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1. Diagnosing & Managing Sepsis Syndrome:  The Emerging Role of Bedside Analyte Testing
2. Continuing Education Credit Date of Release: 6/29/2011Date of Expiration: 6/29/2013 Estimated time to complete this educational activity: 1 hour
3. Continuing Education Credit Physicians - This program has been reviewed and is acceptable for up to one (1.0) prescribed credit hour by the American Academy of Family Physicians. AAFP prescribed credit is accepted by the AMA as equivalent to AMA PRA Category I for the AMA Physicians‘Recognition Award. When applying for the AMA PRA, prescribed hours earned must be reported as prescribed hours, not as Category I. Nursing - Educational Review Systems is an approved provider of continuing education in nursing by ASNA, an accredited provider by the ANCC/Commission on Accreditation. Provider #5-115. This program is approved for one (1.0) hour. Educational Review Systems is also approved for nursing continuing education by the State of California and the District of Columbia. Respiratory Therapy - This program has been approved for 1 contact hours Continuing Respiratory Care Education (CRCE) credit by the American Association for Respiratory Care, 9425 N. MacArthur Blvd. Suite 100 Irving TX 75063 Course # 213078000
4. Statement of Need Sepsis kills more than 210,000 Americans each year and is becoming more common, especially in the hospital. Sepsis is a medical emergency that can be difficult to define, diagnose, and treat, but every minute counts in the effort to save lives. This learning activity will describe how bedside analyte testing could aid therapeutic decision making and improve the prognosis for patients with sepsis.
5. Intended Audience The primary audience for this learning activity are health care professionals (physicians and nurses) who are involved in the testing, diagnosis, treatment, and management of sepsis and are interested in the role of biomarkers to improve the care forthese patients.
6. Learning Objectives After completing this activity, the participant should be able to: Discuss the scope of sepsis morbidity and mortality. Describe the role of sepsis biomarkers in screening, diagnosis, risk stratification, and monitoring of response to therapy in sepsis. List factors to be considered when evaluating sepsis testing and results. Identify situations where point-of-care analyte testing might benefit patients with a suspected or confirmed diagnosis of sepsis. Apply information to assist in the identification and treatment of patients with sepsis and improve patient outcomes.
7. Medical Advisement We would like to acknowledge the following medical expertswho served as advisors to this educational program: Emanuel P. Rivers, MD, MPH Arthur P. Wheeler, MD Thomas Ahrens, DNS, RN, PhD Jill A. Sellers, BSPharm, PharmD
8. Disclosures
9. Introduction to Sepsis Definition, Etiology, Morbidity and Mortality
10. Definition of Sepsis Sepsis Systemic response to infection Manifested by two or more SIRS criteria as a result of proven or suspected infection Temperature ≥ 38C or ≤ 36C HR ≥ 90 beats/min Respirations ≥ 20/min WBC count ≥ 12,000/mm3 or ≤ 4,000/mm3 or > 10% bands PaCO2 < 32 mmHg ACCP/SCCM Consensus Conference. Crit Care Med. 1992;20(6):864-74.
11. Case Study
12. Case Study: Mr. Z He tells you “My tooth is killing me! You can pull it if you need to. I feel like it is going to explode.” Mr. Z is a 47 year-old male who was admitted to the emergency department. He is complaining of a toothache that has been present for 7 days. His tooth pain is severe and he cameto the emergency department since he could not see his dentist until themorning. He has drainage from tooth #20, for which a culture has been obtained and sent to the lab.
13. Case Study: Mr. Z He is started on Cefoxitin (Mefoxin®) 2 g IV q6h. Mr. Z is alert and oriented. He has a history of hypertension and had a hemorrhagic stroke 10 years ago but has had no major health issues since this time. His heart and lung sounds are normal and his skin is cool and moist. He has good capillary refill, abdomen soft and non-tender.
14. Case Study: Mr. Z SPO2: Pulse oximetry oxygen saturation; NIBP: Non-invasive blood pressure Questions Does Mr. Z have signs of sepsis? Yes 2) What is a blood test that would be useful? Lactate
15. Case Study: Mr. Z
16. Case Study: Mr. Z A decrease in lactate shows improved perfusion. If the lactate had remained elevated, more fluids could have been given. The use of the lactate allowed the clinician to better evaluate the seriousness of the situation. Often, vital signs are normal when lactates are elevated.
17. Sepsis is Serious. Sepsis is a serious medical condition caused by an overwhelming immune response to infection. Complex chain of events: Inflammatory and anti-inflammatory processes Humoral and cellular reactions Circulatory abnormalities Results in impaired blood flow, which damages organs by depriving them of nutrients and oxygen. http://www.nigms.nih.gov/Education/factsheet_sepsis.htm
18. The Intracellular Immune Response to Infection Adapted from Holmes CL, Russell JA, Walley KR. Chest. 2003;124:1103-15.
19. Symptoms of Sepsis Sepsis can begin in different parts of the body and can have many different symptoms. Rapid breathing and a change in mental status, may be the first signs of sepsis. Other symptoms include: Fever Chills/hypothermia Decreased urination Tachycardia Nausea and vomiting
20. Clinical Manifestations of Shock  Platelets  PT/APTT  Protein C  D-dimer Delirium and Encephalopathy Hyperpyrexia or Hypothermia Adrenal Dysfunction Acute Lung Injury or ARDS Oliguria Anuria  Creatinine Metabolic acidosis Jaundice  Enzymes  Albumin  PT Gut Dysfunction Altered Glucose Metabolism ARDS: Acute respiratory distress syndrome; PT: Prothrombin time; APTT: Activated partial thromboplastin time
21. The Sepsis Continuum SIRS with a presumed or confirmed infection Local or systemic infection or traumatic injury A clinical response arising from a nonspecific insult, including ≥ 2 of the following: Temperature > 38ºC or < 36ºC Heart rate > 90 beats/min Respiratory rate > 20 breaths/min or PaCO2 < 32 Torr WBC > 12,000 cells/mm3, < 4,000 cells/mm3, or > 10% immature Sepsis with ≥ 1 sign of organ failure: Cardiovascular (refractory hypotension) Renal Respiratory Hepatic Hematologic CNS Unexplained metabolic acidosis Adapted from Bone RC, Balk RA, Cerra FB et al. Chest. 1992;101:1644-55.
22. The Relationship Between SIRS, Sepsis, and Severe Sepsis Other Sepsis Severe Sepsis Pancreatitis Infection SIRS Trauma Burns Bone RC, Balk RA, Cerra FB et al. Chest. 1992;101:1644-55.

23. Locations for Common Infection

    Skin and soft tissue Lungs Vascular Catheters(endovascular) Abdomen Appendix http://www.nigms.nih.gov/Education/factsheet_sepsis.htm Urinary Tract
24. Microbes Many different types of microbes can cause sepsis: Bacteria (most common) Fungi Viruses Severe cases often result from a localized infectionbut sepsis can also spread throughout the body. Staphylococcus sp. (Bacteria) Aspergillus sp. (Fungi) Influenza (Virus) CDC/ Matthew J. Arduino CDC/ Robert Simmons CDC/ Erskine. L. Palmer, PhD; M. L. Martin http://www.nigms.nih.gov/Education/factsheet_sepsis.htm
25. Mortality Rates Sepsis remains the leading cause of death in critically ill patients in the United States. Each year 750,000 people will develop sepsis. AIDS Breast Cancer Severe Sepsis National Center for Health Statistics, 2001. American Cancer Society, 2001. Angus DC, Linde-Zwirble WT, Lidicker J et al. Crit Care Med. 2001;29(7):1303-10.
26. Sepsis Incidence in Men and Women 300 200 100 0 Men Women Population-Adjusted Incidence of Sepsis (No./100,000) 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 Martin GS et al. N Engl J Med. 2003;348:1546-54.
27. Sepsis Incidence by Race 500 400 300 200 100 0 Other Black White Population-Adjusted Incidence of Sepsis (No./100,000) 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 Martin GS, Mannino DM, Eaton S et al. N Engl J Med. 2003;348:1546-54.
28. Sepsis Incidence in the United States: 2000 250 200 150 Incidence per 100,000 100 50 0 Sepsis Breast Acute Multiple Lung Colon AIDS Cancer Myocardial Sclerosis Cancer Cancer Infarction Martin GS, Mannino DM, Eaton S et al. N Engl J Med. 2003;348:1546-54. SEER Cancer Statistics Review. National Cancer Institute. www.cancer.gov. 2007. HIV/AIDS Surveillance Report. Centers for Disease Control. 2001;11.Incidence & Prevalence: 2006 Chart Book on Cardiovascular and Lung Diseases. NHLBI, NIH. 2006.Turabelidze G. J Neurol Sci. 2008;269:158-62.
29. Sepsis Morbidity and Mortality In severe cases, one or more organs fail. Worst case scenario: Blood pressure drops Septic shock Multiple organ system failure Death The number of sepsis cases per year has been on the rise: Aging population, the increased longevity of people with chronic diseases, the spread of antibiotic-resistant organisms, an upsurge in invasive procedures and broader use of immunosuppressive and chemotherapeutic agents. http://www.nigms.nih.gov/Education/factsheet_sepsis.htm

30. How Do I Decide Who is Really Sick With an Infection?

31. Sepsis Biomarkers Use in Diagnosis, Risk, and Response
32. Utility of Biomarkers Diagnosis/differentiation Prognostication Value of baseline Value of change over time Following success/failure of therapy
33. Diagnosis of Sepsis Bacteria in the blood or other body fluids Source of the infection A high or low white blood cell count A low platelet count Low blood pressure Too much acid in the blood (acidosis) Altered kidney or liver function  Biomarkers
34. Sepsis Biomarkers: Screening Diagnosis of sepsis and evaluation of its severity is complicated by the highly variable and non-specific nature of signs and symptoms. Distinguishing patients with localized infections or SIRS from those with sepsis is challenging. SIRS is not specific to sepsis and can result from other conditions such as acute pancreatitis and immunodeficiencies. Biomarkers of sepsis may improve diagnosis and therapeutic decision making. Lever A, Mackenzie I. Br Med J. 2007;335:879–83.
35. Sepsis Biomarkers More than 170 biomarkers have been assessed for sepsis prognosis and diagnosis Some common biomarkers include: White blood cell count Interleukins and other cytokines Procalcitonin (PCT) C-reactive protein (CRP) Lactate Procoagulant factors Pierrakos C, Vincent JL. Crit Care. 2010,14:R15.
36. Procalcitonin and Accuracy of Diagnosis 1.00 0.75 0.50 0.25 0.00 Sensitivity Clinical model with PCTAUC: 0.94 Clinical model without PCTAUC: 0.77 0.00 0.25 0.50 0.75 1.00 1 - Specificity Harbarth S, Holeckova K, Froidevaux C et al. Am J Respir Crit Care Med. 2001;164:396-402.
37. Procalcitonin Reference Range ACCP/ Society of Critical Care Medicine Consensus Conference. Crit Care Med. 1992;20:864-74. Harbarth S, Holeckova K, Froidevaux C et al. Am J RespirCrit Care Med. 2001;164:396-402. Christ-Crain M, Jaccard-Stolz D, Bingisser R et al. Lancet. 2004;363:600-7.
38. Survival Curves in Severe Sepsis and Septic Shock: Baseline Cortisol and Post-ACTH 1.00 Basal Plasma Cortisol Level  34 g/dL,  max > 9 g/dL 0.80 Basal Plasma Cortisol Level  34 g/dL,  max  9 g/dL or Basal Plasma Cortisol Level > 34 g/dL,  max > 9 g/dL 0.60 Probability of Survival 0.40 0.20 Basal Plasma Cortisol Level > 34 g/dL,  max  9 g/dL 0 0 7 14 21 28 Time (Days) Annane D, Sebille V, Troche G et al. J Am Med Assoc. 2000;283:1038-45.
39. CRP Levels Correlate With Mortality and Organ Failure in Sepsis CRP Day 0 Survivors * CRP Day 2 Non-Survivors 30 20 10 0 30 20 10 0 p = 0.001 * p < 0.05 p = 0.002 CRP mg/dL CRP mg/dL 0 1 2 3 > 4 Day 0 Day 2 (116/115) (111/110) (56/56) (20/19) (4/4) Number of Organ Failures Survivors vs. Non-Survivors Lobo SM, Lobo FR, Bota DP et al. Chest. 2003;123:2043-9.
40. Anaerobic Glycolysis (Cytoplasm) (Mitochondria) O2 Glycogen Glucose Pyruvate Citric Acid Cycle CO2 H2O Lactate 1 Glu + 2 ADP + 2 Pi 2 Lactate + 2 ATP 1 Glu + 6 O2 + 38 ADP + 38 Pi 6 CO2 + 6 H20 + 38 ATP Lactic Acidosis Aerobic Glycolysis Mizock BA, Falk JL. Crit Care Med. 1992;20:80-95.
41. Diagnostic and Therapeutic Markers 80 60-80% Normal 60 50% Lactic Acidosis (≥ 4 mmol/L) 40 SvO2
42. Serum Lactate as a Predictor of Mortality 30 25 20 15 10 5 0 50 40 30 20 10.0 0.0 28% 38-40% % of Mortality Rate % of Mortality Rate 0-2.4 2.5-3.9 > 4.0 0-2.4 2.5-3.9 > 4.0 N = 827 N = 238 N = 112 Initial Lactate (mmol/L)2 Lactate1 Death within 3 days 28 day in-hospital mortality 1 Trzeciak S, Dellinger RP, Chansky ME et al. Intensive Care Med. 2007;33:970-7. 2 Shapiro NI, Howell MD, Talmor D et al. Ann Emerg Med. 2005; 45:524-8.
43. Prognostic Value of Serum Lactate Jansen TC, van Bommel J, Mulder PG et al. Crit Care. 2008,12:R160.
44. Serum Lactate as a Predictor of Mortality 7 6 5 4 3 2 1 0 Non-survival Survival p = 0.001 p < 0.001 Mean Lactate Level (mmol/L) Arrival Scene (T1) Emergency Department (T2) Jansen TC, van Bommel J, Mulder PG et al. Crit Care. 2008,12:R160.
45. Value of Blood Lactate Levels First Lactate Measurement N = 124 < 3.5 mmol/l ≥ 3.5 mmol/l 11 Missing (0 Died) 7 Missing (4 Died) 8/66 (12%) Mortality 24/58 (41%) Mortality p < 0.001 Second Lactate Measurement N = 55 N = 51 < 3.5 mmol/l ≥ 3.5 mmol/l < 3.5 mmol/l ≥ 3.5 mmol/l 8/54 (15%) Mortality 0/1(0%) Mortality 2/14 (14%) Mortality 18/37(49%) Mortality p = 1.00 p = 0.025 N = 106 Second Lactate Cumulative < 3.5 mmol/l ≥ 3.5 mmol/l 10/68 (15%) Mortality 18/38 (47%) Mortality p < 0.001 Adapted from Jansen TC, van Bommel J, Mulder PG et al. Crit Care. 2008,12:R160.
46. Lactate, SBP, and Mortality 60 50 40 30 20 10 0 Mortality (%) > 3.5 < 100 Lactate (mmol/l) > 100 < 3.5 SBP (mmHg) Jansen TC, van Bommel J, Mulder PG et al. Crit Care. 2008,12:R160.
47. Serum Lactate and Mortality in Severe Sepsis Initial serum lactate evaluated in 839 adults admitted with severe sepsis. p = 0.001 50 45 40 35 30 25 20 15 10 5 0 p = 0.022 p < 0.001 28-Day Mortality (%) High initial serum lactate associated with ↑ mortality regardless of presence of shock or MODS. p = 0.024 Low Int High Low Int High Non-Shock Shock Mikkelsen ME, Miltiades AN, Gaieski DF et al. Crit Care Med. 2009;37:1670-7.
48. Improving Lactate a Good Prognostic Sign 8 6 4 2 0 p < 0.05 Non-survivors Lactate (mmol/L) p < 0.05 p < 0.01 Survivors INITIAL +8h +16h +24h FINAL Time Bakker J, Gris P, Coffernils M et al. Am J Surg. 1996;171:221-6.
49. Sepsis is No Longer Just an ICU Disease Levy MM, Dellinger RP, Townsend SR et al. Crit Care Med. 2010;38:367-74.
50. Hospital Origin and Mortality Origin Mortality ED 52.4% 27.6% ICU 12.8% 41.3% Wards 34.8% 46.8% You have to go to the disease instead of waiting for it to come to you! Surviving Sepsis Campaign. http://ssc.sscm.org.
51. Risk Stratification of Sepsis Hypotension, vasopressors 36.7% Lactate > 4 mmol/L only 30.0% SBP < 90 mmHg and lactate > 4 mmol/L 46.1% Surviving Sepsis Campaign. http://ssc.sscm.org.
52. Sepsis Testing and Results Guidelines, Algorithms, and Protocols
53. Factors to Consider When Evaluating Sepsis Blood gases Electrolytes Glucose Hematocrit Lactate
54. Sepsis Resuscitation Bundle The Sepsis Resuscitation Bundle is published by the Surviving Sepsis Campaign and is used by multiple hospitals across the country. The goal is to perform all indicated tasks 100% of the time within the first 6 hours of identification of severe sepsis. Surviving Sepsis Campaign. http://ssc.sscm.org.
55. Sepsis Resuscitation Bundle Measure serum lactate. Obtain blood cultures prior to antibiotic administration. Administer broad-spectrum antibiotic, within 3 hours of emergency department (ED) admission and within 1 hour of non-ED admission. In the event of hypotension and/or a serum lactate > 4 mmol/L: Deliver an initial minimum of 20 ml/kg of crystalloid or an equivalent. Apply vasopressors for hypotension not responding to initial fluid resuscitationto maintain mean arterial pressure (MAP) > 65 mmHg. In the event of persistent hypotension despite fluid resuscitation (septic shock) and/or lactate > 4 mmol/L: Achieve a central venous pressure (CVP) of > 8 mmHg. Achieve a central venous oxygen saturation (ScvO2) > 70% or mixed venous oxygen saturation (SvO2) > 65%. Surviving Sepsis Campaign. http://ssc.sscm.org.
56. Sepsis Management Bundle Efforts to accomplish these goals should begin immediately, but these items may be completed within 24 hours of presentation for patients with severe sepsis or septic shock. The tasks are: Administer low-dose steroids for septic shock in accordance with a standardized ICU policy. If not administered, document why the patient did not qualify for low-dose steroids based upon the standardized protocol. Administer recombinant human activated protein C (rhAPC) in accordance with a standardized ICU policy. If not administered, document why the patient did not qualify for rhAPC.   Maintain glucose control > 70 mg/dL, but < 150 mg/dL. Maintain a median inspiratory plateau pressure (IPP) < 30 cm H2O for mechanically ventilated patients. Surviving Sepsis Campaign. http://ssc.sscm.org.
57. Point-of-Care Analyte Benefits A 2010 study published in the Journal of Emergency Medicine found that point-of-care testing provided a reliable and feasible way to measure serum lactate at the bedside.1 Base excess (BE) Some studies suggest BE is an accurate marker for the prediction of elevated lactate in the emergency department (ED).2 Some studies also show poor correlation due to effects of other conditions.3 Point-of-care lactate is useful in the diagnosis of sepsis at the bedside Recommended for institutions where clinical decisions are limited by lack of laboratory infrastructure or reliability.4 1 Shapiro NI, Fisher C, Donnino M et al. J Emerg Med. 2010;39:89-94.2 Montassier E, Batard E, Segard J et al. Am J Emerg Med. 2010. Epub ahead of print.3 Martin MJ, FitzSullivan E, Salim A et al. Am J Surg. 2006;191:625-30.4 Moore CC, Jacob ST, Pinkerton R et al. Clin Infect Dis. 2008;46:215-22.
58. Point-of-Care Analyte Testing Single-use, self-calibrating, 100-150 uL WB sample pH, pCO2, pO2, lactate, Na, K, iCa, Glu, Hct (plus calculated values TCO2, HCO3, BE, sO2, Hb) Analytes on a single test card Requires only room temperature storage* Bar-coded for patient safety Cant use expired cards* Smart card technology Low-cost test cards < $ than benchtop systems *unique market features
59. Turnaround Time Serum lactate must be available with rapid turnaround time (within minutes) to effectively treat severely septic patients.  An arterial blood gas analyzer located in the clinical laboratories usually accomplishes this.  Hospitals should invest in adequate equipment in to meet present standards of care for septic patients. If a central analyzer is not efficient in a particular hospital setting, point-of-care analyzers should be evaluated for faster turnaround time. http://www.survivingsepsis.com/bundles/individual_changes/serum_lactate.www.emcrit.org/wp-content/uploads/lactate-faq.pdf.
60. Time and Cost for Measuring Blood Lactate Boldt J, Kumle B, Suttner S et al. Acta Anaesthesiol Scand. 2001;45:194–9.
61. Sepsis Treatment Guidelines Antibiotic therapy Begin intravenous antibiotics as early as possible Always within the first hour of recognizing severe sepsis (1D) and septic shock. (1B) Broad-spectrum One or more agents active against likely bacterial/fungal pathogens and with good penetration into presumed source. (1B) Reassess antimicrobial regimen daily to optimize efficacy, prevent resistance, avoid toxicity, & minimize costs. (1C) Consider combination therapy in Pseudomonas infections. (2D) Surviving Sepsis Campaign. http://ssc.sscm.org.
62. Sepsis Treatment Guidelines Antibiotic therapy Consider combination empiric therapy in neutropenic patients. (2D) Combination therapy no more than 3-5 days and de-escalation following susceptibilities. (2D) Duration of therapy typically limited to 7-10 days Longer if response slow, undrainable foci of infection, or immunologic deficiencies. (1D) Stop antimicrobial therapy if cause is found to be non-infectious. (1D) Surviving Sepsis Campaign. http://ssc.sscm.org.
63. CCF Sepsis Flowchart SIRS + Suspected Infection SIRS 2 Criteria T > 38° or < 36° HR > 90 RR > 20 or PaCO2 32 mmHg WBC > 12 K or < 4 K; Bands > 10% Lactate POC (< 15’) Fluid Bolus Organic Dysfunction SBP < 90 p bolus Lactate > 4 Sepsis Identification No Yes Severe Sepsis/ Septic Shock Abs Labs Cultures Mechanical Ventilation Antibiotics (< 4°) Initiate Sepsis Bundle Central Venous Arterial Line Access (< 2°) Rapid CV CVP < 8 NS 500 cc IVB until CVP > 8 Preload NTG 10-60 mcg/min until CVP < 12 or MAP < 110 CVP > 15 + MAP > 110 MAP < 65 Norepinephrine 2-20 mcg/min or Dopamine 5-20 mcg/kg/min Afterload MAP > 110 NTG 10-60 mcg/min until MAP < 90 or Hydralazine 10-40 mg IV EGDT Central Venous Oxygen Content ScvO2 < 70% Hgb < 10 Hgb Transfuse PRBC ScvO2 < 70% ScvO2 Dobutamine 2.5-20 mcg/kg/min CVP 8-12 MAP 65-110 ScvO2 > 70% Lactate improved Goals Achieved? No Glycemia Control 80-110 Semi-recumbent Position APACHE III > 25 Bundled Therapies Activated Protein C Rivers EP. N Engl J Med. 2001;345:1368-77.
64. Applications Identification, Treatment, and Outcomes
65. Identification of Sepsis If YES, patient has SIRS If YES, patient has Sepsis If YES, patient has Severe Sepsis Daniels R. J Antimicrob Chemother. 2011;66(Suppl 2):ii11–ii23.
66. Treatment of Patients With Sepsis Early goal-directed therapy: standard operating procedure Apply with critical care/sepsis team if patient remains hypotensive or lactate remains high following fluid challenges Site central venous catheter using ultrasound guidance where practicable, according to proper procedures for infection control If central venous pressure (CVP) < 8 mmHg, give further fluid challenges to achieve a target CVP of > 8 mmHg (> 12 mmHgif ventilated) unless the patient shows signs of fluid overload If patient remains hypotensive, start a norepinephrine infusion to target SBP > 90 mmHg or MBP > 65 mmHg. Daniels R. J Antimicrob Chemother. 2011;66(Suppl 2):ii11–ii23.
67. Improve Patient Outcomes Lactate clearance is associated with improved patient outcome. Lactate measurement is associated with increased risk of death independent of other aspects of sepsis bundle guidelines. Point-of-care measurements of lactate are faster than central laboratories. May be beneficial for serial measurements. Nguyen HB, Rivers EP, Knoblich BP et al. Crit Care Med. 2004;32(8):1637-42. Afessa B, Keegan MT, Schramm GE et al. Crit Care Med. 2011;15(Suppl 1): P286. Boldt J, Kumle B, Suttner S et al. Acta Anaesthesiol Scand. 2001;45:194–9.
68. Conclusion