAPACHE-II score in surgical ICU patients [48] emphasizes that platelets are deeply related tooutcome. Correlating platelet count with PCT and mortality may show to what extent theplatelet count reflects inflammation and organ dysfunction respectively.

Interactions of acute systemic inflammation with other physiological systems.Many interactions between the inflammatory system and other (patho)physiological systemshave been uncovered. Except where platelets and coagulation are concerned, these systemswere not subject of this thesis. To illustrate that inflammation has connections with many

1 10 100Hours

Arb

itrar

y un

its

Figure 10.2. Comparison of CRP (dashed line) with ideal inflammatory marker(solid line).

Page 128: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

121

other important physiological processes some examples:− Inflammation ↔ specific immune response:

Example: induction of antigen specific immunoglobulin and T-cell responses− Inflammation ↔ stress system

Examples: Astronauts produce increased levels of IL-6 during launch and landing [79];exercise induces cytokines [80] and acute phase proteins [81]

− Inflammation ↔ central nervous systemExample: inflammation and IL-6 induce sleep [82]; sleep induces IL-6 [83]

− Inflammation ↔ metabolismExample: down-regulation of albumin and induction of cachexia by inflammation[84,85,86]

Systemic inflammation in health and diseaseTo summarize, in this thesis several aspects of acute systemic inflammation were studied.FES is a special form of systemic inflammation in which fat globuli are involved. Why thisresponse occurs only in a small subset of patients with long bone fractures and why it doesnot occur in the vast majority of apparently similar patients has only partly been clarified. SIRS with it’s associated increases in cytokines, PCT and acute phase proteins, is a universalresponse in patients who are critically ill, whether due to trauma, surgery or infection.

1 10 100Hours

Arb

itrar

y un

its

Figure 10.3. Comparison of levels of PCT (dashed line) with an idealinflammatory marker (solid line). PCT rises considerably faster than CRP, but stillhas a relatively long half-life.

Page 129: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

122

Sequestration of platelets accompanies SIRS initially in virtually all patients, and secondarilyin many patients with complicated courses. For a number of reasons, PCT and the plateletcount stand out for their diagnostic and prognostic value compared to many other parameters. Although we do only partly understand why this is the case, further investigation into theseparameters must lead to phenomena central to the outcome of critically ill patients.

REFERENCES

1. Bulger EM, Smith DG, Maier RV, Jurkovich GJ. Fat embolism syndrome. A 10-year review.Arch Surg 1997;132:435-439.

2. Pinney SJ, Keating JF, Meek RN. Fat embolism syndrome in isolated femoral fractures: doestiming of nailing influence incidence? Injury 1998;29(2):131-133.

3. ten Duis. Fat embolism syndrome. Injury 1997;28:77-85.4. Pell AC, Hughes D, Keating J, Christie J, Busuttil A, Sutherland GR. Brief report:

fulminating fat embolism syndrome caused by paradoxical embolism through a patentforamen ovale. N Engl J Med 1993;329:926-999.

5. Forteza AM, Koch S, Romano JG, Zych G, Bustillo IC, Duncan RC, Babikian VL.Transcranial doppler detection of fat emboli. Stroke 1999;30:2687-2691

6. Lechat P, Mas JL, Lascault G, Loron P, Theard M, Klimczac M, Drobinski G, Thomas D,Grosgogeat Y. Prevalence of patent foramen ovale in patients with stroke. N Engl J Med1988;318:1148-1152.

7. Moon RE, Camporesi EM, Kisslo JA. Patent foramen ovale in decompression sickness indivers. Lancet 1989;i:513-514.

8. Peltier LF. Fat embolism, an appraisal of the problem. Clin Orthop 1984;187:3.9. Hulman G. Fat macroglobule formation from chylomicrons and non-traumatic fat embolism.

Clin Chim Acta 1988;177:173-138.10. Christie J, Robinson CM, Pell AC, McBirnie J, Burnett R. Transcardiac echocardiography

during invasive intramedullary procedures. J Bone Joint Surg Br 1995;77:450-455.11. Fonte DA, Hausberger FX. Pulmonary free fatty acids in experimental fat embolism. J

Trauma 1971;11:668-672.12. Tyrrell DJ, Horne AP, Holme KR, Preuss JM, Page CP. Heparin in inflammation: potential

therapeutic applications beyond anticoagulation. Adv Pharmacol 1999;46:151-208.13. Derks CM, Peters RM. The role of shock and fat embolus in leakage from pulmonary

capillaries. Surg Gynecol Obstet 1973;37:945-948.14. Tornabene VW, Fortune JB, Wgner PD, Halasz NA. Gas exchange after pulmonary fat

embolism in dogs. J Thorac Cardiovasc Surg 1979;78:589.15. Jones JG, Minty BD, Beeley JM, Royston D, Crow J, Grossman RF Pulmonary epithelial

permeability is immediately increased after embolisation with oleic acid but not with neutralfat. Thorax 1982;37:169-174.

Page 130: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

123

16. Fabian TC, Hoots AV, Stanford DS, Patterson CR, Mangiante EC. Fat embolism syndrome:prospective evaluation in 92 fracture patients. Crit Care Med 1990;18:42-64.

17. Erdem E, Namer IJ, Saribas O, Aras T, Tan E, Bekdik C, Ziledi T. Cerebral fat embolismstudied with MRI and SPECT. Neuroradiology 1993;35:199-201.

18. Ueo H, Inoue H, Honda M, Uchida I, Nischimura M, Aranaga S, Nakashima H, Akiyoshi T.Production of interleukin-6 at operative wound sites in surgical patients. J Am Coll Surg1994;326-32.

19. Aarden L, Helle M, Boeije L, Pascual-Salcedo D, de Groot E. Differential induction ofinterleukin-6 production in monocytes, endothelial cells and smooth muscle cells. EurCytokine Netw 1991;2:115-20.

20. Krishnaswamy G, Kelley J, Yerra L, Smith JK, Chi DS. Human endothelium as a sourcemultifunctional cytokines: molecular regulation and possible role in human disease. JInterferon Cytokine Res 1999;19:91-104.

21. Hollen CW, Henthorn J, Koziol JA, Burstein SA. Elevated serum IL-6 levels in patients withreactive thrombocytosis. B J Haematol 1991;79:286-290.

22. van Gameren MM, Willemse PHB, Mulder NH, et al.: Effects of recombinant humaninterleukin-6 in cancer patients: a phase I-II study. Blood 1994; 84:1434-1441.

23. Kaushansky K. Thrombopoietin. N Engl J Med 1998;339:746-754.24. Fattori E, Della Rocca C, Costa P, Giorgio M, Dente B, Pozzi L, Ciliberto G. Development

of progressive kidney damage and myeloma kidney in interleukin-6 transgenic mice. Blood1994;83:2570-2579

25. Sati HI, Apperley JF, Greaves M, Lawry J, Gooding R, Russell RG, Croucher PI.Interleukin-6 is expressed by plasma cells from patients with multiple myeloma andmonoclonal gammopathy of undetermined significance. Br J Haematol 1998;101:287-295

26. Nishimoto N, Sasai M, Shima Y, Nakagawa M, Matsumoto T, Shirai T, Kishimoto T,Yoshizaki K. Improvement in Castleman's disease by humanized anti-interleukin-6 receptorantibody therapy. Blood 2000;95:56-61.

27. Talstad I, Haugen HF. The relationship between the erythrocyte sedimentation rate (ESR)and plasma proteins in clinical materials and models. Scand J Clin Lab Invest 1979;39:519-24.

28. Gudmundsson M, Bjelle A. Viscosity of plasma and blood in rheumatoid arthritis.Br J Rheumatol. 1993;32:774-779.

29. Hack CE, Aarden LA, Thijs LG. Role of cytokines in sepsis. Adv Immunol 1997;66:101-195.30. Xing Z,Gauldie J, Cox G, Baumann H, Jordana M, Lei XFR, Achong MK. IL-6 is an anti-

inflammatory cytokine required for controlling local or systemic acute inflammatoryresponses. J Clin Invest 1998;101:311-320.

31. Abraham E. Why immunomodulatory therapies have not worked in sepsis. Intensive CareMed 1999;25:556-566.

32. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-7633. Herrmann W, Ecker D, Quast S, Klieden M, Rose S, Marzi I. Comparison of procalcitonin,

sCD14 and interleukin-6 values in septic patients. Clin Chem Lab Med 2000;38:41-6

Page 131: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

124

34. Schroder J, Staubach KH, Zabel P, Stuber F, Kremer B. Procalcitonin as a marker of severityof septic shock. Langenbecks Arch Surg 1999;384:33-38.

35. Nylen ES, Jeng J, Jordan MH, Snider RH, Thompson KA, Lewis MS, O'Neill WJ, BeckerKL. Late pulmonary sequela following burns: persistence of hyperprocalcitonemia using a 1-57 amino acid N-terminal flanking peptide assay. Respir Med 1995;89:41-46.

36. Nylen ES, Snider RH Jr, Thompson KA, Rohatgi P, Becker KL. Pneumonitis-associatedhyperprocalcitoninemia. Am J Med Sci 1996;312:12-18.

37. Carsin H, Assicot M, Feger F, Roy O, Pennacino I, Le Bever H, Ainaud P, Bohuon C.Evolution and significance of circulating procalcitonin levels compared with IL-6, TNFalpha and endotoxin levels early after thermal injury. Burns 1997;23:218-224.

38. Nijsten MW, Olinga P, The TH, de Vries EG, Koops HS, Groothuis GM, Limburg PC, tenDuis HJ, Moshage H, Hoekstra HJ, Bijzet J, Zwaveling JH. Procalcitonin behaves as a fastresponding acute phase protein in vivo and in vitro. Crit Care Med 2000;28:458-461.

39. Silomon M, Bach F, Ecker D, Graeter T, Grundmann U, Larsen R. Procalcitonin nachextrakorporaler Zirkulation. Synthese im Hepatosplanchnikusgebiet? Anaesthesist1999;48:395-398.

40. Kushner I. The phenomenon of the acute phase response. Ann N Y Acad Sci 1982;389:39-48.41. Nylen ES, Whang KT, Snider RH, et al.Mortality is increased by procalcitonin and

decreased by an antiserum reactive to procalcitonin in experimental sepsis. Crit Care Med1998; 26:1001-1006.

42. Braithwaite SS. Procalcitonin – marker or mediator? Crit Care Med 1998; 26:977-978.43. Braithwaite SS. Procalcitonin: new insights on regulation and origin. Crit Care Med

2000;28:586-587.44. Brigden M. The erythrocyte sedimentation rate. Still a helpful test when used judiciously.

Postgrad Med 1998;103:257-274.45. Mozes G,Friedman N,Shainkin-Kestenbaum R. Serum amyloid A: an extremely sensitive

marker for intensity of tissue damage in trauma patients and indicator of acute response invarious diseases. J Trauma 1989;29:71-74.

46. Snider RH Jr, Nylen ES, Becker KL: Procalcitonin and its component peptides in systemicinflammation: immunochemical characterisation. J Investig Med 1997; 45:552-560.

47. Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, Bohuon C. Procalcitoninincrease after endotoxin injection in normal subjects. J Clin Endocrinol Metab1994;79:1605-1608.

48. Nijsten MWN, ten Duis HJ, Zijlstra JG, Porte RJ, et al. Blunted rise in platelet count incritically ill patients is associated with worse outcome. Crit Care Med 2000;28:3843-3846.

49. Nijsten MWN, Tulleken JE, vd Werf TS. Absence of secondary thrombocytosis is associatedwith prolonged ICU-stay and decreased survival. Intensive Care Med 1997;23 S1:59.

50. Mitterstieler G, Kurz R, Haas H, Desoye G. Thrombopoiesis and blood coagulation inpediatric patients with septicemia. Padiatr Padol 1981;16:257-65.

51. Vincent et al. Use of the SOFA score to assess the incidence of organ dysfunction/failure inintensive care units: results of a multi-center, prospective study. Working group on "sepsis-

Page 132: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

125

related problems" of the European Society of Intensive Care Medicine. Crit Care Med1998;26:1793-1800.

52. Gando S, Nanzaki S, Kemmotsu O. Disseminated intravascular coagulation and sustainedsystemic inflammatory response syndrome predict organ dysfunctions after trauma:application of clinical decision analysis. Ann Surg 1999;229:121-127.

53. Gando S, Kameue T, Nanzaki S, Nakanishi Y. Disseminated intravascular coagulation is afrequent complication of systemic inflammatory response syndrome. Thromb Haemost 1996;75:224-228.

54. Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Platelets induce sinusoidalendothelial cell apoptosis upon reperfusion of the cold ischemic rat liver. Gastroenterology2000;118:183-191.

55. Gando S, Nanzaki S, Morimoto Y, Kobayashi S, Kemmotsu O. Systemic activation oftissue-factor dependent coagulation pathway in evolving acute respiratory distress syndromein patients with trauma and sepsis. J Trauma 1999;47:719-23.

56. Neame PB, Kelton JG, Walker. Stewart IO, Nossel HL, Hirsch J (1980) Thrombocytopeniain septicemia: the role of intravascular coagulation. Blood 56:88-92.

57. Stephan F, Hollande J, Richard O, Cheffi A, Maier-Redelsperger M, Flahault AThrombocytopenia in a surgical ICU. Chest 1999;115:1363-70.

58. Stephan F, Cheffi MA, Kaplan C, Maillet J, Novara A, Fagon J, Bonnet F. Autoantibodiesagainst platelet glycoproteins in critically ill patients with thrombocytopenia. Am J Med2000;108:554-60.

59. Chatzipetrou MA, Tsaroucha AK, Weppler D, Pappas PA, Kenyon NS et al.Thrombocytopenia after liver transplantation. Transplantation 1999;67:702-706.

60. Hoefs JC, Wang FW, Lilien DL, Walker B, Kanel G] A novel, simple method of functionalspleen volume calculation by liver-spleen scan. J Nucl Med 1999;40:1745-1755.

61. Demir G, Derman U, Berkarda B. Haematological effects of pulse steroid therapy. Int JPharmacol Res 1994;14:101-106.

62. Vanderschueren S, de Weerdt A, Malbrain M, Vankersschaever D, Frans E, Wilmer A,Bobbaers H. Thrombocytopenia and the prognosis in intensive care. Crit Care Med2000;28:1871-1876.

63. Buckley MF, James JW, Brown DE, Whyte GS, Dean MG, Chesterman CN, Donald JA. Anovel approach to the assessment of variations in the human platelet count. Thromb Haemost2000;83:480-484.

64. Porte RJ, Blauw E, Knot EAR, et al: Role of the donor liver in the origin of plateletdisorders and hyperfibrinolysis in liver transplantation. J Hepatol 1994;21:592-600.

65. McCaughan GW, Herkes R, Powers B, et al: Thrombocytopenia post liver transplantation.Correlations with pre-operative platelet count, blood transfusion requirements, allograftfunction and outcome. J Hepatol 1992;16:16-22.

66. Kas-Deelen AM, de Maar EF, Harmsen MC, Driessen C, van Son WJ, The TH. Uninfectedand cytomegalic endothelial cells in blood during cytomegalovirus infection: effect of acuterejection. J Infect Dis 2000;181:721-724.

Page 133: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

126

67. Almeida-Porada GD, Ascensao JL. Cytomegalovirus as a cause of pancytopenia. LeukLymphoma 1996;21:217-223.

68. Sigurdsson GH, Christenson JT, el-Rakshy MB, et al: Intestinal platelet trapping aftertraumatic and septic shock. An early sign of sepsis and multiorgan failure in critically illpatients? Crit Care Med 1992;20:458-467.

69. Verheggen PW, de Maat MP, Cats VM, Haverkate F, Zwinderman AH, Kluft C, BruschkeAV. Inflammatory status as a main determinant of outcome in patients with unstable angina,independent of coagulation activation and endothelial cell activation. Eur Heart J1999;20:567-574.

70. Vallance P,Collier J, Bhagat K. Infection, inflammation, and infarction: does acuteendothelial dysfunction provide a link? Lancet 1997;349:1391-1392.

71. Cannon CP. Incorporating platelet glycoprotein IIb/IIIa inhibition in critical pathways:unstable angina/non-ST-segment elevation myocardial infarction. Clin Cardiol1999;22(Suppl):IV30-36.

72. Frenette PS, Denis CV, Weiss L, Jurk K, et al. P-Selectin glycoprotein ligand 1 (PSGL-1) isexpressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med2000;191:1413-1422.

73. Woltjes J, de Jong JC, ten Duis HJ, Wildevuur CR.The priming of extracorporeal circuits:the effect on canine blood elements. Transfusion 1979;19:552-557.

74. de Bont E, Vellenga E, Swaanenburg J, Kamps W. Procalcitonin: a diagnostic marker ofbacterial infection in neutropenic cancer patients with fever? Infection 2000; 28:398-400.

75. Meisner M. Procalcitonin. A new innovative infection parameter. Biochemical and clinicalaspects. Berlin: Brahms Diagnostica 1996. ISBN 3-00-000803-9

76. Knaus WA, Draper EA, Wanger DP, Zimmerman JE. APACHE-II: a severity of diseaseclassification system. Crit Care Med 1985;13:818-829.

77. Baker SP, O'Neill B. The injury severity score: un update. J Trauma 1976;16:882-885.78. Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. Multiple organ

dysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med1995;23:1638-1652.

79. Stein TP, Schluter MD. Excretion of IL-6 by astronauts during spaceflight. Am J Physiol1994;266:448-452.

80. Pedersen BK, Ostrowski K, Rohde T, Bruunsgaard H. The cytokine response to strenuousexercise. Can J Physiol Pharmacol 1998;76:505-511.

81. Shek PN, Shepard RJ. Physical exercise as a model of limited inflammatory response. Can JPhysiol Pharmacol 1998;76:589-597.

82. Spath-Schwalbe E, Hansen K, Schmidt F, Schrezenmeier H, Marshall L, Burger K, FehmHL, Born J. Acute effects of recombinant human interleukin-6 on endocrine and centralnervous sleep functions in healthy men. J Clin Endocrinol Metab 1998;83:1573-1579.

83. Vgontzas AN, Papanicolaou DA, Bixler EO, Lotsikas A, Zachman K, et al. Circadianinterleukin-6 secretion and quantity and depth of sleep. J Clin Endocrinol Metab1999;84:2603-2607.

Page 134: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Chapter 10

127

84. LeGrand EK. Why infection-induced anorexia? The case for enhanced apoptosis of infectedcells. Med Hypotheses 2000;54:597-602.

85. Haupt W, Holzheimer RG, Riese J, Klein P, Hohenberger W. Association of lowpreoperative serum albumin concentrations and the acute phase response. Eur J Surg1999;165:307-13.

86. Michie HR. Metabolism of sepsis and multiple organ failure. World J Surg 1996;20:460-464.

Page 135: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Summary, discussion and lines of further investigation

128

Page 136: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

NEDERLANDSE SAMENVATTING

Page 137: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Acute systemische ontsteking bij gezonden en zieken

130

Acute systemische ontsteking

In dit proefschrift zijn een aantal systemische uitingen van acute ontsteking onderzocht. Dezekunnen grotendeels als generalisaties van de locale ontsteking worden beschouwd. Klassiekworden 5 kenmerken van de locale ontsteking beschreven, te weten calor, tumor, rubor, functiolaesa en dolor. De systemische equivalenten van de eerste vier uitingen zijn respectievelijk koorts,oedeem, erytheem en orgaanfalen. Het begrip acute fase reactie wordt gebruikt om delen van dezegecoördineerde respons te beschrijven. Behalve koorts kenmerkt de acute fase reactie zich doorverhoogde spiegels van een scala aan effector eiwitten, de zogenaamde acute fase eiwitten.Metingen van deze eiwitten, met als prototype het C-reactive protein (CRP), maken het mogelijkde omvang en duur van de acute fase reactie te kwantificeren. De afgelopen jaren is duidelijkgeworden dat cytokinen een centrale rol spelen bij de inductie van de acute fase reactie.

Behalve sterk gestegen cytokine-spiegels en verhoogde spiegels van acute fase eiwitten bestaan ermeer markers in het bloed die wijzen op de aanwezigheid van een systemische ontstekingsreactie.Voorbeelden hiervan zijn het leukocytengetal (aantal witte bloedcellen) en het trombocytengetal(aantal bloedplaatjes).

Om een beter beeld te krijgen van de wijze waarop de diverse responsen met elkaar samenhangen,is het van belang de volgorde waarin de verschillende markers veranderen, te bepalen. Om dezevolgorde zo duidelijk mogelijk waar te nemen is het zinvol ongevalspatiënten te onderzoeken. Bijdeze patiënten, die tevoren in de regel gezond waren, is het moment waarop de ontstekingsprikkelontstaat (het ongeval) duidelijk. Dit in tegenstelling bijvoorbeeld tot een chronisch zieke patiëntdie geleidelijk aan een sepsis ontwikkelt.

Het stereotiepe gedrag van de acute systemische ontsteking bij ongevalspatiënten, heeft alsmethodologisch voordeel dat bij relatief kleine patiëntengroepen reeds significante patronenzichtbaar worden.

Het vetembolie syndroom

De hoofdstukken 2 en 3 betreffen een bijzonder soort ontstekingsreactie, het vetembolie-syndroom (FES). FES ontstaat soms bij patiënten die één of meer lange pijpbeenderen hebbengebroken. Na een klachtenvrij interval ontwikkelt de patiënt oxygenatie-stoornissen, cerebralestoornissen en petechiën. Van deze drie manifestaties is aangetoond dat zij door embolisatie vanbeenmergvet veroorzaakt worden. Aangezien bij de meeste patiënten met fracturen, circulerendbeenmergvet kan worden aangetoond, is het onduidelijk welke factoren ervoor verantwoordelijkzijn dat slechts een minderheid van de patiënten FES ontwikkelt.

In hoofdstuk 2 is gekeken naar de relatie tussen kenmerken van de fractuur en het optreden vanFES. Tevens is gekeken naar de vroege verschijnselen van systemische ontsteking. Het bleek dateen gesloten, en niet direct geopereerde fractuur (de zogenaamde non-decompressie groep)geassocieerd was met verhoogde kans op FES. Ook bleken de patiënten met FES reeds vroeg (dag0) koorts te krijgen, hetgeen suggereert dat systemische ontsteking een rol speelt bij de inductievan FES. Daar bekend is dat CRP vet kan agglutineren, is als mogelijke verklaring voorgesteld

Page 138: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Nederlandse samenvatting

131

dat verhoogde CRP spiegels de agglutinatie en embolisatie van circulerend beenmergvetbevorderen.

In hoofdstuk 3 is getoetst of patiënten die een FES hebben doorgemaakt een open foramen ovalehebben. De aanwezigheid van een open foramen ovale maakt een rechts-links shunt mogelijkzodat embolieën de grote circulatie kunnen bereiken zonder het longvaatbed te passeren. Voorcerebrale luchtembolieën en thrombo-embolieën is aangetoond dat een open foramen ovale hetrisico hierop inderdaad verhoogt. Bij onderzoek met transoesophageale echocardiografie bijpatiënten die een FES hadden doorgemaakt, werden geen relevante shunts gevonden. Derhalve ishet onwaarschijnlijk dat een open foramen ovale een belangrijke rol speelt bij FES. Blijkbaar zijnde vetbollen in staat zich te vervormen en zo de longcapillairen te passeren waarmee ze de grotecirculatie te bereiken.

Interleukine-6 en de acute fase reactie

In hoofdstuk 4 en 5 zijn interleukine-6 (IL-6) spiegels, tezamen met een aantal markers vanontsteking, bij patiënten met brandwonden gemeten. IL-6 bleek gecorreleerd met CRP en vooralmet koorts.

Zoals schematisch op de omslag van dit proefschrift is weergegeven, werd een duidelijkevolgorde van de responsen waargenomen: eerst IL-6↑, daarna koorts, CRP↑, α1-antitrypsine↑,trombocytengetal↑, IgM↑ en IgG↑.

De mogelijkheid dat IL-6 bij deze reacties een causale rol speelt, wordt besproken op basis van dedestijds (±1988) bekende gegevens. Latere onderzoeken, zoals het toedienen van IL-6 aanmensen, hebben de veronderstelling bevestigd dat IL-6 al de bovengenoemde onderdelen van deacute fase reactie kan veroorzaken. Voor de volledigheid moet worden aangetekend dat ookcytokinen zoals tumor necrosis factor α (TNFα) en IL-1 eveneens een belangrijke rol spelen bijde inductie van de acute fase reactie.

Procalcitonine

De afgelopen vijf jaar staat het eiwit procalcitonine (PCT; 13kD) als nieuwe marker vanontsteking toenemend in de belangstelling. Verhoogde spiegels van PCT zijn volgens sommigeauteurs met name geassocieerd met systemische ontsteking als gevolg van bacteriële infectie. Alsdit inderdaad het geval is, zou dat van grote klinische betekenis kunnen zijn bij het differentiërentussen bacteriële infectie en andere oorzaken van systemische ontsteking. Sommige auteurshebben gesuggereerd dat PCT direct door endotoxine zou worden geïnduceerd en niet door detussenkomst van cytokinen, zoals dat voor de acute fase eiwitten het geval is. In hoofdstuk 6hebben wij getoetst of cytokinen in vitro en in vivo PCT kunnen induceren. In een humaan lever-slice model kon met zowel TNFα als met IL-6 significante eiwitproductie van PCT wordengeïnduceerd In twee groepen van patiënten met kanker die respectievelijk TNFα en IL-6 kregentoegediend, werd eveneens duidelijke PCT-productie gemeten. Bij de TNFα behandeldepatiënten bleek tevens dat PCT na circa 8 uur half-maximale waarden bereikte, tegenover 20 uurbij CRP. Op basis van deze observaties is geconcludeerd dat endotoxine geen noodzakelijke

Page 139: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Acute systemische ontsteking bij gezonden en zieken

132

factor is voor PCT-inductie. De aanzienlijk snellere stijging van PCT dan van CRP maakt PCT inieder geval een aantrekkelijke marker bij patiënten bij wie tijdige informatie over de omvang vande systemische ontsteking gewenst is. In hoofdstuk 6 en ook in hoofdstuk 10 worden deeigenschappen van PCT vergeleken met die van cytokinen en acute fase eiwitten, in het licht vande onbekende functie van PCT. De titel van hoofdstuk 6 stelt dat PCT zich gedraagt als een snelacuut fase eiwit. Of het ook een acuut fase eiwit is, zal nog moeten blijken.

Het tijdsverloop van het trombocytengetal

De hoofdstukken 7, 8 en 9 onderzoeken de betekenis van het aantal trombocyten in patiënten meternstige ontstekingsverschijnselen De begrippen stolling en ontsteking werden vroeger als min ofmeer aparte processen beschouwd. Maar het is gaandeweg duidelijk geworden dat het om tweesterk geïntegreerde systemen gaat. Situaties waaraan het stollingssysteem en hetontstekingssysteem het hoofd moeten bieden, dienen zich vaak op dezelfde plaats en tijd aan. Detrombocyten blijken ook allerlei eigenschappen te hebben die de functie als stollingsdeeltje teboven gaan. Er zijn allerlei aanwijzingen (hoofdstuk 1, 7, 8, 9 en 10) dat de sequestratie vantrombocyten de hoofdoorzaak is van dalingen in het trombocytengetal zoals die zowel bijtraumapatiënten als bij intensive care (IC) patiënten wordt gezien. Hoewel met gevoeligemethoden bij veel van deze patiënten de aanwezigheid van enige diffuse intravasale stolling(DIC) kan worden aangetoond, vindt belangrijke sequestratie van trombocyten in allerlei organenplaats, zonder dat er sprake is van duidelijke DIC.

Het trombocytengetal is eenvoudig, betrouwbaar en goedkoop te bepalen. Terwijl veel onderzoeknaar (kwalitatieve) eigenschappen van trombocyten verricht is, is het gedrag van hettrombocytengetal relatief weinig bestudeerd. Afhankelijk van de ernst en van de fase waarin eensystemische ontstekingsreactie zich bevindt, kan het trombocytengetal dalen of stijgen. Derespons van het trombocytengetal na stomp trauma (hoofdstuk 2) of na brandwonden (hoofdstuk5) is al langer bekend. Het trombocytengetal daalt de eerste twee dagen (primaire verandering),om daarna te stijgen (secundaire verandering), en bij die patiënten die verder eenongecompliceerd beloop hebben na ongeveer een week het beeld van trombocytose (>350⋅109/l)te vertonen.

Trombocytengetal - de primaire veranderingen

Hoofdstuk 7 beschrijft verloop van het trombocytengetal gedurende de eerste 2 dagen bijpatiënten die een matig ernstig ongeval hebben gehad waarbij zij de rug hebben gebroken. Hettrombocytengetal daalt gedurende de eerste 48 uur relatief sneller dan het hemoglobine. Dezedaling van het aantal trombocyten is groter dan uit het bloedverlies kan worden verklaard.

Gezien de argumenten dat dalingen in het trombocytengetal met name het gevolg zijn sequestratiesecundair aan systemische ontsteking, is in hoofdstuk 7 gekeken naar het effect van een dosismethylprednisolon van ± 10 000 mg/24 uur (!), gestart binnen 4 uur na het ongeval, op hetdalende trombocytengetal. Bij vergelijkbare groepen patiënten met wervel-trauma, was er geenverschil in daling van het trombocytengetal tussen de methylprednisolon-groep en de

Page 140: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Nederlandse samenvatting

133

controlegroep. Dit betekent dat de processen (endotheel-activatie?) die verantwoordelijk zijn voortrauma-geïnduceerde trombocyten-sequestratie binnen 4 uur refractair zijn voor een van de meestpotente ontstekingsremmende interventies die momenteel beschikbaar zijn.

Trombocytengetal - de secundaire veranderingen

De meeste patiënten (zowel traumapatiënten als IC-patiënten) vertonen eerst een daling van hettrombocytengetal. Zoals reeds aangegeven, stijgt bij patiënten die een ongecompliceerd klinischbeloop hebben het trombocytengetal tot supranormale waarden na 1 week. Deze reactievethrombocytose is onder andere het gevolg van verhoogde IL-6 spiegels, reden waarom rhIL-6vóór de ontdekking van het thrombopoietine getest is als thrombopoeiticum. Ondanks het feit datde meeste patiënten op de IC verhoogde IL-6 spiegels hebben met een adequate aanmaak vantrombocyten, stijgt hun trombocytengetal vaak nauwelijks. Bekend is dat geactiveerd endotheel,zoals dat aanwezig is bij een systemische ontstekingsreactie, trombocyten kan binden zonder dater sprake is van DIC.

Op basis van deze observaties werd de hypothese geformuleerd dat een afwezige secundairestijging van het trombocyten bij ernstig zieke patiënten een uiting is van voortdurendesequestratie bij een persisterende systemische ontsteking. Daar er een bekende relatie vanpersisterende ontsteking met mortaliteit is, hebben wij het beloop van het trombocytengetal bijsurvivors en non-survivors vergeleken.

In hoofdstuk 8 is naar de relatie gekeken bij patiënten opgenomen op de chirurgische IC. Vooriedere patiënt werd tussen IC-dag 2 en 10 met regressie-analyse de dagelijkse verandering in hettrombocytengetal (∆PC/∆t) gekwantificeerd. Bij vergelijking van ∆PC/∆t met de gangbareAPACHE-II score, toonde ∆PC/∆t een groter onderscheid tussen survivors en non-survivors. Bijde survivors bedroeg ∆PC/∆t 30⋅109/l/dag versus slechts 6⋅109/l/dag bij de non-survivors. Ditfenomeen was niet beperkt tot een bepaalde populatie IC-patiënten: in iedere subgroep vertoondende survivors een hogere ∆PC/∆t.

In de literatuur zijn er veel observaties beschreven over de associatie van het laagstetrombocytengetal met mortaliteit, bij een diversiteit van ernstige acute aandoeningen zoalspolytrauma, gebarsten aneurysma van de aorta, malaria, meningitis en septische shock.

In hoofdstuk 9 is de waarde van de het trombocytengetal geverifieerd in een andere groep ICpatiënten. Het betrof ruim 5000 IC-patiënten, zowel medisch als chirurgisch, die warenonderzocht in een Europese multicenter studie. Bij deze gevarieerde groep werd de relatie vanprimaire en secundaire veranderingen in het trombocytengetal met mortaliteit bestudeerd. Uit dedagelijkse metingen van het trombocytengetal werden voor iedere patiënt drie parametersberekend: ∆PC/∆t0→2 de dagelijkse verandering in het trombocytengetal tussen IC-dag 0 en 2;PC2 het trombocytengetal op dag 2 en ∆PC/∆t2→10 de dagelijkse verandering van hettrombocytengetal tussen dag 2 en 10.

PC2 was uitstekend gecorreleerd met het laagste waargenomen trombocytengetal, een maat dieveel auteurs hebben gerelateerd met mortaliteit. Hoewel PC2 enige associatie toonde metmortaliteit, vertoonde ∆PC/∆t2→10 de sterkste associatie met mortaliteit. De relaties van PC2 en

Page 141: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

Acute systemische ontsteking bij gezonden en zieken

134

∆PC/∆t2→10 met mortaliteit waren additief. De relatie van ∆PC/∆t2→10 met mortaliteit werd ook bijde onderscheiden subgroepen van medische en chirurgische patiënten, evenals kinderen,waargenomen.

Ten slotte

In hoofdstuk 10 worden de resultaten van de oudere studies (hoofdstukken 2 t/m 5) aan de actueleliteratuur gespiegeld. Met betrekking tot het vetemboliesyndroom moet worden vastgesteld dat ergeen essentieel nieuwe gezichtspunten zijn ten opzichte van 10 jaar geleden. Een echtrepresentatief diermodel ontbreekt nog steeds. De klinisch manifeste vorm van FES wordt nogslechts zelden gezien; de therapie is ondersteunend - niet causaal - met een redelijk goedeprognose. De inmiddels gangbare vroege stabilisatie van fracturen heeft waarschijnlijk eenbijdrage geleverd aan de verminderde incidentie van FES.

Wat betreft de interleukines is er erg veel vooruitgang geboekt. De rol van IL-6, TNF-α en IL-1bij de inductie van systemische ontsteking is uitgebreid in kaart gebracht. Helaas hebbeninterventiestudies gericht op het remmen van deze mediatoren nog geen succes opgeleverd.

Naar de rol van PCT is vooral veel klinisch onderzoek in de vorm van spiegel-bepalingen bijallerlei patiëntengroepen verricht. Er moet nog veel fundamenteel PCT onderzoek verrichtworden. PCT heeft zowel eigenschappen van een acuut fase eiwit als van een cytokine. Defysiologische functie van PCT is nog onduidelijk. Hoewel wij hebben aangetoond dat de leveraanzienlijke hoeveelheden PCT kan synthetiseren is de cellulaire bron nog een raadsel.

De studies naar het trombocytengetal hebben aangetoond dat een simpele, goedkope parameterdie op iedere IC dagelijks bepaald wordt, meer informatie in zich herbergt dan veelal is gedacht.De hypothese dat het beloop van het trombocytengetal een belangrijke indicator is vansystemische endotheel-activatie kan in de komende jaren hopelijk getoetst worden. Het isinteressant te zien wat de effecten van diverse interventies gericht op het raakvlak van stolling enontsteking (e.g. antithrombine-III, geactiveerd proteine C, P-selectine remmers) op hettrombocytengetal zullen zijn.

Hopelijk zullen zulke studies bijdragen aan een beter begrip van de processen die de outcome vanernstig zieke patiënten bepalen.

Page 142: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

135

DANKWOORD

Graag zou ik allen die hebben bijgedragen aan dit proefschrift willen bedanken.In de eerste plaats gaat mijn dank natuurlijk uit naar mijn promotores. Henk-Jan ten Duis wil ik danken voor zijn nimmer aflatende enthousiasme en inzet voor velegevarieerde onderzoeksonderwerpen, en voor zijn vaste optimisme ten aanzien van een goedeafloop. Maar ook zonder het initiatief, de voortdurende steun en wijze sturing van Hauw The was ditproefschrift niet tot stand gekomen. Het is altijd een bijzonder genoegen om over de vele aspecten van ontsteking in het algemeen envan de bloedplaatjes in het bijzonder met Robert Porte van gedachten te wisselen. De leescommissie, bestaande uit Jan Goris, Reinout van Schilfgaarde en Bert Thijs, dank ik voor demoeite die zij hebben genomen bij de beoordeling van het manuscript. Hans Makkinga en Bart de Smet, jullie kunnen nu tien jaar nadat jullie ervoor gevraagd zijn,optreden als paranymf en weer eens naar Groningen afreizen.Erik Hack, Lucien Aarden, en Bert Feltkamp wil ik hartelijk bedanken voor de gebodengastvrijheid en steun bij het verrichten van onderzoek op het CLB. De sfeer en deonderzoeksmentaliteit op de afdeling Autoimmuunziekten van het CLB hebben tijdens mijn korteverblijf op het CLB een onuitwisbare positieve indruk achtergelaten. Bert, ik neem aan dat deuitvoering van de omslag in ieder geval je instemming kan vinden.Ik hoop dat ik nog met een aantal van jullie in de toekomst onderzoek kan verrichten.

Lieve Anja, je geduld is erg lang op de proef gesteld, maar nu is het toch eindelijk af.

Page 143: ACUTE SYSTEMIC INFLAMMATION · 2016-03-08 · ACUTE SYSTEMIC INFLAMMATION IN HEALTH AND DISEASE Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de

136