LABORATORY ACTIVITY #5

C-REACTIVE PROTEIN

I. Desired learning outcomes

C-reactive protein (CRP) usually appears in the sera of patients in the acute stages of a number of
inflammatory conditions. This procedure will show an immunologic reaction between CRP as an antigen and
the corresponding antibody coated on the surface of biologically inert latex particles.

After performing this activity, students should be able to:

a. perform with accuracy and precision the procedure in detecting the presence of C-reactive protein in
the blood;
b. identify the different factors that could affect the correct reading and interpretation of results, and
c. explain the results and correlate them to clinical conditions.

II. Material
1. Pipettor 5. CRP Test Kit
2. Test tube 6. Test tube rack
3. Centrifuge 7. Non-lipemic serum
4. Glass slide

III. Procedures
QUALITATIVE TEST
1. Bring the reagents and samples to room temperature.
2. Place 50 µL of the sample and 1 drop of the control into separate circles on the card.
3. Re-suspend the latex gently.
4. Add one drop of the latex reagent to each circle next to the sample that is to be tested.
5. Mix with the disposable pipette/stirrer and spread over the entire area enclosed by the ring. Use a new stirrer
for each sample.
6. Rotate the cards at 100 rpm for 2 minutes.

QUANTITATIVE TEST
1. Using a semi-automatic pipette, add 50 µL of 9 g/L saline to circles 2, 3, 4, and 5. Do not spread the saline.
2. Add 50 µL of patient sample to circles 1 & 2.
3. Mixt the saline and sample in circle 2 by drawing the mixture up and down being careful to avoid the
formation of bubbles.
4. Transfer 50 µL from circle 2 to the saline in circle 3.
5. Perform serial dilutions in the same manner until the last circle, discarding 50 µL at the end.
6. Using the pipette/stirrer, spread the diluted samples over the entire area of each circle starting at circle 5 and
working backward to the neat sample in circle 1.
7. Proceed as a qualitative test from step 3.

Reading and Interpretation:

 1. Examine macroscopically for the presence or absence of clumps or agglutination within 1 minute of
removing the card from the rotator.
 Positive results – the presence of agglutination indicates a level of > 6 mg/L
 Negative results – no agglutination would indicate a level of CRP <6 mg/L
2. Normal levels in adults = > 6 mg/L.
3. Positive sera may be titred. To titrate, make serial two-fold dilutions in 9 g/L saline as indicated in the
quantitative test procedure.
4. The serum titer is defined as the highest dilution showing macroscopic agglutination. The approximate
CRP concentration in the sample may be obtained by multiplying the titer by the limit of sensitivity = 6
mg/L.

Dilution CRP mg/L

Neat 6
1:2 12
1:4 24
1:8 48
1:16 96

Limitations of the Procedure:

1. CRP levels in the range of 15 mg/L or above may cause false negative results due to prozone effects.
2. A final diagnosis should not be made based on the result of a single test but should be based on the
correlation of test results with other clinical findings.

Notes:
1. The sensitivity of the test may be reduced at low temperatures. The best results are obtained over 10 0C.
2. A delay in reading the results may result in over-estimation of the CRP level.
 LABORATORY OUTPUT

TITLE OF LABORATORY ACTIVITY: Lab. Act. #5 – C-Reactive Protein DA

I. Observation/Results
Instruction: Paste pictures of the results of the CRP test (both qualitative and quantitative). Label what is seen
in your pictures.

Figure 1 Figure 2
Qualitative C-Reactive Protein Test Result Quantitative C-Reactive Protein Test Result

Note. The absence of agglutination observed within 1 Note. Following the quantitative CRP test, the absence
minute of removing the card from the rotator of macroscopic agglutination at all dilution levels
indicates a non-reactive result, suggesting a CRP level implies a non-reactive result, indicative of a CRP level
below the cutoff of 6 mg/L. This outcome, while below the sensitivity limit of 6 mg/L. This finding
indicating a negative result for acute inflammatory suggests a low concentration of CRP in the sample but
conditions associated with elevated CRP, but still still necessitates clinical correlation with other
requires the importance of considering other clinical diagnostic parameters to assess inflammatory
findings when diagnosing inflammatory states. conditions comprehensively.

II. Discussion
 The interpretation of the results from the qualitative C-Reactive Protein (CRP) test showed negative
agglutination at spot 2, indicating a non-reactive result with a CRP level below the detection threshold of 6 mg/L
according to the package insert of Biosystems’ (2013) CRP Slide test. Conversely, for the quantitative CRP test, at all
dilution levels across the card, no agglutination was observed, further confirming a non-reactive result with a CRP
concentration below the sensitivity limit of 6 mg/L. This outcome suggests the absence of significant inflammation or
acute phase response in the tested sample, which can be correlated to the healthy condition of the patient. In a clinical
context, a non-reactive CRP result may indicate a lower likelihood of recent tissue damage or inflammation, still, it's
essential to consider other clinical findings and complementary tests for a comprehensive assessment, as a single test
result should not be solely relied upon for diagnosis as advised by the package insert. Furthermore, vigilance is still
needed as elevated CRP levels (>15 mg/L) can sometimes yield false-negative results due to prozone effects, as well as,
icterus, hemolysis, and lipemia outside their respective indices; the poor correlation between turbidity and triglyceride
concentration; the possibility of a high-dose hook effect at CRP concentrations over 50 mg/dL; and the potential for
significantly decreased CRP values in patients treated with carboxypenicillins (University of Iowa, 2022). With this
nonspecific test, one should be cautious in result interpretation and clinical correlation to rule out potential underlying
conditions.

III. Guide Questions

1. How can these factors affect CRP results?
A. Prozone
The Prozone phenomenon can affect C-reactive protein (CRP) results by causing a false negative response due to
high antibody titers, which interfere with the formation of the antigen-antibody lattice necessary for visualizing a
positive flocculation test (Sidana et al., 2011). This phenomenon is particularly associated with overwhelming antibody
titers in diseases like secondary syphilis, HIV co-infection, and pregnancy. The incidence of the Prozone phenomenon is
increasing, especially among populations at risk for sexually transmitted diseases such as HIV-positive individuals. In the
context of syphilis, the Prozone effect may become more prevalent due to the current AIDS epidemic, which can lead to
excess antibody production and anomalous B-cell behavior. Laboratories typically do not routinely test for the Prozone
phenomenon, so it's important to notify the laboratory when clinical findings strongly suggest syphilis and when non-
treponemal serological test results are negative. Diluting the patient's serum to bring the antibody concentration into
the zone of equivalence can help detect the Prozone effect.

B. Lipemic specimens
Lipemic specimens may cause false positive results due to non-specific agglutination according to the package
insert of Labtest’s (2015) CRP Latex—even in the absence of a target antibody. Turbid sera can be removed by
centrifuging the specimen at 19000g for 30 minutes.

C. Longer reaction time

Prolonged reaction time in the CRP Latex assay can lead to drying of the reaction mixture, potentially causing
false positive results (Biomed, 2021).

2. List conditions in which CRP is most applicable to help in the diagnosis.

The C-reactive protein (CRP) test is particularly applicable in diagnosing and monitoring various acute and
chronic inflammatory conditions (Schloman, 2022). This test measures the level of CRP, a protein produced by the liver
in response to inflammation. Elevated CRP levels indicate the presence of inflammation, but they do not specify the
cause or location of the inflammation. Conditions where the CRP test is most useful include infections caused by bacteria
or viruses, inflammatory bowel diseases like Crohn's disease and ulcerative colitis, autoimmune disorders such as lupus
and rheumatoid arthritis, and lung diseases like asthma. Additionally, the CRP test aids in evaluating the effectiveness of
treatments for chronic inflammation and helps guide treatment decisions in severe cases like sepsis. Notably, CRP levels
can rapidly increase within hours of tissue injury or inflammation onset and decline as the inflammatory process
resolves. This test is also valuable in predicting outcomes and assessing the severity of conditions like acute coronary
syndrome (ACS), acute aortic dissection, certain cancers (lung, pancreas, hepatocellular, urological), and chronic
obstructive pulmonary disease (COPD) (Kaur, 2017). Higher CRP levels generally correlate with poorer prognosis and
increased risk of adverse outcomes, making it a critical biomarker in clinical management and prognosis prediction
across various diseases and medical contexts.

IV. Conclusion
The objectives were sufficiently achieved as I performed with accuracy and precision the procedure in detecting
the presence of C-Reactive Protein in the blood—all while being vigilant to interfering factors and correlating results to
various clinical conditions. I have learned that interpretation of both qualitative and quantitative C-Reactive Protein
(CRP) tests, showing non-reactive outcomes with CRP levels below the detection threshold, suggests minimal
inflammation; however, caution is warranted due to potential test limitations like prozone effects, lipemic specimens,
and longer reaction times affecting result accuracy, emphasizing the CRP test's significance in diagnosing and monitoring
various inflammatory conditions, including infections, inflammatory bowel diseases, autoimmune disorders, and lung
diseases, where elevated CRP levels often correlate with poorer prognosis and increased risk of adverse outcomes. One
unique misconception that has been clarified for me about the topic is that elevated CRP levels always indicate the
presence of infection, whereas CRP can also be elevated in various non-infectious inflammatory conditions.

V. Reference/s

BioMed. (2021). BioMed-CRP Latex [Package insert]. Retrieved from

https://egy-chem.com/pdf/Latex_Qualitative_Slide_Test/BioMed-CRP%20Latex.pdf
BioSystems. (2013). C-Reactive Protein (CRP) - SLIDE [Package insert]. Retrieved from
https://www.biosystemsne.com.br/files/product/2f3d173b25fd8037b151671f696f323f.pdf
Kaur, M. (2017). C-reactive protein: A prognostic indicator. International Journal of Applied and Basic Medical
Research, 7(2), 83-84.
Labtest. (2015). CRP Latex SD [Package insert]. Retrieved from
https://labtest.com.br/wp-content/uploads/2016/12/CRP_Latex_SD_144K_Eng.pdf
Schloman, B. F. (2022). C-Reactive Protein (CRP) test: Medlineplus medical test. MedlinePlus.
https://medlineplus.gov/lab-tests/c-reactive-protein-crp-test/
Sidana, R., Mangala, H. C., Murugesh, S. B., & Ravindra, K. (2011). Prozone phenomenon in secondary syphilis. Indian
Journal of Sexually Transmitted Diseases and AIDS, 32(1), 47-49.
University of Iowa. (2022). Reactive protein (CRP). University of Iowa Diagnostic Laboratories.
https://www.healthcare.uiowa.edu/path_handbook/rhandbook/test347.html