: CRP Determination [Laboratory] [ORTEGA, JULIANNE]

C-Reactive Protein
 marker of acute inflammation
 Members of the family known as Pentraxin.
 It increases rapidly within 4-6 hours following
infection, surgery, or other trauma to the body
 Reaches peak value within 48 hours
 Plasma half life: 18 hours
 Main substrate: Phosphocholine
 increases faster than ESR in responding to
inflammation, whereas the leukocyte count
may remain within normal limits despite
infection.
 Produced by the liver under the control of IL-6
 Serum concentrations can increase 1000-fold
with an acute inflammatory reaction.
 CRP binds to specific receptors found on
monocytes, macrophages, and neutrophils,
which promotes phagocytosis.
 CRP acts somewhat like an antibody, as it is
capable of opsonization, agglutination,
precipitation, and activation of complement by
the classical pathway.
 Normal levels range from 0.1 mg/dL in newborns
to 0.5 mg/dL in adults.
Clinical Uses of CRP:
1. Most widely used indicator of acute
inflammation
2. Used to monitor patient’s response in antibiotic
therapy
3. Used to monitor patient’s response to
chemotherapy
4. Used to monitor patient’s response to organ
transplantation
5. Used to detect whether the patient has a risk of
developing heart attack or stroke in the future.
1. CRP positive control = 1 drop of positive control
2. Patient’s sample = 1 drop of serum
3. Negative control = 1 drop of negative control
4. Add 1 drop of reagent on all of the circle
5. Mix.
False negative:
 due to high levels of CRP in undiluted specimens
False-positive reactions:
 reaction time longer that 2 minutes
 specimens are lipemic, hemolyzed, or
contaminated with bacteria
Limitations:
 Because the latex slide agglutination test is a
qualitative and semiquantitative procedure,
other methods such as nephelometry should be
used for the quantitative determination of the
CRP level when indicated.
 The strength of the agglutination reaction is not
always indicative of the CRP concentration.
 Weak reactions may be produced in samples
with elevated or low CRP values.
 Results may vary, depending on the patient’s
condition.
 This 2-minute slide latex agglutination test has a
detection level of 1 mg CRP/dL; therefore,
patients with CRP values less than 1 mg/dL may
go undetected.
 The sensitivity of the procedure has been
assessed at 93%.

C-Reactive Protein Rapid Latex Agglutination Test

 The C-reactive protein rapid latex agglutination
test is based on the reaction between patient
serum containing CRP as the antigen and the
corresponding antihuman (CRP) antibody
coated to the treated surface of latex particles.
 The coated particles enhance the detection of
an agglutination reaction when antigen is
present in the serum being tested.

C-Reactive Protein (CRP) Determination

 Principle: Reverse Passive Agglutination –
Antibody is coated with a carrier particle
 Reagent: 1 percent suspension of polystyrene
latex particles coated with anti–human CRP
produced in goats or rabbits
 Preservative: Sodium Azide
 Specimen: SERUM
: CRP Determination [Laboratory] [ORTEGA, JULIANNE]
 : ASO Latex Determination [Laboratory]
Group A Streptococcal Infection
Streptococcus pyogenes (Group A β-haemolytic
Streptococci)
 Frequent infections can lead to sequelae
infections such as __________________________
and __________________________.
BACTERIAL TOXINS
1. Streptolysin O (SLO)
 is an that _________________causes hemolysis by
binding to cholesterol in the RBC membrane
 It is antigenic, and the presence of antibodies to
SLO is an indicator of recent streptococcal
infection.
2. Streptolysin S
 a nonantigenic, _____________________.
 It causes hemolysis by disrupting the selective
permeability of the RBC membrane.
LABORATORY DIAGNOSIS
A. CULTURE
 plated on sheep blood agar and incubated
 Small translucent colonies surrounded by a clear
zone of β hemolysis will be visible
B. Identification
 susceptibility to bacitracin
 testing for L-pyrrolidonyl-β-naphthylamide (PYR)
activity
 Lancefield typing
Detection of Group A Streptococcal Antigens
Lateral flow immunochromatographic assays (LFA)
 used for the detection of bacterial, viral, fungal,
and parasitic antigens in clinical sampl
 strep A antigen extracted from a throat swab
reacts with an enzyme-labeled antibody on a
test membrane
[Ortega, Julianne]
Detection of Group A Streptococcal Antibodies
 Antistreptolysin O (ASO) Testing
 Anti-DNase B Testin
 Streptozyme Testing
Antistreptolysin O testing
 detect antibodies to the streptolysin O enzyme
produced by Group A streptococcus.
ASO titer
 based on the ability of antibodies in the
patient’s serum to neutralize the hemolytic
activity of streptolysin O.
 involved preparing dilutions of patient serum to
which a measured amount of streptolysin O
reagent was added.
 These were allowed to combine during an
incubation period after which reagent RBCs
were added as an indicator.
 If enough antibodies were present, the
streptolysin O was neutralized and no hemolysis
occurred.
 The titer was reported as the reciprocal of the
highest dilution demonstrating no hemolysis.
 This titer could be expressed in either Todd units
ASO TUBE METHOD
 The test estimates the amount of antibody (ASO)
that in the presence of constant dose of SLO
can completely inhibit hemolysis of a constant
given number of red cells
 Principle: Neutralization / Enzyme Inhibition Test
 TODD Unit = Titer
Positive Result: No Hemolysis
Negative Result: With Hemolysis
Elevated if: Children - 320 Todd units; Adults- Aleast 240
Todd units
 : ASO Latex Determination [Laboratory] [Ortega, Julianne]

4. Add one drop of ASO Latex reagent next to the

drop of serum.
5. Spread the reagent and serum sample over the
entire area of the test circle using a separate
stirrer for each sample.
6. Gently tilt the test slide backwards and forwards
for two minutes.
7. Observe and interpret the results.
 Add 0.5 ml strep O Ag on all tubes except tube  NOTE: Positive and Negative Controls should be
13 (red cell control) run at regular intervals
 Tube 14 (strep O control tube)
 Shake incubate for 15min, 37C
 Add 0.5 ml Type O human red cells to all tubes
 Incubate for 30 mins at 37C

 Centrifuge, check for presence of hemolysis

 Positive: absence of hemolysis
 Negative: hemolysis (red supernatant)
Titer: last tube with no hemolysis (clear supernatant)
Tube 13: no hemolysis
Tube 14: with hemolysis
Significant/ diagnosis titer: 166 todd units
ASO SLIDE METHOD
Anti-DNase B Testing
 useful in patients suspected of having
glomerulonephritis preceded by streptococcal
skin infections
 Antibodies to DNase B: may be detected in
patients with acute rheumatic fever who have
a negative ASO test result
 based on a neutralization methodology
Streptozyme Testing

 PRINCIPLE: Passive/ indirect agglutination  A slide agglutination screening test for the
o Antigen is coated with a carrier particle detection of antibodies against streptococcal
 Sample: Serum antigens
 ASO LATEX REAGENT: Suspension of polystyrene  positive in 95% of patients with acute
particles sensitized with Streptolysin O poststreptococcal glomerulonephritis because
 REAGENTS of GAS pharyngitis
o ASO LATEX REAGENTS  Sheep RBCs are coated with streptolysin,
 A suspension of polystyrene latex streptokinase, hyaluronidase, DNase, and
particles sensitized with streptococcal NADase
exoenzyme.
 ASO POSITIVE CONTROL
 A stabilized human serum containing
ASO reactive with ASO latex reagent.
 ASO NEGATIVE CONTROL
 A stabilized human serum containing
ASO non-reactive with ASO latex
reagent.
 All reagents contain 0.1% Sodium azide
as a preservative

Materials:
 ASO latex reagent
 Pipette
 Control sera (Positive and
Negative Controls)
 Applicator stick
 Black test cards
 Mechanical rotator

PROCEDURE

1. Allow each components of the test kit

(reagents, controls) to reach room temperature.
2. Gently shake the latex reagent to disperse the
particles.
3. Place a drop of undiluted serum into a circle of
a test slide.
 : Determination of Anti-Nuclear Antibodies [Laboratory] [ORTEGA, JULIANNE]

SYSTEMIC LUPUS ERYTHEMATOSUS: a chronic systemic inflammatory disease that affects between 40 and more than 200
persons per 100,000, depending on the population

 Women are more likely to be affected that men

Laboratory Diagnosis:

 Screening test for Anti-Nuclear Antibodies

 not specific for SLE

Fluorescent Antinuclear Antibody (FANA) Test - Most widely used and accepted test because it is highly sensitive,
detects a wide range of antibodies, and is inexpensive and easy to perform

SLE Latex Agglutination Test - Detects autoantibody which is present in the serum

 Reagent: SLE Latex Reagent

 Suspended Inert latex particles coated with DNP
 Principle: Passive/ Indirect Agglutination
 [ORTEGA, JULIANNE]
: RF Latex Agglutination [Laboratory]

RHEUMATOID ARTHRITIS

 can be characterized as a chronic, symmetric, and erosive arthritis of the peripheral joints that can also affect
multiple organs such as heart and the lungs.
 Caused by inflammatory process that results in the destruction of bone and cartilage
 Anti-CCP = second major type of antibody associated with RA.

Clinical Signs and Symptoms:

 Initial symptoms involve the joints, tendons, and bursae
 Nonspecific symptoms such as malaise, fatigue, fever, weight loss, and transient joint pain that begin in the
small joints of hands and feet
 Joint pain can lead into muscle spasms and limitation of motion
 If left untreated, may result in permanent joint dysfunction and deformity
 A small percent may develop Felty's Syndrome = a combination of chronic, nodular RA coupled with
neutropenia and splenomegaly
 Most common cause of death= Cardiovascular disease

Laboratory Diagnosis:
 Manual Agglutination tests using charcoal or latex particles coated with IgG
 ELIS
 Chemiluminescence Immunoassay
 Nephelometric methods
RF Latex Agglutination Test
 Principle: Passive/ Indirect Agglutination

Materials and Reagents:

1. RF Latex Reagent: A suspension of uniform polystyrene particles coated with IgG (human) in glycine buffer, pH
8.2; reagent sensitivity is standardized with the World Health Organization RF Standard. MIX WELL BEFORE USING.
2. RF Positive Control Serum: A stabilized, prediluted human serum containing at least 8 IU/mL of RF.
3. RF Negative Control Serum: A stabilized, prediluted human serum containing less than 8 IU/mL of RF.
4. Glycine-Saline Buffer (20x): pH 8.2 ± 0.1M glycine and 0.15M NaCl
5. Reaction Slide.
6. Pipette / Stir Sticks

Procedure: QUALITATIVE METHOD

1. Allow each components of the test kit (reagents, controls) to reach room temperature.
2. Gently shake the latex reagent to disperse the particles.
3. Place a drop of undiluted serum into a circle of a test slide.
4. Add one drop of Rf Latex reagent next to the drop of serum.
5. Spread the reagent and serum sample over the entire area of the test circle using a separate stirrer for each
sample.
6. Gently tilt the test slide backwards and forwards for two minutes.
7. Observe and interpret the results.

NOTE: Positive and Negative Controls should be run at regular intervals

Results: Qualitative Test
 Negative Result: A negative reaction is indicated by a uniform milky suspension with no agglutination observed
with the RF Negative Control.
 Positive Result: A positive reaction is indicated by any observable agglutination in the reaction mixture.
The specimen reaction should be compared to the RF Negative and Positive Controls.
 [ORTEGA, JULIANNE]
: Slide Agglutination [Laboratory]
 3+ Moderate clumping with a fairly clear fluid background
 4+ Large clumping with a clear fluid background

AGGLUTINATION

 process by which particulate antigens such as

cells aggregate to form larger complexes when
a specific antibody is present.

Principle:

 The ABO blood groups (A, B, AB, and O)

represent the antigens expressed on the
erythrocytes (red blood cells, RBCs) of each
group. Reagent typing sera contains specific
antibodies to A antigen and B antigen.
 When an unknown patient’s RBCs are mixed
with known antibody A or antibody B,
agglutination of the RBCs will occur if a specific
antigen-antibody reaction occurs.
STEPS IN AGGLUTINATION  This is called direct blood typing.

 Sensitization: involves antigen–antibody

combination through single antigenic
determinants on the particle surface

 Lattice Formation: representing the sum of

interactions between antibody and multiple
antigenic determinants on a particle
TYPE OF REACTION PRINCIPLE RESULT
Direct Agglutination Antigen is naturally found on Agglutination
a particle indicates the
presence of patient
antibody
Indirect (Passive) Particles coated with Agglutination
Agglutination antigens not normally found indicates the
on their surfaces presence of patient
antibody
Reverse Passive Particles coated with Agglutination
reagent antibody indicates the
presence of patient
antibody
Agglutination Haptens attached to carrier Lack of agglutination
Inhibition particles. Particles compete is a positive test,
with patient antigens for a indicating the
limited number of antibody presence of patient
sites. antigen
Hemagglutination Red blood cells Lack of agglutination
Inhibition spontaneously agglutinate if is a positive test,
viral particles are present. indicating the
presence of patient
antibody.
 1+ Very small clumping with an opaque fluid background
 2+ Small clumping with a slightly opaque fluid background
 [ORTEGA, JULIANNE]
: Febrile Antigens [Laboratory]

Febrile Antigens
 a standard panel of serologic antigens used in
screening patients with unexplained fever.

Febrile diseases
 group of microbial infections characterized by
fever and the production of antibodies known as
febrile agglutinins.

These diseases include:

1. Brucellosis, which is caused by the bacteria
Brucella abortus
2. Paratyphoid fever, which is caused by
Salmonella paratyphi
3. Rocky Mountain spotted fever, which is caused
by rickettsiae
4. Tularemia, which is caused by Francisella
tularensis
5. Q fever, which is caused by rickettsiae 1. Slide Method

WIDAL TEST  Agglutination is a positive test result and if the

 an agglutination test which detects the presence positive reaction is observed with 20 ul of test
of serum agglutinins (H and O) in patients serum sample, it indicates presence of clinically
with _______________ and _______________. significant levels of the corresponding antibody in
 Developed by ____________________in 1896. the patient serum.
 No agglutination is a negative test result and
Antigens used indicates absence of clinically significant levels of
the corresponding antibody in the patient serum.

2. Rapid Slide Titration Technique

 Somatic Antigen or “O” Antigen

 Flagellar Antigen or “H” Antigen
 Capsular Antigen or “K” Antigen or “Vi”
Antigen

 Salmonella antibody starts appearing in serum at

the end of first week and rise sharply during the
3rd week of endemic fever.
 In acute typhoid fever, ______________ can
usually be detected 6–8 days after the onset of
fever and ______________ after 10–12 days.
 [ORTEGA, JULIANNE]
: Febrile Antigens [Laboratory]

3. Tube Agglutination Test

Interpretation
o INC titer on OD Ag:
o INC titer on OA, OB and OC:
o INC titer on HD Ag:
o INC titer on HA, HC, HB:
o INC titer on O and H Ag:

WEIL-FELIX TEST
 allows the diagnosis of Rickettsiosis
 based on the fact that three strains of Proteus
which share somatic antigenic components with
Rickettsia, are agglutinated by antibodies
present in the serum of infected patients.

 Several rickettsiae, such as Rickettsia prowazekii,

Rickettsia mooseri, and R. rickettsii, posses
antigens that cross-react with antigens of OX
strains of Proteus vulgaris.
 Same glycolipid antigenic determinant
 Same polysaccharide

Antigens used
1. Proteus OX-2
2. Proteus OX-19
3. Proteus OX-K

Methods
1. Slide Method
2. Rapid Slide Method
3. Tube Method

Interpretation