PHAGOCYTIC ENGULFMENT 6.
Air dry the slides and stain with Wright’s
PRE-ANALYTICAL PHASE:
The site of the experiment should be sterilized. The
medical technologist should wear proper personal
protective equipment before the experiment
(laboratory gown, laboratory goggles or face-shield,
laboratory mask, and gloves)
No special preparation of the patient is required
before specimen collection. Blood should be drawn
by an aseptic technique. A minimum of 2 ml of
heparin blood (green-top evacuated tube) or 15 to
20 heparinized capillary tubes are required. The
specimen should be centrifuged, and the test should be
performed promptly. For the quality control, a fresh,
heparinized sample of blood from a healthy volunteer
should be tested simultaneously.
The microscope should be placed on a plain, stable
surface. The voltage of the power supply should be
inclined with that of the equipment. Proper handling
and cleanliness of compound microscope should be
observed at all times by the medical technologist. Lens
paper is provided in order to clean the objective and
ocular lenses.
ANALYTICAL PHASE:
Materials
- Broth culture of Bacillus subtilis or
Staphylococcus epidermidis
o 0 mins
o 15 mins
o 30 mins
- Microscope slides
- Pasteur pipettes
- Rubber bulb
- Test tubes
- Wright’s stain
Procedure:
1. Label three test tubes: Patient test tube and
Control test tube
2. Add 4 to 8 drops of the buffer coat from either
the patient’s heparinized blood or from the
normal control to the respectively labeled
tubes.
3. Add 2 to 3 drops of the bacterial broth culture
to each tube.
4. Incubate both tubes at room temperature and
37 C for 30 minutes.
5. Place 1 drop of the incubated specimen on a
glass slide and prepare a smear.
stain.
Wright’s Stain Procedure:
A. Cover each smear generously with filtered
Wright’s stain and allow the stain to remain on
the slide for at least 5 minutes.
B. Slowly add distilled water or buffer to the stain
until the buffer begins to overflow the stain.
Watch for the appearance of a metallic luster.
C. Gently blow on the slide to mix the stain and
buffer.
D. Allow the buffer to remain on the slide for at
least 5 minutes.
E. Gently wash the stain and buffer off the slide
with distilled water.
F. Air dry or carefully blot the slide between two
sheets of bibulous paper
7. Place a drop of immersion on each smear and
examine microscopically with the oil (100x)
immersion objective.
POST-ANALYTICAL PHASE:
All infectious materials should be disposed
appropriately. The working area should be cleansed
with the disinfectant before leaving. The PPE of each
individual should be removed properly. These cannot
be exposed outside the laboratory premises.
Interpretation of Results
POSITIVE - Demonstration of the engulfment of
bacteria
NEGATIVE - No engulfment of bacteria
INTRODUCTION
Phagocytosis
- Process by which specialized cells engulf and
destroy foreign particles such as
microorganisms or damaged cells.
- Macrophages and neutrophils (pmns) are
the most important phagocytic cells
A mixture of bacteria and phagocytes is incubated and
examined for the presence of engulfed bacteria. This
simple procedure may be useful in supporting the
diagnosis of impaired neutrophilic function in
conjunction with clinical signs and symptoms
 with anti-human CRP in
a stabilized buffer with
less than 0.1% sodium
Sources of error azide as preservative
CRP Positive Control: Human serum that
This procedure may produce false-negative results if contains more than
the blood specimen is not fresh or if a coagulase 6mg/L CRP and less
positive Staphylococcus specimen is used. It is than 0.1% sodium azide
important to distinguish between granules and cocci. In as preservative
Addition, the bacteria must be intracellular and not CRP Negative Control: Human serum that has
extracellular for the test to be positive been diluted and
stabilized with buffer
Clinical Applications and contain less than
0.1% sodium azide as
The failure of phagocytes to engulf bacteria can preservative
support the diagnosis of neutrophilic dysfunction; Glycine-saline To be diluted 1:20 with
however, these results must be used in conjunction Buffer: distilled water
with patient signs and symptoms (20X)
Concentrate
Limitations of this Procedure
Disposable pipettes and test slides
This is the simple screening procedure for engulfment.
III. Results
The presence of engulf bacteria does not demonstrate
that the bacteria have been destroyed
- Positive Result: Agglutination
- Negative Result: Smooth milky suspension
- Since negative results may be caused by
C-REACTIVE PROTEIN DETERMINATION
antigen excess, the test should be repeated
C-reactive Protein – acute phase or reactants on using a diluted serum sample in case prozone
which its concentration or its levels on your serum effect is suspected
increases during inflammation
IV. Clinical Significance
-
Non- specific marker of inflammation
-
Increase when you have a fever, TB, viral C-reactive protein (CRP)
infections, bacterial infections
- a trace constituent of serum originally
Reagents must be brought to room temperature
thought to be an antibody to the
and must be shaken well before dispensing.
Polysaccharide of pneumococci
Undiluted serum will be used to react with the CRP - It was discovered by Tillet and Francis in
latex reagent. 1930 when they observed that serum from
patients with Streptococcus pneumoniae
infection precipitated with a soluble extract
LABORATORY DISCUSSION GUIDE: of the bacteria.
- Now CRP is known to have a more
I. Principle of the test generalized role in innate immunity.
- Reverse Passive Agglutination. It is based on - CRP can be thought of as a primitive,
the latex- agglutination method. The principle nonspecific form of an antibody molecule
of this test is based on the immunological that is able to act as a defense against
reaction between CRP as an antigen and the microorganisms or foreign cells until specific
corresponding antibody coated on the surface antibodies can be produced
of biologically inert latex particles
II. Reagents of the test CRP is a relatively stable serum protein with a half-
life of about 18 hours. 1 It increases rapidly within 4
to 6 hours following infection, surgery, or other trauma
MATERIALS AND COMPONENTS to the body. Levels increase dramatically as much as a
Materials provided with the test kits hundredfold to a thousand-fold, reaching a peak value
within 48 hours. Elevated levels are found in
CRP Latex Reagent: Contains polystyrene conditions such as bacterial infections, rheumatic
latex particles coated fever, viral infections, malignant diseases,
tuberculosis, and after a heart attack. The median CRP one drop of CRP Latex to each circle that
value for an individual increases with age, reflecting contains specimens on the slide. Spread the
an increase in subclinical inflammatory conditions resulting mixture by using the paddle end of
the pipette. Do not use the same paddle end to
Because the levels rise and then decline so rapidly,
mix each test serum or control as this will
CRP is the most widely used indicator of acute
cause cross- contamination.
inflammation. Although CRP is a nonspecific indicator
5. Gently tilt and rotate slide by hand for two (2)
of disease or trauma, monitoring of its levels can be
minutes.
useful clinically to follow a disease process and
6. Observe for macroscopic clumping using the
observe the response to treatment of inflammation and
indirect oblique light source.
infection
7. Compare the reaction of the test serum to the
It is a nonsurgical means of following the course of CRP positive and negative control sera
malignancy and organ transplantation because a rise in
the level may mean a return of the malignancy or, in
the case of transplantation, the beginning of organ Method II (Semi-Quantitative)
rejection. CRP levels can also be used to monitor the
1. For each test serum to be titrated, set up a least
progression or remission of autoimmune diseases.
6 test tubes (12 x 75 mm) and label 1:2, 1:4,
Assays for CRP are sensitive, reproducible, and
1:8, 1:16, 1:32, 1:64, etc.
relatively inexpensive. CRP is easily destroyed by
2. To each tube add 0.2 ml of Diluted Glycine-
heating serum to 56°C for 30 minutes. The destruction
Saline Buffer.
of CRP is often necessary in the laboratory because it
3. To Tube No. 1 add 0.2 ml of undiluted test
interferes with some testing for the presence of
serum
antibodies
4. Serially make two-fold dilutions by mixing
The Centers for Disease Control and Prevention contents of Tube No. 1 with pipette and
(CDC) has recommended that a CRP concentration of transferring 0.2 ml to Tube No. 2. Repeat
less than 1 mg/L is associated with a low risk for serial transfers for each tube. For the 6 tubes,
cardiovascular disease; 1 to 3 mg/L is associated with the dilutions range from 1:2 to 1:64. If
an average risk; and greater than 3 mg/L is associated required, additional serum dilutions can be
with a high risk. Normal levels in adults range from added.
approximately 0.47 to 1.34 mg/L. A mean for people 5. Repeat steps 3 to 7 as given in Method I
with no coronary artery disease is 0.87 mg/L. Thus, (Qualitative)
monitoring CRP may be an important preventative
measure in determining the potential risk of heart
PRECAUTIONS
attack or stroke. High-sensitivity CRP testing has the
necessary lower level of detection of 0.01 mg/L, which This product is for In Vitro Diagnostic Use Only. Each
enables measurement of much smaller increases than donor unit used in the preparation of this product has
the traditional latex agglutination screening test been tested by an FDA approved method and found
non-reactive for the presence of HbsAg and antibody
V. And other important information in the test
to HIV Virus. Because no known test method can offer
insert
complete assurance that hepatitis B virus, HIV Virus,
Method I (Qualitative) or other infectious agents are absent, all human blood
based products should be handled in accordance with
1. Bring all test reagents and serum specimens to
good laboratory practices. The preservative sodium
room temperature.
azide may react with metal plumbing to form explosive
2. Gently shake the CRP latex vial to disperse
metal oxides. In disposal, flush with a large volume of
and suspend latex particles.
water to prevent metal azide build up
3. Positive and negative controls should be tested
with each series of test. STORAGE & STABILITY
4. Using the disposable pipette provided, place
When not in use, store reagents and controls at 2 - 8
one drop of test serum onto a circle on the
degree Celsius. DO NOT FREEZE. Prior to use, allow
slide. Use a separate disposable pipette for
reagents and controls to warm up to room temperature.
each test serum. Important: The Cortez CRP
Expiration date is specified on the kit label and on each
Latex Reagent must be agitated well for about
vial. Biological indication of product instability is
10 seconds prior to using on each day’s
evidenced by inappropriate reaction of the latex
testing. Do not use a vortex mixer. Deliver
reagent with the corresponding positive and negative
control sera