IMMUNOLOGY SEROLOGY

A.Y. 2020 - 2021

LABORATORY EXERCISE # 1
C-REACTIVE PROTEIN

INTRODUCTION:
CRP was named because it was first discovered as a substance in the serum of patients with acute inflammation that reacted
with the C-(capsular) polysaccharide of pneumococcus. Upon electrophoresis, this group of three proteins migratesto an area
between the Beta and Gamma regions. They are considered to among the ‘acute phase proteins’ whose concentrations increase in
a non-specific response to inflammation and cellular necrosis.Increased C-Reactive Protein (CRP) is an indicator inflammation or
necrosis in the tissues.It is manufactured by the liver and released into the plasma during many necrotic, inflammatory or infectious
diseases including myocardial infarction (MI), active rheumatic fever, and pneumococcal pneumonia. CRP disappears rapidly from
the blood after recovery from disease and is not present in the serum from healthy people. The presence of CRP in a patient with
MI or rheumatic fever is considered to be the most sensitive indicator of necrosis and inflammation. CRP detection is considered to
be of greater practical significance than all other indices of inflammation in assessing inflammatory diseases. CRP has several
advantages over the erythrocyte sedimentation rate (ESR) including:

It is abnormally elevated immediately whereas ESR determinations may be borderline and may, in some cases, remain elevated in
the absence of inflammation. Results are not influenced by anemia, ESR will be falsely elevated. Results are not altered by abnormal
serum proteins which affect the ESR.Although it is not diagnosis of any particular disease, the level of CRP in serum indicates the
intensity of the disease, the response of the patient to treatment, and can be used to monitor patient progress. Tests for the
detection of CRP may give a false negative in cases where CRP is present in a large amount. In this test system CRP is the antigen;
anti-CRP is coated on the latex particles. If all antigen binding sites of the antibody are bound to a single CRP molecule there are
no sites left to bind to an antigen bound on an adjacent antibody molecule. Lattice formation cannot occur. This phenomenon is
called “postzone”. To prevent a false negative due to postzone some manufacturers recommend testing a sample diluted AND
undiluted. If either sample gives a positive result the result is “positive”. Review the illustration below.

PRINCIPLE:
When latex particles coated with human anti-CRP are mixed with a patient’s serum containing C -reactive proteins, an agglutination
reaction will take place. Agglutination indicates the presence of CRP, no agglutination indicates CRP is not present or is not present
in a sufficient quantity to be detected by the test.

OBJECTIVES:
1.State the clinical significance of elevated C-reactive protein (CRP)levels.
2.List three conditions in which elevated levels of CRP are found.
3.State three advantages that the CRP test has over the erythrocyte sedimentation rate.
4.State why some manufacturer’s recommend testing patient samples both diluted and undiluted.
5.Describe “postzone” as it applies to the test for CRP.
6.Select and evaluate specimens for acceptability for CRP testing.
7.Perform a serological test for the detection of CRP to obtain control and patient results that match instructor values with 100%
accuracy.
8.After reviewing the procedure and package insert state:
a.the substance being analyzed,
b.samples which are acceptable for testing,
c.storage temperature and time for samples,
d.the principle of the procedure,
e.the expected value,
f.significance of abnormal results,
g.limitations of the procedure, and
h.troubleshooting procedures to follow when control results are unacceptable.

9.Accurately record kit information, patient information and record results as instructed.
10.List five limitations of the procedure.

MATERIALS:
1.C -Reactive Protein test kit.
Reactive control
Non-Reactive control
CRP latex reagent
Glycine-saline solution
2.Patient serum specimens.
3.Digital/Electronic Timer
4.Disposable test cards
5.Disposable stirrer pipets
PROCEDURE:

INTERPRETATION:
Agglutination of latex particles is considered a positive reaction, indicating the presence of C-Reactive Protein at a significant and
detectable level. Specimens which do not contain human CRP will not cause agglutination. Consult the reagent product insert(s) for
specific information.Results are reported out in a qualitative manner. If agglutination of the latex particles occurs report out as
“positive”, if no agglutination of latex particles occurs report out as “negative”, if controls do not give expected reactions the test is
invalid and must be repeated.

PROCEDURE:
1. Allow all reagents and samples to warm to room temperature (20-30°C) before use. Remove
reagents from foam holders. Do not heat reagents in a water bath.
2. All reagents are ready for use as supplied. Gently mix the reagents before use; avoid foaming.
3. Gently mix the LATEX REAGENT before each use to ensure homogeneity.
4. Using the stirrer pipets, dispense one free-falling drop (0.05 ml) of each serum sample onto a
separate circle on the test card. Use a fresh stirrer pipet for each sample. When using the stirrer
pipet, keep it in a vertical position to ensure accurate delivery. Repeat by adding one free-falling drop
of REACTIVE or NONREACTIVE CONTROL from the dropper vials supplied. Note the location of
each sample by using the numbers located below and to the right of each circle.
5. Expel the contents of the LATEX REAGENT dropper and refill. Add one drop of the reagent to each
serum specimen and to each control.
6. Using the flat end of the stirrer pipets, mix each specimen and control serum with the LATEX
REAGENT, in a circular manner, over the entire area in the circles of the card.
7. Gently tilt and rotate the card for two (2) minutes and observe for agglutination. All test results should
be compared to both REACTIVE and NONREACTIVE CONTROLS.

EXPECTED RESULTS:
Agglutination indicates a CRP concentration of greater than or equal to 6 mg/L in the serum sample. Sera
that elicit a positive result should be retested and titered using the “Semiquantitative Assay Protocol”.
LIMITATIONS:
1. CRP levels in the range of 15 mg/L or above can cause false negative results due to the phenomenon
known as prozone.
2. Serum CRP levels increase in the first trimester of pregnancy. Women on oral contraceptives and
utilizing intrauterine devices may also have elevated serum CRP levels
3. CRP levels are higher among men who suffer MI or ischemic stroke later in life
4. Contaminated, lipemic, or grossly hemolyzed sera should not be used because of the possibility of
nonspecific results.
5. Plasma samples should not be used because of the possibility of nonspecific results.
6. Temperature of the reagents and samples is crucial to test outcome. It should be between 20- 30°C.
7. Reaction times longer than specified might cause false positive results due to a drying effect.
8. In accord with all diagnostic methods, a final diagnosis should not be made on the result of a single
test, but should be based on a correlation of test results with other clinical findings
 LABORATORY WORKSHEET #1: CRP agglutination test

SCHEMATIC DIAGRAM:
Create a schematic diagram (graphic illustration of the procedure with the use of pictures (may be download using internet) or
drawn images connected with arrows to depict flow of instruction) of the procedure so that it can be easily followed.

OBSERVATIONS AND RESULTS:

POST LABORATORY QUESTIONS:

2.What are high levels of CRP an indication of? (1 point)

3.List 3 diseases (not conditions) where elevated levels ofCRP are found. (1.5 points)
a.
b.
c.

4.What are the latex particles coated with?(1 point)

5.State three (3) advantages that the CRP test has over ESR. (3points)
a.
b.
c.

6.According to the product insert list the type(s)of sample(s)which may be used for this test.(1 point)

7.Why do some manufacturers recommend testing specimens diluted and undiluted?(1 point)

8.Describe the phenomenon known as “postzone”? (1point)

9.List two limitations of this procedure. (2 points)

10.After reviewing the product insert state the principle of this test kit including the appearance of positive and negative reactions
and what each indicates.(3points)

REFERENCES:
Indicate the sources of your work.