Copyright and License Notice for PDF Courses

LabCE courses are provided in PDF format for the sole use of LabCE subscribers. Distribution to non-subscribers is prohibited in every form, including electronic and print. Do not make multiple copies of this PDF file. If you are an individual subscriber, you are the only person authorized to use this PDF file. Please do not redistribute it to others inside or outside your organization. Instead, please contact LabCE about obtaining an institutional subscription. This Copyright and License Notice is part of the Terms of Service for LabCE. If you have any questions, please contact us. LabCE retains all copyright to this course and all material contained therein.

Laboratory Methods to Aid in the Detection of Sepsis

Author: Lynne Brodeur, MA, MLS(ASCP) Reviewer: DeRhonda Crawford, MT(ASCP)

Course Instructions
Please proceed through the course by clicking on the blue arrows or text links. Use the table of contents to monitor your progress. Your progress will be saved automatically as you proceed through the course, and you may later continue where you left off even if you use a different computer. You may encounter practice questions within the course, which are not graded or recorded.

Course Info
This course carries the following continuing education credits:
l

P.A.C.E. Contact Hours: 1.00 hour(s) Course Number: 578-041-12 Florida Board of Clinical Laboratory Science CE - General (Clinical Chemistry/UA/Toxicology): 1.00 hour(s)

Development and Progression of Sepsis

Sepsis Definition
Sepsis is an overreaction by the immune system to infection (usually bacterial, but could be viral, fungal, or parasitic). It is a systemic inflammatory response, which can be life-threatening. A weakened immune system, certain chronic disorders, an artificial joint or heart valve, and certain heart valve abnormalities increase the risk for sepsis. Sepsis has been reported to be the most common cause of death in the noncoronary intensive care unit. Note that it is sepsis (the immune system's response) that is usually the cause of death and not the infection. Therefore, it is crucial that the recognition of sepsis be made as quickly as possible. Delay in identifying sepsis limits the effectiveness of treatment. Sepsis includes two or more of the following symptoms:
l

l l l

A body temperature >38C (100.4F) or <36C (96.8 F) A heart rate >90 beats/minute Respiratory rate >20 breaths/minute An alteration in the white blood cell (WBC) picture, such as a count >12.0 x 10 /L or <4.0 x 10 /L or >10% immature neutrophils
9 9

However, these inflammatory responses may also occur in the absence of infection, a condition termed systemic inflammatory response syndrome (SIRS). When the cause of the systemic inflammatory response is infection, the condition is defined as sepsis.

Development and Progression of Sepsis

Additional Criteria for Diagnosis of Sepsis

Blood cultures are important for diagnosis of sepsis, but they take time to grow. This time can mean the difference between life and death for a patient. Although sepsis cannot be definitively diagnosed until it is determined that there is a blood or tissue infection, the diagnosis may still be made if other criteria strongly suggests its presence. If the patient exhibits altered mental status and/or edema in addition to the SIRS criteria, or if one or more of the laboratory test results shown below are obtained along with evidence from the clinical assessment, the physician may choose to start antibiotic treatment before the results of the cultures are available:

l l l l

Hyperglycemia in the absence of diabetes Elevated C-reactive protein (CRP) Elevated procalcitonin Elevated lactic acid (lactate)

Development and Progression of Sepsis

Severe Sepsis

If sepsis progresses to severe sepsis, organs begin to shut down. Multiple organ (lung, liver, and kidney) dysfunction is possible, which may result in death. Severe sepsis is characterized by organ dysfunction, hypoperfusion, and/or hypotension.

Development and Progression of Sepsis

Septic Shock
A patient progresses to septic shock from severe sepsis if the hypotension due to the systemic infection does not respond to fluid resuscitation. Septic shock is life-threatening with a mortality rate of 40 - 60%.

Development and Progression of Sepsis

Sepsis and Bacterial Toxins

Sepsis occurs when toxins produced by the bacteria cause cells in the body to release cytokines. Cytokines then produce inflammation. Even though cytokines aid the immune system in fighting infection, they also can have detrimental side effects that may include vasoconstriction (restriction of blood flow) and hypercoagulation in capillaries that supply blood to organs. This in turn can lead to a series of severe complications such as:

l l

l l l

Continuous cycle of inflammation and coagulation Weakening of the heart caused by the strain of increased pumping in an attempt to compensate for the decreased blood flow Cardiovascular insufficiency Tissue hypoxia Multiple organ failure

Development and Progression of Sepsis

Ungraded Practice Question

Which of the following are indicators of sepsis? More than one answer is correct. Please select all correct answers c Erythrocytosis d e f g c Increased heart rate d e f g c Leukocytosis d e f g

c Increased body temperature d e f g

Development and Progression of Sepsis

Ungraded Practice Question

Which of the following are indicators of sepsis? More than one answer is correct. Please select all correct answers c Erythrocytosis d e f g c Increased heart rate d e f g c Leukocytosis d e f g c Increased body temperature d e f g

Feedback Sepsis results in an immune response by the body. This response includes increasing body temperature and leukocytes in order to help fight off the infection, and increased heart rate in order to get more blood and oxygen to the tissues. Erythrocytosis is not part of the immune response.

Laboratory Tests Used in the Detection of Sepsis

Biomarkers
An ideal biomarker for diagnosis of disease has these properties:

l l l l

High sensitivity (accurately identifies the presence of disease and has few false-negatives) High specificity (accurately detects the absence of disease and has few false-positives) Relates to the extent of disease Changes as the clinical condition evolves

A biomarker that is able to identify sepsis, or determine which patients with sepsis are likely to develop severe sepsis, would be very useful. However, an ideal marker for sepsis is still not available. C-reactive protein (CRP), procalcitonin (PCT), and lactic acid (lactate) are currently the tests that are most often used to aid in the detection of sepsis. However, test specificities and sensitivities are not high and if these tests are used to presumptively diagnose sepsis prior to availability of the blood culture or other culture reports, the results must be interpreted along with the clinical assessment. As mentioned earlier in the course, sepsis may include these symptoms:
l l l l l

A body temperature >38C or <36C A heart rate >90 beats/minute Respiratory rate >20 breaths/minute An alteration in the white blood cell (WBC) picture, such as a count >12.0 x 109 /L or < 4.0 x 109 /L or >10% immature neutrophils Altered mental status

l l l l l

Edema Hyperglycemia in the absence of diabetes Elevated CRP Elevated PCT Elevated lactic acid (septic shock)

Laboratory Tests Used in the Detection of Sepsis

Glucose
Hyperglycemia commonly develops in sepsis; it is a part of the body s inflammatory response as well as a common response to stress. However, hyperglycemia does not always develop with sepsis, and in some patients, it may develop even in mild disease. Therefore, it cannot be considered a reliable biomarker as it lacks both specificity and sensitivity. It may be used along with other indicators to identify sepsis.

Laboratory Tests Used in the Detection of Sepsis

C-Reactive Protein (CRP)

CRP begins to rise within 4-6 hours after stimulus from an inflammation/infection. The level doubles every eight hours and peaks at 36-50 hours. CRP is a sensitive marker of inflammation and tissue damage. However, it has a low specificity. Conditions other than sepsis that can cause a rise in CRP levels include:

l l l

Rheumatic diseases Systemic lupus erythematosus Systemic sclerosis Sjogren syndrome Inflammatory bowel disease Leukemia Transfusion associated graft-vs-host disease

Once the determination has been made that sepsis is present and therapy has been initiated, CRP is useful for monitoring response to antibiotics and predicting prognosis.

Laboratory Tests Used in the Detection of Sepsis

Procalcitonin (PCT)
PCT increases early in infection and has a greater specificity for infection than CRP. Increased PCT can be observed within 3-6 hours of infection. PCT enables the differentiation between a severe bacterial infection and other clinical conditions that may be causing a systemic inflammatory response, allowing antibiotic treatment to begin sooner. Elevated PCT values generally correlate well

with positive blood and other culture results. Depending on the clinical assessment, a PCT concentration >0.1 ng/mL (reference value <0.05 ng/mL) can indicate clinically relevant bacterial infection. PCT levels are usually low in viral disorders, chronic inflammatory conditions, or autoimmune conditions.

Laboratory Tests Used in the Detection of Sepsis

Procalcitonin (PCT) as a Sepsis Biomarker

PCT significantly increases during severe sepsis and septic shock, making it a valuable marker for determining prognosis and monitoring antibiotic therapy. PCT concentrations increase considerably in patients with septic shock and decrease with successful treatment of septic infection. As the PCT increases, so does the risk for severe sepsis and septic shock. In healthy individuals, PCT is < 0.05 ng/mL. Moderately elevated levels (up to 2.0 ng/mL) indicate that sepsis is possible, and there is a slight risk of progression to severe sepsis. PCT levels in sepsis are generally greater than 2 ng/mL and often can reach values between 10 and 100 ng/mL in severe sepsis, or considerably higher in septic shock. However, the role of PCT in the discrimination between SIRS and sepsis is still controversial. Although the majority of studies have shown higher values in patients with sepsis, a recent randomized trial did not support its use and patients in the PCT group actually had longer hospital stays.* Reference: Faix J. Sepsis: New approaches to diagnosis and treatment. Clin Lab News. July 2012:38(7);12-14.

Laboratory Tests Used in the Detection of Sepsis

Lactic Acid (Lactate)

Blood lactic acid concentration may be measured to detect and monitor impaired circulation and tissue oxygenation in critically ill patients, such as patients experiencing septic shock. When cells do not receive enough oxygen because they are not receiving enough blood, they release excess lactic acid into the bloodstream. Organ failure as a result of septic shock may be indicated by unexplained metabolic acidosis (low blood pH and low bicarbonate level) and extremely elevated lactic acid, where blood pH is <7.30 and plasma lactic acid is >1.5 times the upper limit of the laboratory s established reference values. Lactic acid can be produced in all tissues, especially skeletal muscle, brain, erythrocytes, and kidneys. In homeostasis, lactic acid clears very quickly through liver metabolism and reconversion of lactate back to pyruvate. This process results in minimal lactic acid levels in normal arterial and venous blood samples.

Laboratory Tests Used in the Detection of Sepsis

Lactic Acid (Lactate), continued

Hyperlactemia in patients with sepsis is an indicator of the severity of stress response. Hyperlactemia may possibly develop as a byproduct of overall acceleration in glycolysis in severe sepsis. This may well be an adaptive host mechanism designed to provide for efficient generation of energy in response to severe stress. However, some investigators have observed that patients with sepsis have decreased lactate clearance rather than increased lactate production. Skeletal muscle and lung tissue have been shown to produce lactate during sepsis. Therefore, hyperlactemia may be caused by increased lactate production in the gut, liver, lungs, and skeletal muscles, decreased lactate clearance in the liver, or a combination of both.

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Which of the following statements regarding a biomarker with high sensitivity is true? Please select the single best answer j Accurately identifies the presence of disease and has few false-negatives k l m n j Accurately identifies the presence of disease and has few false-positives k l m n j Accurately detects the absence of disease and has few false-negatives k l m n j Accurately detects the absence of disease and has few false-positives k l m n

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Which of the following statements regarding a biomarker with high sensitivity is true? Please select the single best answer j Accurately identifies the presence of disease and has few false-negatives k l m n j Accurately identifies the presence of disease and has few false-positives k l m n j Accurately detects the absence of disease and has few false-negatives k l m n j Accurately detects the absence of disease and has few false-positives k l m n

Feedback A biomarker with high sensitivity accurately identifies the presence of disease and has few false-negatives. A biomarker with high specificity accurately detects the absence of disease and has few false-positives.

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

C-reactive protein (CRP) is more useful for monitoring response to antibiotics and predicting prognosis than for actual diagnosis of sepsis. Select true or false j True k l m n j False k l m n

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

C-reactive protein (CRP) is more useful for monitoring response to antibiotics and predicting prognosis than for actual diagnosis of sepsis. Select true or false j True k l m n j False k l m n

Feedback CRP is a sensitive marker for detection and monitoring progression of inflammation and tissue damage. However, CRP lacks specificity. Once a diagnosis of sepsis has been made and therapy has been initiated, CRP is useful for monitoring response to antibiotics and predicting prognosis.

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Of the three laboratory tests that are listed, which has proven to be most effective for early differentiation of systemic inflammatory response syndrome (SIRS) from sepsis due to its increase following infection and higher specificity? Please select the single best answer j C-reactive protein (CRP) k l m n j Procalcitonin (PCT) k l m n j Lactic acid k l m n

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Of the three laboratory tests that are listed, which has proven to be most effective for early differentiation of systemic inflammatory response syndrome (SIRS) from sepsis due to its increase following infection and higher specificity? Please select the single best answer j C-reactive protein (CRP) k l m n j Procalcitonin (PCT) k l m n j Lactic acid k l m n

Feedback PCT usually rises within 3-6 hours of infection. CRP also increases rapidly following infection, but is not as specific for infection as PCT. A rise in CRP could also occur with SIRS. Lactic acid is usually used to detect and monitor impaired circulation and tissue oxygenation in critically ill patients. .

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Blood lactic acid concentration is an indicator of impaired circulation and tissue oxygenation in critically ill patients. If circulation and tissue oxygenation are impaired, blood lactic acid concentration will decrease below the lower end of the established reference range. Select true or false j True k l m n j False k l m n

Laboratory Tests Used in the Detection of Sepsis

Ungraded Practice Question

Blood lactic acid concentration is an indicator of impaired circulation and tissue oxygenation in critically ill patients. If circulation and tissue oxygenation are impaired, blood lactic acid concentration will decrease below the lower end of the established reference range. Select true or false j True k l m n j False k l m n

Feedback The statement is false. Lactic acid will increase. When cells do not receive enough oxygen because they are not receiving enough blood, they release excess lactic acid into the bloodstream. Organ failure as a result of septic shock may be indicated by unexplained metabolic acidosis (low blood pH and low bicarbonate level) and extremely elevated lactic acid, where blood pH is <7.30 and plasma lactic acid is >1.5 times the upper limit of the laboratory s established reference values.

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Mechanism of C-Reactive Protein (CRP)

CRP is an acute phase reactant that is synthesized in the liver. Serum CRP levels increase following a variety of proinflammatory events such as infection, tissue necrosis, trauma, surgery, and malignancy. CRP levels can increase quickly and dramatically (often 100-fold) during inflammation. CRP can activate complement, bind Fc receptors and can function as an opsonin, enhancing phagocytosis with certain infections.

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Mechanism of Procalcitonin (PCT)

PCT is a precursor of the hormone calcitonin and is synthesized physiologically by thyroid C cells. PCT levels are low in homeostatic conditions. In bacterial infections, PCT is synthesized in various extrathyroidal neuroendocrine tissues. Overall, PCT secretion is a component of the inflammatory response that appears to be relatively specific to systemic bacterial infections. PCT is a 116-amino acid polypeptide precursor to the calcium regulatory hormone calcitonin. It is composed of three sections (see image on the right):

l l l

The amino terminus (N-ProCT) Immature calcitonin Katacalcin

Synthesis of PCT is regulated by the Calc-1 gene located on chromosome 11. In healthy individuals, production of PCT, and subsequently calcitonin, is restricted to the thyroid C cells.

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Mechanism of Lactic Acid (Lactate)

Pyruvate is the normal end-product of glycolysis (glucose metabolism). In

the presence of oxygen, pyruvate is converted to acetyl-coenzyme A (CoA), which ultimately produces energy in the form of ATP. However, in an oxygen-deficient environment, CoA is not produced and pyruvate is converted to lactate through anaerobic metabolism. The quantitative measurement of lactic acid in plasma indicates the severity of oxygen deprivation. An elevated lactate is known to be associated with increased mortality rates. If the lactate can be cleared, prognosis improves. Patients who develop severe sepsis or septic shock commonly demonstrate hyperlactemia and lactic acidosis. Increased lactate production during anaerobic metabolism and decreased lactate clearance are likely contributors to hyperlactemia.

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Other Causes of Increased Lactic Acid (Lactate) Concentration

Lactate can also be increased, even when there is no evidence of oxygen deprivation. Increased lactate can occur if patients have decreased activity of the enzyme pyruvate dehydrogenase or impaired clearance of lactate as a result of hepatic dysfunction. Plasma lactate concentration can be falsely increased if the plasma is not separated from the cells shortly after sample collection (Centrifugation and separation of plasma from cells is recommended within 15 minutes of collection). Blood cells continue to metabolize glucose following collection, resulting in the production of lactic acid. For this reason, a gray-top tube containing sodium fluoride, which inhibits glycolysis, is usually recommended for plasma lactate sample collection. Other preanalytic errors that may also produce falsely increased lactate concentrations include: tourniquet usage during specimen collection combined with patient clenching and unclenching his/her fist and specimen hemolysis. It is also recommended that the blood specimen is placed on ice immediately after collection to further inhibit glycolysis and lactic acid formation.

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Ungraded Practice Question

In healthy individuals, procalcitonin is synthesized by which cells in the body? Please select the single best answer j Hepatocytes k l m n

j Leukocytes k l m n j Thyroid C cells k l m n j Red blood cells k l m n

Mechanisms of C-Reactive Protein, Procalcitonin, and Lactic Acid

Ungraded Practice Question

In healthy individuals, procalcitonin is synthesized by which cells in the body? Please select the single best answer j Hepatocytes k l m n j Leukocytes k l m n j Thyroid C cells k l m n j Red blood cells k l m n

Feedback In healthy individuals, procalcitonin is only synthesized by the thyroid C cells. In bacterial infections, PCT is synthesized in various extrathyroidal neuroendocrine tissues.

Future Perspectives

Increase in Sepsis Cases

The incidence of sepsis is reportedly increasing by 8% annually (adjusting for population). The highest increases in cases of severe sepsis are reported in older adults and the nonwhite population. The rise in the number of cases is believed to be caused by:

l l l l l l l

Immunosuppresive conditions, including HIV/AIDS, cancer, and solid organ tumors Increased use of invasive procedures Immunosuppressive drugs Chemotherapy Organ transplantation Prosthetic implants Antimicrobial resistance

Age is a risk factor in itself, even without an underlying medical condition. With the aging of the "baby boomer" generation, the United States will soon have a larger group of people over the age of 65 than it has every had before in the history of the country. The number of people over the age of 65 in 2030 is predicted to be double what it was in 2000. Oltermann has dubbed this potential occurrence the "sepsis boom."*

*Reference: Oltermann MH. The coming "sepsis boom...". MLO online. Available at: http://www.mlo-online.com/articles/201202/thecoming-and-ldquosepsis-boom-and-rdquo.php. Accessed October 30, 2012.

Future Perspectives

Novel Biomarkers That Focus on Immunosuppression

With the rise in occurrence of sepsis, it is more important than ever to identify biomarkers that can be used for early detection of severe sepsis. Some researchers have been focusing on the transition that occurs from sepsis (overreaction by the immune system) to severe sepsis (immunosuppression) and the alterations in monocytes and T cells that are characteristic of the immunosuppressive phase of severe sepsis. Research has determined that circulating monocytes from patients with severe sepsis have decreased amounts of major histocompatibility complex (MHC) class II proteins on their surfaces and circulating T cells have significantly increased CTLA-4 ligand. It is yet to be determined if these facts can translate to useful biomarkers for early identification of severe sepsis. Flow cytometry may be the laboratory method of choice, if changes in circulating monocytes and T cells provide the ideal biomarkers for early detection of severe sepsis.

References

References
Angus DC, Linde-Zwirble WT, Lidicker J, et al. Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med. 2001;29:1303-1310. Castelli GP, Pognani C, Meisner M, et al. Procalcitonin and C-reactive protein during systemic inflammatory response syndrome, sepsis, and organ dysfunction. Available at: http://ccforum.com/content/8/4/R234. Accessed October 30, 2012. Faix J. Sepsis: New approaches to diagnosis and treatment. Clin Lab News. July 2012;38(7):12-14. Harbarth S, Holeckova K, Froidevaux C, et al. Geneva Sepsis Network. Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis. Am J Respir Crit Care Med. 2001;164:396-402. LaRosa SP. Sepsis. Cleveland Clinic website. Available at: http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectious-disease/sepsis/#s0050. Accessed October 30, 2012. Mller B, Becker KL, Schchinger H, et al. Calcitonin precursors are reliable markers of sepsis in a medical intensive care unit. Crit Care Med. 2000;28(4):977-983. . Oltermann MH. The coming "sepsis boom...". MLO online. Available at: http://www.mlo-online.com/articles/201202/the-comingand-ldquosepsis-boom-and-rdquo.php. Accessed October 30, 2012.