The Role of C-Reactive Protein in the Evaluation and Management of Infants With Suspected Sepsis

Joan M. Hengst, RNC, MSN, ARNP

Adv Neonatal Care. 2003;3(1)

Laboratory Methods to Measure CRP

The 3 clinical laboratory methods used to measure serum CRP levels are as follows:

Qualitative
Semiquantitative
Quantitative

All 3 tests are based on the ability of CRP to bind to a variety of biologic ligands forming CRP-ligand complexes. When a reagent containing anti-human CRP antibodies is added to a serum sample containing CRP, the CRP binds to the antibodies forming insoluble CRP-ligand complexes that clump and precipitate, which can then be visualized and measured. Table 2 provides a comparison of these methods.

The qualitative latex agglutination test is the first laboratory method developed to measure CRP.^[12,29,43] This method measures the presence or absence of agglutination and precipitation, indicating only whether CRP is present or absent in the serum sample.^[12,29] A positive test result indicates the presence of CRP-ligand complexes formed when CRP binds to cause agglutination and precipitation, whereas a negative test result occurs when no agglutination is present.^[12,44] Positive test results indicate a CRP level greater than 6 mg/L or more than 10 mg/L, depending on the specific testing kit and reagent being used.^[44] Qualitative tests can be performed rapidly at the bedside within 10 to 15 minutes.^[44]

Purely qualitative CRP testing methods typically are not used to measure CRP levels because they have a low sensitivity, with positive results occurring with any condition involving inflammation or tissue destruction.^[12,29,35] A positive qualitative CRP test should always be followed by a semiquantitative test, which is a more sensitive measuring method to quantify the concentration of CRP.

The semiquantitative latex agglutination assay involves the use of serial dilutions of serum and saline. Each is mixed with a latex reagent and observed for the presence of agglutination.^[29,44,45] The highest dilution in which agglutination is visualized corresponds to an approximate titer or concentration of CRP-ligand complexes.

Like the qualitative test, the semiquantitative test can be performed in 15 to 30 minutes and has a reported upper detection level between 6 and 10 mg/L.^[35,45] Results are reported as a ratio and/or an approximate mg/L concentration of CRP.^[44,45] The reported cost of a Murex semiquantitative testing kit (Murex Diagnostic, Norcross, GA) is approximately $21.00.^[44]

Quantitative immunoassay is the most rapid, sophisticated, and sensitive method of detecting and measuring CRP.^{[12,43,46,47]} The enzyme-linked immunoabsorbent assay (ELISA) and immunofluorescence quantitative test uses monoclonal anti-CRP antibodies marked with an enzyme or fluorescent tracer fixed to microwells located within a microtiter plate or test tube. When diluted human serum is added, CRP binds to the immobilized marked anti-CRP antibody forming bound CRP-ligand complexes.^[43] Unbound antibodies are then washed from the test tube. The fluorescent-marked CRP-ligand complexes can be visualized and measured under a fluorescence microscope. A substrate is added to the enzyme-marked CRP-ligand complexes, which reacts with the enzyme causing a color reaction. The specimen is read with a spectrophotometer, which calculates the net color absorbency. This method of testing can be performed within 10 to 15 minutes and is capable of detecting CRP levels between 1 to 40 mg/L.^[43,46,47]

Laser and rate nephelometry quantitative immunoassays use infrared light-emitting diodes and detectors passed through test tubes containing fixed amounts of anti-CRP monoclonal antibody mixed with human serum, resulting in the formation of CRP-ligand complexes.^{[29,46,47,48]} Because the antibody concentration remains constant, the extent of light scatter is determined from the amount of CRP-ligand complexes. This method can be performed within 30 to 60 minutes. When using a fully automated analyzer, however, it takes 10 minutes.^[29,47] The cost of the fully automated quantitative CRP test is reported to be around $50.00.^[47]

Qualitative and semiquantitative methods of measuring CRP, although less expensive, are not accurate enough to be used for infants.^[12,27,29] The limited detection level of 6 mg/L in some kits could easily miss infants with true sepsis and result in longer antibiotic therapy.^[47] It is imperative that practitioners be aware of which CRP testing method is used in their own institution and the sensitivity and specificity of that test when evaluating measured CRP levels in the clinical setting.

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Table 1. Essential terms used in describing neonatal sepsis
Term	Definition
Infection	The body's response to the invasion of any microorganism, including bacterial, fungal, protozoan, or viral agent, that causes clinical signs of infection.^[3]
Clinical signs of infection	Generalized signs and symptoms seen with any infectious process, including respiratory distress, lethargy or irritability, poor muscle tone, feeding intolerance, apnea, bradycardia, tachycardia, pallor, cyanosis, poor perfusion, and temperature instability.^[3,6]
Maternal risk factors	Factors, when present, that increase the risk for infection in the infant. These include prolonged rupture of membranes (PROM) >18 hr, maternal fever, chorioamnionitis, maternal vaginal culture positive for group B streptococcus, history of a previous infant with group B streptococcus disease, and premature delivery <37 weeks gestation, and are associated with an increased risk of early-onset sepsis in the infant.^[2,3,5]
Sepsis	A bacterial bloodstream infection identified by one or more positive blood cultures in the presence of clinical signs of infection.
Early-onset sepsis	Sepsis that occurs within the first 3 days of life. It is associated with birth canal organisms acquired in utero or during delivery. Group B streptococcus remains the primary causative agent. One or more maternal risk factors are usually present. Term and preterm infants often present with respiratory distress, which can progress quickly to multisystem involvement within the first 24 hours.^[2,3,5,6]
Late-onset sepsis	Sepsis that occurs after the first 3-5 days of life and is associated with nosocomially acquired microorganisms. It occurs more frequently in premature and LBW infants and often includes meningitis. Coagulase-negative staphylococci are the primary causing bacteria.^[9] Presentation of signs of infection are slow and insidious.^[3,4,6]

Table 2. Comparison of CRP testing methods
Test Type	What Is Measured	How the Test Is Performed	How the Results Are Reported
Qualitative	The presence or absence of CRP-antibody complexes within a given amount of serum sample.	A measured amount of serum is added to a slide or test tube, mixed with a latex reagent, and examined for any signs of agglutination.	Positive: Presence of any agglutination indicates a concentration of CRP >6 mg/L or >10 mg/L
Qualitative		A latex reagent usually consists of a uniform-sized polystyrene or plastic beads coated with anti-human CRP antibodies. CRP binds to the antibody forming a CRP-antibody complex, which precipitates.	Negative: Absence of agglutination indicates a concentration of CRP <6 mg/L or <10 mg/L
Semiquantitative	An approximate amount of CRP-antibody complexes within a given amount of serum sample.	Serial serum/saline dilutions are mixed with a latex reagent and examined for the presence of agglutination. The highest dilution in which agglutination is visualized corresponds to the approximate amount of CRP.	The highest dilution in which agglutination occurs is reported as an approximate titer of CRP in mg/dL or mg/L 1:6 ratio = 6 to 12 mg/L 1:12 ratio = 12 to 24 mg/L 1:24 ratio = 24 to 48 mg/L 1:48 ratio >48 mg/L
Quantitative	The amount of CRP-antibody complexes within a serum sample.	Diluted serum samples are mixed with a reagent containing monoclonal antibodies that react specifically to CRP. The resulting CRP-antibody complexes are then measured.	Both quantitative methods are reported as an amount in mg/L or mg/dL
	Directly measures CRP-antibody complexes marked with an enzyme or fluorescent tracer.	Enzyme-linked multiplied immunoassay (ELISA), immunofluorescence CRP binds to fixed monoclonal anti-CRP-antibodies marked with an enzyme or fluorescent tracer. Enzyme marked CRP-antibody complexes are measured by a handheld or fully automated analyzer spectrophotometer. Fluorescent marked CRP-antibody complexes are visualized and measured via a fluorescent microscope.
	Directly measures the amount of agglutination or precipitation caused by the presence of CRP-antibody complexes.	Immunoturbidity, nephelometry, or radial immunodiffusion A beam of light from an infrared light emitting diode or detector is passed through a test tube containing CRP-antibody complexes. The amount of light scattered is measured from different angles using a nephelometer device or handheld turbidimeter or automated analyzer. The amount of CRP is proportional to the amount of light scattered.

Table 3. Studies evaluating the sensitivity and specificity of CRP in infants
Author(s) and Sample	Testing Method	Cutoff Value	Samples Collected	Test and Timing	Sensitivity	Specificity	Comments
Russel et al.^[25] n = 56 premature and term infants	Quantitative rate nephelometry	>8 mg/L	Serial: daily	CRP	71%	72%
				I:T Ratio	34%	73%
Pourcyrous et al.^[15] n = 187 premature and term infants	Quantitative immuno-nephelometry	≥9 mg/L	Serial: at least 2 initial and 12 to 24 hours later			91%
Kawamura et al.^[23] n = 108 term and n = 240 preterm infants with suspected sepsis	Quantitative laser nephelometry	>10 mg/L	Serial: at least 3 levels		Preterm: 61.5%	Preterm: 95%	Infected infants' levels peaked at 48 hours
					Term: 75%	Term: 97.8%
Franz et al.^[20] n = 223 preterm infants	Quantitative nephelometry	≥10 mg/L	Serial: 2 levels initial and 12-60 hours later	CRP and IL-8	93%	100%
				CRP alone	41%	41%
Benitz et al.^[11] n = 1,002 EOS and n = 184 LOS preterm and term infants	Qualitative rate nephelometry	>10 mg/L	Serial: 3 levels First level on admission, and the next 2 mornings (at least 8 hours apart between level 1 and level 2)	Initial CRP 1 for EOS	35%	90%
				Initial CRP 1 for LOS	61.5%	92.5%
				CRP 2 and 3 for EOS	88.9%	97.6%
				CRP 2 and 3 for LOS	96.4%	71.8%
Berger et al.^[26] n = 195 term and preterm infants	Qualitative immunoassay	>20 mg/L	Serial: 3 levels Initial level and then every 12-24 hours	CRP	75%	86%	Infected infants' levels peaked 24 hours after positive culture
				I:T Ratio	78%	73%

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The Role of C-Reactive Protein in the Evaluation and Management of Infants With Suspected Sepsis

Laboratory Methods to Measure CRP

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