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Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova Servizio di Medicina di Laboratorio
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Page 1: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Markers di flogosi in chirurgia

Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio

Azienda Ospedaliera – Università di Padova

Servizio di Medicina di Laboratorio

Page 2: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

INITIAL INJURY RESPONSE

Initial sterile “hit”(stress) as trauma or surgery includes activation of inflammation-responsive system

Patients with surgical injuries or trauma exhibit alterations in hemodynamic, metabolic, and immune responses that are largely orchestrated by activation of cytokine cascade.

Proinflammatory cytokine production in the intraoperative and early postoperative periods is initiated by macrophages and monocytes at the initial site of injury as part of the acute-phase response.

Page 3: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

No evidence of a systemic response with mild or modest injury after surgery.

An insult of sufficient magnitude induces systemic response that encompass many features of a proinflammatory state.

As the systemic inflammatory response continues, patient management and pathogen virulence (exogenous factors) assume a more prominent influence on the balance of systems activity. The interactions between endogenous and exogenous factors lead to uncertainty (gray area) over time about systems activity and overall responsiveness to secondary insults, such as infection.

INDUCTION of SYSTEMIC RESPONSE

Page 4: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Cytokines Tumor necrosis factor-α Earliest and most potent mediators of subsequent host responses. Half-life is less than 20 minutes. The primary sources of TNF-α synthesis include monocytes/macrophages and T cells, which are abundant in the peritoneum and splanchnic tissues. Surgical or traumatic injuries to the abdominal viscera undoubtedly have profound influences on the generation of inflammatory mediators and homeostatic responses such as acute-phase protein production.IL-1Half-life approximately 6 minutes. Local inflammatory mediator. Induces the classic inflammatory febrile response. Pain perception after surgery can be mediated by IL-1 by promoting the release of β-endorphins from the pituitary gland and increasing the number of central opioid like receptors.Interleukin-6TNF-α and IL-1 are potent inducers of IL-6 production from virtually all cells and tissues, including the gut. Levels are detectable by 60 minutes, peak between 4 and 6 hours, and can persist for as long as 10 days. Circulating IL-6 levels appear to be proportional to the extent of tissue injury during an operation, more than to the duration of the surgical procedure itself. Evidences have demonstratedboth a proinflammatory role and an antiinflammatory role for IL-6. [Tilg H et al, 1994]

Page 5: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Pleiotropic actions of interleukin-6: induction of Acute-Phase Proteins

Increase of 10-100 times Increase of 2-10 times < 2 times Decrease

C-reactive protein alfa1-Proteinaseinhibitor Caeruloplasmin alfa-antitrypsin

Serum amyloid A alfa1-Acid glycoprotein C3 of complement Transferrin

  alfa1-Antichymotrypsin Alfa2-Antiplasmin alfa1-Lipoprotein

  Fibrinogen C1-inactivator Prealbumin

  Haptoglobin   Albumin

Page 6: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Changes in plasma CRP concentration after elective surgery

Changes in plasma concentrations of (a) CRP, and (b) IL-6 in patients undergoing total

abdominal hysterectomy in two anesthesia groups

Akintola, D. F. et al. Clin Chem 1997;43:845-847

Acute phase response of adult human hepatocytes stimulated with cytokines. (a) Dose-dependence; (b) time course Heinrich P.C. et a. Biochem J 1990; 265: 621-636

General anesthesia

Extradural blockade

Page 7: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

BIOMARKERS of SIRS

Page 8: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

C-Reactive Protein

Discovered in 1930 by Tillet W. and Francis T. Term derived from the the reaction between the protein and C polysaccharide of the cell wall of the pneumococcus

115,000 dalton, cyclic pentameric protein made of five protomers, each of 206 AA; synthesised in liver; IL-6 (and IL-1 and TNFa) stimulate synthesis via action on promoter (NF-kB)

Half-life of 19 h but clinical half-life after a single stimulus such a trauma or surgery is requiring several days until returning to baseline

Binds phosphocoline and phospholipid constituents of damaged cells, recognizes foreign pathogens

Activates the complement system, acts as “opsonine”

Induces inflammatory cytokines and tissue factor in monocytes, decreases the adhesion of PMN to endothelial cells, inhibits superoxide production, stimulates IL-1ra by mononuclear cells.Thus, its net effect is antiinflammatory

The biology, utilization, and attenuation of C-reactive protein in cardiovascular disease: Part I Annemarie Armani, MD,a and Richard C. Becker, MD,b New York, NY, and Durham, NC American Heart Journal Volume 149, Number 6

Page 9: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

ACUTE-PHASE RESPONSE in SURGERY

TIME AFTER SURGERY CRP LEVELS

4 to 12 hours Increase

24 to 72 hours Peak

4 to 15 days Steady state

After 15 days Decrease

Page 10: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

CRP CONCENTRATIONS AND CLINICAL APPLICATIONS

In surgical trauma markedly elevated levels of CRP are associated with the increased levels of IL-6 and neutrophil elastase [Hildebrandt U et al, 1999]

CRP is particularly useful in monitoring recovery from an operative procedure in uncomplicated cases [Fischer Cl et al,1976; Gonheim ATM et al, 1982; Shentag JJ et al,1984,].

The increase of CRP is related to the extent of the surgical trauma, because laparoscopic performance of identical operating procedures is capable of reducing the acute phase response [Hildebrandt U et al, 2003]

CRP is elevated by surgery, trauma, malignancy, acute pancreatitis, myocardial infarction

Because of clinical half-life, serial CRP measurements often provide only a limited information to differentiate of critical conditions (SIRS vs Sepsis) [Vigushin, 1993]

For the diagnosis of sepsis the optimal cut-off value of CRP has yet to be established. Studies suggest as reasonable the value of 50-100 mg/L [Luzzani A et al, 2003]

CRP concentrations did not reflect the severity of sepsis and were not significantly different between survivor and nonsurvivor [Ciccarelli et al, 2008]

Page 11: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Characteristic Patterns of Change in Plasma Concentrations of Some Acute-Phase Proteins after a

Moderate Inflammatory Stimulus

Page 12: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Serum Amyloid A

C. M. Uhlar and A. S. Whitehead Eur. J. Biochem.,1999

Multifunctional proteins is an apolipoproteinthat rapidly binds to high-density lipoprotein after their synthesis, influencing cholesterol metabolism during inflammatory states, causing adhesion and chemotaxis ofphagocitic cells and lymphocyte.

Overexpression caused by chronic inflammation produces damaging effects, e.g. amyloidosis and possibly arteriosclerosis.

Regulated mainly by IL-1, TNF-a, IL-6 and glucocorticoids at the transcriptional and post-transcriptional levels. [Patewl et al, 1998; Gabay et al 1999] In postoperative septic shock CRP and SAA strongly correlated up to the fifth day of observation but were not good predictors of mortality in septic shock. [Ciccarelli et al 2008]

Page 13: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

SYSTEMIC INFLAMMATORY RESPONSE SYNDROME ( SIRS):

DEFINIZIONE

The systemic inflammatory response to a variety of severe clinical insults. The response is manifested by two or more of the following conditions:

1. temp above 38°C or below 36°C2.

2. heart rate >90 beats/min

3. respiratory rate >20/min or PaCO2 < 32 mm Hg4.

4. WBC: > 12,000 cells/mm3, < 4,000 cells/mm3 or immature (band) forms >10%

(ACCP/SCCM; 1992 Consensus Statement on definitions. CritCare Med; 20:864)

Page 14: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

INFLAMMATORY VARIABLES: WBC bands and IG

BAND CELLS:

wide variation in results both between individuals and laboratories

Poor clinical utility

IMMATURE GRANULOCYTES:automated immature granulocyte measurements reflect a biologically and clinically relevant phenomenon but are not sensitive enough to be used as screening assays for prediction of infection or bacteremia.

Traditional markers of infection, such as neutrophilia, lack sufficient sensitivity among hospitalized patients to be of value in distinguishing sepsis from noninfective inflammatory processes. (Cohen, Crit Care Med 2004 Vol. 32, No. 11 (Suppl.)

Page 15: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

SYSTEMIC HOST RESPONSE: PROCALCITONIN

Procalcitonin (PCT): the promoter of the PCT gene (Calc-1) has site for NFkb and AP-. The Calc-1 gene produces two different transcripts with very different functions.

In the course of a study on pediatric burn patients →increased serum PCT was shown to be a specific marker of sepsis in a pediatric population (Assicot et al, 1993; Lancet, 341:515)

PCT mRNA is markedly stimulated in mononuclear cells by proinflammtory cytokines and LPS. PCT has been shown to inhnibit prostaglandin and thromboxane synthesis.

Page 16: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

PROCALCITONIN and SURGERYReference Surgery Without

postoperative complications

With postoperative complications

Reith et al. 1998

Abdominal 0.9- 1.2 ng/ml 0.36 – 6.9 ng/ml

Meisner et al. 1998

Different types < 1 ng/ml

<2 ng/ml in cardiac, thoracic (8%) and abdominal surgery (25% of patients)

> 10 ng/ml (92% of patients)

Karpel et al, 2000

Abdominal 0.44 ng/ml 4.79 ng/ml

Clinton et al, 2003

Cardiac 0.25 ng/ml 1.19 ng/ml

Falcoz et al, 2005

Thoracic 0.63±0.62 ng/ml 3.6±5.5 ng/ml

Sponholz et al, 2006

Cardiac < 5 ng/ml 4.0 to 30 ng/ml

Novonty et al, 2007

PCT and the APACHE II score

Calculation of the prognosis-score allowed for an early prediction of the septic course with high sensitivity and specificity.

Novonty et al, 2009

Abdominal sepsis

Ratio > 1.03 (1/2d ) Ratio < 1.03 (1/2d)

Page 17: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

PCT as a marker of infection following surgery

Serum PCT levels increase after uncomplicated surgery, reaching a peak level within 24 hours postoperatively. In the first 4 days levels may rise to < 10 ng/mL. Levels higher than this suggest infection. During the subsequent days values should fall to < 0.1 ng/mL. A PCT increase during this period strongly suggests bacterial infection

The dynamics of PCT levels, rather than absolute values, may be more important for identifying patients with infectious complications after surgery.

Intestinal surgery and major operations more often increase PCT, whereas it is normal in the majority of patients after minor and primarily aseptic surgery. PCT can thus be used postoperatively for diagnostic means only when the range of PCT concentrations during the normal course of a certain type of surgery is considered and concentrations are followed up. [Meisner M et al 1998]

PCT seems to be superior to CRP in discriminating infection after surgery. [Rothenburger M et al, 1999]

The diagnostic accuracy of PCT as a marker for postoperative complications is not yet sufficiently clarified [Boysen A et al 2005]

Page 18: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

PROCALCITONIN“Normal” values are in general < 0.5 ng/mL

Cut-off values for clinical decision-making are essential and must be context-specific for:

the clinical setting (eg primary care, emergency room, intensive care unit, post-operative or trauma patients)

the site and extent of the infection (eg RTI, endocarditis, meningitis, others)

co-morbidites (eg impaired pulmonary reserve, immunosuppression)

the clinical implications drawn (ie diagnosis, prognosis, antibiotic stewardship) For the majority of infections optimal cut-off ranges remain still to be determined in observational studies and validated in intervention studies

Page 19: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

PCT and CRP for sepsis in critical patients after surgery or trauma

Summary receiver operating characteristics curves for procalcitonin (circle, solid line) and Creactive protein (triangle, dashed line); n =15 studies.

Global diagnostic accuracy odds ratios for procalcitonin (PCT, circle, solid line) and C-reactive protein (CRP, triangle, dashed line); n=15 studies. OR, odds ratio; CI, confidence interval.

Uzzan B et al. Crit Care Med 2006; 34: 1996-2003

Diagnostic accuracies were performed significantly better by PCT than CRP (Q* values for PCT=0.78, for CRP=0.71)

Q values

Page 20: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

DETERMINANTS of SIRS

Page 21: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Infection, a documented or suspected, and some of the following:

Metabolic variablesLeukocytosis (WBC count >12 109/L)Leukopenia (WBC count < 4 109/L)Normal WBC count with >10% immature formsHyperglycemia (plasma glucose >7.7 mmol/L) in the absence of diabetes

Inflammatory variables

Plasma C-reactive protein >2 SD above the normal valuePlasma procalcitonin >2SD above the normal valueCoagulation abnormalities (INR >1.5 or aPTT >60 secs)

Organ dysfunction variablesAcute oliguria (urine output <0.5 mL/kg/1hr)Creatinine increase >44.2 mmol/LThrombocytopenia (platelet count <100 109L)Hyperbilirubinemia (plasma total bilirubin >70 mmol/L (4 mg/dL )

Diagnostic criteria for SIRS/SEPSIS

Tissue perfusion (haemodinamic)variablesHyperlactatemia (>1 mmol/L) 

2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference

Page 22: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Infections, SIRS and Sepsis

The ACCP/SCCM Consensus Conference Committee. The ACCP/SCCM Consensus Conference Committee. Chest. Chest. 1992;101:1644 1992;101:1644-1655.

MARKERS

Page 23: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Candidate biomarkers for the diagnosis of SIRS/ Sepsis

Page 24: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

BIOCHEMICAL MARKERS in SIRS

Biochemical features in patients meeting the 1992 SIRS criteria:

•Interleukin (IL)-6

•Adrenomedullin

•Soluble(s)CD14

•sELAM-1

•MIP-1

•Extracellular phospholipase A2

Page 25: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

Considerations to be addressed in sepsis biomarker

SPR, surface plasmon resonance; QCM, quartz crystal microgravimetry

Carrigan SD et al. Clin Chem, 2004

Page 26: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

a) CONCLUSIONS

The manifestation of SIRS criteria is the common clinical phenotype of stressed surgical patients. It reflects the presence of consequential systemic inflammation and suggests incresing risk for complications and an adverse outcome. [Lowry, 2009]

Page 27: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

SIRS/SEPSIS

A single marker cannot reflect the entire spectrum of the disease in every biological

aspects

Measures, markers, and mediators: Toward a staging system for clinical sepsis. A Report of the Fifth Toronto Sepsis Roundtable,Toronto, Ontario, Canada, October 25–26, 2000 John C. Marshall, MD; Jean-Louis Vincent, MD, PhD; Mitchell P. Fink, MD; Deborah J. Cook, MD, MSc;Gordon Rubenfeld, MD, MPH; Debra Foster, BSc, RN; Charles J. Fisher Jr; Eugen Faist, MD; Konrad Reinhart, MD Crit Care Med 2003 Vol. 31, No. 5

Page 28: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

BIOCHEMICAL MARKERS in SIRS

Carrigan et al. Clin Chem, 2004

Page 29: Markers di flogosi in chirurgia Elisa Piva, Mario Plebani Dipartimento di Medicina di Laboratorio Azienda Ospedaliera – Università di Padova.

b) CONCLUSIONS

Thus, a unique marker cannot differentiate sepsis from other causes of systemic inflammatory response syndrome; rather, markers are a part of a systematic evaluation that includes clinical examination . Sequential measurement of the inflammatory markers is likely to add more value for the diagnosis of infection than one-off measurements.

Cohen, Crit Care Med 2004 Vol. 32, No. 11 (Suppl.)


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