LABORATORY SAFETY AND QUALITY ASSURANCE STANDARD PRECAUTIONS

PROPER HANDWASHING
1. Wet your hands with clean, running water (warm or cold), turn
Safety Standards and Agencies off the tap, and apply soap.
OSHA Occupational Safety and Health Administration 2. Lather your hands by rubbing them together with the soap.
Lather the backs of your hands between your fingers, and under
CLSI Clinical and Laboratory Standards Institute your nails.
CDC Centers for Disease Control and Prevention 3. Scrub your hands for at least 20 seconds. Need a timer? Hum
the “Happy Birthday” song from beginning to end twice.
JCAHO Joint Commission on Accreditation of Healthcare Organization 4. Rinse your hands well under clean, running water.
DOH Department of Health 5. Dry your hands using a clean towel or air dry them.
DOLE Department of Labor and Employment

General Safety Practices:

1. Staff must wear laboratory coats and be additionally
protected from contamination by infectious agents.

2. Food and drinks should not be consumed in work area or

stored in the same area as specimens. Container, refrigerators
or freezers used for specimen should be marked as containing
a biohazard.

3. Specimens needing centrifugation are capped and place into

a centrifuge with a sealed dome.

4. A gauze square is used when opening rubber-stopped test

tubes to minimize aerosols (introduction of substances into the
air).

5. Auto-dilutors or safety bulbs are used for pipetting. Pipetting

of any clinical material by mouth is strictly forbidden. Biosafety - is the application application of safety precautions
precautions that reduce a laboratorian's risk of exposure to a
UNIVERSAL PRECAUTIONS potentially infectious microbe and limit contamination of the
Instituted by the CDC in 1985 to protect health-care workers from
the exposure to bloodborne pathogens, primarily HBV and HIV.
work environment and, ultimately, the community.
GUIDELINE:
1. Wearing gloves when collecting or handling blood and body Types of Safety Hazards
fluids contaminated with blood. Type Source Possible Injury
2. Wearing face shields when there is danger of blood splashing Biologic Infectious agents Bacterial, fungal, viral
on mucous membranes. or parasitic infections
3. Disposing of all needles and sharp objects in puncture Sharps Needles, lancets, broken glass Cuts, punctures or
resistant containers without recapping. blood-borne pathogen
exposure
Chemical Preservatives and reagents Exposure to toxic,
carcinogenic or
caustic agents
BODY SUBSTANCE ISOLATION
Radioactive Equipment and radioisotopes Radiation exposure
A guideline stating that all moist body substances are capable of
Electrical Undergrounded or wet Burns or shock
transmitting disease.
equipment; frayed cords
GUIDELINE:
Fire/Explosive Open flames, organic chemicals Burns or
1. Precaution is now not limited to bloodborne pathogens and
dismemberment
considers all body fluid and moist body substances to be
Physical Wet floors, heavy boxes, patients Falls, sprains or
potentially infectious.
strains
2. Recommends handwashing only after removing gloves if visual
contamination is present.

STANDARD PRECAUTIONS
In 1996, the CDC expanded the concept and changed the term to
standard precautions, which integrated and expanded the
elements of universal precautions to include contact with all
body fluids (except sweat), regardless of whether blood is
present.
GUIDELINE:
1. Hand hygiene.
2. Use of personal protective equipment (e.g., gloves, masks,
eyewear).
3. Respiratory hygiene / cough etiquette.
4. Sharps safety (engineering and work practice controls).
5. Safe injection practices (i.e., aseptic technique for parenteral
medications).
BIOHAZARD
6. Sterile instruments and devices. Infectious agents or hazardous biologic materials
7. Clean and disinfected environmental surfaces. that present a risk or potential risk to the health of
humans, animals, or the environment.
 BIOLOGICAL WASTE DISPOSAL
1. Incineration
2. Autoclaving
3. Pickup by a certified hazardous waste company.

NOTE:
Urine may be discarded by pouring it into a laboratory sink.
Disinfection of the sink using a 1:5 or 1:10 dilution of sodium
hypochlorite should be performed daily

SHARPS HAZARD
All sharp objects must be disposed in punctureresistant, leak-
proof container with the biohazard symbol.

CHEMICAL HAZARD
When skin contact occurs, the best first aid is to flush the area
with large amounts of water for at least 15 minutes, then seek
medical attention.
MEANS OF TRANSMISSION
1. Direct contact: the unprotected host touches the patient, CHEMICAL HANDLING
specimen, or a contaminated object (reservoir)
Acid should always be added to water to avoid the possibility
2. Airborne: inhalation of dried aerosol particles circulating on of sudden splashing caused by the rapid generation of heat
air currents or attached to dust particles in some chemical reactions.
Wearing goggles and preparing reagents under a fume hood
3. Droplet: the host inhales material from the reservoir (e.g., are recommended safety precautions.
aerosol droplets from a patient or an uncapped centrifuge tube,
or when specimens are aliquoted or spilled)

4. Vehicle: ingestion of a contaminated substance (e.g., food, RADIOACTIVE HAZARD

water, specimen) Radioactivity may be encountered in the clinical laboratory
when procedures using radioisotopes are performed.
5. Vector: from an animal or insect bite

MSDS (Material Safety Data Sheet)

BLOOD BORNE PATHOGENS:
• HIV 1. Physical and chemical characteristics
• HBV
• HCV 2. Fire and explosion potential
• Syphillis
• Malaria 3. Reactivity potential
• Ebola
4. Health hazards and emergency first aid procedures
Transmission-Based Precautions Classifications
Type Possible Conditions PPE 5. Methods for safe handling and disposal
Airborne Tuberculosis, measles, chickenpox, Standard Precautions
herpes zoster/shingles, mumps, Mask or respirator
adenovirus 6. Primary routes of entry
Droplet Infection with Neisseria meningitides, Standard Precautions
Heamophilus sp, pertussis/whooping Mask
cough, Group A stretococcus,
7. Exposure limits and carcinogenic potential
influenza, rhinovirus, scarlet rever,
parvovirus B19, respiratory syncytial
virus and diphtheria
Contact Clostridium difficile, rotavirus, Standard Precautions
draining, wounds, antibiotic-resistant Gown and gloves
infectons, scabies, impetigo, herpes
simplex, respiraotry syncytial virus
and herpes zoster
ELECTRICAL HAZARD PHYSICAL HAZARD
When an accident involving electrical shock occurs, the Physical hazards are not unique to the laboratory,
electrical source must be removed immediately. and routine precautions observed outside the
workplace apply
This must be done without touching the person or the
equipment involved to avoid transferring the current.

Turning off the circuit breaker, unplugging the equipment, or SCREENING TEST FOR PHAGOCYTIC ENGULFMENT
moving the equipment using a nonconductive glass or wood
object are safe procedures to follow. PRE-ANALYTICAL PHASE:
The site of the experiment should be sterilized. The medical
technologist should wear proper personal protective
equipment before the experiment (laboratory gown, laboratory
FIRE/ EXPLOSIVE HAZARD
goggles or face-shield, laboratory mask, and gloves)
Flammable chemicals should be stored in safety cabinets and
explosion-proof refrigerators, and cylinders of compressed No special preparation of the patient is required before
gas should be located away from heat and securely fastened to specimen collection. Blood should be drawn by an aseptic
a stationary device to prevent accidental capsizing. technique. A minimum of 2 ml of heparin blood (green-top
In case of Fire (RACE)
evacuated tube) or 15 to 20 heparinized capillary tubes are
Rescue Rescue anyone in immediate danger required. The specimen should be centrifuged, and the test
Alarm Activate the institutional fire alarm system should be performed promptly. For the quality control, a fresh,
Contain Close all doors to potentially affected areas heparinized sample of blood from a healthy volunteer should
Extinguish/Evacuate attempt to Extinguish the fire, if possible or Evacuate,
closing the door
be tested simultaneously.

The microscope should be placed on a plain, stable surface. The

In case of Fire: How to use fire extinguisher (PASS) voltage of the power supply should be inclined with that of the
Pull Pull pin equipment. Proper handling and cleanliness of compound
Aims Aim at the base of the fire microscope should be observed at all times by the medical
Squeeze Squeeze handles
Sweep Sweep nozzle side to side
technologist. Lens paper is provided in order to clean the
objective and ocular lenses.

NFPA LABELING SYSTEM: ANALYTICAL PHASE:

Materials
o Broth culture of Bacillus subtilis or Staphylococcus
epidermidis
o Microscope slides
o Pasteur pipettes
o Rubber bulb
o Test tubes
o Wright’s stain

Procedure
1. Label three test tubes :
Patient test tube and Control test tube

2. Add 4 to 8 drops of the buffer coat from either the patient’s

heparinized blood or from the normal controlto the respectively
labeled tubes.

3. Add 2 to 3 drops of the bacterial broth culture to each tube.

Types of Fires and Fire Extinguishers
Fire Extinguish Type/Com Extinguisher 4. Incubate both tubes at room temperature or 37 C for 30
Type Material position of
Fire minutes.
Class Wood, paper, Class A Water
A clothing 5. Place 1 drop of the incubated specimen on a glass slide and
Class Flammable Class B Dry chemicals, carbon dioxide, foam prepare a smear.
B organic or halon
chemicals
Class Electrical Class C Dry chemicals, carbon dioxide or 6. Air dry the slides and stain with Wright’s stain.
C halon
Class Combustible None Sand or dry powder
D metals Class ABC Dry chemicals
Class Grease, oils, Class K Liquid designed to prevent splashing Wright’s Stain Procedure:
K fats and cool the fire
A. Cover each smear generously with filtered Wright’s stain and
allow the stain to remain on the slide for at least 5 minutes.
B. Slowly add distilled water or buffer to the stain until the C-REACTIVE PROTEIN DETERMINATION
buffer begins to overflow the stain. Watch for the appearance
of a metallic luster. PRE-ANALYTICAL PHASE:
The work area of the experiment should be sterilized. The
C. Gently blow on the slide to mix the stain and buffer. Students should wear proper personal protective equipment
before the experiment (laboratory gown, eye protector,
D. Allow the buffer to remain on the slide for at least 5 minutes.
laboratory mask, gloves and hair net). Standard precaution is
E. Gently wash the stain and buffer off the slide with distilled applied to every phase of experiment.
water.
LABORATORY DISCUSSION GUIDE:
F. Air dry or carefully blot the slide between two sheets of
I. Principle of the test
bibulous paper.
- Reverse Passive Agglutination. It is based on the latex-
7. Place a drop of immersion on each smear and examine
agglutination method. The principle of this test is based on the
microscopically with the oil (100x) immersion objective.
immunological reaction between CRP as an antigen and the
corresponding antibody coated on the surface of biologically
inert latex particles.
POST-ANALYTICAL PHASE:
II. Reagents of the test
All infectious materials should be disposed appropriately. The
working area should be cleansed with the disinfectant before
leaving. The PPE of each individual should be removed properly.
These cannot be exposed outside the laboratory premises.

Interpretation of Results:
POSITIVE - Demonstration of the engulfment of bacteria.
NEGATIVE - No engulfment of bacteria

Introduction:
Phagocytosis is the process by which specialized cells engulf
and destroy foreign particles such as microorganisms or
damaged cells. Macrophages and neutrophils (PMNs) are the III. Results
most important phagocytic cells. - Positive Result: Agglutination
- Negative Result: Smooth milky suspension
A mixture of bacteria and phagocytes is incubated and - Since negative results may be caused by antigen excess, the
examined for the presence of engulfed bacteria. test should be repeated using a diluted serum sample in case
prozone effect is suspected.
This simple procedure may be useful in supporting the
diagnosis of impaired neutrophilic function in conjunction with IV. Clinical Significance
clinical signs and symptoms. C-reactive protein (CRP) is a trace constituent of serum
originally thought to be an antibody to the Cpolysaccharide of
Sources of Error pneumococci. It was discovered by Tillet and Francis in 1930
This procedure may produce false-negative results if the blood when they observed that serum from patients with
specimen is not fresh or if a coagulase positive Staphylococcus Streptococcus pneumoniae infection precipitated with a soluble
specimen is used. It is important to distinguish between extract of the bacteria. Now CRP is known to have a more
granules and cocci. In Addition, the bacteria must be generalized role in innate immunity. CRP can be thought of as
intracellular and not extracellular for the test to be positive. a primitive, nonspecific form of an antibody molecule that is
able to act as a defense against microorganisms or foreign
cells until specific antibodies can be produced.
Clinical Applications
CRP is a relatively stable serum protein with a half-life of about
The failure of phagocytes to engulf bacteria can support the
18 hours. 1 It increases rapidly within 4 to 6 hours following
diagnosis of neutrophilic dysfunction; however, these results
infection, surgery, or other trauma to the body. Levels increase
must be used in conjunction with patient signs and symptoms.
dramatically as much as a hundredfold to a thousandfold,
reaching a peak value within 48 hours. Elevated levels are
Limitations of this Procedure
found in conditions such as bacterial infections, rheumatic
This is the simple screening procedure for engulfment. The
fever, viral infections, malignant diseases, tuberculosis, and
presence of engulf bacteria does not demonstrate that the
after a heart attack. The median CRP value for an individual
bacteria have been destroyed.
increases with age, reflecting an increase in subclinical
inflammatory conditions.
Because the levels rise and then decline so rapidly, CRP is the Method II (Semi-Quantitative)
most widely used indicator of acute inflammation. Although
CRP is a nonspecific indicator of disease or trauma, monitoring 1. For each test serum to be titrated, set up a least 6 test tubes
of its levels can be useful clinically to follow a disease process (12 x 75 mm) and label 1:2, 1:4, 1:8,
and observe the response to treatment of inflammation and 1:16, 1:32, 1:64, etc.
infection.
2. To each tube add 0.2 ml of Diluted Glycine-Saline Buffer.
It is a nonsurgical means of following the course of malignancy
and organ transplantation because a rise in the level may mean 3. To Tube No. 1 add 0.2 ml of undiluted test serum.
a return of the malignancy or, in the case of transplantation, the
beginning of organ rejection. CRP levels can also be used to 4. Serially make two-fold dilutions by mixing contents of Tube
monitor the progression or remission of autoimmune diseases. No. 1 with pipette and transferring 0.2 ml to Tube No. 2. Repeat
Assays for CRP are sensitive, reproducible, and relatively serial transfers for each tube. For the 6 tubes, the dilutions
inexpensive. CRP is easily destroyed by heating serum to 56°C range from 1:2 to 1:64. If required, additional serum dilutions can
for 30 minutes. The destruction of CRP is often necessary in the be added.
laboratory because it interferes with some testing for the
5. Repeat steps 3 to 7 as given in Method I (Qualitative).
presence of antibodies.
PRECAUTIONS
The Centers for Disease Control and Prevention (CDC) has
This product is for In Vitro Diagnostic Use Only. Each donor unit
recommended that a CRP concentration of less than 1 mg/L is
used in the preparation of this product has been tested by an
associated with a low risk for cardiovascular disease; 1 to 3
FDA approved method and found non-reactive for the presence
mg/L is associated with an average risk; and greater than 3
of HbsAg and antibody to HIV Virus. Because no known test
mg/L is associated with a high risk. Normal levels in adults
method can offer complete assurance that hepatitis B virus, HIV
range from approximately 0.47 to 1.34 mg/L. A mean for people
Virus, or other infectious agents are absent, all human blood
with no coronary artery disease is 0.87 mg/L. Thus, monitoring
based products should be handled in accordance with good
CRP may be an important preventative measure in determining
laboratory practices. The preservative sodium azide may react
the potential risk of heart attack or stroke. High-sensitivity CRP
with metalplumbing to form explosive metal oxides. In disposal,
testing has the necessary lower level of detection of 0.01mg/L,
flush with a large volume of water to prevent metal azide build
which enables measurement of much smaller increases than
up.
the traditional latex agglutination screening test.
STORAGE & STABILITY
V. And other important information in the test insert
When not in use, store reagents and controls at 2 - 8 degree
Method I (Qualitative)
Celsius. DO NOT FREEZE. Prior to use, allow reagents and
1. Bring all test reagents and serum specimens to room
controls to warm up to room temperature. Expiration date is
temperature.
specified on the kit label and on each vial. Biological indication
2. Gently shake the CRP latex vial to disperse and suspend latex of product instability is evidenced by inappropriate reaction of
particles. the latex reagent with the corresponding positive and negative
control sera.
3. Positive and negative controls should be tested with each
series of test.

4. Using the disposable pipette provided, place one drop of test

serum onto a circle on the slide. Use a separate disposable
pipette for each test serum. Important: The Cortez CRP Latex
Reagent must be agitated well for about 10 seconds prior to
using on each day’s testing. Do not use a vortex mixer.

Deliver one drop of CRP Latex to each circle that contains

specimens on the slide. Spread the resulting mixture by using
the paddle end of the pipette. Do not use the same paddle end
to mix each test serum or control as this will cause cross-
contamination.

5. Gently tilt and rotate slide by hand for two (2) minutes.

6. Observe for macroscopic clumping using the indirect oblique

light source.

7. Compare the reaction of the test serum to the CRP positive

and negative control sera.
IMMUNOLOGY & SEROLOGY 311 Laboratory
Dilutions (Exp. 4: Serial Dilution to Detect Cold Reacting Antibodies) Midterms

PRE-ANALYTICAL PHASE

● The site of the experiment should be sterilized. The medical

technologist should wear proper personal protective
equipment before the experiment (laboratory gown,
laboratory goggles or face shield, laboratory mask, and
gloves)
● 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.
Interpretation of Results:
ANALYTICAL PHASE ● The last tube showing agglutination is the endpoint of the
test.
● The titer is reported out as the reciprocal of the last dilution
Materials:
showing a positive result.
● Antibody A (anti-A sera)
● 3% RBC suspension
● 0.85-0.90% saline (NSS) Calculating Dilution
● Ten 12x75 test tubes
● Refrigerator Formula:
● Test tube rack 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 =
● Waterproof marker 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

● Three serological pipettes

● Total volume = amount of solute + diluent
Procedure:
1. Label 10 test tubes 1-10 Serial Dilution
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.
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 from tube #2 to tube #3,
then #4, then tube #5, etc. discard 0.25 mL from tube #5.
6. Use clean serological pipette to add 0.25 mL of 3% RBC
suspension to each tube.
7. Mix well and show your instructor your tubes.
8. Place in refrigerator for 30 minutes.
9. Remove from refrigerator, centrifuge for 20 seconds. Read
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
red blood cell suspension is negative.

Maekaella R. Roca – OLFU Laguna

 Types of Dilution Types of Dilution

● “For many serology tests, it is the serum that is 1. How much diluent needs to be added to 0.2 mL of serum to
concentrated; it may be necessary to dilute it with saline in make a 1:20 dilution
order for a visible reaction to occur.”

Simple Dilution:

2. What is the final dilution of serum obtained from the

following serial dilutions: 1:4, 1:4, 1:4, 1:4, 1:4, 1:4?
Example:
● 2 mL solution of a 1:20 dilution is needed to run a specific
serological test. How much serum and how much diluent
are needed to make this dilution?

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒

● A 1:5 dilution of patient serum is necessary to run a

serological test. There is 0.1 mL of serum that can be
used. What amount of diluent is necessary to make this
dilution using all of the serum?

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 1 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 − 1
= 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡

Compound Dilution:

● For 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
IMMUNOLOGY & SEROLOGY 311 Laboratory
Slide Agglutination Test Midterms

antigen-antibody reaction occurs. This is called direct blood

typing.
PRE-ANALYTICAL PHASE
● Agglutination, like precipitation, is a two-step process that
results in the formation of a stable lattice network. The first
● The work area of the experiment should be sterilized. The reaction, called sensitization, involves antigen–antibody
Students should wear proper personal protective equipment combination through single antigenic determinants on the
before the experiment (laboratory gown, eye protector, particle and is rapid and reversible. The second step, or
laboratory mask, gloves and hair net). Standard precaution is lattice formation, is the formation of cross-links that form
applied to every phase of experiment. the visible aggregates. This represents the stabilization of
antigen–antibody complexes with the binding together of
Materials: multiple antigenic determinants.
● Antibody A ● Sensitization is affected by the nature of the antigens on the
● Red blood cells (#1 and #2) agglutinating particles. If epitopes are sparse or if they are
● Slides obscured by other surface molecules, they are less likely to
● Antibody B interact with antibody. Additionally, red blood cells (RBCs)
● Pipettes and bacterial cells have a slight negative surface charge;
● Applicator sticks because like charges tend to repel one another, it is
sometimes difficult to bring such cells together into a lattice
LABORATORY DISCUSSION GUIDE formation.
● IgM antibodies are strong agglutinins because of their larger
size. Achieving visible reactions with IgG often requires the
I. Principle of the test use of enhancement techniques that vary physicochemical
conditions such as the ionic strength of the solution, the pH,
and the temperature. Antibodies belonging to the IgG class
● Hemagglutination reaction
agglutinate best at 30°C to 37°C, whereas IgM antibodies
react best at temperatures between 4°C and 27°C. Because
II. Reagents of the test naturally occurring antibodies against the ABO blood groups
belong to the IgM class, these reactions are best run at room
● Anti-A and Anti-B antisera temperature. Antibodies to other human blood groups
● Anti-D usually belong to the IgG class; reactions involving these
must be run at 37°C. These latter reactions are the most
important to consider in selecting compatible blood for a
III. Results transfusion because these are the ones that will actually
occur in the body.
● Agglutination (clumping) of the red blood cells is positive. ● If an agglutination reaction involves RBCs, then it is called
No agglutination is negative. It is critical to read the results hemagglutination. The best example of this occurs in ABO
immediately as false positives can occur when the mixture blood group typing of human RBCs, one of the world’s most
begins to dry on the side. frequently used immunoassays. Patient RBCs mixed with
antisera of the IgM type can be used to determine the
presence or absence of the A and B antigens; this reaction is
Anti-A Anti-B Blood Group
usually performed at room temperature without the need for
+ 0 A
any enhancement techniques. Group A RBCs will agglutinate
with anti-A antibody and Group B RBCs will agglutinate with
0 + B anti-B antibody. This type of agglutination reaction is simple
to perform, is relatively sensitive, and is easy to read.
+ + AB ● A titer that yields semi-quantitative results can be performed
in test tubes or microtiter plates by making serial dilutions of
0 0 O
the antibody. The reciprocal of the last dilution still exhibiting
a visible reaction is the titer, indicating the antibody’s
Legend: + → with agglutination
0 → without agglutination strength. Interpretation of the test is done on the basis of the
cell sedimentation pattern. If there is a dark red, smooth
button at the bottom of the microtiter well, the result is
IV. Clinical Significance negative. A positive result will have cells that are spread
across the well’s bottom, usually in a jagged pattern with an
irregular edge. Test tubes also can be centrifuged and then
● The ABO blood groups (A, B, AB, and O) represent the
shaken to see if the cell button can be evenly re-suspended.
antigens expressed on the erythrocytes (red blood cells,
If it is re-suspended with no visible clumping, then the result
RBCs) of each group. Reagent typing sera contains specific
is negative. Positive reactions can be graded to indicate the
antibodies to A antigen and B antigen. When an unknown
strength of the reaction.
patient’s RBCs are mixed with known antibody A or antibody
B, agglutination of the RBCs will occur if a specific

Maekaella R. Roca – OLFU Laguna

 FORWARD TYPING

Blood Type Anti-A Anti-B Anti-D

A+ + - +

A- + - -

B+ - + +
V. Procedure
B- - + -

1. On the section of slide labeled anti-A, placed one drop of AB+ + + +

antibody A.
2. On the section of slide labeled anti-B, placed one drop of AB- + + -
antibody B.
3. Place one drop of cells in each antibody-containing circle. O+ - - +
4. Carefully mix each solution with a separate applicator stick
5. Tilt slowly for one minute. O- - - -
6. Record results.
● Sample: Antigen
Agglutination Reactions:
● Reagent: Antibodies

REVERSE TYPING

Blood Type A1 cells B cells

A - +

B + -

AB - -

O + +

Blood Type Antigens Antibodies ● Sample: Antibodies

● Reagent: Antigen
A+ A,D B

A- A B,D

B+ B,D A

B- B A,D

AB+ A,B,D —

AB- A,B D

O+ D A,B

O- — A,B,D

● Universal recipient: AB+

● Universal donor: O-
IMMUNOLOGY & SEROLOGY 311 Laboratory
Febrile Antigens Midterms

B. Rapid Slide Titration Method

PRE-ANALYTICAL PHASE
1. Using a pipettor, dispense 0.08ml, 0.04ml, 0.02ml, 0.01
and 0.005ml of undiluted serum into a row of 3 cm
● The work area of the experiment should be sterilized. The diameter circles.
Students should wear proper personal protective equipment 2. Shake the reagent bottle well and add 1 drop of the
before the experiment (laboratory gown, eye protector, undiluted antigen suspension to each serum aliquot.
laboratory mask, gloves and hair net). Standard precaution is 3. Mix well using a stirring stick and rotate the slide for 1
applied to every phase of experiment. minute.
Note: Bring all reagents to room temperature before testing. Shake
antigen suspension well before disposing.
Materials:

Widal Test Weil-Felix The degree of agglutination is recorded as follows:

● 4+ – 100% of the organism is agglutinated
● Antigen suspensions S. typhi O ● Proteus OX2
● 3+ – 75% of the organism is agglutinated
● Antigen suspensions S. typhi H ● Proteus OX19 ● 2+ – 50% of the organism is agglutinated
● Pipettes ● Proteus OXK ● 1+ – 25% of the organism is agglutinated
● Glass slides ● Applicator stick ● 1/+ – Less than 25% of the organism is agglutinated
● NSS ● Test tube ● Negative – No agglutination is observed
● Antigen suspensions S. typhi ‘BH’ ● NSS
● Antigen suspensions S. typhi ‘AH’ ● Transparent glass ring Although the slide test is not recommended to established the titer
● Test tubes antigen vortex mixer slide
● Mixing sticks ● Serum pipettes
of the serum, the quantity of the serum giving 2+ or 50 %
agglutination can be used to approximate the titer of the serum. The
serum dilution in the slide test is approximately equivalent to the
test tube dilution shows below:
LABORATORY DISCUSSION GUIDE

Serum Volume Approximate Tube Dilution

● Typhoid fever, simply typhoid, caused by the bacterium
Salmonella enterica subsp. enterica serovar Typhi growing in 0.08 mL 1:20
the intestines and blood. Typhoid is spread by eating or
0.04 mL 1:40
drinking food or water contaminated with the feces of an
infected person. Diagnosis is made by any blood, bone 0.02 mL 1:80
marrow, or stool cultures and with the Widal test
(demonstration of antibodies against Salmonella antigens 0.01 mL 1:160
O-somatic and H-flagellar). In epidemics and less wealthy
countries, after excluding malaria, dysentery, or pneumonia, 0.005 mL 1:320
a therapeutic trial time with chloramphenicol is generally
undertaken while awaiting the results of the Widal test and
cultures of the blood and stool. C. Tube Agglutination Test

All positive results obtained through the slide test should be

WIDAL TEST confirmed using the following technique.
1. Label 8 small test tubes in a rack.
● Quantitatively measures the antibodies in the serum of 2. Using a pipette, dispense 1.9ml of 0.85% Saline into the
patient with Typhoid fever. first test tube, and 1.0 ml into the remaining seven.
3. Using a pipette, dispense 0.1 ml of the patient’s undiluted
3 Kinds of Antigens: serum into the first test tube. Mix well using the larger
1. O Ag – somatic antigen; heat stable pipette volume and tip.
2. H Ag – flagellar antigen; heat labile 4. Using the pipette, dispense 1.0 ml from the first tube into
3. K Ag – Vi antigen capsule) (virulence) the second tube. Mix well.
– thermo labile, unstable, delicate 5. Continue this method of doubling dilutions up to the
– reference laboratories seventh tube. Discard 1.0 ml from the seventh tube. The
eight tube will contain only saline as a control and
3 Types of Methods in Widal’s therefore should not contain any serum.
6. Shake the reagent bottle well. And add 1 drop of the
appropriate antigen suspension into each tube and mix
A. Rapid Slide Screening Method
well.
7. Incubate at the water bath as follows:
1. Using a pipettor, dispense 1 drop (0.08ml) of undiluted
● Salmonella “O” Antigens and Proteus: 500C for
serum into a row of 3 cm diameter circles.
4 hours
2. Shake the reagent bottle well and add 1 drop of the
● Salmonella “H” Antigens: 500C for 2 hours
undiluted antigen suspension to the serum.
8. Examine the tubes after the appropriate incubation time
3. Mix well using a stirring stick and rotate the slide for
and check for agglutination. The titer to be taken is the
1 minute.
last tube to show agglutination.
4. Observe and interpret result.
5. A positive serum of known titer and a negative serum
should be included as controls.

Maekaella R. Roca – OLFU Laguna

 Results ○ Convalescent/ vaccination against typhoid
fever
● Agglutination is a positive test result and indicates the ● INC titer on HA, HC, HB: indicates past infection on
presence of the corresponding antibody in the patient S.paratyphi
serum. No agglutination is a negative test result and it ● INC titer on O and H Ag: indicates mixed infection
indicates the absence of the corresponding antibody in
the patient serum. NON-SPECIFIC TEST

Rapid Slide Screening Test: Agglutination is indicative of serum Weil-Felix Test

titer of 1:20 in a tube test. ● Used to test for antibodies to rickettsial infections of
Typhus fever caused by: (R.prowazekii)
Rapid Slide Titration Method: If agglutination reactions are ● Proteus ox-2 (from P.vulgaris)
observed, the following approximate titers would be observed if a ● Proteus ox-19 (from P.mirabilis)
confirmatory tube test was carried out: ● Proteus ox-k (uses kingsburry strain)
● Cross reaction mechanism
Serum Volume Titer ○ Cross reactivity between rickettsia and certain
serotypes of proteus spp.
0.08 ml 20 ○ Possess the same antigenic determinant

0.04 ml 40

0.02 ml 80

0.01 ml 160

0.005 ml 320

In this way, the rapid slide test provides an approximation to the

expected results from a corresponding tube test.

NOTE: It is necessary to perform all dilutions in the slide test to

obviate the “prozone” effect where higher concentrations of the
serum may give a negative result but further dilutions may give a
positive result.

Tube Agglutination Test: A positive test will show an obvious

floccular agglutination throughout the tube. A negative result and
the saline control should show no change in appearance and should
show a characteristic swirl when flicked. Tube must not be shaken.

Note:
1. A single negative test does not exclude the absence of
infection.
2. A single positive test is of no diagnostic value unless the
titer is sufficiently high.
3. Agglutinin is not always produced in bacterial infections.
4. Cross- reaction may occur in certain pathologies.
5. The considered significant titer in the Widal test is 1:80
while 1:160 in the Weil-Felix Test

Two Types of Agglutination Test in Widal’s

1. Somatic agglutination
● Compact aggregates that are not easily
dispersed
● Stronger agglutination than H Ag
2. Flagellar agglutination
● Fluffy and floccular aggregates that are easily
dispersed

Interpretation

Significant titer: 1:80 (80)

● < 80 = normal
● > 80 = indicative of infection

Interpretation:
● INC titer on OD Ag: indicates acute/ recent typhoid fever
● INC titer on OA, OB and OC: indicates acute/ recent
paratyphoid infection
● INC titer on HD Ag: indicates previous/ past infection with
typhoid fever
IMMUNOLOGY & SEROLOGY 311 Laboratory
ASO Latex Test Midterms

● Reagents:
○ ASO LATEX REAGENTS
PRE-ANALYTICAL PHASE
■ A suspension of polystyrene latex
particles sensitized with streptococcal
● The work area of the experiment should be sterilized. The exoenzyme.
Students should wear proper personal protective equipment ○ ASO POSITIVE CONTROL
before the experiment (laboratory gown, eye protector, ■ A stabilized human serum containing
laboratory mask, gloves and hair net). Standard precaution is ASO reactive with ASO latex reagent.
applied to every phase of experiment. ○ ASO NEGATIVE CONTROL
■ A stabilized human serum containing
Materials: ASO non-reactive with ASO latex
● ASO latex reagent reagent.
● Pipette ● All reagents contain 0.1% Sodium azide as a preservative.
● Control sera (Positive and Negative Controls)
● Applicator stick Procedure:
● Black test cards 1. Allow each components of the test kit (reagents, controls) to
● Mechanical rotator reach room temperature.
2. Gently shake the latex reagent to disperse the particles.
LABORATORY DISCUSSION GUIDE 3. Place a drop of undiluted serum into a circle of a test slide.
4. Add one drop of ASO Latex reagent next to the drop of
serum.
● In infections caused by B-hemolytic streptococci, 5. Spread the reagent and serum sample over the entire area of
Streptolysin-O is one of the two hemolytic exotoxins librated the test circle using a separate stirrer for each sample.
from the bacteria that stimulates production of ASO 6. Gently tilt the test slide backwards and forwards for two
antibodies in the human serum. The presence and the level minutes.
of these antibodies in serum may reflect the nature and 7. Observe and interpret the results.
severity of infection. The ASO TEST is a stabilized buffered
suspension of polystyrene latex particles that have been NOTE: Positive and Negative Controls should be run at regular intervals
coated with Streptolysin O. When the latex reagent is mixed
with serum containing ASO, agglutination occurs
B. Semi-quantitative Determination

A. ASO Slide Method

● The semi-quantitative test can be performed in the same
way as the qualitative test using dilutions of the serum in
● PRINCIPLE: Passive/ indirect agglutination saline, phosphate buffer saline or glycine saline as follows:
● Sample: Serum
● ASO LATEX REAGENT: Suspension of polystyrene particles
sensitized with Streptolysin O 1st tube 2nd tube 3rd tube 4th tube 5th tube
● Analytical sensitivity:
Dilutions 1/2 1/4 1/8 1/16 1/32
○ 200 IU/mL
○ Positive: if titer is > 200 IU/mL
Sample 100 ul
○ Negative: if titer is < 200 IU/mL serum

Materials and Components: Saline 100 ul 100 ul 100 ul 100 ul 100 ul

(diluent)
ASO latex reagent Contains polystyrene latex particles
coated with Streptolysin O in a stabilized → 100 ul 100 ul 100 ul 100 ul
buffer with less than 0.1% sodium azide → → → →
as preservative
Volume 50 ul 50 ul 50 ul 50 ul 50 ul
ASO positive control Human serum containing more than 200 of sample
IU/mL ASO with less than 0.1% sodium
azide as preservative 200 x 200 x 2 200 x 4 200 x 8 200 x 16 200 x 32
Dilution
factor
ASO negative control Human serum that has been diluted and
stabilized with buffer and contains less IU/ml 400 800 1600 3200 6400
than 0.1% sodium azide as preservative
Disposable pipettes and test slides
NOTE: If titer is 2 multiply to 200 = 400 IU/mL (1st circle)

Normal levels (adults): less than 200 IU/Ml

Maekaella R. Roca – OLFU Laguna

CALCULATION: In the semi-quantitative test, the titer is expressed as
OTHER INFORMATION
the reciprocal of the highest dilution showing macroscopic
agglutination. For example, if agglutination occurs in dilution 3, the titer
is 1600 Sensitivity
● The sensitivity of the latex reagent has been adjusted to yield
agglutination when the level of ASO is greater than 200
C. ASO Tube Test IU/ml, a level determined to be indicative of disease by
epidemiological and clinical studies.
● Diagnosis of the probability for sequelae S. pyogenes ● Positive: if titer is > 200 IU/mL
infection ● Negative: if titer is < 200 IU/mL
● Compound dilution
● Principle: Neutralization/ enzyme inhibition test Results
○ If Ab ( ASO) is present in patient serum , it will bind ● Positive Result: Agglutination
to SO is not free to react with indicator RBC’s, the ● Negative Result: Smooth milky suspension
result appears as no hemolysis. ● Since negative results may be caused by antigen excess, the
○ If Ab is absent , Streptolysin O is free to react with test should be repeated using a diluted serum sample in
RBC’s resulting in hemolysis case prozone effect is suspected.

Interfering substances:
● Heavy bacterial contamination - may cause false positive
agglutination.
● Markedly lipemic sera - should not be tested because of the
possibility of non-specific reactions

Clinical Significance
● Materials:
○ 14 test tubes ● Streptococci are gram-positive cocci that are spherical,
○ Tube 13 and tube 14 (control tubes) ovoid, or lancet-shaped organisms often arranged in pairs or
○ Streptolysin O reagent chains when observed on Gram stain. GAS is one of the
○ Streptolysin O buffer most common and ubiquitous pathogenic bacteria and
○ 5% human type O red cell suspension causes a variety of infections. The M protein is the major
○ Serological pipettes virulence factor of GAS and has a net-negative charge at the
● Procedure: amino-terminal end that helps to inhibit phagocytosis. In
○ Add strep O Ag on all tubes except tube 13 (red addition, the presence of the M protein limits deposition of
cell control) C3 on the bacterial surface, thereby diminishing complement
○ Tube 14 (strep O control tube) activation. The M protein, along with lipoteichoic acid and
○ Shake incubate for 15 min, 37C protein F, help GAS attach to host cells. Immunity to GAS
○ Centrifuge, check for presence of hemolysis appears to be associated with antibodies to the M protein.
● RESULTS: There are more than 100 serotypes of this protein and
○ Positive: absence of hemolysis immunity is serotype-specific. Therefore, infections with one
○ Negative: hemolysis (red supernatant) strain will not provide protection against another strain.
● NOTE: ● Additional virulence factors include various exotoxins that
○ Titer: last tube with no hemolysis (clear may be produced during the course of an infection.
supernatant) Pyrogenic exotoxins A, B, and C are responsible for the rash
○ Tube 13: no hemolysis; positive control seen in scarlet fever and also appear to contribute to
○ Tube 14: with hemolysis; negative control pathogenicity. Additional extracellular substances include
● Significant/ diagnosis titer: 166 todd units the enzymes streptolysin O, deoxyribonuclease B (DNase B),
hyaluronidase, nicotinamide adenine dinucleotide (NAD), and
streptokinase. Antibodies produced against these
substances are useful in the diagnosis of infection and for
the potential development of complications or sequelae
associated with GAS infection.
● S pyogenes can be responsible for infections ranging from
pharyngitis (a throat infection) to life-threatening illnesses
such as necrotizing fasciitis and streptococcal toxic shock
syndrome. The two major sites of infections in humans are
the upper respiratory tract and the skin, with pharyngitis
(“strep throat”) and streptococcal pyoderma (a skin
infection) being the most common clinical manifestations.
The reason GAS receives so much attention is the potential
for the development of two serious sequelae, acute
rheumatic fever and poststreptococcal glomerulonephritis.
● Diagnosis of acute streptococcal infections typically is made
by culture of the organism from the infected site. The
 specimen is plated on sheep blood agar and incubated. If the classic hemolytic method described previously. When
Group A streptococcus is present, small translucent colonies using the nephelometric method, individual laboratories
surrounded by a clear zone of β hemolysis will be visible. As must establish their own upper limits of normal for
an alternative to culture, rapid assays have been populations of different ages. ASO titers typically increase
commercially developed to detect Group A streptococcal within 1 to 2 weeks after infection and peak between 3 to 6
antigens directly from throat swabs. The Group A antigens weeks following the initial symptoms (e.g., sore throat).
are extracted by either enzymatic or chemical means and the However, an antibody response occurs in only about 85% of
process takes anywhere from 2 to 30 minutes, depending on acute rheumatic fever patients within this period.
the particular technique. Additionally, ASO titers usually do not increase in individuals
● Culture or rapid screening methods are extremely useful for with skin infections.
diagnosis of acute pharyngitis. However, serological
diagnosis must be used to identify acute rheumatic fever
OTHER TESTS
and poststreptococcal glomerulonephritis because the
organism is unlikely to be present in the pharynx or on the
skin at the time symptoms appear. The antibody response to Streptozyme Test
these streptococcal products is variable because of several ● Multi-enzyme test to detect Antibody to the multiple
factors, such as age of onset, site of infection, and exoenzymes of S. pyogenes
timeliness of antibiotic treatment. The most diagnostically ● PRINCIPLE: Passive Hemagglutination
important antibodies are the following: anti-streptolysin O ● REAGENT: Formalinized/ aldehyde fixed sheep RBCs coated
(ASO), anti-DNase B, anti-NADase, and anti-hyaluronidase with exoenzymes of S. pyogenes
(AHase). Assays for detection of these antibodies can be Exoenzymes
performed individually or through use of the streptozyme ● Streptolysin
test which detects antibodies to all these products. ● Streptokinase
● Let us focus on ASO testing. ASO tests detect antibodies to ● Hyaluronidase
the streptolysin O enzyme produced by Group A ● DNAse B
streptococcus. This enzyme is able to lyse RBCs. Presence ● NADase
of antibodies to streptolysin O indicates recent streptococcal
infection in patients suspected of having acute rheumatic Quality control procedure:
fever or poststreptococcal glomerulonephritis following a ● Positive and Negative controls should be included in each
throat infection. The classic hemolytic method for test series. The Positive control should produce visible
determining the ASO titer was the first test developed to agglutination; and Negative control should produce no
measure streptococcal antibodies. This test was based on agglutination.
the ability of antibodies in the patient’s serum to neutralize
the hemolytic activity of streptolysin O. The traditional ASO Limitations of procedure:
titer involved preparing dilutions of patient serum to which a 1. The results of this test SHOULD NOT be used as a single
measured amount of streptolysin O reagent was added. diagnostic tool to make a clinical diagnosis. Instead, the test
These were allowed to combine during an incubation period results must be evaluated together with other clinical
after which reagent RBCs were added as an indicator. If findings and observed symptoms to aid in the final
enough antibodies were present, the streptolysin O was diagnosis.
neutralized and no hemolysis occurred. The titer was 2. This test is designed to be performed by hand rotation. The
reported as the reciprocal of the highest dilution use of a mechanical rotator could yield false
demonstrating no hemolysis. This titer could be expressed in positive/negative results.
either Todd units (when the streptolysin reagent standard is 3. Serum samples showing gross hemolysis, lipemia, turbidity,
used) or in international units (when the World Health or contamination should not be used since false positive
Organization international standard is used). results may occur. Both elevated Beta-lipoprotein and
● The range of expected normal values varies, depending on cholesterol level may suppress a rise in ASO titer.
the patient’s age, geographic location, and season of the 4. The test reaction must be read immediately following the
year. ASO titers tend to be highest in school-age children and two (2) minutes rotation. Delayed readings may result in
young adults. Thus, the upper limits of normal must be false positive results.
established for specific populations. Typically, however, a 5. When not in use, store reagents and controls at 2 - 8 degrees
single ASO titer is considered to be moderately elevated if Celsius. DO NOT FREEZE. Prior to use, allow reagents and
the titer is at least 240 Todd units in an adult and 320 Todd controls to warm up to room temperature. Expiration date is
units in a child. specified on the kit label and on each vial. Biological
● Because of the labor-intensive nature of the traditional ASO indication of product instability is evidenced by inappropriate
titer test and because the streptolysin O reagent and the reaction of the latex reagent with the corresponding positive
RBCs used are not stable, ASO testing is currently performed and negative control sera.
by nephelometric methods. Nephelometry has the advantage
of being an automated procedure that provides rapid,
quantitative measurement of ASO titers.
● 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
IMMUNOLOGY & SEROLOGY 311 Laboratory
RF Latex Test Study Finals

3. Place a drop of undiluted serum into a circle of a test

PRE-ANALYTICAL PHASE slide.
4. Add one drop of Rf Latex reagent next to the drop of
serum.
★ The work area of the experiment should be sterilized. The 5. Spread the reagent and serum sample over the entire area
Students should wear proper personal protective equipment of the test circle using a separate stirrer for each sample.
before the experiment (laboratory gown, eye protector, 6. Gently tilt the test slide backwards and forwards for two
laboratory mask, gloves and hair net). Standard precaution is minutes.
applied to every phase of experiment 7. Observe and interpret the results.

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

intervals
RF Latex suspension Positive and Negative Controls
Black test cards Disposable stirrers Semi-Quantitative Determination
Mechanical rotator ★ The semi-quantitative test can be performed in the same
way as the qualitative test using dilutions of the serum in
saline, phosphate buffer saline or glycine saline as
LABORATORY DISCUSSION GUIDE follows:

Dilutions 1/2 1/4 1/8 1/16 1/32

I. Principle of the Test
Sample 100 uL
serum
★ The RF reagent is a suspension of polystyrene latex particles
sensitized with specially prepared human IgG. The reagent is Saline 100 uL 100 uL 100 uL 100 uL 100 uL
based on an immunological reaction between human IgG
bound to biologically inert latex particles and rheumatoid → 100 uL 100 uL 100 uL 100 uL
→ → → →
factors in the test specimen. When serum containing
rheumatoid factors is mixed with the latex reagent, visible Volume 50 uL 50 uL 50 uL 50 uL 50 uL
agglutination occurs. The RF latex reagent sensitivity has of
been adjusted to detect a minimum of 8 IU/mL of Sample
rheumatoid factors according with the WHO International
8x 8x2 8x4 8x8 8 x 16 8 x 32
Standard without previous sample dilution.
Dilution
★ Sensitivity: 8 IU/ml or above. factor

IU/mL 16 32 64 128 256

II. Reagents of the Test

Normal levels (adults): less than 8 IU/ml

RF Latex Reagent A suspension of uniform polystyrene particles
coated with IgG (human) in glycine buffer, pH 8.2 CALCULATION: In the semi-quantitative test, the titer is expressed
as the reciprocal of the highest dilution showing macroscopic
Reagent sensitivity is standardized with the WHO agglutination. For example, if agglutination occurs in dilution 3, the
RF Standard. titer is 64.
Mix well before using

RF Positive Control A stabilized, prediluted human serum containing IV. Results

Serum at least 8 IU/mL of RF

RF Negative Control A stabilized, prediluted human serum containing Qualitative Test:

Serum less than 8 IU/mL of RF
★ Negative Result: A negative reaction is indicated by a
uniform milky suspension with no agglutination observed
Glycine-Saline Buffer pH 8.2 ± 0.1M glycine and 0.15M NaCl
with the RF Negative Control.
(20x)
★ Positive Result: A positive reaction is indicated by any
observable agglutination in the reaction mixture. The
Reaction Slide
Pipette/Stir Sticks
specimen reaction should be compared to the RF
Negative and Positive Controls

Semi-quantitative test:
III. Procedure
★ The titer of the serum is the reciprocal of the highest
dilution, which exhibits a positive reaction. An estimate of
the RF concentration in the specimen can be expressed in
Qualitative Method IUml by using the following equation:
1. Allow each components of the test kit (reagents, controls) IU/ml of specimen = IU/ml control x specimen titer
to reach room temperature. ★ The approximately RF level (IU/ml) present in the sample
2. Gently shake the latex reagent to disperse the particles. may be obtained by the multiplying the titer by the

Maekaella R. Roca – OLFU Laguna

 positive test result is not specific for RA because RF is also
analytical sensitivity of the Rf Latex reagent (8 IU/ml)
present in about 5% of healthy individuals and in 10% to 25%
of those over the age of 65.
★ In addition, RF can be found in patients with other connective
tissue diseases such as SLE, Sjögren’s syndrome,
scleroderma, and mixed connective tissue disease, as well
as in people with some chronic infections. Manual
agglutination tests using charcoal or latex particles coated
V. Clinical Significance with IgG have been used for many years to detect RF. These
tests, however, only detect the IgM isotype, found in about
75% of patients, and have been largely replaced by ELISA,
★ RA is another example of a systemic autoimmune disorder. It
chemiluminescence immunoassay, and nephelometric
affects about 0.5% to 1.0% of the adult population, but
methods, which are automated, have greater precision and
prevalence varies with ethnicity and geographic location.
sensitivity, and can also detect other RF isotypes.
Women are three times as likely to be affected as men; in
★ Once a diagnosis of RA is made, the most helpful tests for
addition, the prevalence of the disease is highest in women
following the progress of the disease are general indicators
who are more than 65 years of age. RA can be characterized
of inflammation, such as measurement of ESR, CRP, and
as a chronic, symmetric, and erosive arthritis of the
complement components. Typically, CRP and ESR are
peripheral joints that can also affect multiple organs such as
elevated and the levels of serum complement components
the heart and the lungs. Associations of RA with more than
are normal or increased because of increased acute-phase
30 genetic regions have been discovered. The strongest
reactivity. CRP levels correlate well with disease activity
associations have been between a subset of patients with
because levels reflect the intensity of the inflammatory
RA and specific HLA-DRB1 alleles or PTPN22 gene
response
polymorphisms. These patients are positive for rheumatoid
factor (RF) or antibodies to CCP (see the section on
Laboratory Diagnosis). The strongest environmental risk Other Important Information
factor for RA is believed to be cigarette smoking, which
doubles the risk of developing the disease. 1. Results should be read two (2) minutes after the mixing or
★ The pathology of RA is caused by an inflammatory process the reagent on the slide.
that results in the destruction of bone and cartilage. The 2. Existence of prozone at high titers has not been
lesions in rheumatoid joints show an increase in cells lining encountered.
the synovial membrane and formation of a pannus, a sheet 3. Increased levels of RF may be round in some diseases other
of inflammatory granulation tissue that grows into the joint than rheumatoid arthritis such as infectious mononucleosis,
space and invades the cartilage. Infiltration of the inflamed sarcodosis, lupus erythematosus, Sjogren's syndrome. 6,7
synovium with T and B lymphocytes, plasma cells, dendritic 4. Certain patients with rheumatoid arthritis will not have the
cells, mast cells, and granulocytes is evidence of RF present in their serum
immunologic activity within the joint.
★ It is not known what role autoantibodies play in the initiation
of the inflammatory response. Two key antibodies found in EXPECTED VALUES
the disease are RF and anti-CCP. RF is an antibody that is
most often of the IgM class and is directed against the FC 1. The diagnosis of rheumatoid arthritis is based largely on
portion of IgG. It has been postulated that RFs may play a clinical examination, but laboratory tests are useful to
role in the pathogenesis of RA by increasing macrophage support the clinical diagnosis and to evaluate the severity
activity and enhancing antigen presentation to T cells by and course or the disease in the individual patient. One of
APCs. In RA, autoantibodies such as RF and anti-CCP are the most useful clinical markers for rheumatoid arthritis is
thought to combine with their specified antigen, and the rheumatoid factor in serum. Rheumatoid factor is a term
resulting immune complexes become deposited in the joints, used to describe a variety of antibodies or immune
resulting in a type III (or immune complex) hypersensitivity complexes or both, that occur with rheumatoid arthritis as
reaction. The complement protein C1 binds to the immune well as in a variety of other diseases. 8
complexes, activating the classical complement cascade. 2. Different studies have shown positive serological reactions
During this process, C3a and C5a are generated, which act for rheumatoid factor in as high as 90 of patients with
as chemotactic factors for neutrophils and macrophages. rheumatoid arthritis compared with less than 5 in control
The continual presence of these cells and their associated groups
cytokines leads to chronic inflammation, which damages the
synovium itself.
EXPECTED VALUES
★ Diagnosis of RA is based on a combination of clinical
manifestations, radiographic findings, and laboratory testing.
RF is the antibody that is most often tested to aid in making 1. Reagents are stable until stated expiration date on bottle
the initial diagnosis. The importance of testing for the label when stored refrigerated (2 - 8°C).
presence of RF is also reflected in the fact that it is one of 2. DO NOT FREEZE.
the classification criteria for RA. 3. The RF latex reagent, once shaken must be uniform without
★ Recall that RF is an autoantibody, usually of the IgM class, visible clumping. When stored refrigerated, a slight
that reacts with the Fc portion of IgG. Approximately 70% to sedimentation may occur and should be considered normal.
90% of patients with RA test positive for RF. Thus, a negative 4. Do not use the latex reagent or controls if they become
result does not rule out the presence of RA. Conversely, a contaminated
IMMUNOLOGY & SEROLOGY 311 Laboratory
Detection of Anti-Nuclear Antibodies Finals

evenly covering the entire diameter of the test circle.

PRE-ANALYTICAL PHASE 5. Tilt the slide back and forth slowly for 3 minutes while
observing for agglutination.
6. Observe and Interpret results.
★ The work area of the experiment should be sterilized. The
Students should wear proper personal protective equipment Semi-quantitative Determination:
before the experiment (laboratory gown, eye protector, ★ Prepare dilutions of the specimens as shown below:
laboratory mask, gloves and hair net). Standard precaution is
applied to every phase of experiment
Dilution Serum Saline
Materials :
1:2 1 part 1 part
RF Latex suspension Positive and Negative Controls
Black test cards Disposable stirrers 1:4 1 part 3 parts
Mechanical rotator
1:8 1 part 7 parts

LABORATORY DISCUSSION GUIDE 1:16 1 part 15 parts

1:32 1 part 31 parts

I. Principle of the Test
1:64 1 part 63 parts

★ Passive or indirect agglutination

★ ELISA and CLIA are assays that can test from a broad range Proceed as in qualitative method
of antibodies if multiple nuclear antigens are coated onto a
single test well, or for a specific ANAs if each well is coated
with a single antigen. These antigens used in commercial V. Results
kits are derived from tissue extracts or produced by
recombinant technology. Because of their advantages, many ★ Agglutination indicates a positive result. If there is no
laboratories are using ELISA methods to screen for presence agglutination, the test result is negative. The test is
of ANAs in addition to identifying specific ANAs considered as NEGATIVE when no difference in agglutination
is observed between the specimen and negative control and
II. Sensitivity of the Test POSITIVE sera must show distinct agglutination within 3
minutes.
★ Agglutination indicates the level of antinuclear antibody
★ There is a large variation in the performance of test (especially anti-DNP) in the range commonly found in the
produced by different manufacturers, which is influenced by SLE.
the antigen preparation used. For example, one study found ★ Findings in the sensitivity studies suggests that
sensitivities of ELISA assays ranging from 69% to 98%, and immunoassays may miss a significant proportion of
specificities from 81% to 98% when they were compared with ANA-positive patients and also yield a significant number of
IIF ANA method. false positive results. Based on such studies, the ACR has
recommended that the IIF test remains the gold standard for
III. Reagents of the Test ANA testing and that clinical laboratories should specify the
method they use when they are reporting the result.

★ ANA reagent
★ Positive and negative control reagents VI. Clinical significance
★ Wells
★ Systemic Lupus Erythematosus is a chronic systemic
IV. Procedure inflammatory disease that affects people usually at the peak
age between 20 and 40 years. Women are much likely more
to be affected than men by a ratio of 9:1. This autoimmune
Qualitative Method: disease appears to originate from complex interaction
1. Bring all reagents to room temperature and mix gently between several factors, such as environmental, genetic
prior to use. susceptibility, and abnormalities within the immune system.
2. Place the following on separate divisions of the same In line with these, over 100 autoantibodies associated with
black test card.
SLE have been discovered. These include antibodies to
Patient’s Serum: 1 drop
Positive Control: 1 drop double stranded DNA (dsDNA), histones, and other nuclear
Negative Control: 1 drop components, as well as autoantibodies to lymphocytes,
3. Add 1 drop of SLE Latex reagent to each sample on the erythrocytes, platelets, phospholipids, ribosomal
black test card. components, and endothelium.
4. Mix with the flat end of pipette/mixer and spread fluid

Maekaella R. Roca – OLFU Laguna

★ Laboratory diagnosis of SLE include CBC, platelet count and
urinalysis. Findings in lupus patients are leukopenia and
possible anemia and thrombocytopenia. Erythrocyte
Sedimentation Rate (ESR) may be elevated even though
C-Reactive Protein tends to be low or normal.
★ When SLE is suspected, the first test typically done is a
screening test for Anti-nuclear Antibodies (ANA). These
antibodies are present in most cases of SLE, antibodies that
are directed against antigens of nuclei of mammalian cells.
ANAs are present in over 95% of patients with active SLE and
is used as a major marker for the disease. However, ANAs
are not specific only to SLE as it may be present in other
connective tissue disease such as Sjogren’s syndrome,
scleroderma, polymyositis-dermatomyositis, and RA. They
can also be found in some individuals with other conditions,
including chronic infections, pregnancy and cancer.
Furthermore, upto 5% of healthy individuals and upto 30% of
elderly people are ANA-positive.
★ ANAs are a heterogeneous group of antibodies that have
different antigen specificities. The nuclear antigens they are
directed against include double stranded and single stranded
DNA, histones, nucleosomes, centromere proteins, and
extractable nuclear antigens. Double stranded DNA
antibodies are the most specific for SLE because they are
mainly seen in patients with lupus and their levels correlate
with disease activity. Antibodies to dsDNA typically produce
a peripheral or homogenous staining pattern on indirect
immunofluorescence (IIF).
★ Methods in detecting ANAs have been developed. These
include IIF, immunoperoxidase staining, enzyme linked
immunosorbent assay (ELISA), microsphere multiplex
immunoassays (MIA), radioimmunoassay (RIA),
immunodiffusion, immunoblotting (Western blot), dot blot,
immunoelectrophoresis, and microarray

VII. Other important information

★ Note the proper procedure and usage of the reagents,

storage temperature and the expiry date of the kit.
IMMUNOLOGY & SEROLOGY 311 Laboratory
Non-treponemal test for Syphilis Finals

2. Hold the dropper over a test card circle and squeeze the teat
to allow one drop (50ul) to fall into the card. It is important to
PRE-ANALYTICAL PHASE
maintain the dropper in a vertical position while dispensing
the sample to be tested.
★ The site of the experiment should be sterilized. The medical 3. Using the flat end of the stirrer, spread the sample to cover
technologist should wear proper personal protective the test circle.
equipment before the experiment (laboratory gown, 4. Attach the dispensing needle to the plastic bottle. Withdraw
laboratory goggles or face-shield, laboratory mask, and sufficient RPR Carbon Antigen (WELL SHAKEN) for the
gloves) number of tests performed. Maintaining the needle in a
★ Reagents must be brought to room temperature and must be vertical position, allow one drop to fall on each test sample.
shaken well before dispensing. Undiluted serum will be used DO NOT RE-STIR!
to react with the RPR Carbon antigen. 5. Rotate the RPR test card manually or using an automatic
★ The microscope should be placed on a plain, stable surface. rotator for 8 minutes at 100 rpm.
The voltage of the power supply should be inclined with that 6. Observe and Interpret results.
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 II. Results
clean the objective and ocular lenses
★ Flocculation indicates a reactive result.
★ If there is no flocculation, the test is reported as non-reactive
LABORATORY DISCUSSION GUIDE

★ The student/s will become familiar in testing

Non-Treponemal test for Syphilis especially Rapid Plasma
Reagin (RPR) using patient serum mixed with RPR Carbon
Antigen. Flocculation must be observed for positive results.
★ Syphilis is a contagious sexually transmitted disease cause
by the spirochete Treponema pallidum. This spirochete
causes an immune response in the body by producing
antibody-like substance in plasma or serum known as
reagin. Cardiolipin, phospholipid derived from the beef heart
reacts with the reagin and this antigenic property of
cardiolipin is used in non-treponemal test.

Rapid Plasma Reagin (RPR)

★ RPR is the most frequently performed non-treponemal test
for syphilis. The RPR card antigen suspension is a
carbon-containing cardiolipin antigen that detects reagin.
★ The specimen serum or plasma is mixed with the RPR card
antigen suspension and allowed to react for 8 minutes. If
reagin is present, it will react to the antigen suspension
forming visible black floccules. This result is reported a
reactive. If reagin is absent, there will be no flocculation. This
result is reported as non-reactive

Materials :

RPR Test slide Positive and Negative Controls

Disposable dispensing pipettes RPR carbon antigen
Needle dropper Mixing sticks
Stopwatch Mechanical rotator

I. Procedure

Qualitative Method:
1. Hold the pipette between the thumb and the forefinger.
Squeeze while inserting the tip into the specimen. Then
release finger pressure to withdraw the sample taking care
not to transfer any cellular elements.

Maekaella R. Roca – OLFU Laguna

IMMUNOLOGY & SEROLOGY 311 Laboratory
HB, Dengue, HIV, ELISA Finals

Results:
LABORATORY DISCUSSION GUIDE

★ Test for Hepatitis B Surface Antigen (HBsAg)

★ Hepatitis B virus is a member of hepadnavirus family. It is

42 nm-eveloped virion, with an icosahedral nucleocapsid
core 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 Reactive:
★ HbsAg is the earliest serological marker that indicate the ★ Two purple bands appear: One in the test region (T) and
presence of acute infection. It is also indicative of chronic one in the Control region I. A positive result indicates the
infection. presence of HbsAg
★ The Hepatitis B virus is also known as serum hepatitis. HbsAg Non-Reactive:
The virus is transmitted via blood transfusion, the sharing ★ One purple band appears in the control region I with no
of needles among the drug users, and via sexual band in the test region (T). A negative result indicates
intercourse. HbsAg is not present in the sample
★ The incubation period is from 60 to 90 days. The virus is Invalid:
found in blood and body secretions. It is carried via ★ A total absence of color in two regions is an indication of
infected blood, saliva, and semen. It can enter to the body procedure error and/or that test reagent deterioration has
with small breaks in the skin or can be transmitted during occurred. Add additional drops of samples to the sample
sexual intercourse if there is a break in the mucous well. If there is still no color band appears, the test should
membrane. be voided
★ The first generation tests were found on Ouchterlony Performance Characteristics:
agar gel plates. The antibody for the HBsAg was put in ★ A correlation study comparing Blue Cross One Step
the plate in one well. Patient’s serum was placed in the HbsAg test had established that relative sensitivity of the
wells surrounding the antibody. A positive result showed a test is 99.27% and that its relative specificity is 98.3%
fine white precipitin line formed between the antigen and
the antibody. The test was said to be not very sensitive.
★ The second-generation test used for HbsAg was counter ★ Test for Dengue IgM/IgG and NS-1 Antigen
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 ★ Dengue viruses transmitted by the mosquitoes, Aedes
form. aegypti and Aedes albopictus, are widely distributed
★ Third-generation tests include radioimmunoassay (RIA) throughout the tropical and subtropical areas of the
and enzyme-linked immunoassay (EIA or ELISA). Other world. There are four known distinct serotypes (dengue
test procedures available were reverse passive virus 1, 2, 3, and 4) . In children, infection is often
hemagglutination and reverse passive agglutination tests subclinical or causes a self-limited febrile disease.
However, if the patient is infected a second time with a
different serotype, a more severe disease, dengue
hemorrhagic fever or dengue shock syndrome is more
Blue Cross R One Step HBsAg Test
likely to occur. Dengue is considered to be the most
★ The immunochromatographic device contains a unique
important arthropod borne viral disease due to the human
set of dye-conjugated and immobilized monoclonal and
morbidity and mortality it causes.
polyclonal antibodies used to produce a distinctive visual
★ Dengue NS-1 Antigen is a glycoprotein expressed by all
pattern indicating presence of HBsAg in the sample
dengue viruses that is found from the first day and up to 9
days after the onset of fever in the sample of primary and
secondary dengue infected patients.
Materials ★ Sd Bioline Dengue IgG/IgM Rapid Test is a solid phase
★ Blue Cross HBsAg Test Pack immunochromatographic assay for the rapid, qualitative
★ Serum sample and differential detection of IgG and IgM antibodies to
★ Timer dengue virus in human serum or plasma.
★ Sample dropper ★ SD Bioline Dengue IgG/IgM Test device has 3 pre-coated
lines, ‘G” (Dengue IgG Test Line), “M” (Dengue IgM Test
Procedure line and “C” ( Control Line) on the surface of the
1. Remove the testing device from the foil pouch by tearing membrane. All three lines in result window are not visible
at the “Notch” and place on a level surface. before applying any samples. The Control Line is used for
2. Check the expiration date. Do not use expired kits. procedural control. Control line should always appear if
3. Holding a sample dropper vertically, add exactly 6 drops the test procedure is performed properly and the test
(approximately 0.2 ml) of the serum to the sample well. reagents of control line are working. When a specimen is
4. Read the results after 10 minutes. Do not read results added to the sample well, anti-Dengue IgGs and IgMs in
after 30 minutes the specimen will react with Recombinant Dengue virus
envelope proteins-colloidal gold conjugates and forms a
complex of antibodies-antigen. As this complex migrate

Maekaella R. Roca – OLFU Laguna

 along the length of the test device by capillary action, it test using a new test device.
will be captured by the relevant anti-human IgG and or
anti-human IgM immobilized in two test lines across the
test device and generate a colored line.
★ The Dengue NS-1 Antigen is an in-vitro
immunochromatographic, one-step 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 pre-coated 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 membrane
chromatographically to the test region (T) and forms a
visible line as the antibody-antigen-antibody gold particle Dengue NS-1 Antigen
complex forms. 1. Negative: The presence of a purple line within the Control
area and the absence of purple line within the Test area.
Materials 2. Positive: The presence of purple lines each at the Control
★ SD Bioline Dengue IgG/IgM test device and the Test area.
★ Assay diluent: 100 mM phosphate buffer (5 mL), sodium 3. Invalid: The absence of purple lines on both the Control
azide (0.01% w/w) and the Test area
★ 5 uL capillary pipette/automatic pipettor
★ Package insert
★ Timer
★ Human Immunodeficiency Virus (HIV) Testing

Procedure
1. Allow all kit components and specimen to room
temperature prior to testing. ★ The etiologic agent of AIDS is a human retrovirus known
2. Remove the test device from foil pouch , place it on a flat, as human immunodeficiency virus(HIV). Retroviruses are
dry surface. defined as viruses that contain a single positive stranded
3. Add 5 ul of serum or plasma specimen into the RNA, which contain the virus genetic information, and a
square-shaped sample well marked “S”. specially enzyme known as “reverse transcriptase” in their
4. Add 3-4 drops (about 90-120 ul) of assay diluent to the core. The enzyme enables the virus to convert viral RNA
assay diluent well (round shaped). to DNA in contrast to the normal process of transcription
5. Interpret test results in 15-20 minutes. where DNA is converted to RNA.
★ Two types of HIV have been classified
Caution: Do not read test results after 20 minutes. Reading too late 1. HIV-1 the causative agent of AIDS is Europe
can give false results. and the United States
2. HIV-2 associated with immunodeficiency and
Dengue NS-1 Antigen clinical syndrome similar to AIDS in West Africa
1. Remove the test device from the foil pouch, and place it ★ HIV was formerly known as lymphadenopathy-associated
on a flat, dry surface. virus (HTLV-III), or AIDS-related virus. It is believe to be a
2. With a disposable dropper, add 3 drops (about 100ul) of member of the group of transforming, cytopathic
specimen into the sample well (S). retroviruses, called lentiviruses, which cause chronic
3. As the test begins to work, you will see purple color move neurodegenerative and wasting diseases in animals
across the result window in the center of the test device. similar to the wasting disease and neurologic disorders
4. Interpret the test results after 15-20 minutes. produced by HIV in humans.
5. A positive result will not change once it has been
established at 15-20 minutes. However, in order to prevent Rapid HIV Testing (SD HIV-1/2 3.0)
incorrect results, the test should not be interpreted after ● Rapid test (which come in both blood and oral version)
20 minutes. are done on-site and give a reading within half an hour. As
with the ELISA, a positive reading on a rapid test requires
Results: a second, confirmatory assay such a Western blot.
● Rapid tests have proved to be as accurate as the ELISA
Dengue IgM/IgG when performed carefully by experienced personnel.
1. Negative – The control line is only visible on the test Technical errors can cause false results with these
device. No IgG and IgM antibodies were detected. Retest assays if the users tend to become careless with these
in 3-5 days if dengue infection is suspected. simple procedures. To deal with this problem, many rapid
2. IgM Positive – The Control Line and IgM Line are visible tests now include a built in control that indicates whether
on the test device . This is positive for IgM antibodies to or not the test was done properly.
Dengue virus. This is indicative of primary dengue
infection. Materials
3. IgG Positive – The control line and IgG Line are visible on ★ Test device
the test device. This is positive for IgG antibodies. This is ★ Timer
indicative of secondary or previous dengue infection. ★ Blood or serum sample
4. IgG and IgM Positive – The control, IgG, IgM lines are ★ 20 uL capillary pipettes or sample dropper
visible on the test device. This is positive for both IgM and ★ Assay diluent
IgG antibodies. This is indicative of a late primary or early
secondary dengue infection. Procedure
5. Invalid – The control line fails to appear. Insufficient 1. Remove the test device from foil pouch, place it in a flat
specimen volume or incorrect procedural techniques are dry surface.
the likeliest reasons for control line failure. Repeat the 2. If capillary pipette will be used, add 20 ul of drawn blood
 into the sample well.
3. If a micropipette is used, add 10 ul of plasma or serum
specimen into the sample well (S).
4. Add 4 drops (about 120 ul) of assay diluent into sample
well (S).
5. As the test begins to work, you will see purple color move
across the result window in the center of the test device.
6. Interpret test results in 5-20 minutes. Do not read results
after 20 minutes. Reading too late can give false results.

Results:

★ HIV Negative
○ Indicated by the presence of only one line
○ A line in the control area within the result
window.
★ HIV-1 Positive
○ Indicated by the presence of two lines
○ One in the control area I and one in the test area
1 (1) within the result window
★ HIV-1 and HIV-2 positive
○ Indicated by the presence of three 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.
★ Invalid
○ Indicated if there are no line seen in the control
area I