chapter 10 - university of groningen · from trauma patients with or without fat embolism (fes),...

CHAPTER 10

SUMMARY, DISCUSSION AND LINES OF FURTHERINVESTIGATION

Summary, discussion and lines of further investigation

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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.

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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


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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

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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


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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.

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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


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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

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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.


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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

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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


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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

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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.

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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.


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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

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

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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.

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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.


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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.

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


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-

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.


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.

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.


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