76
hyaluronic acid
by GAS. (U) M protein, F proteins of pili, and theM-proteins inhibit
AFigure 1. Evasion of innate immunityinvasion: (I) SLO, hyaluronic acid capsule, and
capsule are involved in adhesion and/or phagolysosomes; (||1)) Secreted GAS factors inhibit
within
phagocytosis and help avoid killing of GAS (C5a peptidase, SIC), prevent. phagocyte recruitment (C5a
complement activation and antimicrobial peptides
peptidase), induce apoptosis of phagocytes (SLO), interfere with cytokines or cytokine production (SLS,
neutrophil extracellular trap; SIC, a
SpeCYP), destroy NETs (DNases). AMP, antimicrobial peptide: NET,
streptococcal inhibitor of complement; SLO, streptolysin O; SLS, streptolysin S.
https:/www.irontiersin.orglarticlesit0.3389itcimb.2014.00140/full
Exerise No. 17
ANTI-STREPTOLYSIN O (ASO) TITRATION
Family Name ,First Name M.I: Year and Seotion:
Lab Profssor/ Preceptor: Date: WM P
1. OBJECTIVES:
At the end of the exercise the students will be able to:
know the procedure in performing ASO Titration
know the principle behind ASO Titration
knowhow to read and interpret ASO titers
II. DISCUSSION:
Anti-streptolysin O titration allows the quantitative analysis of the antibody, based on an
internationally recognized unit system (Todd, 1932). The system defines a minimal hemolytic dose
of SLO as that amount of toxin that will completely hemolyze 0.5 ml of a 5 percent suspension
of rabbit red blood cells, measured in Todd units.
III. MATERIALS/EQUIPMENT:
2test tubes
5 (2 mL) pipets
2 (5 mL) pipets
1 (10mL) pipet
1(15 mL) large tube
4 (19 mL)Wasserman test tubes
IV. REAGENTS:
1. Saline
2. Streptolysin O buffer
3. Streptolysin O
4. Group "0" 5% RBC suspension
V. PROCEDURE:
1. Prepare adilution of serum as follows using streptolysin O buffer
1:10 dilution solution as diluent:
0.5 mL serum and 4.5 ml buffer solution
1:100 dilution 1.0 mL of the 1:10 serum dilution and 9 ml of buffer solution
1:500 dilution 2.0mL of the 1:100 serum dilution and add 8 ml of buffer solution
2. Set up 14 Wasserman test tubes in the rack, properly number
the respective materials as indicated by the from 1 to 14. Place in each test tube
following protocol.
77
Protocolfor Antistreptolysin O titration
Serum 1:10 1:100 1:500 Red SLO
dilutions Cel! CTRL
CTRL
Tube m
3 4 5 6 7 8 9 10 11 12 13 14
Add serum 0.8 0.2 1.0 0.8 0.6 0.4 0.3 1.0 0.8 0.6 0.4 0.2
dilution, mL
Add buffer
solution, mL 0.2 0.8 0.2 0.4 0.6 0.7 0.2 0.4 0.6 0.8 1.5 1.0
Shake gentiy ta
mix
Add
Streptolysin
O, mL 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Shake gently to
mix
lncubate at 37 *C
for 15 nins
Add 5% RBC
suspension, 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
mL
Shake gentty to rmix. Ircubate at 37 "C for 45 mins, shaking tubes after the first 15 mins. Following incubation
ot 1,500 rpm.
centrifuge tubes for 1 minute
Todd unit .12 50 100 125 166 250 333 500 625 833 1250 2500
value
*CTRL: control /SLO: Streptolysin O
VI. RESULT:
VII. INTERPRETATION:
VIII, ILLUSTRATION: (must be colored and labeled at all times)
78
IX. STUDY QUESTIONS:
1. What are the complications of S. pyogenes infection?
2. Enumerate some diseases or conditions wherein the ASO titer is increased.
3. What do you mean by Todd units?
4. How long does an ASO titer remain elevated?
5. What is the clinical significance of the ASO test?
6. Does the presence of ASO by itself readily signifies adisease is going on? Why or why not?
90
Exercise No. 14
Name, First Name M,:
Abbott Bioline TM HBsAg Rapid Test
Year and Seotion:
ofessor/ Preceptor: Date: M
OBJECTIVES:
At the end of the exercise the students will be able to:
knowthe procedure in performing the HBsAg rapid test
know the principle behind the HBsAg rapid test
interpret the positive and negative results of the HBSAg rapid test
DISCUSSION:
The Bioline TM HBSAg test is an in vitro
determination of HBsAg in human serum or immunochromatographic
plasma. The Bioline TM
assay designed for the qualitative
HBsAg is intended only for professional
USe as an aid to diagnosis. Reactive specimens should be confirmed by a
Vitro diagnostic use only. This product is suitable for use in supplemental assay. This kit is for in
pregnant women,
haven't yet performed the test. This test may not be suitable for the diagnosis of but in the case of neonates, we
eary infection or blood donation
screening.
Test Principle
This test device contains a membrane strip, which is pre-coated with mouse
on the test band region. The mouse monoclonal monoclonal anti-Hbs Ab
anti-HBS-colloid gold conjugate and the specimen
the membrane chromatographically to the test region (T) and forms a visible line as move along
complex forms. the Ab-Ag-Ab gold particle
MATERIALS/EQUIPMENT:
Bioline TM HBSAg test kit
Micropipette
Serum or plasma specimen
Red top or EDTA tube
Centrifuge
REAGENTS:
NA
PROCEDURE:
Specimen collection and storage
1.Plasna: collect the whole blood into the EDTAtube by venipuncture and then centrifuge the blood to get the
plasma specimen. (note: Anticoagulants like EDTA, citrate, and heparin do not affect the test results hence any
of the tubes containing such anticoagulants can be used)
2.Serum: Collect the whole blood into the EDTA tube by venipuncture, leave to setle for 30 minutes for blood
coagulation, and then centrifuge blood to get the serum specimen of the supernatant.
3.If plasma or serum samples cannot be used immediately, they should be refrigerated at 2-8C. For
astorage
period longer than 3 days, freezing (below -20°C) is recommended. They should be brought to 15-30°C prior to
Use.
4.Plasma or serum specimens containing a precipitate may yield inconsistent results. Such specimens must be
cdarified prior to assaying.
Test Procedure
1. Allow all kit components and specimens to room temperature (between 15°C and 30°C) prior to testing.
2. Remove the test device from the foil pouch and place it on aflat, dry surface.
3. Dispense 100 pL of plasma or serum specimen into the specimen well (Figure 1: next
4. As the test begins to work, you willsee a purple color move across the result window inpage)
the center of the test
device.
5. Interpret test results at 20 minutes.
91
Specimen
Abott
HBsAg C SP I00p
20 min [Figurel]
SP 100 pl: Serum 100pl or Plasma 100pl
rpretation
Apurple-colored band will appear in the left section of the results window to show that the test is working
roperly.
The right section of the results window indicates the test results. If another purple band appears in the right
ection of the results window, this is the test band.
Wegative result: The presence of only the control band (C) within the results window indicates a negative
esult.
Positive result:the presence of the test line (T) and the control line (C) within the results window, regardless
of which band appears first, indicates a positive result.
aution: The presence of any test line, no matter how faint, the result is considered positive.
T T
Invalid result: If the control band is not visible within the result window after performing the test, the result
sconsidered invalid. Instructions may not have been followed correctly or the test may have
deteriorated
Deyond the expiration date. It is recommended that the specimen be retested using a new test kit.
C T
T
aitations
ve result does not preclude the possibility of infection with HBV. Other clinically available tests are
required if
able results are obtained. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the
fa single test, but should only be made by the physician after all clinical and laboratory
d.
findings have been
ige and Stability
he BiolineTM HBsAg test should be stored between 1°C and 30°C. Do not freeze the kits.
he test device is sensitive to both heat and humidity. Perform the test immediately after removing the test
ievice from the foil pouch.
Jonot use the test kít beyond its expiration date.
he shelf life of the kit is indicated on the outer package.
Jo not use the test kit if the pouch is damaged or the seal is broken.
Vear protective gloves while handling the specimens and wash hands thoroughly afterward.
lecontaminate and dispose of all specimens, reaction kits, and potentially contaminated materials in a
iohazard container as if they were an infectious waste.
or in vitro diagnostic use only. Do not reuse the test device.
A Do not eat or smoke while
handling
5. Avoid splashing or aerosol formationspecimens.
of the specimen (especialy during centrifugation).
l. RESULT:
I. INTERPRETATION:
X. ILLUSTRATION: (must be colored and labeled at all times)
93
X. STUDY QUESTIONS:
1. What ls Hepatitis B?
2. What are the signs and symptoms of Hepatitis B?
3. With a positive HBSAg, transmission routes for infection include:
4. What Is HBsAg?
5.
6. Why do you get an HBsAgtest when you're pregnant?
7. How is HBSAg positive treated?
8. Complete the table. The third row is also done for you.
Serologic markers
HBsAg Total anti IgM anti Anti- HBs interpretation
HBc HBc
negative negative negative negative Susceptible, never been infected
Acute infection, early incubation
Acute infection
Acute resolving infection
Past infection, recovered and
Chronic infection immune
False positive (i.e.,
infection, or Yow level"susceptible),
chronic
past
infection
Immune, titer is> 10 mlU/mL
if
Exercise No. 20
SD Biolinem HIV 1/2 3.0 test
Family NAmo, First Name M.I: Year and Seotion:
Lab Professor/ Preceptor: Date: m
OBJECTIVES:
At the end of the exercise the
students will be able to:
know the procedure in performing the SD Bioline TM HIV 1 12 3.0 test
know the principle behind the SD BiolineM HIV 1 |23.0 test
nterpret the positive and negative results of the SD Bioline TM HIV 172 3.0est
DISCUSSION:
V Human Immunodeficiency Virus) is recognized as the etiologic agent of Acquired
Immune Deficiency Syndrome (AIDS). The virus is transmitted by sexual contact, exposure to
intected blood, certain body fluids or tisSues, and from mother to fetus or child during tne
perinatal period. HIV-1 has been isolated from patients with AlDSand AIDS related complex, and Trom
heathy persons with high potential risk of develooing AIDS. Patients with HIV-2 are found primarHy in
parts of West Africa. Its course is marked by increasing levels of viral replication and the
of more virulent viral strains. emergence
HIV-1 and HIV-2 are similar in their morphology, cel tropism, host interaction and
generic structure. Serological studies have determined that HIV-1 and HIV-2 have multiple common
epitopes in core antigens but much less so in the envelope antigens. This clinical diagnosis of HIV
may include the detection of antibodies to HIV 1/2 in human plasma or serum by
The presence of HIV can be identified by detection of antibodies to HIV 1/I2 in human immunoassay.
serum,
and whole blood by immunoassay. This advanced assay utilizes recombinant antigens targeted plasma,
immunogenic proteins. The major immunoreactive antigens of these proteins are HIV-1 gp41, p24 against
and
HIV-2 gp36.
TEST PRINCIPLE
The Bioline TM HIV 1/2 3.0 test contains a membrane strip, which is
precoated with
recombinant HIV-1 capture antigen (gp41, p24) on test line 1 region and with recombinant HIV-2
capture antigen (gp36) on test line 2region respectively. The recombinant HIV 1/2 antigen (gp41,
p24 and gp36)-colloid gold conjugate and the sample move along the membrane
to the test region (T) and forms. a visible line as the chromatographically
forms with high degree of sensitivity and specificity. antigen-antibody-antigen gold particle complex
INTENDED USE
The Bioline TM HIV 1/2 3.0 kit is a rapid, qualitative test for the
detection of antibodies to all
isotypes(lgG, lgM, lgA)specific to HIV-1 and HIV-2 simultaneously in human
blood. The Bioline TM HIV 1/2 3.0 kit is intended only for professional use and serum, plasma, or whole
This test may not be for in vitro diagnostic use.
suitable for diagnosis of early infection or blood donation screening.
Positive samples should be confirmed by a supplemental
test. The performance of the assay has not been establishedassay such as ELISA or Western Blot
for populations of infants, children, or
adults.
III. MATERIALS/EQUIPMENT: Abbott
the Bioline TM HIV 1/2 3.0 test kit contains the following HIV I/2 3.0
itens to perform the assay:
" Test devices with desiccant in individual foil
pouches
" Assay diluent (1 x4 mlvial)
"Capillary pipettes (20 ul), 25 Sterile lancets, 25 10-20min C
Alcohol swabs
micropipette, 2
protective gloves
timer
biohazard container
SAMPLE WB20plSP10 /
Whole blood and or
Serum and or
98
VI. RESULT:
VIl.
INTERPRETATION:
VIll.
ILLUSTRATION: (must be colored and labeled at all times)
IX. STUDY QUESTIONS:
1. How many days can an HIV
antibody test detect HIV after exposure?
2. What is the principle behind nucleic acid testing (NAT)?
3. What is/are the limitation/s of HIV rapid diagnostic tests?
4. What is the "window period" and how does it impact the screening test results?
0. What does a positive HIV screening test result mean?
6. What is the confirmatory test to detect HIV? Explain its principle.
99
71e the rapid HIV ELISA test as good as
the standard HIVELISA test?
Exercise No. 21
SD Biosensor STANDARD Q Malaria P.f/ Pan Ag
Test
Family NAme, First Name MI Year and Seotion:
Lab Professor/ Precoptor: pate: m
OBJECTIVES:
At the end of the exercise the students will be able to:
know the procedure in performing the HBsAg
know the principle behind the HBsAg rapid testrapid test
interpret the positive and negative results of the HBsAg rapid test
I. DISCUSSION:
Malaria remains an important cause of ilness and death in children and adults in coutrie5
which it is endemic. Globally, an estimated 3.2 billion peoole in 97 countries and territories are at risk or benig
infected with malaria and developing disease. and 1.2 billion are at high risk (>1 in 1,000 chance or gets
malaria inayear). According to the World Malaria Report 2015. there were 210 million cases of malaria globy
in 2015 (uncertainty range 149-303 million) and 438.000 malaria deaths (range 236,000-635,000).
representing a decrease in malaria cases and deaths of 37% and 60% since 2000, respectively.
The protozoal parasites that cause malaria are from Plasmodium falciparum, vivax, ovale
and malariae with the first two species causing the most infections worldwide. Classic symptoms of malaria
incdude fever, headache, chills,vomiting, shívering and convulsions. In some rare forms of P. afciparum
the patient may present with delirium or coma.Severe anemia is often attributed to the cause of death trom
malaria. Accurate and prompt diagnosis of malaria is of utmost importance due to the morbidity assOCIated
with the other malarial forms.
Rapid diagnostic test is an ideal diagnostic tool for malaria diagnosis in that it can provide
arapid determination if the patient is infected with malaria allowing for accurate treatment and improved
outcomes. STANDARD Q Malaria P.f/Pan Ag Test, a reliable and sensitive screening test, would enhance the
accuracy of the diagnosis of malaria infection and thus make clinical treatment decision effectively.
TEST PRINCIPLE
STANDARD Q MalariaP.f/Pan Ag Test contains two pre-coated lines, "P.f" (P. falciparum), Pan"
(Plasmodium species: P. falciparum, vivax, ovale and malariae) as test lines and "C" as control line on
the surface of the nitrocellulose membrane. The test lines and control line in the result window of the test
device are not visible before applying any specimens. Monoclonal anti-P. falciparum HRP-2 is coated on the
P.f test line,monoclonal anti-Malaria pLDH is coated on the Pan test line, and monocional anti-chicken igY
is coated on the control line region. During the test, the P. falciparum specific HRP-2 antigen and/or
Plasmodium species specific plLDH in the specimen react to the gold-conjugated monoclonal anti-Malaria
HRP-2 andlor gold-conjugated monoclonal anti-Malaria pLDH,and then bind to them respectively. Any P.
falciparum specific HRP2 antigen-antbody gold particle complex and/or Plasmodium species specific pLDH
antigen-antibody gold particle complex also migrate with the buffer and are immobilized by monoclonal anti-P.
falciparum HRP-2 and/or monoclonal anti-Malaria pLDH at the two individual test lines to formation of violet test
colored band(s)which confirms a positive result.
Absence of thísviolet-colored band indicates a negative resutt. The control line is used for procedural
control,and should always appear if the test procedure is performed properly and the test reagents of the
control line are working.
INTENDED USE OF THE TEST KIT:
STANDARDO Malara P.Pan Ag Test is a rapid and membrane based immmunochromatography for the
qualitative detection of Plasmodium falciparum (P. falciparum) specific Histidine Rich Protein 2 (HRP-2) and
Plasmodium species (P. falciparum, vivax, ovale and malariae) specific Plasmodium lactate dehydrogenase
(pLDH) in human capillary and venous whole blood specimens of patients suspected of having malaria.
STANDARD Q Malaria P.f/Pan Ag Test is intended to be used by trained healthcare or laboratory professionals
Of other health care workers who have received appropriate training. Ihis product can be used by trained lay
providers operating in point-of-care settings in resource-limited lower- and middle-income cOuntries., This
product is not intended for self-testing.
MATERIALS/EQUIPMENT: blood
Anti-coaqulant tube containing heparin, EDTA or sodium citrate for collection of venouS whole
Micropipette and tip
Timer
PPE (Personal Protective Equipment)
105
VI. RESULT:
VIl. INTERPRETATION:
VIIl.
ILLUSTRATION: (must be colored and labeled at all times)
IX. STUDY QUESTIONS:
1. Can malaria be transferred from one person to another?
2. Is malaria a communicable disease or not?
you mean by exoerythrocytic cycle?
Uoes malaria reproduce in humans? What do
4. What is the gold standard for diagnosing malaria?
106
5. Howis malaria diagnosedd
according to WH0?
6 What is the clinical hallmark of malaria?
7 Give the principle involved in the rapid diagnostic tests for
malaria.
