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Systemic Inflammation and Monocyte

Function after Major Surgery

J.W. Haveman

Haveman, JW

Systemic inflammation and monocyte function after major surgery

Proefschrift Groningen, met literatuur opgave en samenvatting in het Nederlands.

ISBN: 978-90-367-3699-2 (boek)

ISBN: 978-90-367-3700-5 (digitaal)

© 2009 J.W. Haveman

All rights are reserved. No part of this publication may be reproduced, stored in a retrieval

system, or transmitted in any form of by any means, mechanically, by photocopying,

recording, or otherwise, without the written permission of the author.

Cover design and layout: J.W. Haveman

Printed by: Gildeprint Drukkerijen BV, Enschede, The Netherlands

Systemic Inflammation and Monocyte Function after

Major Surgery

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

woensdag 28 januari 2009 om 13.15 uur

door

Jan Willem Haveman

geboren op 10 februari 1974 te Assen

Promotor: Prof. dr. J.H. Zwaveling

Copromotores: Dr. A.P. van den Berg

Dr. M.W.N. Nijsten

Beoordelingscommissie: Prof. dr. C.G.M. Kallenberg

Prof. dr. R.J. Ploeg

Prof. dr. D.F. Zandstra

Paranimfen: Drs. H.C. van Calker

Dr. V.E. Hagens

Financial support for this thesis was granted by the Ambroise Paré

foundation, Groningen, The Netherlands.

Financial support for this thesis was kindly given by: Astellas Pharma BV,

Bauerfeind Benelux BV, Baxter BV, Becton Dickinson Biosciences BV,

Biomet Nederland BV, Bristol-Myers Squibb BV, Boehringer Ingelheim

BV, Cook Nederland BV, Edwards Lifesciences BV, WL Gore &

Associates BV, Graduate School GUIDE, Novartis Pharma BV and

Olympus Nederland BV.

Voor mijn ouders

CONTENTS

Chapter 1 Introduction and scope of the thesis 9

Chapter 2

The central role of monocytes in the pathogenesis of sepsis:

consequences for immunomonitoring and treatment

Netherlands Journal of Medicine 1999;55:132-141

21

Chapter 3

Low HLA-DR expression on peripheral blood monocytes predicts

bacterial sepsis after liver transplantation: relation with

prednisolone dose.

Transplantation Infectious Disease 1999;1:146-152

37

Chapter 4

Changes in laboratory values and their relationship with time after

rupture of an abdominal aortic aneurysm

Surgery Today 2008;38:1091-1101

51

Chapter 5

HLA-DR expression on monocytes and systemic inflammation in

patients with ruptured abdominal aortic aneurysms

Critical Care 2006;10:R119

71

Chapter 6

Results of streamlined regional ambulance transport and

subsequent treatment of acute abdominal aortic aneurysm

Emergency Medicine Journal 2006;23:807-810

85

Chapter 7

Multicentre aneurysm screening study (MASS).

Letter to the editor. Lancet 2003;361:1058

95

Chapter 8

Summary, general discussion and conclusions

99

Chapter 9

Samenvatting

111

Literatuur

119

Dankwoord 137

Bibliografie

141

Curriculum vitae

145

CHAPTER 1

INTRODUCTION AND SCOPE OF THE THESIS

Chapter 1

10

SYSTEMIC INFLAMMATION AFTER MAJOR SURGERY

History

The classic signs of inflammation (rubor, tumour, calor, dolor) were first described by

Celsus in the 1st century A.D. About two centuries later Galen added functio laesa of

the affected area as the fifth symptom. These signs of inflammation are still recognised

in trauma patients with skin wounds, but also appear in a more generalised pattern

following major surgery and severe infections. This is known as the systemic

inflammatory response syndrome (SIRS). Rubor results from the increased blood flow

caused by vasodilatation. In systemic inflammation, vasodilatation presents as a

decrease in systemic blood pressure with compensatory tachycardia and an increased

cardiac output. Tumour develops because of (generalised) oedema resulting from

increased capillary permeability and fluid leak. Calor is a local manifestation of

inflammation presenting as warm skin. Systemically, calor may present with fever,

caused by interleukins and toll-like receptor triggering, which elevates the set point of

the thermoregulatory center in the hypothalamus (1). Dolor is manifested as local or

generalised pain. Functio laesa presents locally as the inability to use the affected body

part. With systemic inflammation, functio laesa presents as multi-organ failure.

Surgery dates back to prehistoric times, and the first successful therapeutic

arm amputation was likely performed in ancient Egypt (2). During the middle ages,

surgical interventions were usually performed by barber-surgeons. Although most of

the surgical procedures they performed were straightforward, complication rates were

high (3). Furthermore, patients suffered considerable pain as anaesthesia was not yet

available. These two factors contributed to the negative image and low social status of

the non-academic barber-surgeons.

Important changes in medicine and in surgery as a profession were required

before major surgery would be feasible. Physicians became increasingly interested in

the field of surgery during the 18th century due in part to the increased knowledge of

pathological anatomy. At the same time, the former barber-surgeons became more

interested in medical research and began to separate themselves from the barber trade.

Introduction

11

As a result, their social standing rose (3). Ultimately, a confrontation resulted at the

University of Paris between the physicians and the surgeons, who demanded the right

to teach the science and art of surgery instead of limiting themselves to wound

treatment, setting bones, letting blood, and trimming beards. This marked the

beginning of the acceptance of surgery as an academic degree by universities.

Figure 1. View of a right foot of a mummy from ancient Egypt. The left panel shows the well healed amputation area of the big toe. The right panel shows a wooden prosthesis attached to the forefoot. (4)

New developments in the 19th century set the foundation for modern surgery.

Painless surgery was first described by Warren and Morton who removed a neck

tumour from a patient under ether anaesthesia at the Massachusetts General Hospital in

Boston. This method was adopted throughout Europe. Soon afterward, chloroform was

introduced as a general anaesthetic, followed by cocaine infiltration as a local

anaesthetic several years later. Minor surgery of the extremities became much easier to

Chapter 1

12

perform when epidural cocaine was available for the blocking of nerve trunks (5).

Although surgery could now be practised without pain, surgery related mortality was

still high due to blood loss and postoperative infection.

In 1848, Semmelweis reported that fever and septicaemia in pregnant women

could largely be prevented by hand scrubbing and washing in chlorine water before

obstetrical examination. It was only after Lister’s publications in 1867 that it became

understood that infections were caused by microbes and that infection was not a normal

stage in the wound healing process. Surgical techniques were also markedly improved

in these years: intestinal suture techniques were first performed by Dieffenbach in 1836

and further refined with sterilised fine silk thread by Kocher in 1888.

These developments resulted in several new major surgical operations in the

late 19th century. Billroth performed his first successful esophagus resection in a dog in

1871, and the first laryngectomy in 1872. He was also the first to attempt

reconstruction after partial gastrectomy by gastroduodenostomy (Billroth I; 1881) and

by gastroenterostomy distal to the ligament of Treitz (Billroth II; 1885). Sauerbruch

became a pioneer in thoracic surgery and performed the first thymectomy in a patient

with myasthenia gravis in 1912.

Many surgical procedures were introduced at the end of the 19th century and at

the beginning of the 20th century. Operations were performed with increasing success

due to a better understanding of anatomy, physiology, increased experience, and the

development of anaesthesia. Other supportive techniques were introduced soon

afterwards, for example the artificial kidney by Willem Johan Kolff in 1944, the heart-

lung machine by Gibbon in 1953, and the treatment of blood loss by blood transfusion.

The incidence of postoperative infections decreased due to the implementation of

antisepsis measures and the invention of Penicillin by Flemming in 1928 (6).

However, despite the development of surgical skills and improved methods in

the treatment of postoperative complications, mortality remained considerable. Many

patients died after the progressive failure of one or more vital organs such as the heart,

the lungs or the kidneys. Treatment aimed at replacing or restoring the function of

individual organs (such as fluid replenishment, mechanical ventilation, haemodialysis

Introduction

13

etc.) often failed to stop the fatal course of events. It became clear that more than

symptomatic treatment was needed, and that insight into the underlying

pathophysiology of the multi-organ failure syndrome would be required for effective

intervention. The central mechanism in the pathophysiology of the development and

progression of multi-organ failure is called systemic inflammation (7).

Systemic inflammation

Theoretically, the inflammatory response which follows surgery facilitates tissue

healing. With a small skin wound, platelets initiate the formation of blood clots,

thereby stopping bleeding. Activated platelets secrete mediators that attract

macrophages and fibroblasts. Chemo-attractants produced by macrophages and

fibroblasts attract and activate neutrophils and monocytes. The wound area is cleaned

by these neutrophils and an extracellular matrix is formed. After the initial phase of

inflammation, the wound is re-epithelialized followed by wound contraction and

extracellular matrix reorganization (8). The inflammatory response in patients with

small skin wounds is principally a local phenomenon, manifested by redness, swelling,

warm skin and mild pain (rubor, tumour, calor, dolor). In patients with extensive

trauma, major surgery, severe burns, pancreatitis or sepsis, the inflammatory response

generally has important systemic manifestations and has been termed the systemic

inflammatory response syndrome (SIRS). SIRS has been defined in a consensus

conference, as shown in table 1 (9).

Table 1. Criteria for SIRS

SIRS is manifested as 2 or more of the following conditions:

- Fever (> 38.0 C) or hypothermia (< 36 C)

- Tachycardia (> 90 beats/min)

- Tachypnea (>20 breaths/min) or hyperventilation (PaCO2 < 4.3 kPa)

- Leukocytes > 12 x109/L or < 4 x109/L or > 10% immature neutrophils

Chapter 1

14

SIRS can progress in an uncontrollable reaction leading to multiple organ

failure (heart, lungs, kidneys, liver and bone marrow). The pathogenesis of multiple

organ failure is complex and involves several mediators and cytokines. Tumour

necrosis factor-alfa (TNF-α) and Interleukin-1 (IL-1) play a key role (10). Activated

monocytes and macrophages are able to release massive amounts of these cytokines.

This activates the coagulation and complement pathways, and the platelets are

triggered by the release of platelet activating factor. The activated endothelium also

releases large amounts of pro-inflammatory cytokines, and the expression of adhesion

molecules is increased. Polymorphonuclear granulocytes become activated, and

through their interaction with the adhesion molecules they transmigrate through the

endothelial cells into the targeted tissue. Subsequently granulocytes release large

amounts of reactive oxygen radicals and proteases to kill the invading micro-

organisms. This induces further endothelial damage and activation resulting in an

uncontrolled cascade (11). The release of prostaglandins and nitric oxide in

combination with the endothelial damage, causes vasodilatation, capillary leakage,

generalised oedema, systemic hypotension, and, finally, multiple organ failure (12). In

the case of an infection, antibiotics should be administered and (surgical) drainage

should be performed whenever possible. Tissue perfusion should be restored by fluid

replacement and the administration of vaso-active medication. In case of respiratory

failure caused by acute respiratory distress syndrome (ARDS), patients should be

mechanically ventilated with high PEEP and low tidal volumes (13). When renal

failure occurs, renal replacement therapy by continuous venovenous hemofiltration

should be started without delay. Despite aggressive treatment, the mortality rate when

multiple organ failure occurs is high (7% for one-organ failure and 48% in three-organ

failure) (14). One reason why supportive therapy often fails to save the patient’s life is

that supportive therapy alone does not interfere with the systemic inflammation which

is driving the whole process. Since this theory was recognised, various efforts have

been made to interfere with the immunological processes in an effort to improve

outcome.

Introduction

15

Intervention in the inflammatory response

The inflammatory response which follows major surgery should facilitate tissue

healing and eliminate the invading organisms, but this mechanism can overshoot,

leading to multiple organ failure and death. Although this hypothesis is plausible,

efforts to interrupt the inflammatory cascade such as administering neutralizing

antibodies against LPS (lipopolysaccharide), anti-TNF-α, soluble TNF receptors, IL-1

receptor antagonist and high dose steroids have not been successful (15-21). There may

be several explanations for these negative results. If the inflammatory response was

already activated, it may no longer have been dependent on the initiating stimuli, such

as TNF-α and IL-1. As only single agents were tested in these trials, just one of the

many active cascades would be affected, allowing the others to continue fuelling the

reaction. A third intriguing explanation is that at the time of the intervention, patients

may no longer have been experiencing an overwhelming pro-inflammatory response

needing counteraction, but instead were exhibiting a form of immunosuppression that

would worsen with anti-inflammatory measures. In this case interventions aimed at

restoring immune capacity would have been more appropriate. A compensatory anti-

inflammatory reaction in response to the initial pro-inflammatory response has been

described to occur and would prevent the excessive tissue destruction resulting from

uncontrolled inflammation (22). Theoretically, this anti-inflammatory response could

promote a state of immunosuppression which would increase the risk of nosocomial

infections. Together with the decreased TNF-α production by monocytes, these factors

make up the compensatory anti-inflammatory response syndrome (CARS) which

follows SIRS (22). This model for systemic inflammation combined with the anti-

inflammatory response would allow for specific treatments which target the

inflammatory cascade. Two different treatment directions are indicated, depending on

the response seen in the patient. Patients with an overwhelming systemic inflammatory

response should be treated by interfering with the pro-inflammatory cascade, whereas

patients exhibiting a dominant anti-inflammatory response should be treated by

stimulating the immune response.

Chapter 1

16

Monocyte function in systemic inflammation

The exact mechanism and timing of the anti-inflammatory response was poorly

understood until recently. Monocytes play a central role in immunity and

inflammation. Hershman et al studied monocyte function in patients after major

surgery and sepsis. They discovered that a decreased expression of HLA-DR on

peripheral blood monocytes was associated with an increased risk of infectious

complications in trauma patients (23). These results were confirmed by other study

groups in patients after major surgery and sepsis (24-29). Low HLA-DR expression

impairs the immune response, which predisposes the host to generalised infections.

Decreased expression of HLA-DR by monocytes is often accompanied by functional

abnormalities, such as a low antigen presentation by monocytes to T-cells (30), a

critical step in the immune response, and by a diminished capacity to release pro-

inflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-8 and interferon-gamma (IFN-

γ) (31). This state is known as monocyte deactivation, and constitutes one of the

mechanisms underlying CARS. Monitoring monocyte function by measuring HLA-DR

expression might be an important parameter to guide intervention in the inflammatory

pathway. We hypothesised that a better understanding of the pro- and counter

inflammatory response is a prerequisite for successful intervention. This thesis reflects

our efforts to better understand these processes.

LIVER TRANSPLANTATION

To investigate monocyte function in patients with sepsis we investigated patients who

underwent orthotopic liver transplantation (OLT). The first successful OLT was

performed by Starzl in 1967 (32). During the following years, the short-term mortality

remained high initially because of uncontrolled haemorrhaging during surgery,

technical problems, and acute rejection. However, due to major improvements in

surgical techniques, anaesthesiology, post-operative care, and the development of

potent immunosuppressive drugs, mortality eventually decreased, and liver

transplantation became an accepted treatment option for end-stage liver disease.

Introduction

17

Unfortunately, the incidence of serious infections remained high and was the principle

cause of death during the first months after transplantation (33,34). Although

immunosuppressive drug therapy is an important cause of these infections, there are

several other factors which contribute as well. For instance: the patient’s poor general

condition and nutritional status before surgery; the presence of intravascular, biliary,

and urinary catheters; mechanical ventilation; a large surgical wound with drains; and

the considerable blood loss that can occur during surgery and afterwards due to poor

clotting and low platelet counts. Antimicrobial drugs are important in the treatment of

these infections, but may not be sufficient when the patient’s natural protective

immune responses fail. In order to enable the immune system to initiate these

protective responses, the dosage of immunosuppressive drugs should be strongly

reduced. This might result in a dilemma, as insufficient suppression of the immune

system predisposes to rejection of the graft. The treatment in these cases depends

largely on clinical intuition as there are no presently recognized clinical parameters

which indicate immune function. Such parameters would be of great value for

calculating dosages of immunosuppressive drugs so that both over-

immunosuppression, with its attendant risk of infection, and under-

immunosuppression, leading to rejection, may be avoided. To address this issue in our

thesis, we note that sepsis was generally preceded by a strong decrease in the

expression of HLA-DR on monocytes. Whereas the return of HLA-DR expression to

normal values accompanies recovery from sepsis and survival, persistently low values

occur in patients who subsequently die from sepsis. As previously discussed, a variety

of conditions impact immunoresponsiveness and monocyte function in OLT patients

including age, pre-transplant illness, nutritional status, co-existing diseases, and the

type and dosage of immunosuppressive drugs. To further investigate the inflammatory

response and monocyte function following major surgery, but without many of the

confounding factors encountered in OLT patients, we studied a second, much more

homogeneous group of patients.

Chapter 1

18

RUPTURED ABDOMINAL AORTIC ANEURYSMS

Patients with ruptured abdominal aortic aneurysms (RAAA) represent a homogeneous

patient group (previously healthy, non-immunosuppressed) with a clearly defined

catastrophic event, followed immediately by surgery. In RAAA patients, haemorrhagic

shock is the primary cause of death both intra-operatively and during the 24 hours

following surgery. After this initial post surgical phase, the most frequent cause of

death is multiple-organ failure. In fact, more patients die from multiple organ failure

than hemorrhagic shock (35,36). Almost all patients who survive surgery develop a

systemic inflammatory response syndrome (SIRS), responsible for the development of

multi-organ failure (37). The aim of our study was to describe HLA-DR expression on

monocytes in RAAA patients after surgery and to establish whether HLA-DR

expression correlated with the occurrence of secondary infections and death. In this

‘clean’ model of post-surgical systemic inflammation, we aimed to establish whether

RAAA patients die from an overwhelming systemic inflammatory response or from

secondary infections with monocyte deactivation and a predominant compensatory

anti-inflammatory response. To achieve this, we analysed several clinical and

immunological variables.

An abdominal aortic aneurysm is a serious medical condition associated with a

high mortality. It is the tenth leading cause of death in men aged 65 to 74 years old,

and its incidence is on the rise (38-40). The management of patients with AAA consists

of surgical repair with an aortic graft. Endovascular treatment (stenting) was more

recently introduced for selected cases. Hospital mortality for the elective repair of

AAAs larger than 5.5 cm is reported to be as low as 2% in some centres (41,42). This

low mortality is in contrast with the in hospital mortality associated with RAAAs,

which ranges from 30% to 70% with surgical repair (43-47).

The advantage of the recently established elective endovascular repair of

abdominal aortic aneurysm (EVAR) is the lower incidence of systemic complications

(48,49) and a lower hospital mortality (50), compared with open repair. Results for

EVAR in symptomatic non-ruptured and ruptured AAAs are promising (51). However,

Introduction

19

the survival benefit of avoiding the trauma of surgery might be off-set by the negative

influence of the time delay that results from waiting for the required CT-scan.

Furthermore, the impact of long-term complications on mortality after EVAR is

unknown and EVAR is frequently not feasible in patients with ruptured AAA (51-53).

AIMS AND OUTLINE OF THE PRESENT THESIS

The concept of systemic inflammation is well established in surgical patients. The aim

of this study was to describe this response in patients after major surgery with special

emphasis on the role of the monocyte. Our studies focused on patients following liver

transplantation and patients with a RAAA. These two groups are highly relevant since

they show a high incidence of secondary systemic complications, with progression to

multiple organ failure and death.

Chapter 2 describes new insights in the pathophysiology of sepsis. The role of

monocytes in the inflammatory response is described in detail. Furthermore, possible

new interventions based on the patient’s immune status are outlined. Liver

transplantation patients are studied in chapter 3. The purpose of this study was to

describe the role of HLA-DR expression on monocytes in infectious complications and

mortality. Our hypothesis was that a low HLA-DR expression on monocytes is

associated with sepsis and mortality. The effect of immunosuppression on monocyte

function was also investigated. In postoperative OLT patients this might be especially

relevant since patients in whom the anti-inflammatory response predominates might

benefit from reduced immunosuppression therapy.

As explained above, patients who have undergone surgical repair of a ruptured

aortic aneurysm are also relevant to the study of the systemic inflammatory response.

Systematic data on the organ responses in terms of biochemical parameters of

patients after RAAA have only rarely been described. Therefore, we studied the typical

biochemical response in RAAA patients in chapter 4. The impact of several parameters

on outcome is also described. Although the role of several different cytokines in

patients after RAAA has been described in a number of studies, the clinical

Chapter 1

20

significance of biochemical parameters is not always clear. In chapter 5 we describe

pro- and anti-inflammatory cytokines and HLA-DR expression on monocytes in

RAAA patients. We hypothesised that a low HLA-DR expression was associated with

a high mortality in these patients. As multiple organ failure is the most frequent cause

of death in our patient population, we evaluated if this was from an ongoing systemic

inflammatory response or from immunosuppresion and secondary infections as is seen

in patients with CARS.

The treatment and outcome of RAAA patients admitted to the University

Medical Center Groningen are described in chapter 6. This study will show how the

streamlined management of patients directly before surgery, during surgery, and after

surgery impacts hospital mortality in patients with symptomatic AAAs.

The Multicentre Aneurysm Screening Study (MASS) has investigated whether

screening men aged 65 to 74 for AAA has an effect on AAA related mortality.

Screening resulted in a significant survival benefit (54). Screening has also been found

to be cost-effective (55). In chapter 7 a letter to the editor in response to the MASS

study is presented, stressing that although screening men seems to improve survival,

measures to improve survival in RAAA patients by prompt and surgery according to

liberal inclusion criteria should not be overlooked.

CHAPTER 2

The central role of monocytes in the pathogenesis of

sepsis: consequences for immunomonitoring and

treatment

JW Haveman1, AC Muller Kobold2, JW Cohen Tervaert2,

AP van den Berg2,3, JE Tulleken4, CGM Kallenberg2,

TH The2

1 Department of Surgery

2 Department of Clinical Immunology 3 Department of Gastroenterology and Hepatology

4 Department of Intensive and Respiratory Care Unit

University Medical Center Groningen, The Netherlands

Netherlands Journal of Medicine 1999;55:132-141

Chapter 2

22

ABSTRACT

Despite important advances in critical care medicine during the last two decades, the

mortality rate of sepsis has remained high, probably because the pathogenesis of sepsis

is still incompletely understood. Recent studies have shown that sepsis is a bimodal

entity. The first phase is characterized by the systemic release of pro-inflammatory

cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-8, and

by activation of the complement and coagulation cascades. In the second phase, anti-

inflammatory mediators such as transforming growth factor-β (TGF-β), IL-10 and

prostaglandin E2 (PGE2) may be released in an effort to counteract ongoing

inflammation. Depending whether the pro- or anti-inflammatory response

predominates, sepsis results in a systemic inflammatory response syndrome (SIRS), or

a compensatory anti-inflammatory response syndrome (CARS). So far, most efforts to

intervene in the immunopathogenesis of sepsis have been directed at the pro-

inflammatory response. None of these interventions has been shown to improve the

prognosis of sepsis, possibly because many patients were already in a state in which

anti-inflammatory responses dominated. Recently, it has been shown that decreased

expression of HLA-DR on monocytes in patients with sepsis constitutes a marker for

CARS. We suggest that HLA-DR expression on monocytes might constitute a useful

indicator of the immunological status of the individual patient with sepsis and a guide

for treatment. Patients with CARS, as manifested by low HLA-DR expression, might

benefit from immunostimulants, while patients with SIRS and normal or high

monocyte HLA-DR expression should receive treatment directed to interfere with pro-

inflammatory pathways.

HLA-DR on monocytes in sepsis

23

INTRODUCTION

Sepsis is a leading cause of acute hospital admissions, and, in addition, often

complicates the clinical course of patients hospitalized for other reasons. Sepsis is

defined by a microbiologically documented infection that is associated with a systemic

response, consisting of hyper- or hypothermia, an increased or decreased white blood

cell count, tachycardia and tachypnea (56). Despite modern antimicrobial therapy and

intensive care facilities, the mortality rate of sepsis remains high at 30–40%, and

morbidity in survivors may be considerable. The incidence of sepsis is rising, because

of increasing numbers of old patients and of patients with complicated conditions,

including major trauma, extensive surgery, and immunosuppressive treatment.

The immune system plays a pivotal role in the pathogenesis of sepsis. In this

review we will discuss new insights in this syndrome, with special emphasis on the role

of HLA-DR expression on monocytes as a prognostic marker and as a guide to direct

im munomodulatory therapy.

PATHOGENESIS OF SEPSIS

Traditionally, sepsis was considered to be an overwhelming and persistent,

inflammatory response of the immune system to a bacterial infection. This response is

initiated by bacterial toxins, such as the endotoxin of gram-negative organisms and the

lipoteichoic acid-peptoglycan complex of gram-positive bacteria. In gram-negative

sepsis, binding of lipopolysaccharide (LPS) to the CD14 receptor on the cell

membrande results in activation of monocytes and macrophages. In addition, LPS may

bind to soluble CD14, and this complex subsequently interacts with, and activates,

endothelial, and smooth muscle cells (57). It is not completely clear how gram-positive

bacteria activate monocytes, but both CD14-dependent mechanisms may play a role

(11).

Chapter 2

24

Figure 1. A simplified scheme of the pro-inflammatory response of sepsis designated as SIRS. After bacterial toxin release monocytes (mono´s) and macrophages (M) produce massive amounts of TNF-α, IL-1, IL-6, IL-8 and reactive oxygen species (ROS), platelet activating factor (PAF), and nitric oxide (NO). Together with endothelial activation this initiates a cascade of reactions, which leads ultimately to endothelial damage, capillary leakage, edema, vasodilatation and systemic hypotension. In order to prevent sepsis, septic shock, multi organ failure (MOF) and eventually death, the left side of the figure shows potentially pharmacologic interventions. Anti LPS; anti TNF; soluble TNF receptor; IL-1-RA (receptor antagonist); PAF antagonist; nitric oxide-inhibitors (N-v-nitro-L-arginine methyl ester) (NO-inhibitors (L-NAME)).


25

Figure 2. The pathogenesis of CARS. After the release of bacterial toxins a pro-inflammatory response follows. The anti-inflammatory response is probably mainly initiated by IL-10, TGF-β and PGE2. Monocyte deactivation follows, manifested by low antigen presenting activity and low release of pro-inflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-8 and IFN-γ. Intervention in the pathophysiology of CARS is possible by administration of IFN-γ and possibly Granulocyte Macrophage–Colony Stimulating Factor (GM–CSF).

Chapter 2

26

Activated monocytes and macrophages release large amounts of TNF-α, which can be

considered to be the principal mediator that sets the septic response in motion. In

addition to TNF-α, monocytes/macrophages produce other pro-inflammatory mediators

including IL-1 and IL-6, eicosanoids, reactive oxygen species (ROS), platelet

activating factor, and nitric oxide (NO); whereas activated endothelial cells release

inflammatory mediators like IL-1, IL-6, IL-8 and NO. Bacterial cell wall products

interact with and activate the complement and coagulation cascades, damaging the

endothelial surface. During activation of endothelial cells the expression of adhesion

molecules is increased. Polymophonuclear granulocytes become activated and, via

interaction with adhesion molecules, adhere to, and transmigrate through the

endothelial monolayer. During this process, the activated granulocytes release oxygen

radicals and proteases, thereby killing micro-organisms. However, these products may

also damage endothelial cells (58). NO, prostaglandins and kinins released by

endothelial cells all cause profound vasodilatation which, in combination with

widespread endothelial damage, results in systemic hypotension, capillary leakage, and

edema. Although the inflammatory response is intended to eliminate the invading

micro-organisms, causing sepsis to deteriorate into septic shock, multiple organ

dysfunction, and death (see Figure 1) (56,59). Numerous efforts have been made to

intervene at various levels of this inflammatory cascade. Neutralising antibodies

against LPS (15), or TNF-α (16,17), soluble TNF receptors (18), IL-1 receptor

antagonists (19), platelet activating factor-antagonist (20), high-dose corticosteroids

(21), and inhibitors of the arachidonic acid pathways were all found to be effective in

animal models, but none of these agents has resulted in a clinically significant survival

benefit in humans so far (for review see (60)). No synthase-inhibitors might also form a

rational intervention in patients with sepsis. However, mortality was not reduced by

treatment of hypotensive septic patients with L-NAME (N-ω-nitro-L-arginine

methylester) (61). This failure to impact on the outcome of sepsis has been thought to

reflect the fact that these immunomodulatory agents had been administered several

hours after the onset of sepsis. At that time, the inflammatory response probably had

been fully activated, and was no longer dependent on its initial stimuli. Only single


27

agents were tested in these trials, each directed at one of the early steps in the

pathogenesis of sepsis. Combination of agents might be required for blocking the septic

response, targeting multiple, probably more distal sites of the inflammatory cascade.

Another explanation, which does not exclude the previous one, suggests that sepsis just

is not as simple as we have always thought. Indeed, the concept that the septic response

consists only of an overwhelming inflammatory reaction, the so-called systemic

inflammatory response syndrome (SIRS), is probably not correct. It has become

evident in recent years that the inflammatory response is followed by counter-

regulatory anti-inflammatory reactions that are intended to prevent undue tissue

destruction from uncontrolled inflammation. This anti-inflammatory reaction may

become the dominant pathophysiologic abnormality, and may result in a state of

immunological anergy with an increased risk of secondary infections. Bone introduced

the term compensatory anti-inflammatory response syndrome (CARS) for this situation

(Figure 2) (22). Therefore, Bone has proposed to consider the immunologic response in

sepsis as a spectrum, with overwhelming inflammation (SIRS) on the one end,

profound immunodepression (CARS) on the other, and the mixed antagonistic response

syndrome (MARS) that has features of both extremes in between (22). Both pro- and

anti-inflammatory responses may give rise to fatal complications. This spectrum

concept of sepsis explains why immuno-interventions directed against the pro-

inflammatory response have been ineffective. Since patients were treated irrespective

of their immune status, inhibition of inflammation might have been beneficial in

patients with SIRS, but deleterious in patients with predominant CARS.

It has to be stressed that the validity of the concepts of CARS and MARS still

has to be proven. However, if this hypothesis of sepsis as a spectrum of pro- and anti-

inflammatory responses is correct, it follows that immunomodulation will be effective

only it is tailored to the immune status of the individual patient.

HLA-DR EXPRESSION ON MONOCYTES IN PATIENTS WITH SEPSIS

Monocytes and macrophages play a central role in the immune response against micro-

organisms and in the pathogenesis of sepsis, as already discussed above. The are

Chapter 2

28

essential for phagocytosis and killing of micro-organisms, production of cytokines, and

presentation of microbial antigens to T cells, thus initiating and regulating both cellular

and humoral immune responses.

It was first shown by Hershman et al. that decreased expression of HLA-DR

on peripheral blood monocytes (Figure 3) was associated with an increased risk of

infectious complications in trauma patients (23). Subsequently, these findings were

extended to various other categories of patients. Low HLA-DR expression was found

to predict sepsis in patients with thermal injury (62), patients undergoing major

abdominal surgery (24,25), neurosurgery (26), or liver transplantation (63), and in

patients on ventricular assist device awaiting cardiac transplantation (27) Table 1.

Moreover, the degree of expression of HLA-DR on monocytes in patienst with

established sepsis was shown tob e of prognostic significance. Low HLA-DR

expression on monocytes on five consecutive days was associated with a mortality rate

of 81% (31). In addition, 11 out of 12 transplant patients with sepsis and low HLA-DR

expression on monocytes, in whom immunosuppression was kept unchanged, died,

whereas all 20 patients with similarly low HLA-DR expression, in whom

immunosuppression was rapidly tapered, survived (28).


29

Table 1. Association between HLA-DR expression and the risk of infection or death

Patient category Number of

patients

Reference Comment

Recipients of

immunosuppression because

of organ transplantation or

autoimmune disease

21 (28) Monocyte HLA-DR expression

was <20% in all patients; 0/13 om

whom immunosuppression was

kept unchanged.

Liver transplant recipients 20 Personal

experience

and (63)

Expression of HLA-DR on <50%

of monocytes preceeded sepsis in

all 7 patients

Trauma patients 69 (23) Low HLA-DR expression

correlated with development of

infections and death.

Patients undergoing elective

laparotomy

24 (25) Five patients with a low

percentage of HLA-DR

expression before and seven days

after the operation developed

surgical wound infections.

36 (24) Twelve patients with low HLA-

DR expression developed sepsis

Patients with a ventricular

assist device awaiting cardiac

transplantation

30 (27) Seven of nine patients who died

of septic MOF had low

percentage HLA-DR expression.

Neurosurgery 46 (26) Nine of ten patients with a low

percentage of HLA-DR

expression developed infectious

complications.

Chapter 2

30

Figure 3. Leukocyte activation in 2 patients with sepsis and in a healthy individual. The course of expression of HLA-DR on monocytes (Fig. 3A and C) and of plasma concentrations of interleukin 10 is shown (Fig. 3B and C). Arrows indicate the length of stay at the ICU for patient 1 and patient 2. The horizontal line represents expression of HLA-DR on monocytes at an intensity of 30% of healthy controls (N=5).


31

Direct comparison between studies is somewhat hampered by the fact that

different parameters were used to quantify HLA-DR expression (either as the

percentage of HLA-DR-positive monocytes, or as the mean fluorescence intensity of

the HLA-DR signal of the monocyte population). Similarly, various cut-off values have

been used for what constitutes pathologically low HLA-DR expression (e.g., 20% (28),

30% (27), 50% (63). Thus, standardization of methodology and cut-off values will

become an important issue. Since many patients with low HLA-DR expression died as

a result of superinfections, Volk et al. coined the term immunoparalysis to denote this

condition. They subsequently demonstrated that the capacity for antigen presentation of

monocytes taken from patients with immunoparalysis is reduced, which is in

agreement with the role of HLA-DR in the presentation by monocytes of antigens to T

cells (30). However, it is difficult to accept that decreased antigen presentation would

predispose to bacterial infections in view of the limited importance of T cells in

antibacterial immunity. The finding that monocytes expressing low amounts of HLA-

DR also produce lower amounts of pro-inflammatory cytokines, such as TNF-α, IL-1,

IL-6, IL-8, and IFN-γ, offers a better explanation for the association between decreased

HLA-DR expression and infections (30). It followed logically that these ´deactivated´

monocytes were a marker for, and probably a mediator of, CARS. Therefore,

elucidation of the pathogenesis of monocyte deactivation, and the development of

strategies to restore normal monocyte function, have become important objectives that

may have a major impact of the treatment of sepsis.

MEDIATORS OF MONOCYTE DEACTIVATION

In cultures of monocytes that have been stimulated with LPS, high levels of the pro-

inflammatory cytokines IL-1, IL-1, IL-6, IL-8, TNF-α, GM-CSF and G-CSF can be

detected within 4–8 h (64). Production of IL-10, a cytokine with potent anti-

inflammatory effects on monocytes and on T 1 lymphocytes, peaks 24–48 h after

stimulation (64). Probably, release of IL-10 is important to downregulate the

inflammatory response. In monocytes that have been stimulated with LPS, reexposure

to LPS leads to low production of pro-inflammatory cytokines, whereas release of anti-

Chapter 2

32

inflammatory mediators such as IL-10 is increased (65). This ‘LPS desensitization’,

which is mediated by IL-10, transforming growth factor-β (TGF-β), and probably by

prostaglandin E2 (PGE2), is an other counter-regulatory mechanism intended to control

the inflammatory response (65). It has been postulated that monocyte deactivation that

occurs during sepsis is caused by IL-10 (65-68) and PGE2 (66,69). IL-10 inhibits the

release of pro-inflammatory cytokines (64), suppresses antigen presentation and

formation of free oxygen radicals (70,71), and down-regulates HLA-DR expression on

monocytes (72). Serum levels of IL-10 are increased in patients with sepsis or septic

shock compared with healthy controls (66). In a series of 32 patients undergoing major

abdominal surgery, IL-10 gene expression correlated inversely with monocyte HLA-

DR expression, which is consistent with a role for IL-10 as a mediator of the

immunosuppression associated with surgical injury (67). We longitudinally monitored

IL-10 levels in plasma and HLA-DR expression on monocytes in two patients with

sepsis, and found that CARS was associated with increased plasma IL-10 levels and

low HLA DR expression, and that normalisation of these parameters coincided with

clinical recovery (see Figure 2) (73).

Other mediators may be involved in the pathogenesis of monocyte

deactivation in addition to IL-10. TGF-β is a cytokine with profound inhibitory effects

on the immune system and on haematopoiesis. It lowers TNF-α production by

monocytes after LPS stimulation (65), and prevents upregulation of HLA-DR

expression on human fibroblasts by IFN-γ (74). The in-vivo sifnificance of TGF-β is

uncertain, however, since serum levels of TGF-β were found to belower in patients

with sepsis and septic shock than in healthy controls (66).

PGE2 has an inhibitory effect on a variety of monocyte functions, and is one of

the mediators of LPS desensitation. It is a potent inhibitor of the expression on Ia, the

rat equivalent of HLA-DR, on rat macrophages (75). PGE2 levels are increased in

patients with sepsis (66,69). Moreover, the production of PGE2 by monocytes in

patients with sepsis is higher than in healthy controls (69). Yet PGE2 does not decrease

the antigen-presenting capacity of monocytes (76,77). These data suggest that IL-10 is


33

the principal mediator of monocyte deactivation, and that TGF-β and PGE2 may be of

secondary importance.

In vitro stimulation by IFN-γ at least partially reverses IL-10 or TGF-β

induced monocyte deactivation (78). Moreover, incubation with IFN-γ of monocytes

taken from patients with sepsis restored the expression of HLA-DR to normal levels,

and partially corrected LPS-stimulated TNF-α secretion (79). Furthermore, it was

recently reported that administration of IFN-γ in vivo in patients resulted in

normalisation of the reduced HLA-DR expression (79). GM-CSF also upregulates the

expression of HLA-DR on monocytes and stimulates the production of TNF-α and IL-1

in vitro (80). These observations suggest that IFN-γ and, probably, GM-CSF may be

valuable in ameliorating the state of immunodeficiency in patients with CARS, thereby

reducing the risks of secondary infections and death.

MONOCYTE HLA-DR EXPRESSION: A GUIDE FOR INDIVIDUALIZING

SEPSIS TREATMENT?

The insight that sepsis is no a homogeneous disorder, but a spectrum of immunological

changes, ranging from overwhelmin inflammation to profound immunodeficiency, has

given rise to calls for a more individualized treatment approach (22,81). Thus,

antimicrobial and general supportive treatment should be supplemented by

immunodulatory interventions that are specifically tailored to correct the

immunological disturbances in the individual patient. This concept is attractive, but

requires the availability of reliable parameters to guide this treatment. Serial measures

of cytokines and other mediators might be an indicator of the pro-/anti-inflammatory

state of the immune system (78). Van Dissel et al measured cytoken levels in patients

admitted because of fever, and found that the IL-10/TNF-α ratio was 1.7 times higher

in patients with a fatal outcome compared to those who survived. Interestingly, the

ratio of these cytokines was more predictive than the level of either cytokine alone

(82).

The expression of HLA-DR on monocytes may be and alternative parameter to

guide immuno-intervention. Monitoring HLA-DR expression has the advantage that it

Chapter 2

34

may provide an extimate of the ‘net’ biological effect of pro- and anti-inflammatory

stimuli. We have previously reported that high plasma levels of IL-10 in patients with

sepsis were associated with low HLA-DR expression, and that clinical recovery was

associated with normalisation of HLA-DR expression and decreases IL-10 levels (73).

More studies will be necessary to determine which marker(s) provide the best

prognostic information.

The validity of the concept of individualized immuno-intervention was

recently tested by Döcke et al (79). IFN-γ was administered for a median 6 days to 10

patients with sepsis and CARS as evidenced by low HLA-DR expression (79,83). The

degree of HLA-DR expression on monocytes was rapidly upregulated in all patients,

increasing from a median of 27% before, to 62% on day 1 after treatment, and it

remained at this level in most patients. Normalisation of HLA-DR expression was

associated with a significant increase in plasma levels of TNF-α and IL-6. The small

number of patients treated, and the uncontrolled nature of the study, make it impossible

to draw conclusions on the clinical efficacy of this intervention. It is clear, however,

that this approach of immuno-intervention is worth pursuing.

CONCLUSIONS AND FUTURE DIRECTIONS

The ultimate goal in the treatment of septic patients is to improve outcome. Recent

advances in antimicrobial therapy and supportive intensive care have, to a large extent,

failed to achieve this goal. Direct intervention in the immunological response is a new

and promising approach, but a variety of immune-modulating agents have not been

effective in reducing mortality. Sepsis is not a homogeneous entity, but can be regarded

as a spectrum of pro- and anti-inflammatory responses. It seems reasonable that

immuno-intervention should be tailored to the immunopathological condition of the

individual patient. The degree of HLA-DR on peripheral blood monocytes appears to

be a good parameter in defining the immune status of patients with sepsis: low HLA-

DR expression is associated with immunological energy due to predominance of anti-

inflammatory responses, that might require intervention with immunostimulants such

as IFN-γ or GM-CSF. Normal HLA-DR expression is observed in patients with an


35

inflammatory response, who might benefit by measures interfering with the pro-

inflammatory cascade. It is now time to test the concept of immuno-intervention as

based on HLA-DR expression in randomized clinical trials. Given the complex

pathophysiology of sepsis it would be surprising if, even in the subgroups of patients

with SIRS or CARS, a single agent would be effective in all patients. Therefore,

intervention at multiple levels of the pro- or anti-inflammatory cascades may be

required for optimal results. Parameters such as HLA-DR expression on monocytes

may be useful to guide this treatment. Ultimately, increased insight in the pathogenesis

of sepsis, and the growing number of new tools for immuno-intervention will lead to

improved outcome for patients with sepsis.

ACKNOWLEDGEMENTS

We would like to thank Mr. Jan Brouwer for his artistic input in figures 1 and 2.

CHAPTER 3

Low HLA-DR expression on peripheral blood monocytes

predicts bacterial sepsis after liver transplantation:

relation with Prednisolone intake

JW Haveman1, AP van den Berg2,3, JMM van den Berk4,

G Mesander2, MJH Slooff1, LHFM de Leij2,

TH The2



University Medical Center Groningen, The Netherlands 4 Department of Internal Medicine, University Hospital Nijmegen, The Netherlands

Transplantation Infectious Disease 1999;1:146-152

Chapter 3

38

ABSTRACT

Bacterial sepsis remains a frequent complication after liver transplantation. We

previously reported the results of a pilot study that suggested that low expression of

HLA-DR on monocytes is a predictive marker for the occurrence of sepsis. We have

studied the value of this marker in an additional cohort of patients, and have analyzed

the relation of HLA-DR expression with the use of immunosuppressive agents. 20

adult liver transplantation patients were prospectively monitored during the first 4

weeks after transplantation. All were treated according to standard protocols. The

percentage of monocytes expressing HLA-DR was measured by flow cytometry. In

addition, the effects of incubation of monocytes with prednisolone in vitro on the

expression of HLA-DR was determined in 7 healthy volunteers. Seven patients

developed bacterial sepsis after a median 15 (range 10–20) days after transplantation.

HLA-DR expression was significantly lower in these patients on days 7, 14, 21, and 28

after transplantation compared with non-septic patients. The percentage of HLA-DR

positive monocytes was 30% or less, 3 (1–8) days before onset of sepsis. On day 7

after transplantation, HLA-DR expression on 50% or less of monocytes had a positive

predictive value for sepsis of 71%, whereas the negative predictive value was 85%.

Patients who developed sepsis received significantly more prednisolone. Incubation

with prednisolone in vitro lowered the expression of HLA-DR in a dose-dependent

manner. We conclude that low HLA-DR expression on monocytes is a marker for a

high risk of subsequent sepsis in liver transplantation patients. This high risk may be

(at least partly) related to the dose of prednisolone.

HLA-DR expression in liver transplantation

39

Sepsis remains an important cause of morbidity and mortality after liver

transplantation, despite improvements in antibiotic treatment and supportive care

(84,85). Immunosuppressive agents probably constitute the most important underlying

cause of the increased susceptibility for infections in these patients, but other factors

such as poor nutritional status, hepatic failure, surgical trauma, uremia, multiple blood

transfusions and viral infections also have a negative influence on the

immunocompetence (86). Drug-induced immunosuppression is the only one of these

factors that can be easily modified in order to prevent over-immunosuppression with its

attendent risk of severe infections. To date, drug dosage still depends on clinical

intuition, as no good parameter is available for identification of patients at high risk of

developing septic complications.

HLA-DR expression on monocytes might be a useful marker of an increased

risk of infection. Low HLA-DR expression on monocytes is associated with an

increased risk of infections in patients with trauma (23) or thermal injury (62), patients

undergoing major abdominal (24,25) or neurosurgery (87), and in patients with a

ventricular assist device awaiting cardiac transplantation (27). We have previously

reported that low HLA-DR expression preceeded clinical signs of sepsis in a small

series of liver-transplant patients (63). Here we present an extension of that study,

confirming the usefulness of HLA-DR expression on monocytes. Moreover, we present

evidence that low HLA-DR expression and sepsis may be related to the dose of

prednisolone.

PATIENTS AND METHODS

Patients

We prospectively monitored 20 adult liver transplant recipients during the first 4 weeks

after transplantation. Results in 9 of these patients have been reported previously (63).

All patients were treated according to standard protocols, without knowledge of the

results of flow-cytometric analysis. Demographic data and details on the operative

procedure are shown in Table 1. The nutritional status was determined before

Chapter 3

40

transplantation by clinical examination by two investigators. This method has been

shown to be as accurate as laboratory testing (88).

Baseline immunosuppression and rejection treatment

Standard immunosuppression consisted of ‘high-dose’ prednisolone (1.25–1.5 mg/kg/d

during the first week, tapering to 0.4–0.5 mg/kg/d at day 28), azathioprine (125 mg/d),

and cyclosporine A (target trough levels 200–250 mg/l), combined with a one-week

induction course of cyclophosphamide, as reported previously (89) in 12 patients. The

remaining patients took part in a randomized trial comparing tacrolimus with

cyclosporin A, both combined with ‘low-dose’ prednisolone (1.5 mg/kg on day 1,

tapered to 0.3 mg/kg at day 7 and 0.2 mg/kg at day 28). Target trough levels of

tacrolimus ranged between 10 and 15 ng/ml (6 patients), whereas trough levels of

cyclosporin A ranged between 200 and 300 ng/ml (two patients). The selection of

patients for this comparative trial did not depend on the risk of infection after

transplantation, or on the expression of HLA-DR on monocytes. In case of abnormal

liver tests a Doppler-ultrasound examination was performed to exclude vascular or

biliary tract problems. A liver biopsy was taken to confirm the diagnosis of rejection.

Firstline rejection treatment consisted of methylprednisolone pulses of 1 g

intravenously on 3 successive days; steroid-resistent rejection was treated with anti-

thymocyte globulin (90).

Infection prophylaxis, monitoring, and treatment

All patients received selective bowel decontamination consisting of colistine,

tobramycine, and amphotericin B until the bile drain had been clamped and full oral

intake had been resumed, as described before (91). In addition, systemic antibiotic

prophylaxis was given for 48h, consisting of cefotaxim with tobramycin, or, in case of

renal failure, imipenem monotherapy. In cases of fulminant liver failure or

retransplantation, amphothericine B was given intravenously in a dose of 0.25 mg/kg

for the first 10 postoperative days as a prophylactic measure against fungal infections.

All patients received low-dose oral acyclovir against herpes simplex virus infections.


41

No prophylaxis was given against CMV or PCP. In case of fever or suspected

infections 2 or 3 blood cultures were taken, in addition to urine and sputum cultures.

Bile, ascites, aspirates from fluid collections or abcesses, and catheter tips were also

cultured. Treatment of infections consisted of antibiotics and surgical drainage when

necessary. In case of severe infections or CMV disease, prednisolone was lowered to

10 mg/d and azathioprine to 50 mg/d.

HLA-DR expression on monocytes

EDTA anticoagulated blood was taken before transplantation, and on days 7, 14, 21,

and 28 post-transplantation. Monoclonal antibodies against CD-14 antigen (anti CD-

14-PE (phycoerythine), Immuno Quality Products, Groningen, The Netherlands) were

used to set a lifegate for monocytes. The percentage of HLA-DR+ monocytes was

determined using anti-HLA-DR FITC (fluoresceine isothiocyanate) (Becton Dickinson

Immunocytometry Systems, San Jose, CA), with an IgG2a isotype control (isotype

control IgG2a FITC, Immuno Quality Products); in the last 11 patients the signal

intensity of fluorescence was also determined. For sequential analysis the flow

cytometer was calibrated by using QC windowsTM microbeads as a standard curve

(Flow Cytometry Standards Corporation (FCSC), San Juan, Puerto Rico).

In vitro monocyte incubation with prednisolone

To determine the effect of prednisolone on HLA-DR expression by monocytes in vitro

we incubated whole blood from 7 healthy volunteers, diluted 1:10 with RPMI, with

increasing concentrations of prednisolone. After 24h of incubation at 37°C, monocytes

were stained by monoclonal antibodies and analyzed by flow cytometry as described

above.

Definitions

Sepsis, septic shock, sepsis syndrome, and multi-organ failure were defined following

the criteria of Bone (92). Sepsis was defined as a systemic response to a

microbiologically confirmed infection consisting of hypothermia or hyperthermia (core

Chapter 3

42

or rectal temperature, <35.5 or >38.0°C), tachypnea (respiration >20 breaths/min; if

mechanically ventilated, minute ventilation >10 l/min), and tachycardia (heart rate >90

beats/min).

Statistical analysis

Data are given as median (range). Patient characteristics were compared with the

Fisher’s exact test or in case of continuous data the Mann–Whitney U-test. HLA-DR

expression was compared with the Mann–Whitney U-test and with the Wilcoxon

signed rank test or Friedman’s test. For detection of correlations we used Spearman’s

rank correlation test. A P-value of 0.05 was considered to indicate statistical

significance.


43

RESULTS

Patients

Seven of the 20 patients experienced an episode of sepsis during the first 4 weeks after

orthotopic liver transplantation (OLT). No significant differences were found regarding

demographic, surgical or postoperative characteristics between patients with and

without sepsis (Table 1).

Table 1. Patient characteristics

Patients with sepsis (n=7)

Patients without sepsis (n=13)

P

Demographic characteristics Sex (male/female) 5/2 8/5 NS Age (yrs) 47 (34-60) 36 (18-61) NS Acute/elective procedure 1/6 1/12 NS Re-transplantation 0 2 NS Child Pugh (A/B/C) 1/1/5 0/5/8 NS Nutritional status (normal/moderate/poor)

5/2/0 5/5/3 NS

SBP 1 5 NS Surgical data Cold ischemic time (min) 760 (369-960) 730 (376-864) NS Warm ischemic time (min) 58 (36-89) 58 (31-76) NS Operative time (min) 520 (440-900) 703 (515-840) NS Blood loss (l) 9 (7-23) 9 (2-35) NS Post operative data Graft function (good/IPF/PNF)

5/2/0 10/3/0 NS

Immune suppressive regimen (1/2/3)

6/0/1 6/6/1 NS

prednisolone dose on day 7 (mg/d)

100 (80-100) 50 (10-100) 0.016

Other infections 6 5 NS CMV infection 2 0 NS Acute rejection 3 2 NS Died 2 0 NS Data represents number of patients or median (range). SBP = spontaneous bacterial peritonitis. Graft function: IPF = immediate poor function, PNF = primary non-function. Immune suppressive regimen: 1 = cyclophosphamide induction therapy, high-dose prednisolone, cyclosporine A and azathioprine, 2 = low-dose prednisolone and tacrolimus, 3 = low-dose prednisolone and cyclosporine A. NS, nonsignificant.

Chapter 3

44

Sepsis was first diagnosed 15 (10–20) days after transplantation. Three

patients developed sepsis with cholangitis, one of them developed sepsis syndrome

caused by Staphylococcus aureus; two had sepsis, one of these caused by S. aureus and

the other by CNS (coagulase-negative Staphylococcus). Two patients had peritonitis,

one with combined infection of S. aureus and Citrobacter, and one developed septic

shock due to infection with Serratia and S. aureus. Two patients died in the sepsis

group. Patient #7 (see Figure 2) died due to a liver rupture resulting in massive blood

loss and catheter sepsis with CNS. Patient #3 died because of fulminant sepsis and

multi-organ failure (MOF) caused by S. aureus.

HLA-DR expression on monocytes and sepsis

Figure 1 shows HLA-DR expression on monocytes in patients with and without sepsis.

Patients who suffered sepsis had a significantly lower HLA-DR expression on

monocytes on day 7 (P=0.013), day 14 (P<0.001), day 21 (P=0.004), and day 28

(P=0.010) after transplantation. In both patients with fatal outcome HLA-DR

expression on monocytes was lower than 30%, and remained so until death, whereas in

all patients who survived sepsis, expression of HLA-DR on monocytes rose during

recovery. On days 21 and 28 it was significantly higher than on day 14 (P=0.039 and

P=0.042, respectively). Figure 2 shows that in all patients with sepsis, low HLA-DR

expression was already present 3 days (1–8) before onset of sepsis. Five of the seven

patients with less than 50% HLA-DR positive monocytes on day 7 developed sepsis

(positive predictive 71%), whereas 11/13 patients with HLA-DR expression on more

than 50% of all monocytes on day 7 remained free from septic complications (negative

predictive value 85%).

The percentage of HLA-DR positive monocytes and the fluorescence intensity

of the positive signal as determined in the 46 samples taken during the second part of

the study were strongly correlated (r=0.60, P<0.001). Signal intensity was low before

onset of sepsis in the 2 patients with this complication.


45

Figure 1. HLA-DR expression on monocytes in patients with and without sepsis during the first month after transplantation. Box plots show the median, 10th, 25th, 75th, and 90th percentiles. Open circles indicate outliers.

Figure 2. HLA-DR expression in seven patients with sepsis. The horixontal axis in the middle of the figure indicates the onset of sepsis. The shaded area indicates mean ±2 SD in non-septic patients.

Chapter 3

46

Prednisolone, white blood cell, and monocytes counts in relation to HLA-DR

expression

Prednisolone dosage was significantly higher at day 7 after transplantation in patients

who developed sepsis compared to those without sepsis (1.33 mg/kg/d (1.25–1.45) vs.

0.60 mg/kg/d (0.18–1.61) respectively, P= 0.022). Differences in prednisolone dosage

were no longer significant 2 weeks or more after transplantation, probably as a result of

our tendency to taper prednisolone in patients with severe infections.

Incubation of monocytes from healthy volunteers with prednisolone in vivo did

not reduce the percentages of HLA-DR positive monocytes (P=0.134). However, the

amount of HLA-DR per monocyte, as reflected by the intensity of HLA-DR

fluorescence signal, decreased in a dose-dependent fashion (Figure 3).

Total numbers of monocytes and white blood cells did not differ in patients

who suffered a septic episode from those in patients without sepsis. Furthermore, there

were no significant correlations between the white blood cell counts or the numbers of

monocytes on the one hand, and HLA-DR expression on monocytes on the other hand

(r=-0.132, P=0.253, and r=-.253, P=0.241, respectively).

HLA-DR expression and other complications

Two patients suffered from a CMV infection. In these patients HLA-DR expression

was comparable with patients not experiencing this complication. Patients with other

infections or technical complications did not show a lower or higher percentage of

HLA-DR positive monocytes. Five patients suffered from acute rejection during the

first 4 weeks after transplantation. Three of these patients also experienced sepsis,

either shortly after (patient #3, Figure 2) or before onset of rejection. One of these

(patient #7) developed rejection 2 days after onset of sepsis when HLA-DR expression

was still very low (16%).


47

Figure 3. Effect of incubation with prednisolone on HLA-DR expression of monocytes taken from healthy volunteers.

DISCUSSION

In this study we show that HLA-DR expression on monocytes is a reliable prognostic

marker for sepsis during the first month after liver transplantation. All seven patients

who suffered from sepsis had a low percentage of HLA-DR positive monocytes (15%,

7–30) before onset of sepsis. On day 7 after transplantation, expression of HLA-DR on

50% or less of monocytes had a positive predictive value for sepsis of 71%, and a

negative predictive value of 85%. These data indicate that monitoring of HLA-DR

expression will identify patients at a high risk of developing septic complications. To

increase the reliability of this parameter, a repeated measurement of HLA-DR

expression on subsequent days might be necessary.

As shown in Figure 1, OLT patients who developed sepsis did not have lower

HLA-DR expression before transplantation. Moreover, demographic and surgical

characteristics did not differ between both groups. Although large studies have shown

that a poor pretransplant nutritional status (86,93,94) and spontaneous bacterial

Chapter 3

48

peritonitis (95) are associated with high post-transplant infection rate and mortality, it

remains difficult to determine the risk of sepsis in the individual patient. Monitoring of

HLA-DR could be helpful in adapting immunosuppression to the needs of the

individual patient. Sepsis occurred in 6 of the 12 patients on the high-dose steroid,

CsA-based regimen, and in only one of the 8 patients on low-dose prednisolone,

generally tacrolimus-based immunosuppression. The baseline conditions, difficulty of

surgery, and graft function were comparable in these groups. This leaves

immunosuppression as the most probable determinant of cause of the difference in the

incidence of sepsis. There is no strong evidence that CsA as a primary

immunosuppressant is, by itself, associated with a higher risk of infection than

tacrolimus (96). The high doses of prednisolone coadministered in the cyclosporin A

regimen may be of greater importance. Patients with sepsis received significantly more

prednisolone on day 7 after transplantation. Moreover, incubation of whole blood with

prednisolone in vitro led to a dose-dependent decrease of HLA-DR expression on

monocytes. Concentrations used in this experiment are comparable with concentrations

in OLT patients, i.e. 10-8 –10-6 (96). This suggests that decreasing of steroids in

patients with low HLA-DR expression could lower risk of sepsis and might improve

prognosis. In this respect data from Oehling et al. are highly relevant. These workers

showed that oral glucocorticoid therapy in bronchial asthma was associated with an

increased frequency of respiratory tract infections. In addition, HLA-DR expression on

monocytes was lower in patients receiving glucocorticoids compared with those who

did not receive oral glucocorticoid therapy (96). Although intensive steroid treatment

undoubtly increases the risks of sepsis, we do not think that low HLA-DR expression

was just a marker for intensive steroid therapy, because half of the patients in the high-

dose steroid group had persistently normal HLA-DR expression, whereas several

patients in the low-dose prednisolone group had low expression.

Volk et al. reported that prednisolone and azathioprine could belowered in

transplant patients with sepsis and low HLA-DR expression without precipiting

rejection (28). The fact that two of our patients suffered from acute rejection shortly

after sepsis, one of them at a time when only 16% of monocytes were HLA-DR


49

positive, suggests that it may not be realistic to expect that a single parameter might be

able to indicate the risks of both infection and rejection.

A causal relationship between low monocyte HLA-DR expression and severe

bacterial infections remains to be elucidated. HLA-DR serves as a scaffold on which

monocytes present peptides derived from exogeneous antigens to CD4+ T cells. T cells

do not play an important role in the defense against bacteria except for those that can

survive intracellularly, and it is difficult to envisage howreduced antigen presentation

would predispose to sepsis. Alternatively, low HLA-DR expression might ‘just’ be a

very early and sensitive marker of as yet subclinical infection. In agreement with this

hypothesis, Randow et al. have shown that repeated LPS stimulation induces monocyte

deactivation and down-regulates HLA-DR expression (65). This theory is difficult to

reconcile with the predictive value of low monocyte HLA-DR expression for sepsis in

patients undergoing elective major surgery (24,25). Thus, we favor the hypothesis that

low HLA-DR expression is a marker of generalized monocyte dysfunction. In

agreement with this, monocytes expressing low amounts of HLA-DR have been shown

to have reduced capacity for synthesis of pro-inflammatory cytokines like TNF-α, IL-1,

IL-6, IL-8, and IFN-γ (30). What causes this monocyte dysfunction and down-

regulation of HLA-DR remains to be determined. Both IL-10 and TGF-β have been

shown to down-regulate HLA-DR expression (72,74). Our study shows that

corticosteroid treatment may be another important factor.

If low HLA-DR expression on monocytes is indeed an indicator of a high risk

of sepsis, prompt reduction of steroids or treatment aimed at improving monocyte

function may prevent development of sepsis. Both IFN-γ and GM-CSF have been

shown to improve monocyte function in vitro and in vivo, but IFN-γ might precipitate

rejection. In contrast, GM-CSF has been administered to liver transplant recipients with

severe infections, apparently without causing rejection, and might therefore an

attractive agent for immunomodulation (97). Randomized studies are required to test

this hypothesis.

CHAPTER 4

Changes in Laboratory Values and their Relationship

with Time after Rupture of an Abdominal Aortic

Aneurysm

JW Haveman1, CJ Zeebregts1, ELG Verhoeven1,

AP van den Berg2,3, JJAM van den Dungen1, JH Zwaveling4,

MWN Nijsten1



University Medical Center Groningen, The Netherlands 4 Department of Intensive Care, University Hospital Maastricht

Surgery Today 2008;38:1091-1101

Chapter 4

52

ABSTRACT

Purpose

Many laboratory values are abnormal after surgery for a ruptured abdominal aortic

aneurysm (RAAA). However, these changes have not been comprehensively evaluated.

We analysed the changes in routine laboratory values and how these changes were

related to outcome in a consecutive series of RAAA patients.

Methods

All patients who underwent surgery for an RAAA between January 1990 and June

2003 at our hospital were included in this study. We analysed laboratory data acquired

during the first week for all patients and at discharge for survivors. We categorised 29

different measurements into six categories based on the related pathological process,

including: haematology and coagulation, metabolism, systemic inflammation, renal

function, liver function, and electrolytes.

Results

A total of 290 patients underwent RAAA surgery, with a hospital mortality of 34%.

Haemorrhage was the most common cause of early death, whereas multiple-organ

failure (MOF) was the most common cause of death several days after surgery. Most

laboratory values deviated from normal at multiple time points and they differed

significantly between survivors and non-survivors.

Conclusions

Both survivors and non-survivors of RAAA surgery displayed characteristic time-

dependent laboratory abnormalities. Awareness of these responses may help us predict

patients prone to complications.

Changes in laboratory values in RAAA

53

INTRODUCTION

Despite major improvements in emergency care, anaesthesiology, surgical equipment,

graft materials and postoperative intensive care management, hospital mortality after

surgical repair of ruptured abdominal aortic aneurysms (RAAAs) remains high and

variable (30%-70%) (43-45,47,98). Multiple-organ failure (MOF) is the major cause of

death in the intensive care unit (ICU) after successful RAAA repair (99,100).

Consequently, there are many associated laboratory abnormalities. However, data on

laboratory values and their specific time course after RAAA repair have not been

comprehensively described in the literature. Identification of common patterns as they

occur in patients who follow a benign course and those who will die could be of great

consequence. As such, one or more of these laboratory values may serve as an indicator

for the development of complications and adverse outcome, whereby unnecessary

diagnostic or therapeutic interventions may be avoided. We conducted this study to

evaluate the time-dependent changes in routine laboratory values during the first week

after RAAA repair. For this purpose we performed two comparisons: First, to find out

which values differed from the normal reference values and at what time; and second,

to find out which values differed between survivors and non-survivors and at what

time. We included essentially all routinely measured laboratory values, which were

arbitrarily grouped into six basic physiologic categories.

Chapter 4

54

PATIENTS AND METHODS

Study Design

We retrospectively analysed all patients who underwent open repair for RAAA in our

tertiary referral centre between January, 1990 and June, 2003. Survival data on this

population was published previously (35). Patients who underwent endovascular

treatment were excluded. From June 2003 onward the number of AAAs treated with

endovascular aneurysm repair (EVAR) increased rapidly. Therefore, no patients who

underwent open repair of an RAAA was included after 2003, since the results might

have been biased by excluding patients who were haemodynamically stable and

anatomically normal (101).

RAAA was diagnosed only if retroperitoneal or intraperitoneal blood, or both,

was identified during the operation. Consequently, all acute non-ruptured aneurysms

without retro- or intraperitoneal blood were excluded from the analysis. Acute

physiology and chronic health evaluation II (APACHE-II) scores were calculated in

patients who survived surgery. The APACHE-II score is based on 12 physiological and

laboratory parameters as well as on age and previous health status, measured in the first

24h of ICU admission. The APACHE-II score ranges from 0 to 71, and mortality rates

increase with an increasing APACHE-II score (102). The number of transfusions of red

blood cells (RBCs) and platelet concentrates were also recorded during the first 24h.

Laboratory Data

The laboratory values studied and their reference values are presented in Table 1.

Laboratory data were measured daily, from day 0 (representing the day of surgery). If

more than one laboratory measurement a day was available, the mean of these values

was calculated before further analysis. In the group of survivors, the laboratory values

available within 5 days of hospital discharge were recorded.

The laboratory values were grouped into the following categories: Haematology and

coagulation: activated partial thromboplastin time (APTT), fibrinogen, haemoglobin,

platelet count, prothrombin time (PT), and white blood cell count. Systemic

inflammation: C-reactive protein (CRP). Metabolic: bicarbonate, pCO2, pH, lactate,


55

glucose and pO2. Renal: creatinine and urea. Liver: albumin, alanine aminotransferase

(ALAT), alkaline phosphatase (AP), aspartate aminotransferase (ASAT), direct

bilirubin, total bilirubin, gamma glutamyl transferase (gGT), and lactate dehydrogenase

(LDH). Electrolytes: calcium, chloride, magnesium, phosphate, potassium and sodium.

Definitions of Outcome Parameters

In addition to ICU-stay and length of stay in hospital, all reoperations were

recorded. The main outcome measure was hospital mortality. In patients who died, the

following major complications were recorded: haemorrhagic shock, sigmoid necrosis,

abdominal infection, infected aortic graft, and organ failure according to the Sequential

Organ Failure Assessment (SOFA)-definitions (103). The SOFA score measures the

failure of six organ systems, respectively defined as the neurological, haemodynamic,

respiratory, renal, liver, and coagulation systems. Organ failure was defined as a SOFA

score ≥ 2 with regard to the relevant organ system (103).

Chapter 4

56

Table 1. Reference values of laboratory measurements

Laboratory measurements Reference values Activated partial thromboplastin time (APTT) 26-36 seconds Alanine-amino transferase (ALAT) 0-30 U/L Albumin 37-47 g/L Alkaline phosphatase (AP) 13-120 U/L Aspartate aminotransferase (ASAT) 0-40 U/L Bicarbonate (HCO3

-) 21-28 mmol/L Bilirubin - direct - total

0-5 mol/L 3-26 mol/L

Calcium 2.25-2.75 mmol/L Chloride 98-107 mmol/L C-reactive protein (CRP) < 5 mg/dL Creatinine 62-106 mol/L Fibrinogen 1.7-3.5 g/L Gamma glutamyl transferase (gGT) 0-65 U/L Glucose 4.0-5.4 mmol/L Haemoglobin (Hb) - male - female

8.7-10.6 mmol/L 7.5-9.9 mmol/L

Lactate 0.5-2.2 mmol/L Lactate dehydrogenase (LDH) 114-235 U/L Magnesium 0.74-1.48 mmol/L pCO2 (arterial) 4.6-6.0 kPa pH (arterial) 7.35-7.45 Phosphate 0.65-1.30 mmol/L Platelet count (PC) 150-350 x 109/L pO2 (arterial) 9.5-13.5 kPa Potassium 3.6-4.8 mmol/L Prothrombin time (PT) 11-16 seconds Sodium 132-144 mmol/L Urea 3.3-6.7 mmol/L White blood cell count (WBC) 4-10 x 109/L


57

Statistics

Data are expressed as means with standard deviation (SD) or median and interquartile

range (IQR) in case of skewed distribution. Differences between categorical variables

were tested with a Chi-square analysis. To calculate differences between continuous

variables, Student’s t-test (normal distribution) or Mann-Whitney U test (skewed

distribution) were used. P values <0.05 were regarded as significant. Since the main

purpose of our study was to define all laboratory abnormalities at different time points,

we did not perform Bonferroni's correction for multiple testing. Receiver operating

characteristic (ROC) curves were made for all laboratory values on postoperative day

(POD) 1. Laboratory values measured on POD 1 after surgery were included in a

multivariate logistic regression analysis when the area under the ROC curve was > 0.6.

Chapter 4

58

RESULTS

Patients

The clinical characteristics of the 290 patients are summarised in Table 2. The mean

age was 71 (±8) years. The mean APACHE-II score was 18 (±7) on ICU admission,

being 23 (±8) for patients who died after surgery vs. 16 (±6) for those discharged from

hospital (p<0.001). Intra-operative mortality was 11% (31/290) and hospital mortality

was 34% (100/290), including the 31 patients who died intra-operatively. Table 3

shows the main causes of death in relation to the times of death of the 100 non-

survivors. Haemorrhagic shock was the most common cause of death during, and in the

first hours after surgery, whereas MOF with or without abdominal sepsis was the most

common cause of death ≥24h after surgery.

Laboratory Data

We analysed 29 laboratory measurements in the 290 RAAA patients, with a total of

49 726 laboratory values. For the survivors, we recorded 261 median laboratory values;

namely, 29 measurements done on 8 days plus the day of discharge. In 122 (47%) of

the 261 medians, these values were not within the reference value range. For the non-

survivors we recorded 126 (54%) of 232 medians; namely, 29 measurements done on 8

days. There were significant differences between the survivors and non-survivors in 95

(41%) of the 232 medians that were compared. These abnormalities were clearly time-

dependent in the survivors and non-survivors. Deviations from the normal range and

differences between survivors and non-survivors were evaluated according to the six

categories defined in figures 1, 2, and 3. Electrolytes and some of the data not

displayed in the figures are shown in Table 4.

The following data are not discussed in this article: chloride and sodium,

APTT, ALAT, AP, ASAT, and direct bilirubin. This is because chloride and sodium

values were not clinically different between survivors and non-survivors, APTT has a

similar pattern to PT (Figure 1), ALAT and ASAT have a similar pattern to LDH

(Figure 3), AP has a similar pattern to gGT (Figure 3), and direct bilirubin has a similar

pattern to total bilirubin.


59

Table 2. Clinical characteristics of the 290 patients who underwent repair of a ruptured

abdominal aortic aneurysm

Survivors

N = 190

Non-survivors

N = 100 P-value

Demographic characteristics

Age (yrs) 70 (±8) 73 (±7) 0.001

Gender (male) 161 (85%) 87 (87%) NS

Risk factors

Platelet count before surgery

(x 109/L) 195 (±95) 161 (±102) 0.024

Serum creatinine before

surgery (μmol/L) 131 (±100) 148 (±129) NS

Suprarenal clamping 6 (3%) 12 (12%) 0.02

No. of RBC transfusions

during surgery 8 (±7) 14 (±12) <0.001

No. of platelet concentrate transfusions during surgery

.17 (±.47) .27 (±.64) NS

APACHE-II 16 (±6) 23 (±8) < 0.001

Postoperative data

Re-operations 34 (18%) 39 (39%) 0.007

Sigmoid resection 11 (6%) 19 (19%) 0.005

Rebleeding 11 (6%) 13 (13%) NS

ICU length of stay (days)

9 (±14)

10 (±14)

NS

Hospital length of stay (days) 26 (±28) 10 (±14) < 0.001

RBC: red blood cell unit, APACHE-II: Acute Physiology and Chronic Health Evaluation, ICU: intensive care unit

Chapter 4

60

Table 3. Main causes of death in relation to time in the 100 non-survivors of a ruptured

abdominal aortic aneurysm

Main cause Time of death

(days)*

Number of deaths according to phase

Intra-operative

<24h at ICU

≥ 24h

Haemorrhagic shock 0.2 (±0.4) 31 5

Brain death 4 1

Sigmoid necrosis with MOF 6 (±7.9) 1 10

Renal failure (no RRT) 6.7 (±3.5) 3

Pneumonia 9 1

Pulmonary embolism 10.5 (±7.8) 2

Gastro-intestinal bleeding 10.5 (±6.4) 2

Multiple-organ failure † 13.5 (±11.1) 5 18

Abdominal sepsis with MOF 17 (±6.2) 7

Cardiogenic shock 17.3 (±5.7) 4

Respiratory failure 21.6 (±8.1) 5

Infected aortic graft with MOF 37 (±33.8) 5

Total 31 11 58

When there were multiple causes of death, the prime cause was selected. RRT: renal replacement therapy. * Mean (± standard deviation) or the day the patient died † Multiple-organ failure without sepsis. MOF: Multiple-organ failure


61

In the haematology and coagulation category, the following differences were

seen: Haemoglobin was decreased in both groups, but it was lower at all times in the

non-survivors. The platelet count dropped in the first days after surgery and failed to

recover in non-survivors. Leukocyte counts were slightly increased, with no clear

time-dependent changes or differences between survivors and non-survivors.

Fibrinogen dropped initially and then increased after 2 days to supra-normal values,

which were slightly higher in the survivors. PT increased immediately after the

operation in the non-survivors. At discharge almost all the laboratory values in the

haematology and coagulation category had returned to normal in the survivors, except

for haemoglobin, which was still low (Figure 1, Table 4).

As a measurement that reflects systemic inflammation, CRP increased

immediately, to much higher levels in the non-survivors peaking on PODs 3 and 4. The

median CRP remained above the reference value until discharge in the survivors

(Figure 1).

In the metabolic category, the following differences were seen: pH and

bicarbonate showed a marked metabolic acidosis, which recovered during the first

week after surgery and was more pronounced in the non-survivors throughout this

period. Analogously, lactate levels increased immediately, to much higher levels in the

non-survivors. Lactate was almost never measured at the time of discharge. The pO2-

levels were initially elevated and returned to normal after 2 days, with no differences

between survivors and non-survivors. Glucose levels were elevated in the survivors

and non-survivors, especially in the first 2 days. No differences or changes were noted

in pCO2 levels (Figure 2, Table 4).

In the renal category, both creatinine and urea were persistently elevated from

POD 1 to POD 7 in the non-survivors, whereas in the survivors, creatinine remained

low and urea was only slightly elevated after surgery, but returned to normal by the

time of discharge (Figure 2, Table 4).

In the liver category, the following changes were seen: LDH was increased in

both groups, both more so in the non-survivors from day 2 onwards. There was a time-

Chapter 4

62

dependent rise in gGT, which was largely within the reference values. Remarkably, the

rise in gGT was higher in survivors than in non-survivors, which persisted until

discharge. In fact, of all the laboratory values analysed, gGT was the only one that

showed a larger deviation from the reference values in survivors than in non-survivors.

Total bilirubin increased gradually above the reference values during the first week,

especially in the non-survivors. Albumin decreased immediately after surgery and

remained unchanged during the first week, with lower levels in the non-survivors

(Figure 3).

In the electrolyte category the following differences were seen: Calcium was

lower in the non-survivors from POD 2 to POD 6. Magnesium was higher in the non-

survivors from POD 5 to POD 7. Phosphate was higher in the non-survivors from day

0 to POD 4 (Table 4).

The area under the ROC curve was more than 0.6 in nine laboratory

measuremernts; namely, APTT, total bilirubin, creatinine, lactate, phosphate, pH,

potassium, PT, and urea. In addition to these laboratory values, age, APACHE-II score,

number of RBC transfusions during surgery and suprarenal clamping and platelet count

before surgery were included in the multivariate analysis because of significant

differences, as outlined in Table 2. After stepwise logistic regression analysis, age,

APACHE-II score, number of RBC transfusions during surgery and serum potassium

level remained significant and were identified as an independent predictor of death

after RAAA. The odds ratios with a 95% confidence interval were as follows: age, 1.07

(1.02-1.13); number of RBC transfusions, 1.05 (1.01-1.10); APACHE-II score, 1.16

(1.09-1.23); and potassium, 2.64 (1.41-4.95)

To classify the various time-dependent laboratory abnormalities in RAAA

patients, we summarised them in Figure 4. Five major pathological processes were

responsible for important laboratory derangements. Important laboratory abnormalities

were seen after RAAA surgery in all patients, but they were more pronounced in the

non-survivors.


63

Figure 1. Haematology and inflammatory measurements in the 290 patients who underwent surgery for a ruptured abdominal aortic aneurysm. * p<0.05, open squares=survivors (n=190) and closed squares=non-survivors (n=100). D, laboratory values in survivors at discharge. Hb, haemoglobin; PT, prothrombin; CRP, C-reactive protein

Chapter 4

64

Figure 2. Renal and metabolic measurements in the 290 patients who underwent surgery for a ruptured abdominal aortic aneurysm. * p<0.05, open squares=survivors (n=190) and closed squares=non-survivors (n=100). D, laboratory values in survivors at discharge.


65

Figure 3. Liver function test measurements in the 290 patients who underwent surgery for a ruptured abdominal aortic aneurysm.* p<0.05, open squares=survivors (n=190) and closed squares=non-survivors (n=100). D, laboratory values in survivors at discharge, but this was not available for lactate. LDH, lactate dehydrogenase; gGT, gamma-glutamyltransferase

Chapter 4

66

Figure 4. Pathological processes in the 290 patients who underwent surgery for a ruptured abdominal aortic aneurysm.Summary of patterns and duration of the different pathological responses leading to abnormalities in laboratory values


67

DISCUSSION

We confirmed distinct laboratory responses in all patients who underwent RAAA. The

laboratory data were more abnormal in the non-survivors than in the survivors,

although they were often outside the reference range in the survivors as well. To our

knowledge, this is the first study to chart these changes comprehensively. The main

goal of our study was to perform univariate analysis of all routine laboratory

measurements at different time-points. Many of the univariate comparisons revealed a

significant difference with a two-sided p-value of < 0.05. When using a p-value of

0.05 as a matter of chance, we would expect 5% or 12 comparisons out of the 232

comparisons between survivors and non-survivors to be significant. In reality, we

found that not 12, but 95 of the 232 comparisons showed a significant difference,

underscoring that many laboratory values differ between survivors and non-survivors.

This study did no aim to identify prospective measurements that predict death, but if

we focus only on day 0 or POD 1, the following variables were differed significantly

between survivors and non-survivors: Hb, fibrinogen, pH, PT, lactate, creatinine, urea,

phosphate, and potassium. The most apparent reasons for the differences in these

values were blood loss and diffuse intravascular coagulation, accounting for Hb,

fibrinogen and PT; lactic acidosis, accounting for pH and lactate; and acute renal

failure, accounting for creatinine, urea, phosphate, and potassium.

Although our main purpose was univariate analysis, we also performed

multivariate analysis, after which age, APACHE-II score, RBC transfusions during

surgery, and potassium remained an independent predictor for outcome. Age,

APACHE-II score, and blood-loss reflected by RBC transfusions during surgery are

well known independent predictors of mortality in many surgical patients. The fact that

potassium remains the only independent biochemical value that predicts outcome also

underscores the limitations of multivariate analysis in this study of 29 biochemical

values. For multivariate analysis, Altman suggests that multiple regressions should not

be applied to small data sets, and that the number of tested variables should be no more

than the square root of the sample size or than the sample size divided by 10 (104).

This explains why only a few values on POD 1 were independent predictors of

Chapter 4

68

outcome with multivariate analysis in our series. It may also underscore the strong

interrelationship of the laboratory values measured. Obviously, our data set did not

allow multivariate analysis at multiple time points after POD 1.

We chose to aggregate the laboratory responses according to the six categories

in this study. Obviously, there will be extensive overlap when artificial classifications

are used. For example, the white blood cell count may be a haematological measure or

an inflammatory measure. Similarly, albumin can be regarded as a measure of

metabolic or inflammatory liver dysfunction. The categories we chose are strongly

related to the causes of death as patients suffer, and subsequently die of massive

haemorrhage, mainly during and within 24h after surgery, whereas inflammatory

response and MOF occur more than 24h after surgery (Table 3).

Haematology and Coagulation

Several laboratory variables showed that non-survivors suffered from more extensive

bleeding than survivors, as underscored by the multivariate relationship between RBC

transfusions and mortality. Severe coagulation disturbances were also seen more

frequently in non-survivors post-surgery, leading to relatively high mortality within the

first 24 hours (Table 3). The pre-operative platelet count was lower in the non-

survivors than the survivors, as reported in other studies (105,106). Fibrinogen, and PT

were also significantly more disturbed in the non-survivors, as reported in several other

studies on RAAA patients (105,107,108). A low postoperative platelet count reflects

intra-operative blood loss and disseminated intravascular coagulation (DIC). The

increasing platelet count seen later in the non-survivors was probably the result of

ongoing systemic inflammation (109). Rebleeding occurred relatively infrequently in

this cohort of RAAA patients, since only 8% (24/290) of our patients required

reoperation for this complication.

Systemic Inflammation

CRP, a commonly used marker of inflammation, was increased in the survivors and

non-survivors, indicating severe systemic inflammation. Non-survivors had a more

severe and longer duration of severe inflammation (Figure 1). Accordingly, CRP was


69

significantly higher in the non-survivors on PODs 3 and 4. The serum concentration of

CRP correlates with the clinical course of infection (110). The platelet count decreased

after surgery and remained low in the non-survivors, primarily as a result of systemic

inflammation (109). Serum albumin is also decreased as a result of redistribution and

down-regulation in severe inflammation and is associated with poor outcome in

critically ill patients (111).

Metabolism

Aortic cross-clamping causes ischaemia and reperfusion injury to distal tissues (112).

The massive blood transfusions and haemorrhage in RAAA patients augment the tissue

damage. The ischaemia reperfusion injury is reflected by lactate acidosis and elevated

enzymes (Figures 2 and 3). Serum lactate is an important prognostic value in critically

ill patients, trauma patients and RAAA patients immediately after the operation (113-

117). Accordingly, the serum lactate levels were higher on day 0 to POD 4 in the non-

survivors, indicating more profound tissue hypoperfusion. These metabolic and acid-

base abnormalities take a long time to recover, as shown by the course of pH post-

surgery.

Acute Renal Failure

Acute renal failure (ARF) characteristically developed within 2 days and was reflected

by a significant increase in serum creatinine and urea. Gordon et al reported an

incidence of ARF of 15% in 91 patients who underwent RAAA surgery (118). ARF is

associated with high mortality, ranging from 38-87%, in RAAA patients (108,118-

121). Preoperative renal dysfunction is also associated with a poor outcome (122-124).

Liver Failure

Liver failure was reflected by a late increase in LDH, gGT, and bilirubin (Figure 3). In

ICU patients in general, liver failure develops late in the MOF cascade (125). Sprung et

al. reported poor outcome in patients undergoing elective AAA repair if they had liver

function test abnormalities (126). In a limited number of acute AAA patients, it is also

associated with high mortality (127). Durrani et al reported a 3% incidence of liver

Chapter 4

70

failure after RAAA repair (128). We cannot explain why gGT was one of the only two

measurements that deviated more from the reference values in the survivors than the

non-survivors. The isolated increase in bilirubin without other liver function

abnormalities in these patients may be the result of haemolysis of transfused blood and

resorption of the retroperitoneal haematoma that exists in almost all patients (129).

Electrolytes

The differences in electrolytes were less pronounced. One possible explanation for this

is that electrolytes depend heavily on infusion rates or renal replacement therapy. The

differences in phosphate and potassium were probably attributable to the higher rate of

renal failure in the non-survivors (130). The fact that only potassium emerged from the

multivariate analysis as an independent laboratory predictor of mortality on POD 1 is

of little consequence since the median potassium levels in survivors and non-survivors

differed by only 0.2 mmol/L and both were within the reference values.

In general, when the surviving patients were discharged from hospital after

about 1 month, most of their laboratory values had normalised, the most notable

exceptions being a decreased haemoglobin value, a mildly increased CRP value, and

discrete liver enzyme abnormalities.

In conclusion, RAAA patients show well-defined laboratory responses after

surgery, starting with haematological and coagulation abnormalities, and followed by

inflammatory, metabolic, renal, and finally liver derangement. These abnormal values

are more prominent in non-survivors, although survivors also show a wide range of

laboratory abnormalities. We believe that recognition of these abnormalities is

important since it may help to identify and predict post-operative complications early.

CHAPTER 5

HLA-DR expression on monocytes and systemic

inflammation with ruptured abdominal aortic aneurysms

JW Haveman1, AP van den Berg2,3, ELG Verhoeven1,

MWN Nijsten1, JJAM van den Dungen1, TH The2,

JH Zwaveling1




Critical Care 2006;10:R119

Chapter 5

72

ABSTRACT

Introduction

Mortality from ruptured abdominal aortic aneurysms (RAAA) remains high. Severe

systemic inflammation, leading to multi-organ failure, often occurs in these patients. In

this study we describe the level of HLA-DR expression in a consecutive group of

patients following surgery for RAAA and compare results between survivors and non-

survivors. A similar comparison is made for IL-6 and IL-10 levels and Sequential

Organ Failure Assessment (SOFA) scores.

Methods

This is a prospective observational study. Patients with RAAA were prospectively

analysed. Blood samples were collected on days 1, 3, 5, 7, 10 and 14. The fraction of

CD-14 positive monocytes expressing HLA-DR was measured by flowcytometry. IL-6

and IL-10 levels were measured by ELISA.

Results

The study included 30 patients with a median age of 70 years, of which 27 (90%) were

men. Six patients died from multiple organ failure, all other patients survived. The

SOFA scores were significantly higher in non-survivors on days 1 through 14. HLA-

DR expression on monocytes was significantly lower on days 3, 5, 7, 10 and 14 in non-

survivors. IL-6 and IL-10 levels were significantly higher in non-survivors on day 1

and days 1 and 3, respectively.

Conclusion

HLA-DR expression on monocytes was decreased, especially in non-survivors. All

patients with RAAA displayed a severe inflammatory and anti-inflammatory response

with an increased production of IL-6 and IL-10. Poor outcome is associated with high

levels of IL-6 and IL-10 and a high SOFA score in the first three days after surgery,

while low levels of HLA-DR expression are observed from day three after RAAA

repair.

Systemic inflammation and HLA-DR in RAAA

73

INTRODUCTION

Mortality in patients following repair of a ruptured abdominal aortic aneurysm

(RAAA) remains high (30% to 70%), despite important advances in emergency

medicine, anaesthesiology, surgery and intensive care (43-47). The postoperative

course of patients after RAAA repair is almost always characterized by systemic

inflammation, sometimes leading to multiple-organ failure, a prolonged intensive care

unit (ICU) stay and a high mortality (99,100,131). Down-regulation of HLA-DR

expression on monocytes has been reported in different groups of surgical patients and

has been associated with septic complications and increased mortality

(12,23,24,29,79). We studied the expression of HLA-DR on monocytes in patients

following surgery for RAAA, taking into account levels of IL-6 and IL-10 and

Sequential Organ Failure Assessment (SOFA) scores. The primary aim of this study

was to describe the level of HLA-DR expression in these patients and to establish, if

possible, whether low HLA-DR expression was associated with increased mortality as

a result of secondary infections.

MATERIALS AND METHODS

Patients and design of the study

Patients with RAAA who survived surgery were prospectively analysed and included

in the study. Patients who underwent endovascular treatment were excluded. Cases

were only classified as RAAA when an aortic aneurysm and retroperitoneal or

intraperitoneal blood were present. The study was approved by our Medical Ethics

Committee. Written informed consent was obtained from a family member.

For each patient, one healthy employee of the laboratory served as normal

control.

On days 1, 3, 5, 7, 10 and 14, 10 ml of EDTA blood was withdrawn from the

patient and HLA-DR expression on monocytes was analysed immediately. For IL-6

and IL-10 measurements blood was kept on ice, centrifuged at 1,655 g at 4°C for 10

minutes and stored at -80°C until analysis.

Chapter 5

74

Acute Physiology and Chronic Health Evaluation (APACHE)-II scores were

calculated on ICU admission (102). The SOFA score was measured daily after surgery

(103). ICU-acquired infections were defined according to the criteria issued by the

Centres for Disease Control and Prevention. All infections were recorded

prospectively. Sepsis was defined according to Bone and colleagues (56).

Laboratory analysis

C-reactive protein (normal value <5 mg/dl) and white blood cell count (normal value 4

to 10 × 109/l) were measured every day.

IL-6 and IL-10 were measured by ELISA in 26 patients (21 survivors and five

non-survivors), using a monoclonal antibody against human IL-6 (Sanquin,

Amsterdam, the Netherlands) or IL-10 (BD Pharmingen, Alphen a/d Rijn, the

Netherlands).

The percentage of CD-14 positive monocytes expressing HLA-DR was

measured by flow-cytometry. Monoclonal antibodies against CD-14 antigen (anti-CD-

14-PE, Immuno Quality Products, Groningen, the Netherlands) were used to set a gate

for monocytes. The percentage of HLA-DR on monocytes was determined using anti-

HLA-DR fluorescein isothiocyante (Becton Dickinson Immunocytometry Systems,

San Jose, CA, USA), with an IgG2a isotype control (IgG2a FITC, Immuno Quality

Products). A live gate was set using forward and sideward scatter characteristics. A

monocyte gate was set by the CD-14+ group. Data were analysed using Cell Quest

software (Becton-Dickinson).

Statistics

Data are given as median with interquartile range. Differences between categorical

variables were tested with Chi-square analysis. The Mann-Whitney U or Kruskal-

Wallis test was performed to calculate differences in continuous variables. For

detection of correlation we used Spearman's rank correlation test. The rank correlation

coefficients were averaged after z-transformation. P values < 0.05 were regarded as

statistically significant.


75

RESULTS

Patients

During the course of the study 46 patients with RAAA were admitted to our Hospital.

All patients were operated upon. Sixteen patients were not included in this study: six

were endovascular treated, five died during surgery, four patients were not included

because of absence of one of the primary investigators (JWH or APvdB), and for one

patient no informed consent was obtained.

Of the remaining 30 patients, the median age was 70 (64 to 75) years and 27

patients (90%) were men. Six patients died and 24 survived until hospital discharge.

Clinical characteristics of survivors and non-survivors are shown in Table 1. The non-

survivors were significantly older, had a higher APACHE-II score and more sigmoid

necrosis was observed. Blood-loss, lowest systolic blood pressure and suprarenal

clamping did not significantly differ between survivors and non-survivors.

Table 2 displays the intra- and postoperative complications of the non-

survivors. The six non-survivors died on days 2, 3, 4, 12, 21 and 30 after RAAA repair.

In three of these patients the sigmoid colon had to be resected because of ischemic

necrosis. Two patients had a culture proven infection. All patients died from multiple-

organ failure.

The SOFA score did not differ significantly between survivors and non-

survivors upon arrival in the ICU, but was significantly higher on days one through 14

in the non-survivors (Figure 1).

C-reactive protein and white blood cell count

The median C-reactive protein level increased postoperatively. In non-survivors and

survivors C-reactive protein (mg/dl) was 84 versus 31 on day 1, 283 versus 190 on day

3, 212 versus 157 on day 5 and 167 versus 159 on day 7. The median white blood cell

count (× 109/l) was 9.6 versus 10.0 on day 1, 7.8 versus 9.5 on day 3, 7.9 versus 9.4 on

day 5 and 12.6 and 10.0 on day 7 in non-survivors and survivors, respectively. All

differences were non-significant.

Chapter 5

76

Table 1. Characteristics of the survivors and non-survivors.

Survivors (N=24)

Non-survivors (N=6)

P

Demographic characteristics Age 68 (64-72) 78 (73-79) <.01 Sex (male) 21 (88%) 6 (100%) NS Intra-operative data

Lowest systolic RR (mmHg)

77 (53-90) 65 (23-79) NS

Blood-loss (litres) 3.8 (1.6-5.4) 4.8 (2.0-12.9) NS Suprarenal clamping (yes)

5 (21%) 2 (33%) NS

Post-operative data APACHE-II score 13 (9-16) 19 (16-23) .01 Re-operations (total) 10 4 NS - Sigmoid resection - Rebleeding - Lower leg amputation

2 (8%) 0

1 (4%)

3 (50%) 0 0

0.04 NS NS

Infectious complications (total patients)

7 2 NS

- Septic shock - Abdominal infection - Pneumonia - CVC infection - Wound infection

(17%) 3 (13%) 4 (17%) 2 (8%)

0

2 (33%) 1 (4%)

0 0

1 (4%)

NS NS NS NS NS

Renal replacement therapy 4 (17%) a 2 (33%) b NS Hydrocortisone treatmentc 3 (13%) 1 (17%) NS ICU length of stay (days)

7 (2-16) 8 (3-23) NS

Hospital length of stay (days)

20 (11-33) 8 (3-23) NS

Data are given as medians with interquartile range, or absolute number of patients with percentage of the total population. NS = non-significant. CVC infection; Central venous catheter related infection. a All patients had a full recovery of renal function at hospital discharge.b In an additional 2 patients renal replacement therapy was indicated but not performed because it was considered futile. c Hydrocortisone treatment was initiated for relative adrenal insufficiency.


77

Table 2. Clinical data in the six non-survivors.

Age Died on day

Resuscitation during surgery

Blood-loss

(L)

Complications Organ failure Infectious complication

79

2 - 7.0 Respiratory and renal

-

80 3 - 9.5 Sigmoid necrosis, ischemic lower legs

Respiratory, cardiovascular and renal

-

74

4 - 2.0 Respiratory, cardiovascular and renal

-

79 12 - 2.5 Sigmoid necrosis Respiratory, hepatic, cardiovascular and renal

Enterococi in blood culture

79 21 - 3.0 Wound infection Respiratory, cardiovascular and renal

Proteus mirabilis in wound and blood

76 30 + 25.0 Sigmoid necrosis

Respiratory, cardiovascular and renal

-

Chapter 5

78

Figure 1. SOFA score after surgery for RAAA patients.The SOFA score was significantly (as indicated by*) higher in non-survivors than survivors from day 1 post-surgery onwards.


79

Cytokine production

IL-6 and IL-10 were elevated in all RAAA patients post-surgery (Figures 2 and 3).

Median IL-6 was significantly higher in non-survivors versus survivors on day 1;

median (interquartile range) 543 pg/ml (90 to 701) versus 122 pg/ml (39 to 137), p =

0.03. IL-10 was significantly higher on days 1 and 3 postsurgery in the non-survivors

(p = 0.03 for both days).

HLA-DR expression on monocytes

On day one after surgery HLA-DR expression on monocytes was comparable in

survivors and non-survivors, and significantly lower than the 76% to 96% observed in

healthy controls. In survivors, HLA-DR expression rose to normal levels, whereas it

decreased further and remained low in non-surviving patients. Percentages of

monocytes expressing HLA-DR were significantly lower on days 3, 5, 7, 10 and 14 in

the patients who died (Figure 4) compared to survivors. No significant differences were

found between HLA-DR expression in the patients who developed infections (two non-

survivors and seven survivors) and those who did not develop infections.

HLA-DR expression on days 1, 3, 5, 7, 10 and 14 had a significant negative

correlation with the SOFA score on these subsequent days. After z-transformation,

mean r = -0.416, 95% confidence interval (CI; -0.56 to -0.25), p < 0.01. The correlation

coefficient between HLA-DR expression and IL-6 was r = -0.055, 95% CI (-0.26 to

0.15), p = 0.60. The correlation coefficient between HLA-DR expression and IL-10

was r = -0.078, 95% CI (-0.28 to 0.13), p = 0.47.

Chapter 5

80

Figure 2. IL-6 after RAAA repair. Levels of IL-6 (normal value < 20 pg/ml) were significantly higher on day one in nonsurvivors. *p < 0.05.

Figure 3. IL-10 after RAAA repair. Levels of IL-10 (normal value < 10 pg/ml) were significantly higher on days one and three in non-survivors. *p < 0.05.


81

DISCUSSION

This study shows that, in the first days after RAAA repair, patients develop a

generalised increase in immunoregulatory cytokines as reflected by elevated levels of

IL-6 and IL-10. HLA-DR expression on monocytes is reduced and remains

consistently low in non-survivors, while it returns to normal levels in survivors. Early

high levels of IL-6 and IL-10 and subsequently reduced HLA-DR were all associated

with multiple-organ failure and death.

Several studies have shown that a severe inflammatory response is associated

with multiple organ failure and poor outcome in RAAA patients (23,132,133). It is

believed that, in RAAA patients, haemorrhagic shock, surgical trauma and ischemia

reperfusion injury all contribute to this overwhelming inflammatory response. Blood-

transfusions and surgery for sigmoid necrosis may also modulate the inflammatory

response (134,135). Our study confirms the presence of such an inflammatory response

by demonstrating an increased production of IL-6. Furthermore, the SOFA score was

significantly higher in the non-survivors from day 1 through day 14, with an increase in

difference compared to survivors from day three (Figure 1). The anti-inflammatory

cytokine IL-10 was significantly higher on days 1 and 3 in the non-survivors. Our

findings are in accordance with a study described by Lekkou and colleagues (136) who

studied 30 patients with severe sepsis and noted that HLA-DR expression was lower in

non-survivors. Furthermore, these authors also found an initial high level of IL- 6 in

non-survivors and high IL-10 on days 3, 10 and 13. The association of an initial high

level of IL-6 with organ failure and poor outcome is confirmed in patients with sepsis,

after trauma and AAA patients (137-140). The initial increase in IL-10 levels is also

described in patients after orthopaedic trauma and pancreatitis (141,142). The initial

hyperinflammatory state followed by immunoparalysis, expressed as a prolonged

increase in IL-10, could not be confirmed in these patientgroups. In patients with septic

shock, Monneret and colleagues described a significantly lower HLA-DR expression

and higher IL-10 in non-survivors (143). Caille and colleagues (144) described that

HLA-DR expression was low in septic shock but not decreased in patients with

haemorrhagic shock. One might conclude that our data from RAAA patients are in

Chapter 5

82

contrast with these findings. However, Caille and colleagues described patients with

trauma and postpartum haemorrhage; these patients do not suffer from an additional

ischemia reperfusion injury. RAAA patients experience haemorrhagic shock and

ischemia reperfusion injury simultaneously.

Figure 4. HLA-DR expression on monocytes after RAAA repair. The expression of HLA-DR on monocytes (normal range 76% to 96%) of patients after RAAA is sharply and significantly decreased from day three post-surgery onwards. a,p = 0.04; b,p = 0.02; c,p < 0.01.

The causal relationship between low HLA-DR expression and poor outcome in

ICU patients remains an interesting point of discussion. In patients with sepsis, low

HLA-DR expression is associated with monocyte deactivation, an anti-inflammatory

cytokine profile, infectious complications and death (22,30). In patients with RAAA

we could only partially confirm these findings. As shown in Figure 4, a decrease in

HLA-DR expression on monocytes is associated with a poor outcome. However, the

presence of a sustained anti-inflammatory response in these patients is difficult to


83

envisage, considering the fact that IL-10 levels are low from day three post-surgery,

following an initial rise. In our series, only two non-survivors developed culture-

proven infection. One of these patients had necrosis of the sigmoid colon and the other

developed a wound infection in the presence of extensive organ failure. The majority of

the patients died from multiple organ failure, not from overwhelming infection due to

functional immunosuppression (Table 2). In theory, early death from multiple organ

failure may have prevented the onset of severe infection, but this remains speculative.

Low HLA-DR expression in the non-survivors might also be the result of their older

age, although this correlation could not be confirmed (145,146). Alternatively, low

HLA-DR expression may not be causally related to mortality. As such, HLA-DR

expression may be no more than a coincidental finding. Bloodloss and sigmoid

resection may also significantly alter the immune response and HLA-DR expression,

although no significant differences were found between these factors in survivors

versus non-survivors.

What can we do to improve the survival of RAAA and other surgical patients?

Since blood-loss and duration of surgery are related to the development of multiple

organ failure, meticulous technique will increase the chances of survival. The recent

advances in endovascular surgery are promising (48,50,51), although long-term

durability is unknown (147-150). Because of the shortcomings of endovascular

surgery, RAAA patients with a severe inflammatory response after open surgery will

continue to be presented to the ICU and a significant number of them will not survive.

In theory, hydrocortisone might also lead to a better outcome in RAAA patients. In

patients with sepsis and relative adrenal insufficiency, hydrocortisone suppletion

improves prognosis (151). In addition Keh and colleagues (152) showed that

hydrocortisone restored haemodynamic stability and modulated the immunological

response. These effects might also benefit RAAA patients since adrenal insufficiency

can be identified in a significant amount of them (153). The clinical effect of

hydrocortisone suppletion in RAAA patients needs to be evaluated further.

Our study has its limitations, mainly because of the small patient numbers and

the relatively low percentage of non-survivors. On the other hand, these low numbers

Chapter 5

84

were enough to reach statistical significance on HLA-DR expression. This is probably

related to the fact that RAAA patients present as an homogenous group with a well-

defined insult, as reflected in similar values for blood-loss, lowest systolic blood

pressure and suprarenal clamping.

Conclusion

Patients with RAAA displayed a severe inflammatory response post-surgery,

with markedly increased immunoregulatory cytokines. HLA-DR expression was low in

non-survivors from the day of surgery onwards. In contrast to survivors, in whom

levels returned to normal values, it remained low. Organ failure was present in non-

survivors from day 1 and was the primary cause of death. A relationship between

impaired monocyte function and death from infectious causes was not apparent in our

series. More likely, the severe initial insult in non-survivors is probably responsible for

both their low HLA-DR expression on monocytes and their onset of fatal multiple

organ failure.

Acknowledgements

The authors thank Geert Mesander and Johan Bijzet for their assistance in laboratory

analysis and Wim J Sluiter for statistical advice. This study was financially supported

by the Ambroise Paré foundation.

CHAPTER 6

Results of streamlined regional ambulance transport and

subsequent treatment of acute abdominal aortic

aneurysm

JW Haveman, A Karliczek, ELG Verhoeven, IFJ Tielliu,

R de Vos, JH Zwaveling, JJAM van den Dungen,

CJ Zeebregts, MWN Nijsten

Department of Surgery


Emergency Medicine Journal 2006;23:807-810

Chapter 6

86

ABSTRACT

Objective

To describe the triage of patients operated for non-ruptured and ruptured abdominal

aortic aneurysms (AAAs) before the endovascular era.

Design

Retrospective single-centre cohort study.

Methods: All patients treated for an acute AAA between 1998 and 2001 and admitted

to our hospital were evaluated in the emergency department for urgent AAA surgery.

All time intervals, from the telephone call from the patient to the ambulance

department, to the arrival of the patient in the operating theatre, were analysed.

Intraoperative, hospital and 1-year survival were determined.

Results

160 patients with an acute AAA were transported to our hospital. Mean (SD) age was

71 (8) years, and 138 (86%) were men. 34 (21%) of these patients had symptomatic,

non-ruptured AAA (SAAA) and 126 patients had ruptured AAA (RAAA). All patients

with SAAA and 98% of patients with RAAA were operated upon. For the patients with

RAAA, median time from telephone call to arrival at the hospital was 43 min

(interquartile range 33–53 min) and median time from arrival at the hospital to arrival

at the operating room was 25 min (interquartile range 11–50 min). Intraoperative

mortality was 0% for SAAA and 11% for RAAA (p = 0.042), and hospital mortality

was 12% and 33%, respectively (p = 0.014).

Conclusions

A multidisciplinary unified strategy resulted in a rapid throughput of patients with

acute AAA. Rapid transport, diagnosis and surgery resulted in favourable hospital

mortality. Despite the fact that nearly all the patients were operated upon, survival was

favourable compared with published data.

Regional transport and treatment of acute AAA

87

As untreated ruptured abdominal aortic aneurysm (AAA) has an almost 100%

mortality, rapid diagnosis and treatment are essential goals. Nevertheless, several

recent series report a hospital mortality >50% for patients with acute AAAs

(44,45,47). These figures include only patients who arrive alive at the hospital.

Therefore, total mortality for acute AAA may approximate 80–90% (43,154,155).

Delay in treatment might significantly influence mortality; therefore, measures to

reduce time to surgery can be valuable in decreasing mortality. Very few studies

describe the delay to surgery (107,156-158). AbuRahma et al describe a mortality of

73% in patients operated after a delay of >2h, whereas patients who were operated

within 2h had a mortality of 48% (p=0.05) (107). Both transportation by ambulance

and intrahospital management protocol have an influence on delays to surgery.

This study aims to present the results of a strategy of direct ambulance

transport to our tertiary referral centre when acute AAA was suspected, followed by

minimal diagnostics and emergency surgery for nearly all patients.

MATERIALS AND METHODS

Patients

This is a retrospective cohort study of consecutive patients undergoing emergency

surgery for acute AAA at a university centre. All patients admitted alive to our hospital

with acute AAAs between 1998 and 2001 were included in the study. Patients were

identified from the ambulance database, hospital and medical records using the

International Classification of Diseases—Ninth Revision codes and the search terms

aneurysm, aorta and aortic surgery.

A rural and stable population provides the referral base for our hospital.

Information on survival was retrieved from hospital databases.

Definitions

Acute AAA was defined as any AAA requiring treatment within 24h. Patients were

classified as having an acute symptomatic, non-ruptured AAA (SAAA) or ruptured

AAA (RAAA), according to the absence or presence of discontinuation of the integrity

of the aortic wall with blood extravasation as shown on computerised tomography or as

Chapter 6

88

found during surgery. Haemorrhagic shock was defined as a systolic blood pressure

≤100 mm Hg before surgery. Hospital mortality was calculated for all patients who

reached the operating theatre alive. Patients treated with emergency endovascular

repair during the same period were excluded.

Transport time was defined as the time from telephone call to the ambulance

service to arrival in the emergency room. Intrahospital delay was defined as time from

arrival of the patient in the emergency department to arrival in the operating theatre.

Total delay to surgery was defined as the sum of transport time and intrahospital delay.

Distance of transport was defined as the distance from the home address of the patient

to our hospital.

Management strategy

When ambulance personnel suspected that the patient had a ruptured aneurysm, our

hospital was alerted, so that an experienced vascular surgeon and a radiologist were

present when the patient arrived. At the same time, the operating room was alerted. The

ambulance personnel inserted two intravenous lines and initiated fluid resuscitation

only in the case of shock (ie, systolic blood pressure ≤100 mm Hg) with altered mental

state. On arrival, the diagnosis was confirmed by physical examination, ultrasound and,

more recently, computerised tomography to evaluate emergency endovascular repair as

a treatment option. For a patient in haemodynamic shock with a palpable aneurysm, no

ultrasound was carried out and the patient was immediately transported to the operating

theatre. Until aortic clamping was carried out in the operating theatre, we accepted a

systolic blood pressure of ≤100 mm Hg, to reduce retroperitoneal blood losses or to

prevent free rupture in the peritoneal cavity. Patients who had a cardiac arrest during

transport or in the emergency room but were successfully resuscitated were also

offered surgery. Exclusion criteria for surgery were prolonged cardiac arrest despite

resuscitation, advanced Alzheimer’s disease and a poor Karnofsky performance score

(159) (≤40; ie, the patient is disabled and requires special care and help), or severe

cardiovascular disease associated with a New York Health Association-IV performance

score. If the clinical picture was unclear or unknown, the patient was still offered

surgery.


89

Surgery was performed with at least one experienced vascular surgeon present.

In the operating theatre, a rapid sequence induction of anaesthesia and intubation was

carried out after prepping and draping the abdomen. After surgery, all patients

remained intubated and mechanically ventilated and were transferred to a tertiary

intensive care unit (ICU). This ICU is staffed with nurses in a 1.5:1 patient–nurse ratio,

with onsite doctors dedicated solely to the ICU for 24h a day and supervised by

certified intensivists.

Statistics

Data are reported as mean and standard deviation (SD), or as median with interquartile

ranges for skewed data. The Student’s t test was used to compare normally distributed

continuous variables, and the Mann–Whitney U test was used for continuous variables

with a skewed distribution. Differences between categorical variables were tested by χ2

analysis. Cumulative survival was calculated using Kaplan–Meier analysis. The log

rank test was used to compare survival curves between patients with SAAA and those

with RAAA. p Values <0.05 were regarded as significant.

RESULTS

Patient population

During the study period, 160 patients with acute aneurysms were operated upon: 34

with SAAA and 126 with RAAA. An additional 15 patients who received emergency

endovascular treatment for their acute AAA were excluded. Table 1 describes the

demographics of this population. Age and sex did not differ between patients with

SAAA and RAAA; nor did comorbidity profiles between these two groups.

All 34 patients presenting to the emergency department with a SAAA were

operated upon. In total, 126 patients with RAAA were evaluated for surgery, of which

only three (2.4%) patients did not undergo surgery, because of severe cardiovascular

disease with a New York Health Association-IV performance score.

Chapter 6

90

Table 1. Demographic data of the patients operated for symptomatic aneurysms. All patients

N=160

SAAA

N=34

RAAA

N= 126

p

Demographic characteristics

Age (y), mean ± SD 71 ± 8 71 ± 7 71 ± 8 NS

Male sex, n (%) 138 (86%) 29 (85%) 109 (87%) NS

Cerebrovascular disease 18 (11%) 2 (6%) 16 (12%) NS

Chronic obstructive pulmonary disease

37 (23%) 7 (21%) 30 (24%) NS

Chronic renal failure 12 (8%) 3 (9%) 9 (7%) NS

Congestive heart failure 17 (11%) 4 (12%) 13 (10%) NS

Coronary artery disease 62 (39%) 15 (44%) 47 (37%) NS

Diabetes mellitus 15 (9%) 3 (9%) 12 (10%) NS

Hypertension 50 (31%) 11 (32%) 39 (31%) NS

SD = Standard Deviation; SBP = Systolic blood pressure in the emergency department; IQR = Interquartile Range

Transport time and intrahospital delay

Median transport time for the 34 patients with SAAA was 43 min (interquartile range

33–52 min). The median distance of transport (between patient pick-up and hospital)

was 22 km (16–31 km). Median intrahospital delay was 160 min (53–452 min).

Median transport time of the 126 operated patients with RAAA was 43 min

(33–53 min). The median distance of transport was 22 km (4–30 km). Median

intrahospital delay was 25 min (11–50 min). Median total delay to surgery was 67 min

(52–102 min; table 2). Of these patients, 66% were referred by a general practitioner or

directly by the ambulance services, and 34% were referred from another hospital.

The diagnostic investigation was different in patients with SAAA and RAAA:

an ultrasound or computed tomography scan was used in 76% and 51%, respectively

(p=0.007).


91

Table 2. Blood pressure at arrival emergency department, delay to surgery, diagnostic approach, level of aortic clamping and length of stay of patients operated for symptomatic aneurysms. All patients

N=160

SAAA

N=34

RAAA

N= 126

p

Systolic blood pressure at arrival

Cardiac arrest

RR 20-60 mmHg

RR 60-100 mmHg

RR >100 mmHg

8 (5%)

31 (19%)

65 (41%)

56 (35%)

0

0

7 (21%)

27 (79%)

8 (6%)

31 (25%)

58 (46%)

29 (23%)

NS

<.01

NS

NS

Delay to surgery (minutes)

Transport time 42 (33-49) 43 (33-52) 43 (33-53) NS

Intrahospital delay 29 (12-61) 160 (53-452) 25 (11-50) <.001

Diagnostic work-up

Physical examination only 70 (43%) 8 (24%) 62 (49%) .007

Ultrasound 40 (25%) 8 (24%) 32 (25%) NS

Computerised tomography 65 (41%) 23 (68%) 42 (33%) <.001

Intra-operative data

Suprarenal clamping (yes) 8 (5%) 0 8 (6%) NS

Length of stay (days), median (IQR)

Intensive care 3 (1-8) 2 (1-3) 4 (2-10) .001

Hospital 12 (8-18) 9 (7-11) 14 (9-21) .001

IQR, interquartile range; NS, not significant; RAAA, ruptured AAA; SAAA, symptomatic, non-ruptured AAA.

Outcome

Overall intraoperative mortality was 0 for patients with SAAA and 14 (11%) for

patients with SAAA (p=0.042). Thirty-day and hospital mortality was 3 (9%) and 4

(12%), respectively, for SAAA. Thirty-day and hospital mortality for patients with

RAAA was 38 (30%) and 42 (33%), respectively (p=0.011 and 0.014). Haemorrhagic

Chapter 6

92

shock was observed in 7 (21%) patients with SAAA versus 97 (77%) patients with

RAAA. None of the patients with SAAA and eight with RAAA had a cardiac arrest

during ambulance transport or in the emergency department. Four of these patients died

intraoperatively, two died in the ICU and another two patients survived until discharge

from hospital. Of these two patients, one died after 4 months; the other is still alive

after >5 years of follow-up. One-year mortality was 6 (18%) for SAAA and 54 (43%)

for RAAA, see figure 1 (p=0.008; log rank). Follow-up was complete in all patients.

Figure 1. Kaplan-Meier curve of the 1-year survival in patients with SAAA and RAAA.

DISCUSSION

This study shows that a unified strategy for the treatment of patients with acute AAAs

is associated with a hospital mortality of 12% and 35%, for SAAA and RAAA,

respectively, despite offering surgery to >95% of all patients. We infer that the

combined transport time and intrahospital delay of only 67 min has contributed to this

relatively favourable outcome.

The survival curves for SAAA and RAAA suggest that longer-term survival is

similar, which reflects the similar effect of associated comorbidities.


93

In the literature, hospital mortality for patients operated for RAAA ranges

from 20% to 70% (44,45,160-162). This wide range is partly explained by selection

bias, with some centres having restrictive and others liberal indications for surgery.

This, however, is not the only explanation, as in some series (160,161,163-165) in

which surgery was offered to an average of 73% (95% confidence interval (CI) 72% to

74%) of the patients with

ruptured aneurysms, the cumulative hospital mortality was 40% (95% CI 38% to 41%).

The average time of 43 min between the emergency call and arrival of patients

at our emergency department reflects the efficiency of our transport system and the size

of our catchment area in the northern Netherlands. In addition, the fact that the

paramedics alert the hospital before their arrival and that the vascular surgeon and the

radiology staff are in hospital at the time of the patients’ arrival reduces intrahospital

delay.

Many other factors affect outcome after acute AAA. Another important

determinant of outcome is the experience and the specialty of the operating surgeon,

with worse survival reported for less experienced surgeons or for general surgeons than

for vascular surgeons (123,166,167). Postoperative treatment also affects outcome.

Sandison et al (168) observed marked differences in mortality between two ICUs that

received patients operated by the same surgical team. Thus, the availability of a well-

staffed surgical ICU may also have been beneficial for the prognosis of our patients.

Comparison of results between hospitals and surgeons is confounded by

patient selection, modality of patient transport, distances, surgical experience,

postoperative care, definition of symptomatic aneurysms and of haemodynamic

stability. The retrospective design of our study limits the inferences that may be drawn.

However, our extensive search for patients with symptomatic aneurysms (including

hospital, doctor and emergency services records), the regional pattern of referral and

the completeness of our follow-up provide an insight to the results that may be

obtained with a unified strategy of aggressive management of patients with

symptomatic aneurysms.

Chapter 6

94

The clinical implications of our findings may vary depending on the setting in

which treatment of acute AAAs is performed. As geographical conditions that

determine the distance that patients should travel vary widely, the effectiveness of

measures to limit transport delay may also vary. Faster means of transportation and a

protocol for the management of these patients are strategies to improve results.

An additional approach may be the implementation of an endovascular

programme for the treatment of symptomatic aneurysms. In elective AAA repair,

endovascular treatment has shown reduced organ dysfunction and lower 30-day

mortality than in open AAA repair (48,50), although long-term survival is comparable

(149,150). Endovascular treatment for symptomatic AAAs showed promising results;

however,

suitability was low (169). Furthermore, a potential hazard of this approach is the

inevitable delay to surgery caused by the necessary computed tomography. Therefore,

the effectiveness of emergency endovascular procedures in the treatment of RAAA

needs confirmation in prospective clinical trials.

Further studies on the management of acute AAA should include a description

of the protocols used, and report the delays involved as well as the number and type of

patients who were denied surgical treatment.

CONCLUSION

In an environment and an ambulance system that has short delays of transportation, a

unified strategy of treatment of acute AAA provides hospital survivals comparable to

the best reported in the literature, despite a policy of offering surgery to most patients.

ACKNOWLEDGEMENTS

We thank CS Cinà for his valuable comments on this article.

CHAPTER 7

Letter to the editor

With regard to:

The Multicentre Aneurysm Screening Study Group.

The Multicentre Aneurysm Screening Study (MASS) into

the effect of abdominal aortic aneurysm screening on

mortality in men: a randomised controlled trial.

Lancet 2002;360:1531–39.

JW Haveman, A Karliczek, ELG Verhoeven,

JJAM van den Dungen, MWN Nijsten

Department of Surgery, University Medical Center Groningen, The Netherlands

Lancet 2003;361:1058

Chapter 7

96

Sir

The Multicentre Aneurysm Screening Study (MASS) Group do not address the

potential effect of measures to improve survival in patients with ruptured abdominal

aortic aneurysm (54). If surgery is withheld from these patients they will die.

Nevertheless, surgery is frequently withheld from these patients: P Basnyat and

colleagues found that 100 of 233 patients with ruptured abdominal aortic aneurysm

who reached hospital alive were denied surgery (160).

It is difficult to predict the outcome of surgery for ruptured abdominal aortic

aneurysm when a patient is still at the emergency department, whereas, as Kniemeyer

and coworkers found, a much more realistic prognosis can be made for those patients

who are alive 48h after surgery (170). Since 1990 we have implemented a policy of

immediate surgery in nearly all patients with suspected ruptured abdominal aortic

aneurysm. When we are notified of such a patient, a vascular surgeon is immediately

called. Surgery is initiated in almost every patient, even those in shock. Surgery is not

done in patients who are moribund or in those with a poor performance score. In 265

consecutive patients (mean age 71 [SD 8] years), surgery was started 25 (IQR 11–50)

min after arrival at hospital. 30-day mortality was 33%, which is similar to 38% in

MASS, whereas most published studies report mortality rates that range from 30% to

70%. We believe these differences are likely to be related to different treatment

policies, including variation in delay to surgery (107).

Although the required effort for screening seems considerable, the MASS

Group previously reported that prevention of ruptured abdominal aortic aneurysm by

screening is cost-effective (55). Now that cost-effectiveness arguments favour

preventive screening for abdominal aortic aneurysm, it would be relevant to see any

data the MASS Group have about the cost-effectiveness of emergency treatment.

When an integral programme for prevention of death from ruptured abdominal

aortic aneurysm is designed, guidelines for prompt treatment should be included.

Patients who develop ruptured abdominal aortic aneurysm despite the presence of a

screening programme should not suffer because of an overly fatalistic view about their

Letter to the editor of the lancet

97

outcome. We believe that prompt and liberal treatment for ruptured abdominal aortic

aneurysm could be as cost effective as screening.

Chapter 7

98

CHAPTER 8

Summary, general discussion and conclusions

Chapter 8

100

SUMMARY

Chapter 1 introduces the concept of systemic inflammation after major

surgery. The understanding of (systemic) inflammation in the context of surgery

throughout previous centuries is described. Chapter 1 also describes the two patient

groups studied: patients who had undergone orthotopic liver transplantation (OLT) on

the one hand, and patients with ruptured abdominal aortic aneurysms (RAAA) on the

other. The concept of the Systemic Inflammatory Response Syndrome (SIRS) is

reviewed, and the role of monocyte function after OLT and RAAA is explored.

In chapter 2 the pathogenesis of sepsis is described with a focus on the role of

monocytes, immunomonitoring and treatment. In this review article, the two phase

theory of sepsis is presented. The first phase represents the inflammatory response

leading to the activation of monocytes with a systemic release of pro-inflammatory

cytokines like TNF-, IL-1 and IL-6. The first phase is generally described as the

Systemic Inflammatory Response Syndrome (SIRS). The second phase represents the

anti-inflammatory phase with the release of anti-inflammatory cytokines as IL-10, and

is referred to as the Compensatory Anti-inflammatory Response Syndrome (CARS).

Despite enormous effort, most randomised controlled trials investigating a single drug-

intervention in sepsis did not show an improvement in survival. The intervention drugs

used in these trials were almost always designed to counteract the inflammatory

response, which is the first phase. The anti-inflammatory response may already have

been in effect at the time the drugs were administered, which may account for the lack

of successful intervention seen in these studies. In this chapter we elaborated on the

role of HLA-DR expression on monocytes as a parameter for the inflammatory

response. A normal or high HLA-DR expression on monocytes corresponds with SIRS

while a low HLA-DR indicates CARS. The expression of HLA-DR may be used as an

indicator for immunomodulation. In patients exhibiting a predominantly systemic

inflammation, anti-inflammatory interventions may improve prognosis, while patients

with sepsis and low HLA-DR expression may benefit from immunostimulation.

Sepsis following orthotopic liver transplantation (OLT) is a complication that

causes significant morbidity and mortality. Chapter 3 describes a prospective

Summary, discussion and conclusions

101

observational study of 20 adult OLT patients. HLA-DR expression on monocytes was

monitored during the first 4 weeks after transplantation. The seven patients who

developed sepsis had a significantly lower HLA-DR expression on monocytes, both

before and after they developed sepsis. In the two patients with sepsis who died, HLA-

DR expression remained low, while in the five septic patients who recovered, HLA-DR

expression returned to normal values. The patients who developed sepsis had received

significantly more prednisolone than the patients who did not. Incubation with

prednisolone in vitro lowered the expression of HLA-DR in a dose-dependent manner.

Low HLA-DR expression on monocytes appears to be a reliable risk marker for

impending sepsis in OLT patients. The level of risk may be, at least in part, related to

the dose of prednisolone. If HLA-DR is treated as a risk indicator for sepsis, treatment

aimed at improving monocyte function or a reduction in steroids should be promptly

initiated in those patients who show a low expression of HLA-DR to prevent the

development of sepsis.

Laboratory values are routinely determined after the surgical repair of a

ruptured abdominal aortic aneurysm (RAAA). In chapter 4 we present the results of a

retrospective analysis of the course of all routinely determined laboratory values in 290

RAAA patients. The purpose of the study was to describe the ‘normal or benign’

course of RAAA patients who survive until hospital discharge and to identify factors

that distinguished them from the non-survivors. Both the survivors and the non-

survivors showed a wide range of laboratory abnormalities after surgery. The

laboratory results were grouped into six categories (haematology and coagulation,

systemic inflammation, metabolic, renal, liver, and electrolytes). The categories we

chose are strongly related to the causes of death. For example, the patients who die of

massive haemorrhage usually do so within the first 24 hours following surgery,

whereas systemic inflammation and MOF develop more than 24 hours after surgery.

We believe that recognition of the associated laboratory abnormalities is important as it

may help in the early prediction and identification of post-operative complications.

Chapter 5 describes systemic inflammation and HLA-DR expression on

monocytes in RAAA patients in a prospective observational study. Thirty patients were

Chapter 8

102

included and analysed consecutively. The primary outcome parameter was hospital

mortality. Mortality was higher in older patients, patients with a higher APACHE-II

(Acute Physiology and Chronic Health Inquiry) score, and the vital need for suprarenal

clamping. The Sequential Organ Failure Assessment score (SOFA) was significantly

higher in non-survivors from day 1 through day 14 after surgery. IL-6 and IL-10 were

transiently higher on days 1 and 3 in non-survivors. HLA-DR expression on monocytes

dropped after surgery in both survivors and non-survivors. However, in non-survivors

HLA-DR expression was significantly lower from day 3 through day 14 after surgery

compared with the survivors. Low HLA-DR expression was associated with multiple

organ failure and death, although death from secondary infections was not seen in our

patients. We conclude that shock and surgery lead to an overwhelming inflammatory

response which sets the stage for fatal multiple organ failure.

Rapid surgical intervention is one of the most important principles in the

clinical management of patients with RAAA. Chapter 6 describes the importance of a

multidisciplinary approach with clear protocols and dedication to the treatment of these

patients. Our treatment protocol consisted of rapid transport by ambulance to the

emergency department while accepting a low systolic blood pressure. During transport,

the hospital was notified of imminent arrival of a patient with a possible RAAA. Only

three of the 126 patients with ruptured abdominal aortic aneurysms were denied

surgery. The average intrahospital delay was 25 minutes. Hospital mortality was 33%.

Despite our liberal policy of offering surgery to almost every patient, hospital mortality

was comparable to the best reported in the literature.

The multicentre aneurysm screening study group has investigated the potential

benefit of screening men aged 65-74 years for an abdominal aneurysm. In chapter 7,

we emphasize that guidelines for the prompt treatment of ruptured aortic aneurysms

should also be included when screening programs are implemented. Patients who

develop a ruptured AAA despite the presence of a screening program should not be

denied treatment due to an overly fatalistic view on their outcome. In our opinion,

immediate surgery for RAAA patients is as cost effective as a screening program.


103

GENERAL DISCUSSION AND CONCLUSIONS

Our results show that both OLT and RAAA patients develop an intense

inflammatory response and depressed HLA-DR expression after surgery which is

associated with high morbidity and mortality. Therefore, understanding the

inflammatory response after major surgery is relevant and may provide opportunities to

improve outcome in these patients.

The implications for understanding the pathophysiology of SIRS

The previously postulated SIRS/CARS sequence of events is depicted in the

left panel of figure 1. A catastrophic event causes an inflammatory reaction that is

followed by a counter-inflammatory response that theoretically should restore the

balance of health. We also see these two phases in the OLT patients’ inflammatory

response, but here they occur against the background of immunosuppression present as

a result of pre-existing illness and immunosuppressive drugs.

Figure 1. left panel the ‘normal’ pro- and anti-inflammatory response, illustrated by a mildly enhanced pro- and subsequently a counter-inflammatory response which re-establishes equilibrium. The right panel shows the response in a patient with an overwhelming counter-inflammatory response which can be counteracted by early intervention (i.e. decreasing immunosuppressants).

Chapter 8

104

In OLT patients, the intensity of the pro-inflammatory phase may be less

intense as a result, and the counter-inflammatory phase becomes more pronounced than

in other patients. This might explain the high incidence of sepsis related complications

in our patient cohort. A timely reduction in immunosuppression (indicated by the arrow

in the right panel of figure 1) might be effective in preventing these infections, as

discussed below.

To complicate matters, our observations in RAAA patients seriously challenge

the SIRS/CARS concept of events. We did not see the development of any

opportunistic infections which are the classical clinical manifestation of

immunodeficiency. Instead, patients died from the complications of multiple organ

failure which developed almost immediately after surgery. Additionally, our data (and

those of others) show that the pro and counter inflammatory responses co-exist from

the start (136,171,172). This is represented in figure 2.

Whether these two types of inflammatory response contribute equally to the

development of multiple organ failure is not clear. This warrants further research as it

has important consequences for intervention.

Figure 2.The co-existing pro- and anti-inflammatory response.

Diagnostic and prognostic aspects of monocyte HLA-DR expression

Previously, HLA-DR expression on monocytes was measured to predict septic

complications after major surgery, trauma, severe burns, and for patients on a


105

ventricular assist device awaiting cardiac transplantation (23-27,62). Most studies

showed that a persisting low HLA-DR expression was associated with poor outcome

(79,136,142,173-176), however a few others could not confirm these results (177,178).

These latter 2 studies measured HLA-DR expression on monocytes only during the

first 2 post-operative days. Our studies on OLT and RAAA patients confirmed that

HLA-DR expression on monocytes is an accurate predictor of outcome. Low HLA-DR

expression on monocytes was present 1-8 days before the onset of clinical symptoms in

all 7 patients who developed sepsis, and remained low in the 2 patients who died from

sepsis. RAAA patients with a low HLA-DR expression on monocytes had a very poor

prognosis. Non-survivors could be identified by a significantly lower intensity of HLA-

DR expression on monocytes as early as day 3, and preceding death by a median of 8

days.

The usefulness of monocyte HLA-DR expression, at least in RAAA patients,

is somewhat limited by our finding that 3 days were required before a distinction

between survivors and non-survivors could be made with any degree of statistical

significance. This observation was confirmed by Monneret et al, who showed that

HLA-DR expression on monocytes is significantly lower in the non-survivors after 3-4

days (175).

Laboratory markers such as lactate levels, platelet counts, CRP, pro-calcitonin,

or cytokine levels also show marked differences between survivors and non-survivors.

It remains to be determined which of these will serve as the most useful prognostic

markers, and whether different markers will be required for different disease processes.

Mechanism(s) of decreased HLA-DR expression

It is not known which pathophysiological mechanism is responsible for the (low) HLA-

DR expression on monocytes; for example signal transduction or mRNA secretion

might be important. A study by Fumeaux et al showed that IL-10 decreases HLA-DR

expression. In patients with septic shock, this is probably due to re-endocytosis of

HLA-DR and intracellular sequestration (179).

Chapter 8

106

Our studies did not clarify whether low HLA-DR expression is “just” a

clinically useful epiphenomenon or whether it constitutes a relevant element in the

pathophysiologic chain that underlies SIRS. In the first theory, monocytes might be

useful as surrogate markers in (immuno-)intervention studies (which by itself is

important given the large numbers of patients required in randomised studies having

survival as outcome parameter). In the second theory, monocytes (or their HLA-DR

expression) might constitute a target for intervention.

Therapeutic implications of monocyte HLA-DR expression

Our observations in OLT patients strongly suggest that it is the combination of

chronic illness, major surgery and immunosuppression that predisposes the patients to

septic complications. While the first two of these factors cannot be changed, the

intensity of immunosuppression can be easily adapted. The potential impact of such

changes is illustrated by our observation that in vitro, prednisolone decreases HLA-DR

expression on monocytes in a dose-dependent way.

Figure 3. The abrogated pro- and anti-inflammatory response by early intervention.

Consequently, monitoring HLA-DR expression provides us with the

opportunity to decrease immunosuppression pre-emptively in OLT patients at a high

risk of developing sepsis in an effort to prevent this complication and improve

outcome. Our findings justify undertaking a prospective, randomised study to

determine whether the lowering of immunosuppression guided by low monocyte HLA-

DR expression decreases the incidence of septic complications. Indeed, our


107

observations on the effect of steroids on HLA-DR expression provides an argument for

low-dose prednisolone or even steroid-free immunosuppression regimens that may be

associated with a lower incidence of sepsis (180,181).

The therapeutic consequences of low HLA-DR expression in other categories

of patients such as those with RAAA are more difficult to envisage. As pro- and

counterinflammatory responses co-exist, immuno-intervention should probably be

aimed at lowering both these two responses. Intuitively it might be disadvantageous to

“block” inflammation, thereby leaving counter-inflammation unopposed. Measures

directed at both components of the response might be more beneficial (Figure 3).

After Ronco et al. showed that a high ultrafiltration rate leads to a better

prognosis in ICU patients with renal failure (182), the early initiation of renal

replacement therapy in septic ICU patients became more accepted (183-186). Other

investigators showed that high cutoff haemofiltration leads to lower levels of IL-6 and

IL-1ra, which had a beneficial effect on leukocyte proliferation, polymorphonuclear

function, and the amount of norepinephrine administered (187-189). Furthermore,

selective extracorporeal immunoabsorption lowers LPS, IL-6, and C5a levels, and

leads to an increase in HLA-DR expression on monocytes. However, the effect on

outcome in patients with sepsis has not yet been established (190).

Hydrocortisone suppletion might also be beneficial, as a significant number of

RAAA patients have relative adrenal insufficiency (153). Hydrocortisone restores

haemodynamic stability and modulates the immune response and probably improves

prognosis in patients with septic shock (151,152,191-193). One can speculate that this

effect also holds for RAAA patients. In a subgroup of RAAA patients even high dose

steroids might be beneficial. However, after the negative results of high dose steroids

in patients with sepsis and the significant increase in secondary infections in the group

of patients receiving methylprednisolone no more trials with high dose steroids were

conducted (194). Nevertheless, hypothetically there may be a two group phenomenon.

One group would have a relatively low pro- and anti-inflammatory response, and the

second group would display a high inflammatory response. The first group would have

Chapter 8

108

a good prognosis anyway, and would not benefit from the administration of

methylprednisolone as they are less susceptible to multiple organ failure to begin with.

The second group, which has the massive immune response may benefit from the

administration of high dose steroids to mitigate that response. To our knowledge, these

two groups have not been separately explored in the literature. It would be worthwhile

to investigate a two group model, although more evidence supporting this theory would

first be necessary before a study of high dose steroid treatment for patients with sepsis

can be re-initiated.

Prevention of RAAA

In order to improve prognosis for RAAA patients, the benefits of screening for

AAA need to be evaluated. The MASS study showed that screening elderly men for

AAA reduced AAA-related mortality (54). However, it is unclear if screening remains

beneficial when all the family members of an AAA patient are screened. Furthermore,

the prevention of AAA related deaths may just change the cause of death in elderly

patients without delaying it, i.e. a lower AAA related mortality may lead to more death

from myocardial infarction and cancer (195). Meticulous surgical technique and

increasing experience with endovascular repair for RAAA patients will likely also

improve outcome. Hopefully this will be confirmed in a randomised controlled trial

(196). In the meantime liberal and immediate surgery for RAAA patients is probably

the best treatment for these patients (35).

HLA-DR expression on monocytes is an important and relevant parameter in

patients following liver transplantation and ruptured abdominal aortic aneurysm repair.

Persistent low HLA-DR expression is associated with a high mortality in several

patient groups. In this situation, OLT patients may benefit from a reduction in

immunosuppressive medications, especially corticosteroids. In patients who have

undergone RAAA repair, the low HLA-DR expression on monocytes is the result of an

overwhelming pro- and anti-inflammatory response. These patients might benefit from

hydrocortisone administration in the case of relative adrenal cortical insufficiency or


109

early high-volume venovenous haemofiltration. More knowledge on the intracellular

mechanism responsible for modulating HLA-DR expression on monocytes is necessary

to make individualised immunointervention treatment a success.

CHAPTER 9

Samenvatting

Chapter 9

112

Dit proefschrift: ‘Systemic Inflammation and Monocyte Function after Major Surgery’

beschrijft de ontstekingsrespons en monocyt functie na grote chirurgie. Een

ontstekingsrespons is het beste uit te leggen door te bedenken wat er gebeurt als

iemand een (snij)wond oploopt. Om te zorgen dat de wond snel geneest treden er een

aantal processen op. De bloedvaten rond de wond gaan openstaan zodat witte

bloedcellen en weefselcellen (fibroblasten) naar de wond gaan. Witte bloedcellen

ruimen onder andere het aangemaakte stolsel op en de fibroblasten zorgen voor de

wondgenezing. Doordat de bloedvaten openstaan, treedt er lokaal een verhoogde

doorbloeding op; dit is te zien aan de rode kleur rond de wond en de warme huid.

Daarnaast treedt er vaak ook enige zwelling op rond de wond omdat er ook vocht uit de

bloedvaten treedt. Door prikkeling van nabijgelegen zenuwen geeft de patiënt pijn aan.

Verder is er verminderde functie van dit gebied doordat bijvoorbeeld een gewricht stijf

wordt. Al deze mechanismen zijn erop gericht een snel herstel van de wond te

bevorderen.

Systemische inflammatie (ontsteking in het hele lichaam) heeft eigenlijk dezelfde

ontstaanswijze als een lokale ontsteking, en is er ook op gericht om sneller herstel te

bevorderen. Soms kan zo´n ontstekingsrespons echter doorschieten en maakt zo de

patiënt alleen maar meer ziek, er treedt orgaanfalen op en patiënt kan zelfs overlijden.

Een systemische onsteking treedt na elke ingrijpende gebeurtenis van het lichaam op.

Enkele voorbeelden zijn: een groot ongeval waarbij de patiënt bijvoorbeeld een

bovenbeenfractuur en letsel van de buikorganen heeft, een alvleesklierontsteking, grote

brandwonden of grote operaties zoals het uitschakelen van een aneurysma (abnormale

verwijding) van de abdominale aorta (grote buikslagader). Ook bij een infectie na

bijvoorbeeld een levertransplantatie kunnen deze processen een rol spelen. Het doel

van het onderzoek was het beschrijven van de systemische inflammatoire respons en

monocyt functie. Ons onderzoek beschrijft deze respons bij patiënten na een

levertransplantatie en patiënten die geopereerd zijn in verband met een gebarsten

aneurysma van de abdominale aorta (RAAA). Deze twee patiëntenpopulaties zijn zeer

relevant omdat zij relatief vaak systemische complicaties ontwikkelen wat kan leiden

tot multi-orgaan falen en overlijden.

Samenvatting

113

Hoofdstuk 1 is het inleidende hoofdstuk waarin kort de geschiedenis van de

heelkunde en de ontwikkeling van grote en complexere operaties wordt weergegeven.

De systemische inflammatoire respons (ontstekingsreactie) wordt beschreven, evenals

de inzichten in de huidige behandeling van patiënten na een levertransplantatie.

Doordat deze patiënten afweerremmende medicamenten (immunosuppressiva)

gebruiken om afstoting te voorkomen zijn zij zeer vatbaar voor infecties. Tenslotte

wordt in deze inleiding de behandeling van patiënten met een geruptureerd aneurysma

van de abdominale aorta besproken met daarbij de problemen en complicaties tijdens

de postoperatieve periode.

Hoofdstuk 2 bevat een overzichtsartikel over de monocytfunctie bij patiënten

met sepsis. Hierbij spelen twee belangrijke immuunreacties een rol. Ten eerste SIRS

(Systemic Inflammatory Response Syndrome): dit treedt vaak vanaf het begin op

wanneer alle afweersystemen geactiveerd zijn. Dit kenmerkt zich door koorts,

verhoogde witte bloedcellen, versnelde ademhaling en een versnelde hartslag. De

tweede respons is CARS (Compensatory Anti-inflammatory Response Syndrome): dit

treedt vaak later op en kenmerkt zich door een verminderde monocytfunctie waarbij de

HLA-DR expressie op het oppervlak van de cel verlaagd is. Als complicatie treden

hierbij vaak infecties met gisten en schimmels (opportunische infecties) op die moeilijk

te behandelen zijn en een hoge mortaliteit hebben.

Er zijn in de jaren voorafgaand aan het verschijnen van dit artikel vele

gerandomiseerde studies verricht naar geneesmiddelen die erop gericht zijn de SIRS

respons te onderdrukken. De resultaten van deze studies waren teleurstellend. Er werd

geen overlevingsvoordeel gezien van patiënten die deze middelen kregen in

vergelijking met de placebogroep. In dit artikel geven wij hier een aantal verklaringen

voor. De belangrijkste verklaring is dat de SIRS reactie vaak al enige tijd bezig is.

Hierbij kan het zo zijn dat de toegediende geneesmiddelen niet op het optimale

moment gegeven waren en dat patiënt al een onderdrukt immuunsysteem had (CARS):

het geven van geneesmiddelen die deze respons onderdrukken is dan logischerwijs niet

zinvol. In het artikel stellen wij voor om aan de hand van de HLA-DR expressie op de

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114

monocyt te beslissen of patiënten afweeronderdrukkende middelen moeten krijgen (bij

SIRS) of juist afweerondersteunende geneesmiddelen (bij CARS). Eerder onderzoek

suggereert namelijk dat een verlaagde expressie van HLA-DR op monocyten (lager dan

30%) geassocieerd is met een slecht werkend immuunsysteem (CARS) met daarbij een

hoog risico op opportunische infecties die een hoge mortaliteit hebben. Een normale

HLA-DR expressie (70-100%) zou geassocieerd zijn met een goed werkend

immuunsysteem. Deze patiënten zouden dus wel baat kunnen hebben bij

afweeronderdrukkende geneesmiddelen waar voorheen al veel onderzoek naar verricht

is. Deze hypothese zou echter nog wel in een gerandomiseerde studie onderzocht

moeten worden.

Hoofdstuk 3 beschrijft een studie bij 20 levertransplantatiepatiënten.

Levertransplantatiepatiënten worden postoperatief behandeld met hoge doseringen

immunosuppressiva. Deze worden gegeven om afstoting van de donorlever te

voorkomen. Bij deze patiënten werd door middel van bloedafname en laboratorium

analyse de HLA-DR expressie op monocyten bepaald; direct voor de transplantatie en

op dag 7, 14, 21 en 28 na transplantatie. Deze studie laat zien dat 7 patiënten na de

operatie een infectie opliepen waarbij zij voldeden aan de criteria voor sepsis, dat wil

zeggen een bewezen infectie met een systemische ontstekingsreactie. Wanneer de

patiënten met en zonder sepsis worden vergeleken blijkt dat de patiënten met sepsis een

significant lagere HLA-DR expressie op monocyten hadden 3 tot 8 dagen voordat zij

septisch werden. Twee van de zeven patiënten met sepsis kwamen te overlijden. Verder

kregen de patiënten met sepsis een significant hogere dosering prednisolon. In vitro

incubatie van monocyten met prednisolon liet een dosis gerelateerde daling van de

HLA-DR expressie op monocyten zien. Deze resultaten suggereren dat het verlagen

van de immuunsuppressiva bij patiënten met een lage HLA-DR expressie op

monocyten, zou kunnen leiden tot minder infecties met sepsis.

Hoofdstuk 4 beschrijft het verloop van 29 laboratoriumbepalingenwaarden bij

290 patiënten met een RAAA in de eerste week na de operatie en bij ontslag. Van deze

290 patiënten overleden er 100 tijdens de ziekenhuisopname (34%). Bijna alle

laboratoriumwaarden waren postoperatief afwijkend. Er werden diverse verschillen

Samenvatting

115

tussen patiënten die overleefden en patiënten die niet overleefden gevonden, waarbij de

laatstgenoemde groep altijd het sterkste afweek van de referentiewaarde. De 29

laboratorium waarden werden verder onderverdeeld in 5 categorieën: hematologie en

stolling, systemische inflammatie, metabool, nierfalen en leverfalen. Daarnaast werden

alle elektrolyten geanalyseerd. De laboratoriumwaarden passend bij hematologie en

stolling waren direct postoperatief het meest afwijkend, waarna deze bij de patiënten

die overleefden het snelst normaliseerden. Hierna volgden de laboratoriumwaarden

passend bij systemische inflammatie, nierfalen, metabool, en leverfalen. Kennis van

deze respons postoperatief kan belangrijk zijn voor het vroegtijdig herkennen van

complicaties.

Hoofdstuk 5 beschrijft de systemische inflammatie en monocytfunctie bij

geopereerde RAAA patiënten. Bij 30 RAAA patiënten werd postoperatief op dag 1, 3,

5, 7, 10 en 14 de HLA-DR expressie op monocyten en het interleukine-6 en

interleukine 10 bepaald. Interleukine 6 is een cytokine dat fors verhoogd is bij

systemische inflammatie. Interleukine-10 is verhoogd bij een anti-inflammatoire

respons (CARS). De ziekenhuismortaliteit was 20% (6 patiënten). De resultaten van

deze studie laten zien dat de HLA-DR expressie op monocyten significant lager was bij

de overleden patiënten op dag 3, 5, 7, 10 en 14. Interleukine-6 was significant hoger bij

de overleden patiënten op dag 1. Interleukine-10 was signifant hoger bij de overleden

patiënten op dag 1 en 3 na de operatie. Patiënten ontwikkelden geen opportunische

infecties. Hieruit concludeerden wij dat de overleden RAAA patiënten een forse

systemische ontstekingsrespons ontwikkelden, zich uittend in een verlaagde HLA-DR

expressie op monocyten en een verhoogd interleukine-6 en interleukine-10 na de

operatie. Deze patiënten overleden voornamelijk ten gevolge van multi-orgaan falen.

Hoofdstuk 6 beschrijft de resultaten van 160 patiënten met een acuut

aneurysma van de abdominale aorta, waarbij 34 patiënten een symptomatisch niet-

geruptureerd AAA (SAAA) hadden en 126 een RAAA. De hele zorgketen vanaf het

telefoontje naar de ambulance tot aan het ontslag naar huis of overlijden is in kaart

gebracht en geanalyseerd. Wanneer een patiënt met een verdenking RAAA wordt

aangekondigd komt direct de vaatchirurg naar de spoedeisende hulp, net als de

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116

dienstdoende radioloog voor een echo of CT-scan en tot slot de anaesthesist. Daarnaast

wordt de operatie kamer in gereedheid gebracht. Alle patiënten met een SAAA en 98%

van de patiënten met een RAAA werden geopereerd. De ziekenhuismortaliteit was

12% vs 33% voor respectievelijk SAAA en RAAA. De mediane totale tijd tussen het

telefoontje naar de ambulance en start van de operatie was 67 minuten voor patiënten

met een RAAA. Onze studie laat een significant lagere mortaliteit zien in de SAAA

groep ten opzichte van de RAAA patiënten. SAAA patiënten hebben weer een hogere

mortaliteit dan electief geopereerde patiënten. Verder is in deze studie de mortaliteit

van RAAA patiënten in vergelijking met de literatuur goed.

Hoofdstuk 7 is een ingezonden brief naar aanleiding van de Multicentre

Aneurysm Screening Study (MASS). De MASS is een onderzoek bij ruim 67.000

mannen tussen de 65 en 74 jaar die middels een echografie gescreend werden op een

AAA. Wanneer de diameter van het aneurysma groter was dan 5.5 cm werden zij

verwezen naar de vaatchirurg om geopereerd te worden om zo een geruptureerd

aneurysma te voorkomen. De andere helft van de studiepopulatie werd niet gescreend.

De resultaten van deze studie laten zien dat aneurysma-gerelateerd overlijden lager was

in de gescreende groep ten opzichte van de niet gescreende controle groep. In onze

ingezonden brief die gepubliceerd is in the Lancet, hebben wij benadrukt dat ondanks

het feit dat er gescreend wordt er nog steeds patiënten met een RAAA naar het

ziekenhuis verwezen zullen worden. Ondanks het feit dat de mortaliteit bij RAAA

patiënten hoog is (literatuur 30-70%) pleiten wij ervoor om al deze patiënten te

opereren tenzij de patiënt een zeer slechte performance score heeft. Deze benadering is

gebaseerd op het feit dat het in de acute setting erg lastig is de postoperatieve

mortaliteit voor de individuele patiënt te voorspellen. De literatuur geeft weinig inzicht

in hoeveel patiënten met een RAAA niet geopereerd worden en dus overlijden. Zoals

ook in hoofdstuk 6 is beschreven zien wij in onze serie waarbij bijna alle patiënten

geopereerd werden goede resultaten met weinig overleden patiënten.

De belangrijkste resultaten van dit proefschrift worden samengevat in

Hoofdstuk 8. Dit proefschrift laat zien dat patiënten na een levertransplantatie en met

een geruptureerd aneurysma postoperatief een forse ontstekingsrespons ontwikkelen.

Samenvatting

117

Een verlaagde HLA-DR expressie op monocyten was in beide groepen geassocieerd

met een hoge mortaliteit. Er is echter ook een belangrijk verschil tussen beide groepen:

patiënten na een levertransplantatie krijgen immunosuppressiva en hebben hierbij een

verhoogde kans op het ontwikkelen van opportunistische infecties. Dit geldt niet voor

patiënten met een geruptureerd aneurysma. Hoewel deze patiënten wel een verlaagde

HLA-DR expressie op monocyten hebben, konden wij geen opportunistische infecties

bij deze patiënten waarnemen. RAAA patiënten komen te overlijden ofwel in de eerste

fase ten gevolge van het ernstige bloedverlies ofwel na enkele dagen door de uit de

hand gelopen inflammatoire respons en multi-orgaan falen.

Levertransplantatiepatiënten met een lage HLA-DR expressie op monocyten en/of

sepsis hebben waarschijnlijk baat bij het verminderen of stoppen van de

immunosuppressiva.

Chapter 9

118

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DANKWOORD

Dankwoord

138

Bijna 10 jaar na de eerste publicatie is het dan zover, het proefschrift is af! Gedurende

zo´n lange periode hebben natuurlijk vele mensen bijgedragen aan 1 of meerdere

hoofdstukken uit dit boekje, die wil ik dan ook allemaal heel erg bedanken. Zonder

compleet te zijn wil ik hier een aantal personen expliciet vermelden.

Mijn promotor, prof dr JH Zwaveling. Beste Jan Harm, jij motiveerde mij om op de

chirurgische intensive care (CHIC) mijn onderzoek weer op te pakken. Ik heb altijd

veel vertrouwen van je gekregen en je hebt me altijd onvoorwaardelijk gesteund.

Daarnaast heb je mij de mogelijkheid gegeven het klinische werk op de CHIC, te

combineren met onderzoek. Na je vertrek naar Maastricht hebben we niet heel frequent

contact gehad over het onderzoek, maar je bleef je inzetten om het proefschrift tot een

goed einde te brengen.

Dr AP van den Berg. Beste Aad, als initiator van dit onderzoek en als directe

begeleider bij de lab studies ben ik je veel dank verschuldigd. Ik heb enorme

waardering voor je analytische vermogen en hulp bij het schrijven van de diverse

hoofdstukken.

Dr MWN Nijsten. Beste Maarten, dankzij jouw enthousiasme en niet aflatende energie

hebben we enkele mooie hoofdstukken geschreven. We konden bijna alles uit het ZIS

halen, wat er te halen viel. Wanneer we dit konden combineren met de juiste toon in

het manuscript hadden we zo een kraakheldere letter geschreven (hoofdstuk 7).

Prof dr CGM Kallenberg, prof dr RJ Ploeg en prof dr DF Zandstra wil ik bedanken

voor hun deelname in de leescommissie en voor hun goedkeuring van dit proefschrift.

Prof dr TH The wil ik graag bedanken voor de goede begeleiding tijdens het onderzoek

bij de klinische immunologie. Ik heb veel geleerd van uw heldere kijk op de patiënten

in relatie met de laboratorium afwijkingen.

Dankwoord

139

Dr JJAM van den Dungen, drs IFJ Tielliu, dr ELG Verhoeven en dr CJAM Zeebregts

wil ik bedanken voor hun enthousiasme in de behandeling van de RAAA patiënten en

de bijdrage aan de artikelen. Clark Zeebregts wil ik bedanken voor zijn onuitputtelijke

energie en hulp bij het schrijven van enkele lastige hoofdstukken. Als initiator van het

retrospectieve deel bij RAAA patiënten wil ik dr Anne Karliczek nog graag bedanken

voor haar inzet bij dit onderzoek.

Alle levertransplantatie chirurgen, fellow´s en hepatologen wil ik graag bedanken voor

hun enthousiasme en betrokkenheid bij de behandeling van de OLT patiënten en de

bijdrage aan dit onderzoek.

De collega´s, verpleegkundigen en staf van de CHIC wil ik enorm bedanken voor de

mooie tijd die ik daar gehad heb. Ik kreeg de mogelijkheid het klinische werk te

combineren met het onderzoek. Deze tijd heeft mij een goede basis gegeven tijdens de

opleiding en ook nog hierna.

Alle medewerkers van het laboratorium klinische immunologie wil ik graag bedanken

omdat ik daar altijd mijn gang kon gaan en van alle apparatuur en dergelijke gebruik

kon maken. Johan Bijzet en Geert Mesander hebben mij geweldig geholpen tijdens de

ELISA en flow-cytometrie.

Mijn opleiders uit de Isala klinieken in Zwolle. Eigenlijk zijn dit alle werkzame

chirurgen, echter de formele opleiders dr JE de Vries en dr EGJM Pierik wil ik graag

nog kort bijzonder bedanken. Beste Hans, het was nogal een overgang om vanuit

Groningen in Zwolle bij de chirurgie te beginnen, maar dankzij jouw begeleiding en

sturing is het een mooie en leerzame periode geworden. Beste Robert, jouw

voortvarendheid en enthousiasme werken enorm inspirerend voor de opleiding in

Zwolle.

Dr GA Patijn: beste Gijs superleuk dat je in de oppositie plaatsneemt.

Dankwoord

140

Prof dr HJ ten Duis wil ik graag bedanken voor het warme onthaal in het UMCG, de

laatste anderhalf jaar van m´n opleiding zie ik met veel vertrouwen tegemoet.

Al mijn collega´s uit Zwolle wil ik graag bedanken voor de geweldige tijd die ik daar

gehad heb. De altijd gezellige borrels en feestjes leverden altijd weer mooie momenten

op en maakte deze periode tot een onvergetelijke tijd.

Collega´s uit het UMCG: de overgang naar Groningen en de laatste loodjes voor mijn

promotie verliepen zeer soepel dankzij de uitstekende samenwerking en collegialiteit.

Mijn paranimfen Vincent Hagens en Rienk van Calker. We kennen elkaar alweer vanaf

1994, dankzij jullie is mijn studietijd een fantastische periode geweest. Daarna is het

goede contact gebleven en onze te weinige afspraken zijn altijd gezellig en lopen altijd

enkele drankjes uit. Super dat jullie m´n paranimfen willen zijn.

Lieve pap en mam, zonder jullie was het nooit zover gekomen. De basis van dit boekje

is door jullie gelegd.

Ook wil ik graag mijn schoonouders, andere familieleden en vrienden bedanken voor

hun belangstelling en steun voor mijn onderzoek.

Lieve lieve Helen, wat ben ik toch een geluksvogel!

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infusion with an implantable pump. Langenbecks Arch Surg 2008: Epub

ahead of print.

16. Haveman JW, Jörning PJG. Een man met een verbreed mediastinum na een

trauma. Ned Tijdschr Geneeskd. In press.

17. Haveman JW, Blomme AM. Entrapment van de nervus peroneus superficialis.

Submitted.

Bibliografie

144

CURRICULUM VITAE

Curriculum vitae

146

Jan Willem Haveman werd geboren in Assen op 10 februari 1974. Hij volgde de lagere

school aan de Meester Andrea school in Diever. Na de HAVO behaalde hij zijn VWO

examen aan het Reestdalcollege in Meppel in 1994. Hierna begon hij met de studie

geneeskunde aan de Rijksuniversiteit Groningen. Tijdens zijn studie was hij actief in de

jaarvertegenwoordiging en was student-lid van de Stuurgroep Klinische Fase. In 1999

begon hij met een onderzoeksproject bij de vakgroep Chirurgisch Oncologie o.l.v. prof. dr.

J.Th. Plukker naar gedifferentieerd schildkliercarcinoom. Zijn wetenschappelijke stage

deed hij bij de vakgroep Klinische Immunologie bij dr. A.P. van den Berg en prof. dr. T.H.

The. In november 2000 werd hij bevorderd tot arts. Hierna werkte hij als ANIOS bij de

afdeling heelkunde in het Universitair Medisch Centrum Groningen en vanaf april 2001 op

de Chirurgische Intensive Care. Naast zijn werkzaamheden op de Intensive Care, pakte hij

zijn onderzoek bij de afdeling Klinische Immunologie weer op, wat uiteindelijk leidde tot

dit proefschrift.

Vanaf september 2004 begon hij met de opleiding heelkunde in de Isala klinieken in

Zwolle; opleider dr. J.E. de Vries en later dr. E.G.J.M. Pierik. Vanaf september 2008

vervolgde hij zijn opleiding in het Universitair Medisch Centrum Groningen; opleider prof.

dr. H.J. ten Duis.

Jan Willem is getrouwd met Helen Lutgers.

university of groningen systemic inflammation and monocyte

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