TYPES OF SAFETY HAZARDS

IMMUNOLOGY AND SEROLOGY 311 A. BIOLOGICAL HAZARDS

LABORATORY SAFETY
PRELIM: LABORATORY
o Source: Infectious agents (Pathogens)
o Chain of Infection
PROPER WEARING AND REMOVAL OF PPE
- Explains how microorganisms are transmitted
DONNING (Wearing) DOFFING (Removing)
- Understating it is essential in preventing infection
o Gown o Gloves
- Requires a continuous link between:
o Hair net/cap o Goggles
o Agent, Host, Environment
o Mask o Gown
o Source: location of potentially harmful microorganisms
o Goggles o Hair net/cap
- Contaminated clinical specimen
o Gloves o Mask
Note: Protective footwear should be placed and removed after hair net/cap and before mask.
- Infected patient
o Mode of transmission – how microorganisms from the source
ELEMENTS OF RESPIRATORY PROTECTION PROGRAM are transmitted to susceptible host
o Medical evaluation - Direct contact
o Fit testing – detect air leaks - Inhalation of infected material
o Training - Ingestion of a contaminated substance
o Fit checking before use - From an animal or insect vector bite
o Fit check o Susceptible host
o Seal check - Agent – pathogens
a. Positive seal check – Fit mask → snug → block exhalation - Host – susceptible host
valve → breathe out → pull - Environment – any entity that could nurture the pathogens
b. Negative seal check – Fit mask → block air inlets →
breathe in → collapse B. SHARP HAZARDS
o anything that cause opening in an intact skin
PROPER GLOVE REMOVAL o Sharp objects including:
- Needles, lancets, broken glassware
o Present a serious threat particularly for the transmission of
blood-borne pathogens
o All sharp objects must be disposed in puncture-resistant
containers labeled with biohazard symbol and must be leakproof
o Recapping – is permitted only by use of one-handed method or
a resheating device. STRICTLY NO RECAPPING.

C. CHEMICAL HAZARDS
o Water – universal solvent
o MSDS – Material Safety Data Sheet
o Chemical Spills
- Best first aid is to flush the area with large amounts of
PROPER HANDWASHING PROCEDURE
water for AT LEAST 15 MINUTES and the seek medical
attention
o Chemical Handling
- Acid should always be added to water (Acid → water)
- Wear goggles when preparing chemical reagents
- Reagents should always be prepared under a fume hood

NATIONAL FIRE PROTECTION

ASSOCIATION HAZARDOUS
MATERIAL CLASSIFICATION
(HAZMAT DIAMOND/NFPA
704)
o Diamond-shaped, color-
coded symbol that
CDC GUIDELINES FOR HAND HYGIENE
contains information
A. Wash hands with anti-microbial soap
relating to health,
✓ When hands are visibly soiled
flammability, reactivity
✓ After gloves are removed
and personal protection,
✓ Before eating
✓ After using the bathroom special precautions.
o These symbols are placed
✓ Prior to leaving the work area
B. Apply an alcohol-based hand rub (if possible handwash) on doors, cabinets and
containers
- Note: Replace gloves every after patient
✓ Before having direct patient contact DEGREE OF HAZARDS Remember!!!
✓ Before and after removal of gloves 0 NO HAZARD Degree of Hazards:
✓ After contact with body fluids (as long as hands are NOT 1 SLIGHT HAZARD “No-SMS-Ex”
VISIBLY SOILED) 2 MINIMAL HAZARD
✓ When moving from a contaminated body site to a clean 3 SERIOUS HAZARD Yellow: SUVSM
body site 4 EXTREME HAZARD Blue: NSHED
✓ After contact with contaminated objects and equipment
D. ELECTRICAL HAZARDS
o Equipment SHOULD NOT be operated with WET HANDS
o All electrical equipment must be grounded with THREE-
PRONGED PLUGS
o If electrical shock occurs, NEVER touch the person or the
equipment involved; instead:
- Turn off the circuit breaker
- Unplug the equipment
- Move the equipment using a non-conductive glass or wood
object

[SARANILLO, KA.]
E. FIRE/EXPLOSIVE HAZARDS
o Flammable chemical should be stored in SAFETY CABINETS and
EXPLOSION-PROOF REFRIGERATORS Remember:
o Persons with burning clothes should be wrapped blanket to ✓ Water (A)
smother the flames ✓ Dry Chemicals (ABC)
✓ Carbon Dioxide (BC)
When a fire is discovered Use of fire extinguisher ✓ Halon (BC)
“R-A-C-E” “P-A-S-S”
o RESCUE o PULL the pin
o ALARM o AIM at the base of the fire
o CONTAIN o SQUEEZE handles TYPE OF WASTES
o EXTINGUISH/EVACUATE o SWEEP nozzle from side to RED Sharps, needles
side YELLOW Infectious wastes
F. PHYSICAL HAZARDS YELLOW with BLACK BAND Chemical wastes (tiger bag)
o General precautions are: ORANGE Radioactive wastes
- Avoid running in rooms and hallways GREEN Non-infectious wet wastes,
- Watch for wet floors Biodegradable wastes
- Bend knees when lifting heavy objects BLACK Non-infectious dry wastes,
- Keep long hair pulled back Non-biodegradable wastes
- Avoid dangling jewelry WHITE Pharmaceutical wastes
- Maintain clean and organized working area
- Wear closed-toe shoes UNIVERSAL PRECAUTION
o Defined as a method for controlling infection in which all blood
LABORATORY WASTE MANAGEMENT and certain body fluids are treated as if infected with hepatitis B,
o All biological waste must be placed in appropriate containers HIV, or other disease producing blood-borne pathogens.
labeled with the biohazard symbol o The reason for universal precaution is that all patients infected
o Sinks should be disinfected daily using 1:5 or 1:10 dilution of with blood borne pathogens cannot be readily identified.
SODIUM HYPOCHLORITE - Skin and mucous membrane exposure to blood and other
o NOTE: fluids must be prevented
- Hypochlorites are the most widely used of the chlorine - Glove must be worn when there is any possibility of coming
disinfectants in contact with blood or other body fluids. When removing
- Most prevalent chlorine products in the US are aqueous gloves, they must be disposed of in biohazardous waste.
solutions od 5.25% to 6.15% sodium hypochlorite, usually Gloves must never be reused.
called HOUSEHOLD BLEACH - Any skin surfaces that become contaminated with blood or
body fluids should be washed immediately. Hands must be
DILUTION
washed upon removal of gloves.
1:10 Large spills of blood
Spot disinfection of countertops and floors
- It is of utmost important that any cuts or scratches be well
1:100 Small spills of blood and non-critical surfaces protected from contamination with blood or body fluids.
Hard smooth surfaces - During phlebotomy, used needles should not be recapped,
bent, or broken by the technologist. The unsheathed
HOW TO PREPARE? needle should be placed directly into an appropriately
RATIO VOLUME OF BLEACH VOLUME OF WATER labelled puncture-resistant biohazard container for
1:10 1 mL 9 mL disposal.
- All specimen for centrifugation must be centrifuged in a
SHELF-LIFE OF SODIUM HYPOCHLORITE closed tube (a top must be on every tube)
Room temperature (diluted with water) 1 week - All laboratory work benches must be decontaminated with
Closed brown bottle/Plastic bottles 1 month the appropriate germicide (10% Clorox) when work has
Recommended Contact Time 20 minutes been completed.
Hepatitis B Virus (HBV) 10 minutes - All materials coming in contact with blood or body fluids
Human Immunodeficiency Virus (HIV) 2 minutes must be placed in biohazardous waste containers when
finished with and disposed of according to the institution’s
TYPES OF FIRE AND THEIR CORRESPONDING EXTINGUISHERS infective waste disposal policy.
TYPE SOURCES TYPE OF EXTINGUISHER - “Treat all specimens as potentially infectious”
A Ordinary combustibles: ✓ Water
✓ Paper ✓ Dry Chemicals
✓ Cloth ✓ Loaded Steam
✓ Rubbish
✓ Plastics
✓ Wood
B Flammable Liquids: ✓ Dry Chemicals
✓ Grease ✓ Carbon Dioxide
✓ Gasoline ✓ Halon Foam
✓ Paints
✓ Oil
C Electrical equipment ✓ Dry Chemicals
and motor switches ✓ Carbon Dioxide
✓ Halon Foam (Recommended)
D Flammable metals: ✓ Metal X
✓ Sodium ✓ Sand
✓ Magnesium
✓ Mercury NOTE: fought by fire fighters only
✓ Lithium
E Detonation/Arsenal Fire Allowed to burn out and nearby
materials are protected
K Cooking Media: Liquid designed to prevent
✓ Grease splashing and cool the fire
✓ Oils
✓ Fats

[SARANILLO, KA.]
 o The Centers for Disease Control and Prevention (CDC) has
IMMUNOLOGY AND SEROLOGY 311 recommended that a CRP concentration of less than 1 mg/L is
C-REACTIVE PROTEIN DETERMINATION
PRELIM: LABORATORY
associated with a low risk for cardiovascular disease; 1 to 3 mg/L
is associated with an average risk; and greater than 3 mg/L is
PRE-ANALYTICAL PHASE
associated with a high risk. Normal levels in adults range from
o The work area of the experiment should be sterilized. The
approximately 0.47 to 1.34 mg/L. A mean for people with no
Students should wear proper personal protective equipment
coronary artery disease is 0.87 mg/L. Thus, monitoring CRP may
before the experiment (laboratory gown, eye protector,
be an important preventative measure in determining the
laboratory mask, gloves and hair net). Standard precaution is
potential risk of heart attack or stroke. High-sensitivity CRP
applied to every phase of experiment.
testing has the necessary lower level of detection of 0.01 mg/L,
which enables measurement of much smaller increases than the
PRINCIPLE OF THE TEST
o Reverse Passive Agglutination. It is based on the latex- traditional latex agglutination screening test.
agglutination method. The principle of this test is based on the
immunological reaction between CRP as an antigen and the METHOD I (QUALITATIVE)
corresponding antibody coated on the surface of biologically 1. Bring all test reagents and serum specimens to room
inert latex particles. temperature.
2. Gently shake the CRP latex vial to disperse and suspend latex
REAGENTS OF THE TEST particles.
o Materials and Components provided with the test kits 3. Positive and negative controls should be tested with each series
o Disposable pipettes and test slides of test.
CRP Latex Reagent Contains polystyrene latex particles 4. Using the disposable pipette provided, place one drop of test
coated with anti-human CRP in a serum onto a circle on the slide. Use a separate disposable
stabilized buffer with less than 0.1% pipette for each test serum. Important: The Cortez CRP Latex
sodium azide as preservative. Reagent must be agitated well for about 10 seconds prior to
CRP Positive Control Human Serum that contains more than using on each day’s testing. Do not use a vortex mixer. Deliver
6mg/L CRP and less than sodium azide as one drop of CRP Latex to each circle that contains specimens on
preservative. the slide. Spread the resulting mixture by using the paddle end
CRP Negative Control Human serum that has been diluted and of the pipette. Do not use the same paddle end to mix each test
stabilized with buffer and contain less serum or control as this will cause cross-contamination.
than 0.1% sodium azide as preservative. 5. Gently tilt and rotate slide by hand for two (2) minutes.
Glysine-saline Buffer: To be diluted 1:20 with distilled water 6. Observe for macroscopic clumping using the indirect oblique
(20x) Concentrate light source.
7. Compare the reaction of the test serum to the CRP positive and
negative control sera.
RESULTS
o Positive Result: Agglutination
METHOD II (SEMI-QUALITATIVE)
o Negative Result: Smooth milky suspension
1. For each test serum to be titrated, set up a least 6 test tubes (12
o Since negative results may be caused by antigen excess, the test
x 75 mm) and label 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, etc.
should be repeated using a diluted serum sample in case prozone
2. To each tube add 0.2 ml of Diluted Glycine-Saline Buffer.
effect is suspected.
3. To Tube No. 1 add 0.2 ml of undiluted test serum.
4. Serially make two-fold dilutions by mixing contents of Tube No.
CLINICAL SIGNIFICANCE
1 with pipette and transferring 0.2 ml to Tube No. 2. Repeat
o C-reactive protein (CRP) is a trace constituent of serum originally
serial transfers for each tube. For the 6 tubes, the dilutions range
thought to be an antibody to the Cpolysaccharide of
from 1:2 to 1:64. If required, additional serum dilutions can be
pneumococci. It was discovered by Tillet and Francis in 1930
added.
when they observed that serum from patients with
5. Repeat steps 3 to 7 as given in Method I (Qualitative).
Streptococcus pneumoniae infection precipitated with a soluble
extract of the bacteria. Now CRP is known to have a more
PRECAUTIONS
generalized role in innate immunity. CRP can be thought of as a
o This product is for In Vitro Diagnostic Use Only. Each donor unit
primitive, nonspecific form of an antibody molecule that is able
used in the preparation of this product has been tested by an
to act as a defense against microorganisms or foreign cells until
FDA approved method and found non-reactive for the presence
specific antibodies can be produced.
of HbsAg and antibody to HIV Virus. Because no known test
o CRP is a relatively stable serum protein with a half-life of about
method can offer complete assurance that hepatitis B virus, HIV
18 hours. 1 It increases rapidly within 4 to 6 hours following
Virus, or other infectious agents are absent, all human blood
infection, surgery, or other trauma to the body. Levels increase
based products should be handled in accordance with good
dramatically as much as a hundredfold to a thousandfold,
laboratory practices. The preservative sodium azide may react
reaching a peak value within 48 hours. Elevated levels are found
with metal plumbing to form explosive metal oxides. In disposal,
in conditions such as bacterial infections, rheumatic fever, viral
flush with a large volume of water to prevent metal azide build
infections, malignant diseases, tuberculosis, and after a heart
up.
attack. The median CRP value for an individual increases with
age, reflecting an increase in subclinical inflammatory
STORAGE AND STABILITY
conditions.
o When not in use, store reagents and controls at 2 - 8 degree
o Because the levels rise and then decline so rapidly, CRP is the
Celsius. DO NOT FREEZE. Prior to use, allow reagents and
most widely used indicator of acute inflammation. Although CRP
controls to warm up to room temperature. Expiration date is
is a nonspecific indicator of disease or trauma, monitoring of its
specified on the kit label and on each vial. Biological indication of
levels can be useful clinically to follow a disease process and
product instability is evidenced by inappropriate reaction of the
observe the response to treatment of inflammation and
latex reagent with the corresponding positive and negative
infection.
control sera.
o It is a nonsurgical means of following the course of 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 rejection. CRP levels can also be used to
monitor the progression or remission of autoimmune diseases.
Assays for CRP are sensitive, reproducible, and relatively
inexpensive. CRP is easily destroyed by heating serum to 56°C for
30 minutes. The destruction of CRP is often necessary in the
laboratory because it interferes with some testing for the
presence of antibodies.

[SARANILLO, KA.]
 MATERIALS
o Broth culture of Bacillus subtilis or Staphylococcus epidermidis
IMMUNOLOGY AND SEROLOGY 311 o Microscope slides
SCREENING TEST FOR PHAGOCYTIC ENGULFMENT
PRELIM: LABORATORY
o Pasteur pipettes
o Rubber bulb
INTRODUCTION
o Test tubes
o Phagocytosis is the process by which specialized cells engulf and
o Wright’s stain
destroy foreign particles such as microorganisms or damaged
cells. Macrophages and neutrophils (PMNs) are the most
PROCEDURE
important phagocytic cells.
1. Label three test tubes : Patient test tube and Control test tube
o A mixture of bacteria and phagocytes is incubated and examined
2. Add 4 to 8 drops of the buffer coat from either the patient’s
for the presence of engulfed bacteria. This simple procedure may
heparinized blood or from the normal control to the respectively
be useful in supporting the diagnosis of impaired neutrophilic
labeled tubes.
function in conjunction with clinical signs and symptoms.
3. Add 2 to 3 drops of the bacterial broth culture to each tube.
4. Incubate both tubes at room temperature or 37 C for 30 minutes.
GLOSSARY OF TERMS
5. Place 1 drop of the incubated specimen on a glass slide and
Phagocytic cells A cell that ingests microorganisms and foreign
prepare a smear.
particles.
6. Air dry the slides and stain with Wright’s stain.
Bacteria Extremely small, relatively simple prokaryotic
WRIGHT’S STAIN PROCEDURE:
microorganisms traditionally classified with
- Cover each smear generously with filtered Wright’s stain
the fungi as Schizomycetes
and allow the stain to remain on the slide for at least 5
Signs Something that suggests the presence or
minutes.
existence of a fact, condition, or quality.
- Slowly add distilled water or buffer to the stain until the
Symptoms Any indication of a disease or injury perceived buffer begins to overflow the stain. Watch for the
by the patient appearance of a metallic luster.
- Gently blow on the slide to mix the stain and buffer.
PROCESS OF PHAGOCYTOSIS - Allow the buffer to remain on the slide for at least 5
1. INITIATION – Activated phagocytes have increased surface minutes.
receptors that allow for adherence to the bacterium; - Gently wash the stain and buffer off the slide with distilled
Phagocytosis is initiated as a result of tissue damage (either water.
trauma or as a result of microorganism multiplication) - Air dry or carefully blot the slide between two sheets of
bibulous paper.
2. CHEMOTAXIS – process in which cells tend to move in a certain 7. Place a drop of immersion on each smear and examine
direction under the stimulation of chemical substances; microscopically with the oil (100x) immersion objective.
Leukocytes show positive chemotaxis; Chemotactic agents
include starch and certain bacteria, as well as antigen-antibody CLEANING AND DISINFECTION
complexes, and dead tissues o All infectious materials should be disposed appropriately. The
- Positive Chemotaxis – toward the stimulating substances working area should be cleansed with the disinfectant before
- Negative Chemotaxis – away from the stimulating substances leaving. The PPE of each individual should be removed properly.
These cannot be exposed outside the laboratory premises.
3. ENGULFMENT – By active ameboid motion; final structure is
known as vacuole or phagosome;bacteria must be hydrophobic INTERPRETATION OF RESULTS
than the phagocyte o POSITIVE - Demonstration of the engulfment of bacteria.
- Opsonins (antibodies and complement components) o NEGATIVE - No engulfment of bacteria
interact with surfaces of bacteria, rendering them
acceptable to the phagocyte. SOURCES OF ERROR
- Phagosome – engulfment of antigen o This procedure may produce false-negative results if the blood
- Phagolysosome –fusion of phagosome and lysosome specimen is not fresh or if a coagulase-positive Staphylococcus
specimen is used. It is important to distinguish between granules
4. DIGESTION – Minute cell particles that contain certain hydrolytic and cocci. In Addition, the bacteria must be intracellular and not
enzymes and peroxides approach the phagosome, fuse with it, extracellular for the test to be positive.
rupture, and discharge content into it.
- Oxygen dependent (oxidative burst) – formation of CLINICAL APPLICATION
superoxide ⟶ hydrogen peroxide ⟶ hypochlorite. o The failure of phagocytes to engulf bacteria can support the
- Oxygen independent – defensins and cathepsin-G diagnosis of neutrophilic dysfunction; however, these results
must be used in conjunction with patient signs and symptoms.
PRE-ANALYTICAL PHASE
o The site of the experiment should be sterilized. The medical LIMITATIONS OF THE PROCEDURE
technologist should wear proper personal protective equipment o This is the simple screening procedure for engulfment. The
before the experiment (laboratory gown, laboratory goggles or presence of engulf bacteria does not demonstrate that the
face-shield, laboratory mask, and gloves) bacteria have been destroyed.
o 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.
o 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.

[SARANILLO, KA.]
 IMMUNOLOGY AND SEROLOGY 311 TYPES OF DILUTION
DILUTIONS
o “For many serology tests, it is the serum that is concentrated; it
PRE-ANALYTICAL PHASE may be necessary to dilute it with saline in order for a visible
o The site of the experiment should be sterilized. The medical reaction to occur.”
technologist should wear proper personal protective equipment
before the experiment (laboratory gown, laboratory goggles, or
face shield, laboratory mask, and gloves)
SIMPLE FORMULA
o Reagents must be brought to room temperature and must be
shaken well before dispensing. EDTA anticoagulated tube is
needed for the preparation of 3% red blood cell suspension.
SIMPLE DILUTION
MATERIALS EXAMPLE
o Antibody A (anti-A antisera) o Refrigerator
2 mL solution of a 1:20 dilution is needed to run a specific serological
o 3% Red blood cell suspension o Test tube rack
o 0.85-0.90% Saline (NSS) o Water proof marker test. How much serum and how much diluent are needed to make this
o Ten 12 x 75 Test tubes o Three serological pipettes dilution?

PROCEDURE
1. Label ten test tubes 1-10
2. Place 0.25 ml of saline in each of the ten tubes
3. Use a clean serological pipette to draw up 0.25 ml antibody A.
Add the antibody to tube #1 by carefully lowering and raising the
solution into the pipette three times to mix, being careful to
avoid creating bubbles in the mixture
EXAMPLE

4. Draw up to 0.25 ml from tube #1 and transfer to tube #2. Again,

raise and lower to solution into the pipette three times to mix
5. Repeat step 4, transferring 0.25 ml tube #2 to tube #3, then #4,
then #5, etc. Discard 0.25 ml from tube #5.
6. Use clean serological pipette to add 0.25 ml of 3% red blood cell
suspension to each tube. EXAMPLE
7. Mix well and show your instructor your tubes A volume of 500 mL of a 10% solution of acetic acid is needed. How
8. Place in refrigerator for 30 minutes much glacial acetic acid is needed and how much diluent should be
9. Remove from refrigerator, centrifuge for 20 seconds. Read used?
immediately for agglutination by gently shaking the tube to
dislodge the red blood cell button. If the tubes are shaken too
roughly false negative reaction can occur. Clumping of the red
blood cell suspension is negative.

COMPOUND DILUTION
EXAMPLE
if a 1:500 dilution is
necessary, it would take
49.9 mL of diluent to
accomplish this in one step
with 0.1 mL of serum

INTERPRETATION OF RESULTS
o The last tube showing agglutination is the endpoint of the test. PRACTICE PROBLEMS
The titer is reported out as the reciprocal of the last dilution
showing a positive result.

CALCULATING DILUTION

DILUTION FORMULA

SERIAL DILUTION
 CLINICAL SIGNIFICANCE
IMMUNOLOGY AND SEROLOGY 311 o RA is another example of a systemic autoimmune disorder. It affects about
RHEUMATOID FACTOR (RF) LATEX AGGLUTINATION TEST 0.5% to 1.0% of the adult population, but prevalence varies with ethnicity and
geographic location. Women are three times as likely to be affected as men;
MATERIALS in addition, the prevalence of the disease is highest in women who are more
o Rf latex suspension o Positive and Negative Controls than 65 years of age. RA can be characterized as a chronic, symmetric, and
o Black test cards o Disposable stirrers erosive arthritis of the peripheral joints that can also affect multiple organs
o Mechanical rotator such as the heart and the lungs. Associations of RA with more than 30 genetic
PRINCIPLE OF THE TEST regions have been discovered. The strongest associations have been between
a subset of patients with RA and specific HLA-DRB1 alleles or PTPN22 gene
o The RF reagent is a suspension of polystyrene latex particles sensitized
polymorphisms. These patients are positive for rheumatoid factor (RF) or
with specially prepared human IgG. The reagent is based on an
antibodies to CCP (see the section on Laboratory Diagnosis). The strongest
immunological reaction between human IgG bound to biologically inert environmental risk factor for RA is believed to be cigarette smoking, which
latex particles and rheumatoid factors in the test specimen. When serum doubles the risk of developing the disease.
containing rheumatoid factors is mixed with the latex reagent, visible o The pathology of RA is caused by an inflammatory process that results in the
agglutination occurs. The RF latex reagent sensitivity has been adjusted destruction of bone and cartilage. The lesions in rheumatoid joints show an
to detect a minimum of 8 IU/mL of rheumatoid factors according with increase in cells lining the synovial membrane and formation of a pannus, a
the WHO International Standard without previous sample dilution. sheet of inflammatory granulation tissue that grows into the joint space and
Sensitivity: 8 IUml or above. invades the cartilage. Infiltration of the inflamed synovium with T and B
lymphocytes, plasma cells, dendritic cells, mast cells, and granulocytes is
REAGENTS OF THE TEST evidence of immunologic activity within the joint.
1. RF Latex Reagent: A suspension of uniform polystyrene particles coated o It is not known what role autoantibodies play in the initiation of the
with IgG (human) in glycine buffer, pH 8.2; reagent sensitivity is inflammatory response. Two key antibodies found in the disease are RF and
standardized with the World Health Organization RF Standard. MIX WELL anti-CCP. RF is an antibody that is most often of the IgM class and is directed
against the FC portion of IgG. It has been postulated that RFs may play a role
BEFORE USING.
in the pathogenesis of RA by increasing macrophage activity and enhancing
2. RF Positive Control Serum: A stabilized, prediluted human serum
antigen presentation to T cells by APCs. In RA, autoantibodies such as RF and
containing at least 8 IU/mL of RF. anti-CCP are thought to combine with their specified antigen, and the
3. RF Negative Control Serum: A stabilized, prediluted human serum resulting immune complexes become deposited in the joints, resulting in a
containing less than 8 IU/mL of RF. type III (or immune complex) hypersensitivity reaction. The complement
4. Glycine-Saline Buffer (20x): pH 8.2 ± 0.1M glycine and 0.15M NaCl protein C1 binds to the immune complexes, activating the classical
5. Reaction Slide. complement cascade. During this process, C3a and C5a are generated, which
6. PipetteStir Sticks act as chemotactic factors for neutrophils and macrophages. The continual
presence of these cells and their associated cytokines leads to chronic
PROCEDURE inflammation, which damages the synovium itself.
Qualitative Method o Diagnosis of RA is based on a combination of clinical manifestations,
1. Allow each components of the test kit (reagents, controls) to reach room radiographic findings, and laboratory testing. RF is the antibody that is most
temperature. often tested to aid in making the initial diagnosis. The importance of testing
2. Gently shake the latex reagent to disperse the particles. for the presence of RF is also reflected in the fact that it is one of the
3. Place a drop of undiluted serum into a circle of a test slide. classification criteria for RA. Recall that RF is an autoantibody, usually of the
IgM class, that reacts with the Fc portion of IgG. Approximately 70% to 90%
4. Add one drop of Rf Latex reagent next to the drop of serum.
of patients with RA test positive for RF. Thus, a negative result does not rule
5. Spread the reagent and serum sample over the entire area of the test
out the presence of RA. Conversely, a positive test result is not specific for RA
circle using a separate stirrer for each sample. because RF is also present in about 5% of healthy individuals and in 10% to
6. Gently tilt the test slide backwards and forwards for two minutes. 25% of those over the age of 65.
7. Observe and interpret the results. o In addition, RF can be found in patients with other connective tissue diseases
o NOTE: Positive and Negative Controls should be run at regular intervals such as SLE, Sjögren’s syndrome, scleroderma, and mixed connective tissue
disease, as well as in people with some chronic infections. Manual
Semi-Quantitative Determination agglutination tests using charcoal or latex particles coated with IgG have been
o The semi-quantitative test can be performed in the same way as the used for many years to detect RF. These tests, however, only detect the IgM
qualitative test using dilutions of the serum in saline, phosphate buffer isotype, found in about 75% of patients, and have been largely replaced by
saline or glycine saline as follows: ELISA, chemiluminescence immunoassay, and nephelometric methods, which
are automated, have greater precision and sensitivity, and can also detect
other RF isotypes.
o Once a diagnosis of RA is made, the most helpful tests for following the
progress of the disease are general indicators of inflammation, such as
measurement of ESR, CRP, and complement components. Typically, CRP and
ESR are elevated and the levels of serum complement components are normal
or increased because of increased acute-phase reactivity. CRP levels correlate
well with disease activity because levels reflect the intensity of the
inflammatory response.
And other important information
1. Results should be read two (2) minutes after the mixing or the reagent
on the slide.
2. Existence of prozone at high titers has not been encountered.
3. Increased levels of RF may be round in some diseases other than
rheumatoid arthritis such as infectious mononucleosis, sarcodosis, lupus
erythematosus, Sjogren's syndrome. 6,7
o Normal levels (adults): less than 8 IU/ml 4. Certain patients with rheumatoid arthritis will not have the RF present in
o CALCULATION: In the semi-quantitative test, the titer is expressed as the their serum.
reciprocal of the highest dilution showing macroscopic agglutination. For
example, if agglutination occurs in dilution 3, the titer is 64. EXPECTED VALUES
1. The diagnosis of rheumatoid arthritis is based largely on clinical
RESULTS
examination, but laboratory tests are useful to support the clinical
Qualitative Test:
diagnosis and to evaluate the severity and course or the disease in the
o Negative Result: A negative reaction is indicated by a uniform milky
individual patient. One of the most useful clinical markers for rheumatoid
suspension with no agglutination observed with the RF Negative Control.
arthritis is rheumatoid factor in serum. Rheumatoid factor is a term used
o Positive Result: A positive reaction is indicated by any observable
to describe a variety of antibodies or immune complexes or both, that
agglutination in the reaction mixture. The specimen reaction should be
occur with rheumatoid arthritis as well as in a variety of other diseases.
compared to the RF Negative and Positive Controls
2. Different studies have shown positive serological reactions for
Semi-quantitative test: rheumatoid factor in as high as 90 of patients with rheumatoid arthritis
o The titer of the serum is the reciprocal of compared with less than 5 in control groups.
the highest dilution, which exhibits a
positive reaction. An estimate of the RF STORAGE
concentration in the specimen can be 1. Reagents are stable until stated expiration date on bottle label when
expressed in IUml by using the following stored refrigerated (2 - 8°C).
equation: 2. DO NOT FREEZE.
3. The RF latex reagent, once shaken must be uniform without visible
o IUml of specimen = IUml control  specimen titer
clumping. When stored refrigerated, a slight sedimentation may occur
o The approximately RF level (IU/ml) present in the sample may be
and should be considered normal.
obtained by the multiplying the titer by the analytical sensitivity of the Rf
4. Do not use the latex reagent or controls if they become contaminated
Latex reagent (8 IU/ml)
 CLINICAL SIGNIFICANCE
IMMUNOLOGY AND SEROLOGY 311 o Systemic Lupus Erythematosus is a chronic systemic
DETECTION OF ANTI-NUCLEAR ANTIBODIES (ANA)
inflammatory disease that affects people usually at the peak age
MATERIALS between 20 and 40 years. Women are much likely more to be
o Rf latex suspension o Positive and Negative Controls affected than men by a ratio of 9:1. This autoimmune disease
o Black test cards o Disposable stirrers appears to originate from complex interaction between several
o Mechanical rotator factors, such as environmental, genetic susceptibility, and
abnormalities within the immune system. In line with these, over
PRINCIPLE OF THE TEST 100 autoantibodies associated with SLE have been discovered.
o ELISA and CLIA are assays that can test from a broad range of o These include antibodies to double stranded DNA (dsDNA),
antibodies if multiple nuclear antigens are coated onto a single histones, and other nuclear components, as well as
test well, or for a specific ANAs if each well is coated with a single autoantibodies to lymphocytes, erythrocytes, platelets,
antigen. These antigens used in commercial kits are derived from phospholipids, ribosomal components, and endothelium.
tissue extracts or produced by recombinant technology. Because o Laboratory diagnosis of SLE include CBC, platelet count and
of their advantages, many laboratories are using ELISA methods urinalysis. Findings in lupus patients are leukopenia and possible
to screen for presence of ANAs in addition to identifying specific anemia and thrombocytopenia. Erythrocyte Sedimentation Rate
ANAs (ESR) may be elevated even though C-Reactive Protein tends to
be low or normal.
SENSITIVITY OF THE TEST o When SLE is suspected, the first test typically done is a screening
o There is a large variation in the performance of test produced by test for Anti-nuclear Antibodies (ANA). These antibodies are
different manufacturers, which is influenced by the antigen present in most cases of SLE, antibodies that are directed against
preparation used. For example, one study found sensitivities of antigens of nuclei of mammalian cells. ANAs are present in over
ELISA assays ranging from 69% to 98%, and specificities from 81% 95% of patients with active SLE and is used as a major marker for
to 98% when they were compared with IIF ANA method. the disease. However, ANAs are not specific only to SLE as it may
be present in other connective tissue diseas such as Sjogren’s
REAGENTS OF THE TEST syndrome, scleroderma, polyomyositis-dermatomyositis, and
o ANA reagent, positive and negative control reagents, wells. RA. They can also be found in some individuals with other
conditions, including chronic infections, pregnancy and cancer.
PROCEDURE Furthermore, upto 5% of healthy individuals and upto 30% of
Qualitative Method elderly people are ANA-positive.
1. Bring all reagents to room temperature and mix gently prior to o ANAs are a heterogeneous group of antibodies that have
use. different antigen specificities. The nuclear antigens they are
2. Place the following on separate divisions of the same black test directed against include double stranded and single stranded
card. DNA, histones, nucleosomes, centromere proteins, and
➢ Patient’s Serum: 1 drop extractable nuclear antigens. Double stranded DNA antibodies
➢ Positive Control: 1 drop are the most specific for SLE because they are mainly seen in
➢ Negative Control: 1 drop patients with lupus and their levels correlate with disease
3. Add 1 drop of SLE Latex reagent to each sample on the black test activity. Antibodies to dsDNA typically produce a peripheral or
card. homogenous staining pattern on indirect immunofluorescence
4. Mix with the flat end of pipette/mixer and spread fluid evenly (IIF).
covering the entire diameter of the test circle. o Methods in detecting ANAs have been developed. These include
5. Tilt the slide back and forth slowly for 3 minutes while observing IIF, immunoperoxidase staining, enzyme linked immunosorbent
for agglutination. assay (ELISA), microsphere multiplex immunoassays (MIA),
6. Observe and Interpret results radioimmunoassay (RIA), immunodiffusion, immunoblotting
(Western blot), dot blot, immunoelectrophoresis, and
Semi-Quantitative Determination microarray.
o Prepare dilutions of the specimens as shown below:
Dilution Serum Saline OTHER IMPORTANT INFORMATION
1:2 1 part 1 part o Note the proper procedure and usage of the reagents, storage
1:4 1 part 3 parts temperature and the expiry date of the kit.
1:8 1 part 7 parts
1:16 1 part 15 parts
1:32 1 part 31 parts

RESULTS
o Agglutination indicates a positive result. If there is no
agglutination, the test result is negative. The test is considered
as NEGATIVE when no difference in agglutination is observed
between the specimen and negative control and POSITIVE sera
must show distinct agglutination within 3 minutes.
o Agglutination indicates the level of antinuclear antibody
(especially anti-DNP) in the range commonly found in the SLE.
o Findings in the sensitivity studies suggests that immunoassays
may miss a significant proportion of ANA-positive patients and
also yield a significant number of false positive results. Based on
such studies, the ACR has recommended that the IIF test remains
the gold standard for ANA testing and that clinical laboratories
should specify the method they use when they are reporting the
result.
 o A titer that yields semiquantitative results can be performed in
IMMUNOLOGY AND SEROLOGY 311 test tubes or microtiter plates by making serial dilutions of the
SLIDE AGGLUTINATION TEST
antibody. The reciprocal of the last dilution still exhibiting a
MATERIALS visible reaction is the titer, indicating the antibody’s strength.
o Antibody A o Antibody B Interpretation of the test is done on the basis of the cell
o Red blood cells (#1 and #2) o Pipettes sedimentation pattern. If there is a dark red, smooth button at
o Slides o Applicator sticks the bottom of the microtiter well, the result is negative. A
positive result will have cells that are spread across the well’s
PRINCIPLE OF THE TEST bottom, usually in a jagged pattern with an irregular edge. Test
o Hemagglutination reaction tubes also can be centrifuged and then shaken to see if the cell
button can be evenly re-suspended. If it is re-suspended with no
REAGENT OF THE TEST visible clumping, then the result is negative. Positive reactions
o Anti-A and Anti-B antisera can be graded to indicate the strength of the reaction.
o Anti-D

RESULTS
o Agglutination (clumping) of the red blood cells is positive. No
agglutination is negative. It is critical to read the results
immediately as false positives can occur when the mixture
begins to dry on the side.
Anti-A Anti-B Blood Group
Positive (+) Negative (0) A
Negative (0) Positive (+) B
Positive (+) Positive (+) AB
Negative (0) Negative (0) O
Legend: + → with agglutination
0 → without agglutination

CLINICAL SIGNIFICANCE
o 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 PROCEDURE
mixed with known antibody A or antibody B, agglutination of the 1. On the section of slide labeled anti-A, placed one drop of
RBCs will occur if a specific antigen-antibody reaction occurs. antibody A.
This is called direct blood typing. 2. On the section of slide labeled anti-B, placed one drop of
o Agglutination, like precipitation, is a two-step process that antibody B.
results in the formation of a stable lattice network. The first 3. Place one drop of cells in
reaction, called sensitization, involves antigen–antibody each antibody-containing
combination through single antigenic determinants on the circle.
particle and is rapid and reversible. The second step, or lattice 4. Carefully mix each solution
formation, is the formation of cross-links that form the visible with a separate applicator
aggregates. This represents the stabilization of antigen–antibody stick
complexes with the binding together of multiple antigenic 5. Tilt slowly for one minute.
determinants. 6. Record results
o Sensitization is affected by the nature of the antigens on the
agglutinating particles. If epitopes are sparse or if they are
obscured by other surface molecules, they are less likely to AGGLUTINATION REACTIONS
interact with antibody. Additionally, red blood cells (RBCs) and Type AB
bacterial cells have a slight negative surface charge; because like (contains
charges tend to repel one another, it is sometimes difficult to agglutinogens A
bring such cells together into a lattice formation. and B)
o IgM antibodies are strong agglutinins because of their larger size.
Achieving visible reactions with IgG often requires the use of
enhancement techniques that vary physicochemical conditions
such as the ionic strength of the solution, the pH, and the Type B
temperature. Antibodies belonging to the IgG class agglutinate (contains
best at 30°C to 37°C, whereas IgM antibodies react best at agglutinogen B)
temperatures between 4°C and 27°C. Because naturally
occurring antibodies against the ABO blood groups belong to the
IgM class, these reactions are best run at room temperature.
Antibodies to other human blood groups usually belong to the Type A
IgG class; reactions involving these must be run at 37°C. These (contains
latter reactions are the most important to consider in selecting agglutinogen A)
compatible blood for a transfusion because these are the ones
that will actually occur in the body.
o If an agglutination reaction involves RBCs, then it is called
hemagglutination. The best example of this occurs in ABO blood Type O
group typing of human RBCs, one of the world’s most frequently (contains no
used immunoassays. Patient RBCs mixed with antisera of the IgM agglutinogens)
type can be used to determine the presence or absence of the A
and B antigens; this reaction is usually performed at room
temperature without the need for any enhancement techniques.
Group A RBCs will agglutinate with anti-A antibody and Group B
RBCs will agglutinate with anti-B antibody. This type of
agglutination reaction is simple to perform, is relatively sensitive,
and is easy to read.
 TUBE AGGLUTINATION TEST
IMMUNOLOGY AND SEROLOGY 311 All positive results obtained through the slide test should be confirmed using
FEBRILE ANTIGENS the following technique.
MATERIALS 1. Label 8 small test tubes in a rack.
Widal Test Weil-Felix 2. Using a pipette, dispense 1.9ml of 0.85% Saline into the first test tube,
▪ Antigen suspension S. typhi O ▪ Proteus OX2 and 1.0 ml into the remaining seven.
▪ Antigen suspension S. typhi H ▪ Proteus OX19 3. Using a pipette, dispense 0.1 ml of the patient’s undiluted serum into the
▪ Pipettes ▪ Proteus OXK first test tube. Mix well using the larger pipette volume and tip.
▪ Glass slides ▪ Applicator stick 4. Using the pipette, dispense 1.0 ml from the first tube into the second
▪ NSS ▪ Test tube tube. Mix well.
▪ Antigen suspension S. typhi ‘BH’ ▪ NSS 5. Continue this method of doubling dilutions up to the seventh tube.
▪ suspension S. typhi ‘AH’ ▪ Transparent glass ring slide Discard 1.0 ml from the seventh tube. The eight tube will contain only
▪ Test tubes Antigen Vortex mixer ▪ Serum pipettes saline as a control and therefore should not contain any serum.
▪ Mixing sticks 6. Shake the reagent bottle well. And add 1 drop of the appropriate antigen
suspension into each tube and mix well.
LABORATORY DISCUSSION GUIDE 7. Incubate at the water bath as follows:
o Typhoid fever, simply typhoid, caused by the bacterium o Salmonella “O” Antigens and Proteus: 500C for 4 hours
Salmonella enterica subsp. enterica serovar Typhi growing in the o Salmonella “H” Antigens: 500C for 2 hours
intestines and blood. Typhoid is spread by eating or drinking food 8. Examine the tubes after the appropriate incubation time and check for
or water contaminated with the feces of an infected person. agglutination. The titer to be taken is the last tube to show agglutination.
Diagnosis is made by any blood, bone marrow, or stool cultures
and with the Widal test (demonstration of antibodies against RESULTS
o Agglutination is a positive test result and indicates the presence of the
Salmonella antigens O-somatic and H-flagellar). In epidemics and
corresponding antibody in the patient serum. No agglutination is a
less wealthy countries, after excluding malaria, dysentery, or negative test result and it indicates the absence of the corresponding
pneumonia, a therapeutic trial time with chloramphenicol is antibody in the patient serum.
generally undertaken while awaiting the results of the Widal test o Rapid Slide Screening Test: Agglutination is indicative of serum titer of
and cultures of the blood and stool 1:20 in a tube test.
o Rapid Slide Titration Method: If agglutination reactions are observed,
Widal Test the following approximate titers would be observed if a confirmatory
o Quantitatively measures the antibodies in the serum of patient tube test was carried out:
with Typhoid fever Serum volume Titer
0.08 ml 20
0.04 ml 40
3 Kinds of Antigen 0.02 ml 80
O Ag Somatic antigen; heat stable 0.01 ml 160
H Ag Flagellar Antigen, heat labile 0.005 ml 320
K Ag Vi Antigen (capsule) (virulence) o In this way, the rapid slide test provides an approximation to the
Thermo labile, unstable, delicate expected results from a corresponding tube test.
o NOTE: It is necessary to perform all dilutions in the slide test to obviate
Reference laboratories
the “prozone” effect where higher concentrations of the serum may give
a negative result but further dilutions may give a positive result.
3 TYPES OF METHODS IN WIDAL’S o Tube Agglutination Test: A positive test will show an obvious floccular
A. Rapid Slide Screening Method agglutination throughout the tube. A negative result and the saline
1. Using a pipettor, dispense 1 drop (0.08ml) of undiluted serum into control should show no change in appearance and should show a
a row of 3cm diameter circles. characteristic swirl when flicked. Tube must not be shaken.
2. Shake the reagent bottle well and add 1 drop of the undiluted
antigen suspension to the serum. NOTE:
3. Mix well using a stirring stick and rotate the slide for 1 minute. 1. A single negative test does not exclude the absence of infection.
4. Observe and interpret result. 2. A single positive test is of no diagnostic value unless the titer is
5. A positive serum of known titer and a negative serum should be sufficiently high.
included as controls. 3. Agglutinin is not always produced in bacterial infections.
B. Rapid Slide Titration Method 4. Cross- reaction may occur in certain pathologies.
1. Using a pipettor, dispense 0.08ml, 0.04ml, 0.02ml, 0.01 and 5. The considered significant titer in the Widal test is 1:80 while 1:160
0.005ml of undiluted serum into a row of 3 cm diameter circles. in the Weil-Felix Test
2. Shake the reagent bottle well and add 1 drop of the undiluted
antigen suspension to each serum aliquot.
3. Mix well using a stirring stick and rotate the slide for 1 minute. TWO TYPES OF AGGLUTINATION TEST IN WIDAL’s:
1. Somatic agglutination
o Compact aggregates that are not easily dispersed
Note: Bring all reagents to room temperature before testing. Shake o Stronger agglutination than H Ag
antigen suspension well before disposing 2. Flagellar Agglutination
o Fluffy and floccular aggregates that are easily dispersed
The degree of agglutination is recorded as follows:
4+ 100% of the organism is agglutinated Significant titer: 1:80 (80)
< 80 = normal
3+ 75% of the organism is agglutinated
> 80 = indicative of infection
2+ 50% of the organism is agglutinated
1+ 25% of the organism is agglutinated Interpretation:
1/+ Less than 25% of the organism is agglutinated o INC titer on OD Ag: indicates acute/ recent typhoid fever
Negative No agglutination is observed o INC titer on OA, OB and OC: indicates acute/ recent paratyphoid infection
o INC titer on HD Ag: indicates previous/ past infection with typhoid fever
- Convalescent/ vaccination against typhoid fever
o Although the slide test is not recommended to established the
o INC titer on HA, HC, HB: indicates past infection on [Link]
titer of the serum, the quantity of the serum giving 2 + or 50 % o INC titer on O and H Ag: indicates mixed infection
agglutination can be used to approximate the titer of the serum.
The serum dilution in the slide test is approximately equivalent NON-SPECIFIC TEST
to the test tube dilution shows below: WEIL- FELIX TEST
o Used to test for antibodies to rickettsil infections of Typhus fever caused
Serum volume Approximate Tube Dilution by: ([Link])
0.08 ml 1:20 o Proteus ox-2 (from [Link])
o Proteus ox-19 (from [Link])
0.04 ml 1:40
o Proteus ox-k (uses kingsburry strain)
0.02 ml 1:80
o Cross reaction mechanism
0.01 ml 1:160 o Cross reactivity between rickettsia and certain serotypes of
0.005 ml 1:320 proteus spp.
o Possess the same antigenic determinant
 o CALCULATION: In the semi-quantitative test, the titer is
IMMUNOLOGY AND SEROLOGY 311 expressed as the reciprocal of the highest dilution showing
ASO LATEX TEST
macroscopic agglutination. For example, if agglutination occurs
MATERIALS in dilution 3, the titer is 1600
o ASO latex reagent o Pipette
o Control sera (+ and - Controls) o Applicator sticks ASO TUBE TEST
o Black test cards o Mechanical rotator o Diagnosis of the probability for sequelae S. pyogenes infection
o Compound dilution
LABORATORY DISCUSSION GUIDE o Principle: NEUTRALIZATION/ enzyme inhibition test
o In infections caused by B-hemolytic streptococci, Streptolysin-O - If Ab (ASO) is present in patient serum, it will bind to SO is
is one of the two hemolytic exotoxins librated from the bacteria not free to react with indicator RBC’s, the result appears as
that stimulates production of ASO antibodies in the human no hemolysis.
serum. The presence and the level of these antibodies in serum - If Ab is absent, Streptolysin O is free to react with RBC’s
may reflect the nature and severity of infection. The ASO TEST is resulting in hemolysis
a stabilized buffered suspension of polystyrene latex particles o Materials:
that have been coated with Streptolysin O. When the latex - 14 test tubes
reagent is mixed with serum containing ASO, agglutination - Tube 13 and tube 14 → control tubes
occurs - Streptolysin O reagent
- Streptolysin O buffer
ASO SLIDE METHOD - 5% human type O red cell suspension
o PRINCIPLE: Passive/ indirect agglutination - Serological pipettes
o Sample: Serum o Procedure:
o ASO LATEX REAGENT: Suspension of polystyrene particles - Add strep O Ag on all tubes except tube 13 (red cell control)
sensitized with Streptolysin O - Tube 14 (strep O control tube)
- Shake incubate for 15min, 37C
REAGENTS - Centrifuge, check for presence of hemolysis
ASO LATEX ▪ Contains polystyrene latex particles coated o RESULTS:
REAGENT with Streptolysin O in a stabilized buffer with ✓ Positive: absence of hemolysis
less than 0.1% sodium azide as preservative ✓ Negative: hemolysis (red supernatant)
▪ A suspension of polystyrene latex particles o NOTE: Titer: last tube with no hemolysis (clear supernatant)
sensitized with streptococcal exoenzyme. o Tube 13: no hemolysis
ASO POSITIVE ▪ Human serum containing more than 200 o Tube 14: with hemolysis
CONTROL IU/ml ASO with less than 0.1% sodium azide as o Significant/ diagnosis titer: 166 todd units
preservative
▪ A stabilized human serum containing ASO SENSITIVITY
reactive with ASO latex reagent. o The sensitivity of the latex reagent has been adjusted to yield
ASO NEGATIVE ▪ Human serum that has been diluted and agglutination when the level of ASO is greater than 200 IU/ml, a
CONTROL stabilized with buffer and contains less than level determined to be indicative of disease by epidemiological
0.1% sodium azide as preservative and clinical studies.
▪ A stabilized human serum containing ASO - Positive: if titer is > 200 IU/mL
non-reactive with ASO latex reagent. - Negative: if titer is < 200 IU/mL
All reagents contain 0.1% Sodium azide as a preservative.
RESULTS
PROCEDURE o Positive Result: Agglutination
1. Allow each components of the test kit (reagents, controls) to o Negative Result: Smooth milky suspension
reach room temperature. o Since negative results may be caused by antigen excess, the test
2. Gently shake the latex reagent to disperse the particles. should be repeated using a diluted serum sample in case prozone
3. Place a drop of undiluted serum into a circle of a test slide. effect is suspected.
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 INTERFERING SUBSTANCES
test circle using a separate stirrer for each sample. o HEAVY BACTERIAL CONTAMINATION - may cause FALSE
6. Gently tilt the test slide backwards and forwards for two POSITIVE AGGLUTINATION.
minutes. o MARKEDLY LIPEMIC SERA - SHOULD NOT BE TESTED because of
7. Observe and interpret the results. the possibility of nonspecific reactions
NOTE: Positive and Negative Controls should be run at regular intervals
CLINICAL SIGNIFICANCE
SEMI-QUANTITATIVE DETERMINATION o Streptococci are gram-positive cocci that are spherical, ovoid, or
o The semi-quantitative test can be performed in the same way as lancet-shaped organisms often arranged in pairs or chains when
the qualitative test using dilutions of the serum in saline, observed on Gram stain. GAS is one of the most common and
phosphate buffer saline or glycine saline as follows: ubiquitous pathogenic bacteria and causes a variety of
infections. The M protein is the major virulence factor of GAS and
has a net-negative charge at the amino-terminal end that helps
to inhibit phagocytosis. In addition, the presence of the M
protein limits deposition of C3 on the bacterial surface, thereby
diminishing complement activation. The M protein, along with
lipoteichoic acid and protein F, help GAS attach to host cells.
Immunity to GAS appears to be associated with antibodies to the
M protein. There are more than 100 serotypes of this protein and
immunity is serotype-specific. Therefore, infections with one
strain will not provide protection against another strain.
o Additional virulence factors include various exotoxins that may
be produced during the course of an infection. Pyrogenic
exotoxins A, B, and C are responsible for the rash seen in scarlet
fever and also appear to contribute to pathogenicity. Additional
extracellular substances include the enzymes streptolysin O,
deoxyribonuclease B (DNase B), hyaluronidase, nicotinamide
o NOTE: If titer is 2 multiply to 200 = 400 IU/mL (1st circle) adenine dinucleotide (NAD), and streptokinase. Antibodies
o Normal levels (adults): less than 200 IU/Ml produced against these substances are useful in the diagnosis of
 infection and for the potential development of complications or infection and peak between 3 to 6 weeks following the initial
sequelae associated with GAS infection. symptoms (e.g., sore throat). However, an antibody response
o S pyogenes can be responsible for infections ranging from occurs in only about 85% of acute rheumatic fever patients
pharyngitis (a throat infection) to lifethreatening illnesses such within this period. Additionally, ASO titers usually do not
as necrotizing fasciitis and streptococcal toxic shock syndrome. increase in individuals with skin infections.
The two major sites of infections in humans are the upper
respiratory tract and the skin, with pharyngitis (“strep throat”) OTHER TESTS
and streptococcal pyoderma (a skin infection) being the most Streptozyme test
common clinical manifestations. The reason GAS receives so o Multi-enzyme test to detect Antibody to the multiple
much attention is the potential for the development of two exoenzymes of S. pyogenes
serious sequelae, acute rheumatic fever and poststreptococcal o PRINCIPLE: Passive Hemagglutination
glomerulonephritis. o REAGENT: Formalinized/ aldehyde fixed sheep RBCs coated with
o Diagnosis of acute streptococcal infections typically is made by exoenzymes of S. pyogenes
culture of the organism from the infected site. The specimen is
plated on sheep blood agar and incubated. If Group A QUALITY CONTROL PROCEDURE
streptococcus is present, small translucent colonies surrounded o Positive and Negative controls should be included in each test
by a clear zone of β hemolysis will be visible. As an alternative to series. The Positive control should produce visible agglutination;
culture, rapid assays have been commercially developed to and Negative control should produce no agglutination.
detect Group A streptococcal antigens directly from throat
swabs. The Group A antigens are extracted by either enzymatic LIMITATIONS OF PROCEDURE:
or chemical means and the process takes anywhere from 2 to 30 1. The results of this test SHOULD NOT be used as a single
minutes, depending on the particular technique diagnostic tool to make a clinical diagnosis. Instead, the test
o Culture or rapid screening methods are extremely useful for results must be evaluated together with other clinical findings
diagnosis of acute pharyngitis. However, serological diagnosis and observed symptoms to aid in the final diagnosis.
must be used to identify acute rheumatic fever and 2. This test is designed to be performed by hand rotation. The use
poststreptococcal glomerulonephritis because the organism is of a mechanical rotator could yield false positive/negative
unlikely to be present in the pharynx or on the skin at the time results.
symptoms appear. The antibody response to these streptococcal 3. Serum samples showing gross hemolysis, lipemia, turbidity, or
products is variable because of several factors, such as age of contamination should not be used since false positive results
onset, site of infection, and timeliness of antibiotic treatment. may occur. Both elevated Beta-lipoprotein and cholesterol level
The most diagnostically important antibodies are the following: may suppress a rise in ASO titer.
anti-streptolysin O (ASO), anti-DNase B, anti-NADase, and anti- 4. The test reaction must be read immediately following the two (2)
hyaluronidase (AHase). Assays for detection of these antibodies minutes rotation. Delayed readings may result in false positive
can be performed individually or through use of the streptozyme results.
test which detects antibodies to all these products. 5. When not in use, store reagents and controls at 2 - 8 degrees
o Let us focus on ASO testing. ASO tests detect antibodies to the Celsius. DO NOT FREEZE. Prior to use, allow reagents and
streptolysin O enzyme produced by Group A streptococcus. This controls to warm up to room temperature. Expiration date is
enzyme is able to lyse RBCs. Presence of antibodies to specified on the kit label and on each vial. Biological indication of
streptolysin O indicates recent streptococcal infection in patients product instability is evidenced by inappropriate reaction of the
suspected of having acute rheumatic fever or poststreptococcal latex reagent with the corresponding positive and negative
glomerulonephritis following a throat infection. The classic control sera.
hemolytic method for determining the ASO titer was the first test
developed to measure streptococcal antibodies. This test was
based on the ability of antibodies in the patient’s serum to
neutralize the hemolytic activity of streptolysin O. The traditional
ASO titer 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 (when the streptolysin
reagent standard is used) or in international units (when the
World Health Organization international standard is used).
o The range of expected normal values varies, depending on the
patient’s age, geographic location, and season of the year. ASO
titers tend to be highest in school-age children and young adults.
Thus, the upper limits of normal must be established for specific
populations. Typically, however, a single ASO titer is considered
to be moderately elevated if the titer is at least 240 Todd units in
an adult and 320 Todd units in a child.
o Because of the labor-intensive nature of the traditional ASO titer
test and because the streptolysin O reagent and the RBCs used
are not stable, ASO testing is currently performed by
nephelometric methods. Nephelometry has the advantage of
being an automated procedure that provides rapid, quantitative
measurement of ASO titers.
o The antigen used in this technique is purified recombinant
streptolysin. When antibody-positive patient serum combines
with the antigen reagent, immune complexes are formed,
resulting in an increased light scatter that the instrument
converts to a peak rate signal. All results are reported in
international units, which are extrapolated from the classic
hemolytic method described previously. When using the
nephelometric method, individual laboratories must establish
their own upper limits of normal for populations of different
ages. ASO titers typically increase within 1 to 2 weeks after
 IMMUNOLOGY AND SEROLOGY 311 IMMUNOLOGY AND SEROLOGY 311
NON-TREPONEMAL TESTS FOR SYPHILIS (RPR) TESTS FOR HUMAN IMMUNODEFICIENCY VIRUS (HIV)
FINALS: LABORATORY FINALS: LABORATORY

PRE-ANALYTICAL PHASE PRE-ANALYTICAL PHASE

o The site of the experiment should be sterilized. The medical o The work area of the experiment should be sterilized. The
technologist should wear proper personal protective equipment Students should wear proper personal protective equipment
before the experiment (laboratory gown, laboratory goggles or before the experiment (laboratory gown, eye protector,
face shield, laboratory mask, and gloves) laboratory mask, gloves and hair net). Standard precaution is
o Reagents must be brought to room temperature and must be applied to every phase of experiment. Students to perform the
shaken well before dispensing. Undiluted serum will be used to said experiment are required to have Anti-Hepatitis B vaccine.
react with the RPR Carbon antigen.
o The microscope should be placed on a plain, stable surface. The HUMAN IMMUNODEFICIENCY VIRUS (HI)
voltage of the power supply should be inclined with that of the o The etiologic agent of AIDS is a human retrovirus known as
equipment. Proper handling and cleanliness of compound human immunodeficiency virus (HIV). Retroviruses are defined
microscope should be observed at all times by the medical as viruses that contain a single positive stranded RNA, which
technologist. Lens paper is provided in order to clean the contain the virus genetic information, and a specially enzyme
objective and ocular lenses. known as “reverse transcriptase” in their core. The enzyme
enables the virus to convert viral RNA to DNA in contrast to the
LABORATORY DISCUSSION GUIDE normal process of transcription where DNA is converted to RNA.
NON-TREPONEMAL TESTS FOR SYPHILIS
o The student/s will become familiar in testing Non-Treponemal TWO TYPES OF HIV HAVE BEEN CLASSIFIED
test for Syphilis especially Rapid Plasma Reagin (RPR) using 1. HIV-1 the causative agent of AIDS is Europe and the United States
patient serum mixed with RPR Carbon Antigen. Flocculation 2. HIV-2 associated with immunodeficiency and clinical syndrome
must be observed for positive results. similar to AIDS in West Africa
o Syphilis is a contagious sexually transmitted disease cause by the
spirochete Treponema pallidum. This spirochete causes an o HIV was formerly known as lymphadenopathy-associated virus
immune response in the body by producing antibody-like (HTLV-III), or AIDS-related virus. It is believed to be a member of
substance in plasma or serum known as reagin. Cardiolipin, the group of transforming, cytopathic retroviruses, called
phospholipid derived from the beef heart reacts with the reagin lentiviruses which cause chronic neurodegenerative and wasting
and this antigenic property of cardiolipin is used in non- diseases and wasting diseases in animals similar to the wasting
treponemal test. disease and neurologic disorders produced by HIV in humans.

RAPID PLASMA REAGIN (RPR) RAPID HIV TESTING (SD HIV-1/2 3.0)
o RPR is the most frequently performed non treponemal test for o Rapid test (which come in both blood and oral version) are done
syphilis. The RPR card antigen suspension is a carbon-containing onsite and give a reading within half an hour. As with the ELISA,
cardiolipin antigen that detects reagin. a positive reading on a rapid test requires a second, confirmatory
o The specimen serum or plasma is mixed with the RPR card assay such a Western blot.
antigen suspension and allowed to react for 8 minutes. If reagin o Rapid tests have proved to be as accurate as the ELISA when
is present, it will react to the antigen suspension forming visible performed carefully by experienced personnel. Technical errors
black floccules. This result is result is reported a reactive. If reagin can cause false results with these assays if the users tend to
is absent, there will be no flocculation. This result is reported as become careless with these simple procedures. To deal with this
nonreactive. problem, many rapid tests now include a built-in control that
indicates whether or not the test was done properly.
MATERIALS
o RPR Test Slide o Positive and Negative Control MATERIALS
o Disposable dispensing o RPR Carbon antigen o Test device o 20 ul capillary pipettes/sample dropper
pipettes o Mixing sticks o Timer o Blood/Serum sample
o needle dropper o Mechanical rotator o Assay diluent
o Stopwatch
PROCEDURE
PROCEDURE 1. Remove the test device from foil pouch, place it in a flat dry
QUALITATIVE METHOD surface.
1. Hold the pipette between the thumb and the forefinger. Squeeze 2. If capillary pipette will be used, add 20 ul of drawn blood into the
while inserting the tip into the specimen. Then release finger sample well.
pressure to withdraw the sample taking care not to transfer any 3. If a micropipette is used, add 10 ul of plasma or serum specimen
cellular elements. into the sample well (S).
2. Hold the dropper over a test card circle and squeeze the teat to 4. Add 4 drops (about 120 ul) of assay diluent into sample well (S).
allow one drop (50ul) to fall into the card. It is important to 5. As the test begins to work, you will see purple color move across
maintain the dropper in a vertical position while dispensing the the result window in the center of the test device.
sample to be tested. 6. Interpret test results in 5-20 minutes. Do not read results after
3. Using the flat end of the stirrer, spread the sample to cover the 20 minutes. Reading too late can give false results.
test circle.
4. Attach the dispensing needle to the plastic bottle. Withdraw RESULTS
sufficient RPR Carbon Antigen (WELL SHAKEN) for the number of o HIV NEGATIVE: is indicated by the presence of only one line: a
tests performed. Maintaining the needle in a vertical position, line in the control area within the result window.
allow one drop to fall on each test sample. DO NOT RE-STIR! o HIV-1 POSITIVE: is indicated by the presence of two lines: one in
5. Rotate the RPR test card manually or using an automatic rotator the control area I and one in the test area 1 (1) within the result
for 8 minutes at 100 rpm. window.
6. Observe and Interpret results. o HIV-2 POSITIVE: is indicated by the presence of two lines: one in
the control area I and one in the test area 2 (2) within the result
RESULTS window.
o Flocculation indicates a REACTIVE result. If there is no o HIV-1 and HIV-2 POSITIVE: is indicated by the presence of three
flocculation, the test result is reported as NONREACTIVE lines: one in the control area I, one in the test area 1 (1) and
another one in the test area 2 (2) within the result window.
o INVALID: is indicated if there are no line is seen in the control
area I

[SARANILLO, KA.]
 MATERIALS
IMMUNOLOGY AND SEROLOGY 311 o Blue Cross HBsAg Test Pack o Timer
TESTS FOR HEPATITIS B SURFACE ANTIGEN (HbsAg)
FINALS: LABORATORY o Serum sample o Sample dropper
INTENDED LEARNING OUTCOMES
o Perform and interpret HbsAg test correctly. PROCEDURE
o Explain Hepatitis B with respect to its characteristics, etiology 1. Remove the testing device from the foil pouch by tearing at the
and serologic tests. “Notch” and place on a level surface.
2. Check the expiration date. Do not use expired kits.
o Perform and interpret Dengue test correctly.
3. Holding a sample dropper vertically, add exactly 6 drops
o Explain Dengue with respect to its characteristics, etiology and
(approximately 0.2 ml) of the serum to the sample well.
serologic tests Perform and interpret HIV test correctly.
4. Read the results after 10 minutes. Do not read results after 30
o Explain HIV with respect to its characteristics, etiology and
minutes
serologic tests.
o Correctly execute and interpret Enzyme Linked Immunosorbent
RESULTS
Assay to detect hepatitis b surface antibodies
o HBsAg REACTIVE: Two purple bands appear: One in the test
region (T) and one in the Control region I. A positive result
PRE-ANALYTICAL PHASE
indicates the presence of HbsAg.
o The work area of the experiment should be sterilized. The
o HbsAg NON-REACTIVE: One purple band appears in the control
Students should wear proper personal protective equipment
region I with no band in the test region (T). A negative result
before the experiment (laboratory gown, eye protector,
indicates HbsAg is not present in the sample.
laboratory mask, gloves and hair net). Standard precaution is
o INVALID: A total absence of color in two regions is an indication
applied to every phase of experiment. Students to perform the
of procedure error and/or that test reagent deterioration has
said experiment are required to have Anti-Hepatitis B vaccine.
occurred. Add additional drops of samples to the sample well. If
there is still no color band appears, the test should be voided.
LABORATORY DISCUSSION GUIDE
o PERFORMANCE CHARACTERISTIC: A correlation study
TEST FOR HEPATITIS B SURFACE ANTIGEN (HBSAG)
comparing Blue Cross One Step HbsAg test had established that
o Hepatitis B virus is a member of hepadnavirus family. It is 42
relative sensitivity of the test is 99.27% and that its relative
nmeveloped virion, with an icosahedral nucleocapsid core
pecificity is 98.3%
containing a partially double stranded circular DNA genome. The
envelope contains a protein called the surface antigen (HBsAg),
which is important to the laboratory diagnosis and
immunization. HbsAg is the earliest serological marker that
indicate the presence of acute infection. It is also indicative of
chronic infection.
o The Hepatitis B virus is also known as serum hepatitis. The virus
is transmitted via blood transfusion, the sharing of needles
among the drug users, and via sexual intercourse.
o The incubation period is from 60 to 90 days. The virus is found in
blood and body secretions. It is carried via infected blood, saliva,
and semen. It can enter to the body with small breaks in the skin
or can be transmitted during sexual intercourse if there is a break
in the mucous membrane.
o The first generation tests were found on Ouchterlony agar gel
plates. The antibody for the HBsAg was put in the plate in one
well. Patient’s serum was placed in the wells surrounding the
antibody. A positive result showed a fine white precipitin line
formed between the antigen and the antibody. The test was said
to be not very sensitive.
o The second-generation test used for HbsAg was counter
electrophoresis. The antibody is placed on wells of an agar plate
across from wells containing patient serum. The plates were
subjected to electric current. If the antigen is on the serum, a fine
white precipitin line would form.
o Third-generation tests include radioimmunoassay (RIA) and
enzymelinked immunoassay (EIA or ELISA). Other test
procedures available were reverse passive hemaglutination and
reverse passive agglutination tests.

BLUE CROSS R ONE STEP HBSAG TEST

o The immunochromatographic device contains a unique set of
dyeconjugated and immobilized monoclonal and polyclonal
antibodies used to produce a distinctive visual pattern indicating
presence of HBsAg in the sample.

[SARANILLO, KA.]
 DENGUE NS-1 Antigen
IMMUNOLOGY AND SEROLOGY 311 1. Remove the test device from the foil pouch, and place it on a flat,
TESTS FOR DENGUE IGM/IGG AND NS-1 ANTIGEN
FINALS: LABORATORY dry surface
DENGUE VIRUS 2. With a disposable dropper, add 3 drops (about 100ul) of
o Dengue viruses transmitted by the mosquitoes, Aedes aegypti specimen into the sample well (S).
and Aedes albopictus are widely distributed throughout the 3. As the test begins to work, you will see purple color move across
tropical and subtropical areas of the world. There are four known the result window in the center of the test device.
distinct serotypes (dengue virus 1, 2, 3, and 4). In children, 4. Interpret the test results after 15-20 minutes.
infection is often subclinical or causes a self-limited febrile 5. A positive result will not change once it has been established at
disease. However, if the patient is infected a second time with a 15-20 minutes. However, in order to prevent incorrect results,
different serotype, a more severe disease, dengue hemorrhagic the test should not be interpreted after 20 minutes.
fever or dengue shock syndrome is more likely to occur. Dengue
is considered to be the most important arthropod borne viral RESULTS (DENGUE IgM/IgG)
disease due to the human morbidity and mortality it causes. 1. Negative- The control line is only visible on the test device. No
o Dengue NS-1 Antigen is a glycoprotein expressed by all dengue IgG and IgM antibodies were detected. Retest in 3-5 days if
viruses that is found from the first day and up to 9 days after the dengue infection is suspected.
onset of fever in the sample of primary and secondary dengue 2. IgM Positive- The Control Line and IgM Line are visible on the
infected patients. test device. This is positive for IgM antibodies to Dengue virus.
o Sd Bioline Dengue IgG/IgM Rapid Test is a solid phase This is indicative of primary dengue infection.
immunochromatographic assay for the rapid, qualitative and 3. IgG Positive- The control line and IgG Line are visible on the test
differential detection of IgG and IgM antibodies to dengue virus device. This is positive for IgG antibodies. This is indicative of
in human serum or plasma. SD Bioline Dengue IgG/IgM Test secondary or previous dengue infection.
device has 3 pre-coated lines, ‘G” (Dengue IgG Test Line), “M” 4. IgG and IgM Positive- The control, IgG, IgM lines are visible on
(Dengue IgM Test line and “C” (Control Line) on the surface of the test device. This is positive for both IgM and IgG antibodies.
the membrane. All three lines in result window are not visible This is indicative of a late primary or early secondary dengue
before applying any samples. The Control Line is used for infection.
procedural control. Control line should always appear if the test 5. Invalid- The control line fails to appear. Insufficient specimen
procedure is performed properly and the test reagents of control volume or incorrect procedural techniques are the likeliest
line are working. When a specimen is added to the sample well, reasons for control line failure. Repeat the test using a new test
anti-Dengue IgGs and IgMs in the specimen will react C T S P O S device
ITIVE C T S N E G A T IV E C T S IN V A L ID with Recombinant
Dengue virus envelope proteins-colloidal gold conjugates and
forms a complex of antibodies-antigen. As this complex migrate
along the length of the test device by capillary action, it will be
captured by the relevant anti-human IgG and or antihuman IgM
immobilized in two test lines across the test device and generate
a colored line.
o The Dengue NS-1 Antigen is an in-vitro
immunochromatographic, onestep assay designed for the
qualitative determination of dengue NS-1 antigen in human
serum, plasma or whole blood for the diagnosis of early acute
dengue infection. This test device contains membrane strip,
which is precoated with anti-dengue NS-1 Ag capture on the test
region. The anti-dengueNS-1 Ag colloid gold conjugate and
serum, plasma or whole blood sample move along the DENGUE NS-1 Antigen
membrane chromatographically to the test region (T) and forms 1. Negative: The presence of a purple line within the Control area
a visible line as the antibody-antigen-antibody gold particle and the absence of purple line within the Test area.
complex forms. 2. Positive: The presence of purple lines each at the Control and
the Test area.
MATERIALS 3. Invalid: The absence of purple lines on both the Control and the
o SD Bioline Dengue IgG/IgM o 5 ul capillary pipette / Test area.
Test Device automatic pipettor
o Assay diluent: 100 mM o Package insert
Phosphate buffer (5 ml), o Timer
Sodium Azide (0.01% w/w)

PROCEDURE
1. Allow all kit components and specimen to room temperature
prior to testing.
2. Remove the test device from foil pouch, place it on a flat, dry
surface.
3. Add 5 ul of serum or plasma specimen into the square-shaped
sample well marked “S”.
4. Add 3-4 drops (about 90-120 ul) of assay diluent to the assay
diluent well (round shaped).
5. Interpret test results in 15-20 minutes.

Caution: Do not read test results after 20 minutes.

Reading too late can give false results.

[SARANILLO, KA.]