IMMUNOLOGY AND SEROLOGY (LABORATORY)

Laboratory Safety
SAFETY o E.g. mouth, nose, or mucous
- To work safely in this environment, membrane
laboratory personnel must learn what ⮚ Mode of Transmission – how an
hazards exist, the basic safety infectious agent can be transferred from
precautions associated with them, and one person, object, or animal, to another.
how to apply the basic rules of o E.g. direct contact (skin to skin
common sense required for everyday contact), airborne (inhalation of
safety for patients, co-workers, and dried aerosol particles), droplets
themselves. (through inhalation too.), vehicle
borne (Contaminated
TYPES OF SAFETY HAZARDS substances: food, water or
specimen.), or vector borne
(infections transmitted by the bite
of infected arthropod species)
⮚ Portal of Entry – the manner in which a
pathogen enters a susceptible host.
E.g. mouth, nose, or mucous
membrane
⮚ Susceptible Host – the organism that
will feel the effects of the infectious
disease that has traveled through the
chain of infection.
o E.g. patients, elderly, newborns,
immunocompromised,
healthcare workers

** Biological Hazard – exposure to infectious agents:

possible agents that can be found in the specimen.
** Sharps Hazard – we are prone in this, as a
MedTech we are always using needles.
** Chemical Hazard – reagents used to perform
laboratory procedures. Carcinogenic: able to cause
cancer.

BIOLOGIC HAZARDS
** Biologic Hazard exposure to biological
substances.
- According to the CDC concept of
Standard Precautions, “All human
blood and other body fluids are
treated as potentially infectious for
human immunodeficiency virus (HIV),
hepatitis B virus (HBV), and other
blood borne microorganisms that can - Proper hand hygiene, correct disposal of
cause disease in human beings.” contaminated materials, and wearing
** As a MedTech working inside the laboratory, personal protective equipment (PPE)
before you deal with the patient specimen you are of major importance in the laboratory.
have to wear your complete PPE.
- Understanding how microorganisms are Handwashing
transmitted (chain of infection) is 1. Stand in front of the sink. Do not lean on
essential to preventing infection. the sink with clothes.
2. Use paper towel to cover the water
Chain of Infection control and turn on the water.
⮚ Infectious Agents – organisms that are 3. Wet hands thoroughly. Allow the water to
capable of producing infection or flow from arms to fingertips.
infectious disease 4. Apply soap to hands.
o E.g. bacteria, viruses, fungi, or 5. Wash the palm, back, and wrist of each
parasites hand using strong, frictional, circular
⮚ Reservoir – Where the microorganisms movements.
live and multiply 6. Interlace fingers and thumbs and move
** Typically a place kung san sila nag papadami hands back and forth for ten seconds.
o E.g. humans, animals, or 7. Rub nails against the palm
environment 8. Rinse hands thoroughly
⮚ Portal of Exit – the path by which a 9. Dry hands well
pathogen leaves its host 10. Use paper towel to turn the water off.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
Soiled Glove Removal Documentation
1. Pinch and hold the outside of the glove ● Documenting annual training of
near the wrist area. employees in safety standards.
2. Peel downwards, away from the wrist, ● Documenting evaluations and
turning the glove inside-out. implementation of safer needle devices.
3. Pull the glove away until it is removed ● Involving employees in the selection and
from the hand, holding the inside-out evaluation of new devices and
glove with the gloved hand maintaining a list of those employees and
4. With your ungloved hand, slide your the evaluations.
finger/s under the wrist of the remaining ● Maintaining a sharps injury log including
glove. Do not touch the outside surface the type and brand of safety device,
of the glove. location and description of the incident,
5. Peel downward, away from the wrist, and confidential employee follow up.
turning the glove inside out.
6. Continue to pull the glove down and over Biologic Waste Disposal
the inside out glove being held in your - All biologic waste, except urine
gloved hand. (discarded in the sink along with running
water), must be placed in appropriate
containers labeled with the biohazard
Standard Precautions symbol (yellow bag)
● Hand hygiene includes both hand - Disinfection of the sink using a 1:5 or 1:10
washing and the use of alcohol based dilution of sodium hypochlorite should be
antiseptic cleansers. performed daily.
● Personal protective equipment o Sodium hypochlorite dilutions
● Patient care equipment stored in plastic bottles are
● Environmental control effective for 1 month if protected
● Prevent injuries when using needles, from light after preparation.
scalpels, and other sharp instruments or
devices. SHARP HAZARDS
● Respiratory hygiene/cough etiquette - includes needles, lancets, and broken
** When to perform Handwashing and when to glassware
use alcohol? - All sharp objects must be disposed in
** Perform Handwashing if your hands are visibly puncture resistant, leak proof container
soiled. with the biohazard symbol.
** Use alcohol if your hands are not visibly soiled. - The biohazard sharp containers should
not be overfilled and must always be
Engineering Controls replaced when the safe capacity mark is
● Providing sharps disposal containers and reached.
needles with safety devices.
● Requiring discarding of needles with the CHEMICAL HAZARDS
safety device activated and the holder
attached. - Every chemical in the workplace should
● Labeling all biohazardous materials and be presumed hazardous.
containers. - When skin contact occurs, the best first
** Inside the laboratory good example of aid is to flush the area with large amounts
engineering controls is that we have proper waste of water for at least 15 minutes, then seek
disposal. medical attention.
** Never ever dispose needles in yellow bag.
** Example: you got contacted with chemicals.
Work Practice Controls First you have to flash the area with running water
for 15 minutes then ask for medical attention.
● Requiring all employees to practice
Standard Precautions and documenting Material Safety Data Sheets
training on an annual basis.
● Prohibiting eating, drinking, smoking, and Information contained in an MSDS includes the
applying cosmetics in the work area. following:
● Establishing a daily work surface ● Physical and chemical characteristics
disinfection protocol. ● Fire and explosion potential
● Reactivity potential
Personal Protective Equipment ● Health hazards and emergency first aid
procedures
● Providing laboratory coats, gowns, face ● Methods for safe handling and disposal
shields, and gloves to employees and ● Primary routes of entry
laundry facilities for non-disposable
● Exposure limits and carcinogenic
protective clothing.
potential
** Every chemicals we see inside the laboratory
Medical
must have material safety data sheets (MSDS).
● Providing immunization for the hepatitis This is required by the OSHA.
B virus free of charge.
● Providing medical follow up to employees Chemical Hygiene Plan
who have been accidentally exposed to
The purpose of the plan is to detail the following:
blood borne pathogens. ● Appropriate work practices
● Standard operating procedures
● Personal protective equipment
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
● Engineering controls, such as fume System for the Identification of the Fire
hoods and safety cabinets for Hazards of Materials, NFPA 704.14
flammables - This symbol system is used to inform
● Employee training requirements firefighters of the hazards they may
● Medical consultation guidelines encounter with fires in a particular area.
** The OSHA requires all facilities that are using - The diamond shaped, color coded
hazardous chemical must have a written symbol contains information relating to
chemical hygiene plan. health, flammability, reactivity, and
personal protection/special precautions.
RADIOACTIVE HAZARDS
- Radioactivity may be encountered in the
clinical laboratory when procedures
using radioisotopes are performed.
- Exposure to radiation during pregnancy
presents a danger to the fetus; personnel
who are pregnant or think they may be
should avoid areas with this symbol.

ELECTRICAL HAZARDS
- Equipment should not be operated with
wet hands.
- Designated hospital personnel monitor
electrical equipment closely; however,
laboratory personnel should continually
observe for any dangerous conditions,
such as frayed cords and overloaded
circuits, and report them to the
supervisor.
- Equipment that has become wet should
be unplugged and allowed to dry
completely before reusing. Equipment
also should be unplugged before
cleaning.
- When an accident involving electrical
shock occurs, the electrical source must
be removed immediately.
- 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 moving the
equipment using a nonconductive glass
or wood object are safe procedures to AGENCIES REGULATING THE LABORATORY
follow. ⮚ Clinical Laboratory Improvement
Amendments (CLIA) – provide
FIRE/EXPLOSIVE HAZARDS requirements for the employees working
When a fire is discovered, all employees are inside the laboratory
expected to take the actions in the acronym ⮚ Clinical and Laboratory Standards
RACE: Institute (CLSI) – create standards and
● Rescue – rescue anyone in immediate guidelines for sample collection and
danger laboratory testing
● Alarm – activate the institutional fire o Starting from specimen
alarm system collection, handling and
● Contain – close all doors to potentially processing, laboratory testing,
affected areas up until reporting
● Extinguish/Evacuate – attempt to ⮚ The Joint Commission (TJC) – provide
extinguish the fire, if possible or accreditation and certification of
evacuate, closing the door healthcare organizations
⮚ College of American Pathologists
(CAP) – provides laboratory
accreditation and proficiency testing

- The National Fire Protection Association

( NFPA) has developed the Standard
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Serial Dilutions
** Antibody A – served as solute: in all serological test, always
dilute the antibody.
** For example: gagamit tayo ng serum; serum + saline, so
PRE-ANALYTICAL PHASE yung dinidilute natin is yung serum. Remember usually asa
** We are performing serial dilution in order to detect the cold serum makikita yung antibody.
reacting antibodies – they are capable of reacting in a cold
environment – EXAMPLE: ABO blood group – is an example 4. Draw up to 0.25 mL from tube #1 and transfer
of IgM antibodies to tube 2. Again raise and lower to solution into
- The site of the experiment should be sterilized. the pipette three times to mix
The medical technologist should wear proper ** Slowly raising and lowering the solution in the use of pipette
personal protective equipment before the to mix the solution ---- aspirate, dispense, aspirate, dispense.
experiment (laboratory gown, laboratory ** Avoid creating bubbles.
goggles or face shield, laboratory mask, and
gloves) 5. Repeat step 4, transferring 0.25 mL tube #2 to
- Reagents must be brought to room temperature tube #3, then #4, then #5, until #10. Discard
and must be shaken well before dispensing. 0.25 mL from tube #10
EDTA anticoagulated tube is needed for the 6. Use clean serological pipette to add 0.25 mL of
preparation of 3% red blood cell suspension. 3% red blood cell suspension to each tube
** Wear PPE and collect blood – Specifically type A blood and ** RCS – were going to add this in order to determine the
placed in EDTA tube strength of the anti a.
** RCS usually not included on the computation, but needed
to be add to have reaction.
ANALYTICAL PHASE
Materials 7. Mix well and place in refrigerator for 30 minutes
• Antibody A (anti-A antisera) 8. Remove from refrigerator, centrifuge for 20
• 3% red blood cell suspension seconds. Read immediately for agglutination by
• 0.85-0.90% Saline (Normal Saline gently shaking the tube to dislodge the red
Solution/NSS) blood cell button. If the tubes are shaken too
• Ten 12 x 75 test tubes roughly, false negative reaction can occur.
• Refrigerator Clumping of the red blood cell suspension is
• Test tube rack negative.
• Waterproof marker
• Three serological pipettes

** Antibody A – reagent used in blood typing; blue reagent.

Usually agglutinate/clumping if it has a contact with type A
blood.
** Note: the concept of antigen and antibody binding should
be specific. There will be a presence of agglutination if the
antigen is specific for that particular antibody. It follows the
concept of LOCK AND KEY.

Procedures
1. Label ten test tubes 1-10 (test tube no., group
no. and section)
2. Place 0.25 mL of saline (diluent) in each of the
ten tubes.
3. Use a clean serological pipette to draw up 0.25
mL antibody A (solute). Add the antibody to
tube #1 by carefully lowering and raising the
solution into the pipette 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

** Usually the grading of agglutination reactions should be red

under the direct light source to see the clumping of red blood
cell.
** 4+ = one large clump seen; 3+ = presence of one large
clump and medium size clumps; 2+ = medium size clumps; 1+
= small clumps and 0 = no agglutination/negative
** Saline – served as diluent: medium making up the rest of ** In 4+ usually the solution is still color blue.
the dilution.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

• TT3 = ID(TT3) x FD(TT2) = ½ x ¼ = 1/8

• TT4 = ID(TT4) x FD(TT3) = ½ x 1/8 = 1/16
• TT5 = ID(TT5) x FD(TT4) = ½ x 1/16 = 1/32
• TT6 = ID(TT6) x FD(TT5) = ½ x 1/32 = 1/64
• TT7 = ID(TT7) x FD(TT6) = ½ x 1/64 = 1/128
• TT8 = ID(TT8) x FD(TT7) = ½ x 1/128 = 1/256
• TT9 = ID(TT9) x FD(TT8) = ½ x 1/256 = 1/512
• TT10 = ID(TT10) x FD(TT9) = ½ x 1/1024 =
1/1024

- Zone of equivalence – equal amount of ** TT = Test Tube

antigen and equal amount of antibody ** ID = Initial Dilution
** FD = Final Dilution
Interpretation of Results
- The last tube showing agglutination is the end
endpoint of the test.
- The titer is reported out as the reciprocal of the
last dilution showing a positive result.

Dilution Formula

𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝑜𝑙𝑢𝑡𝑒
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = “For many serology tests, it is the serum that is
𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒
concentrated; it may be necessary to dilute it with
• Where Total Volume = amount of solute + saline in order for a visible reaction to occur.”
diluent
• Note:
o Solute: material being diluted (Anti-sera SIMPLE DILUTION
A)
o Diluent: the medium that is used for 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝑜𝑙𝑢𝑡𝑒
dilution (NSS) 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 =
** 0.25mL saline = diluent; 0.25mL antibody a = solute; 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒
0.25mL RCS = not included in computation.
** Dilution = amount of solute: 0.25mL / total volume: 0.25mL A 1:20 dilution implies 1 part of solute
+ 0.25mL = 0.50mL and 19 parts of diluent

Serial Dilution ** Dilution:

Solute – material being diluted
Diluent – medium making up the rest of the solution

Example 1
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?
Initial Dilution:
Given:
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝑜𝑙𝑢𝑡𝑒 0.25 𝑚𝐿 TV = 2mL
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = = Dilution = 1:20
𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 0.50 𝑚𝐿 Amount of serum = ?
5 𝟏 Amount of diluent = ?
0.50 𝑚𝐿 = = 𝒐𝒓 𝟏: 𝟐
10 𝟐 1 𝑥
= = 𝟎. 𝟏 𝒎𝑳 𝒔𝒆𝒓𝒖𝒎
20 2 𝑚𝐿

𝑇𝑉 = 𝑎𝑚𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 + 𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡

𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 = 𝑇𝑉 − 𝑎𝑚𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 = 2 𝑚𝐿 − 0.1 𝑚𝐿 = 𝟏. 𝟗 𝒎𝑳

Final Dilution:
• TT1 = ID(TT1) = FD(TT1) = ½
• TT2 = ID(TT2) x FD(TT1) = ½ x ½ = ¼
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Given:
2mL = total volume 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑆𝑜𝑙𝑢𝑡𝑒 0.1 𝑚𝐿
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = =
1:20 = dilution 𝑇𝑜𝑡𝑎𝑙 𝑉𝑜𝑙𝑢𝑚𝑒 50 𝑚𝐿
Serum/solute = ?
Diluent = ? 2 𝟏
0.002 𝑚𝐿 = =
1000 𝑚𝐿 𝟓𝟎𝟎 𝒎𝑳
Amount of solute:
1 = ? --- Cross multiply = ? (20) = 1(2mL)
20 2mL ? = 2 / 20 = 0.1mL amt of solute

Amount of diluent:
Total Volume = amt of solute + amt of diluent
Amt of diluent = TV – amt of solute
= 2mL – 0.1 mL = 1.9 mL

Example 2
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?
• 4 – large clump
Given: • 3 – large clump with small clumps
Dilution = 1:5
• 2 – medium clumps
Amount of solute = 0.1 mL
• 1 – small clumps
Amount of diluent = ?
• 0 – no agglutination
1 𝑎𝑚𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
=
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 − 1 𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 PRACTICE DAW:

1 0.1 𝑚𝐿
=
5−1 𝑥

1 0.1 𝑚𝐿
= = 𝟎. 𝟒 𝒎𝑳 𝒅𝒊𝒍𝒖𝒆𝒏𝒕
4 𝑥 Tube no. ID FD Result
Tube 1 1/6 1/6 4+
Tube 2 1/6 1/36 2+
Example 3 Tube 3 1/6 1/216 1+
A volume of 500 mL of a 10% solution of acetic acid is
Tube 4 1/6 1/1296 No. aggt.
needed How much glacial acetic acid is needed and
Tube 5 1/6 1/7776 No. aggt.
how much diluent should be used?
Given:
Given:
Diluent = 10mL
TV = 500 mL
Solute (serum) = 2mL
Dilution = 1/10
RCS = 2mL ---- not included
Glacial acetic acid = ?
Diluent = ?

1 𝑥 Dilution = 2mL = 2mL = 1/6 - ID

= = 𝟓𝟎 𝒎𝑳 𝒈𝒍𝒂𝒄𝒊𝒂𝒍 𝒂𝒄𝒆𝒕𝒊𝒄 𝒂𝒄𝒊𝒅 10mL + 2mL 12mL
10 500 𝑚𝐿

𝑇𝑉 = 𝑎𝑚𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 + 𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 • ID tt1 = FD tt1 = 1/6

• ID tt2 x FD tt1 = 1/36
𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 = 𝑇𝑉 − 𝑎𝑚𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 • ID tt3 x FD tt2 = 1/216
• ID tt4 x FD tt3 = 1/1296
𝑎𝑚𝑡 𝑜𝑓 𝑑𝑖𝑙𝑢𝑒𝑛𝑡 = 500 𝑚𝐿 − 50 𝑚𝐿 = 𝟒𝟓𝟎 𝒎𝑳 • ID tt5 x FD tt4 = 1/7776

TITER = 216
COMPOUND DILUTION DILUTION FACTOR = 6 (nanggaling sa 1/6 = yung
denominator)
Example 1
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 AND SEROLOGY (LABORATORY)

Screening Test for Phagocytic Engulfment

2. Add 4 to 8 drops of the buffer coat from
- engulfment of cells and particulate matter either the patient’s heparinized blood or
by leukocytes, macrophage, and other cells from the normal control to the respectively
- “cell-eating” labeled tubes.
- Most important phagocytic cells: 3. Add 2 to 3 drops of the bacterial broth
o Neutrophils culture to each tube.
o Macrophage 4. Incubate both tubes at room temperature
** So what happen is when the WBC such as for 37 C for 15 mins and 30 minutes.
neutrophils and macrophage recognize a foreign 5. Place 1 drop of the incubated specimen on
object inside our body, they will phagocyte it. a glass slide and prepare a smear.
6. Air dry the slides and stain with Wright’s
stain.

Wright’s Stain Procedure

- Principle: A drop of whole blood is mixed 1. Cover each smear generously with
with a drop of a bacterial culture and filtered Wright’s stain and allow the stain
incubated at room temperature to to remain on the slide for at least 5
demonstrate engulfment of bacteria by minutes.
leukocytes. 2. Slowly add distilled water or buffer to the
- Extracted blood on a heparin tube stain until the buffer begins to overflow
- Common bacterial culture: Bacillus subtilis the stain. Watch for the appearance of a
and Staphylococcus epidermidis metallic luster.
- Incubate for 0min, 15min, and 30min at 3. Gently blow on the slide to mix the stain
room temperature and buffer.
- Prepare a blood smear and stain it using 4. Allow the buffer to remain on the slide for
Wright stain, then observe under the at least 5 minutes.
microscope 5. Gently wash the stain and buffer off the
- Will check the engulfment of bacteria by slide with distilled water.
leukocyte 6. Air dry or carefully blot the slide between
two sheets of bibulous paper.
PRE-ANALYTICAL PHASE 7. Place a drop of immersion on each smear
 Disinfect the working area and examine microscopically with the oil
 Personal Protective Equipment (100x) immersion objective.
** For the patient, no special preparation
will be needed before the specimen Results
collection  Positive: demonstration of the engulfment
 Minimum of 2ml Heparinized blood (green of bacteria
top evacuated tube)  Negative: no engulfment of bacteria
** Centrifuge the specimen and collect the  False negative results:
buffy coat layer. o if the blood specimen is not fresh
o if a coagulase-positive S. aureus
ANALYTICAL PHASE specimen is used.
Materials ** This test is only use for screening test for
phagocytic engulfment
 Broth culture of Bacillus subtilis (look for the
presence of bacilli, elongated bacteria) or Note: if the phagocyte failed to engulf the bacteria,
Staphylococcus epidermidis (look for the this can support the diagnosis of neutrophilic
presence of cocci, circular bacteria) dysfunction. However, those results must be
 Microscope slides (blood smear) correlated with patient’s signs and symptoms
 Pasteur pipettes
 Rubber bulb
 Test tubes
 Wright’s stain 1. PHYSICAL CONTACT BETWEEN THE WBC
AND FOREIGN PARTICLES
Procedure Adhesion
1. Label three test tubes: Patient test tube - At least five steps appear to be necessary
(0min, 15min, and 30min) and Control test for effective leukocyte recruitment to the
tube site of injury capture (the neutrophil will
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

bind to the P-selectin), rolling, slow 3. FORMATION OF PHAGOLYSOSOME

rolling, firm adhesion, and - fusion between cytoplasmic granules and
transmigration/diapedesis (movement of the phagosome
WBC across the blood vessel wall).
- P-selectin – is the primary adhesion
molecule for capture and the initiation of
rolling
- Chemotaxis – movement of WBC towards
the site of inflammation
** Remember our neutrophils has granules. What
will happen is that granules will coat the bacterial
then they will be fuse

- These granules contain degradatory

enzymes of the following three types:
o Primary or azurophilic granules
(contains enzymes)
o Secondary or specific granules
(contains substances such as
lactoferrin)
o Tertiary granules (contains
substances such as gelatinase)
- Elastase, one of several substances that
can damage host tissues, is also release
- The myeloperoxidase granules are
responsible for the action of the oxygen-
dependent, myeloperoxidase mediated
** If nagkaroon ng signal kay neutrophil na merong system.
bacteria na nakapasok sa host: - Hydrogen peroxide (H2O2) and an
1. Neutrophil undergo capture, will bind to p- oxidizable cofactor serve as major factors
selectin they will initiate the rolling, slow in the actual killing of bacteria within the
rolling then arrest then transmigration vacuole.
2. It will now start chemotaxis:
4. DIGESTION AND RELEASE OF DEBRIS
- Physical contact occurs as neutrophils roll TO THE OUTSIDE
along until they encounter the site of injury - The granules then release their contents,
or infection. and digestion occurs.
- Any undigested material is excreted from
the cells by exocytosis

2. FORMATION OF A PHAGOSOME
- cellular cytoplasm flows around the foreign
particle and fuses with it
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

o Neutrophils having giant granules

display, impaired chemotaxis, and
NONINFECTIOUS NEUTROPHIL MEDIATED
delayed killing of ingested bacteria.
INFLAMMATORY DISEASE
o Abnormal so makikita sa smear na
- Prolonged activation of NADPH oxidase merong giant granules
- This process occurs when phagocytes  Chronic Granulomatous Disease –
attempt to engulf particles that are too genetically heterogeneous group of
large. disorders of oxidative metabolism affecting
- The phagocyte releases oxygen radicals the cascade of events required for H2O2
and granule contents onto the particle, but production by phagocytes.
these escape into the surrounding tissues, o Can have infection with catalase
generating tissue damage. positive bacteria and fungi
- This is often observed in response to dust o They can engulf but they cannot
inhalation and smoking (e.g. nicotine) and destroy
in persistent infections such as cystic  Complement Receptor 3 Deficiency –
fibrosis. presents as a leukocyte adhesion
deficiency
** Non-infectious – hindi nakakahawa o Leukocyte adhesion deficiency
Example: autoimmune diseases type 1(LAD 1) is caused by a
deficiency of CD18
o Abnormal neutrophils (poor
adherence)
 Myeloperoxidase Deficiency – autosomal
recessive trait on chromosome 17.
o In this disorder, azurophilic
granules are present, but
myeloperoxidase is decreased or
absent.
 Specific Granule Deficiency – caused by
a failure to synthesize specific granules and
some contents of other granules during
differentiation of neutrophils in the bone
marrow.
o Patients have increased risk for
pyogenic infections
ABNORMAL NEUTROPHIL FUNCTION
 Leukocyte mobility may be impaired in
some diseases (e.g., rheumatoid arthritis,
cirrhosis, CGD)
 Leukocyte immobility can also be seen in
patients receiving steroids and in those with
lazy leukocyte syndrome.
o A marked defect in the cellular
response to chemotaxis can be
seen in patients with diabetes
mellitus, Chédiak Higashi anomaly
(syndrome), or sepsis, as well as in
those with high levels of antibody
immunoglobulin E (IgE), as in Job’s
syndrome.
o Since hindi makakapunta ng
mabilis si leukocyte sa site of
inflammation.

CONGENITAL NEUTROPHIL ABNORMALITIES

 Chédiak Higashi Syndrome – a rare
familial disorder inherited as an autosomal
recessive trait and expressed as an
abnormal granulation of neutrophils.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Laboratory Tests
C-REACTIVE PROTEIN (CRP) DETERMINATION
- Members of the family known as Pentraxin.
- Marker of acute inflammation - Pertaining to inflammation
- It increases rapidly within 4-6 hours following - CRP is an indicator of acute inflammation
infection, surgery, or other trauma to the body - The C reactive protein rapid latex agglutination test
- Reaches peak value within 48 hours is based on the reaction between patient serum
- Plasma half life: 18 hours containing CRP as the antigen and the
- Main substrate: phosphocholine corresponding antihuman (CRP) antibody coated
- increases faster than ESR (Erythrocyte to the treated surface of latex particles (to enhance
Sedimentation Rate test in hematology) in agglutination).
responding to inflammation, whereas the ** we will be using a latex antigen which CRP will act as
leukocyte count may remain within normal limits antigen and they are coated with latex particles
despite infection. - The coated latex particles enhance the detection
** for marker of acute inflammation: Serology – CRP of an agglutination reaction when antigen is
DETERMINATION while for hematology – ESR present in the serum being tested.
- Produced by the liver under the control of IL-6 - Observe the presence of agglutination
- Serum concentrations can increase 1000 fold with
an acute inflammatory reaction. Important Information
- CRP binds to specific receptors found on
- Principle: Reverse Passive Agglutination –
monocytes, macrophages, and neutrophils, which
antibody is coated with a carrier particle (can be
promotes phagocytosis
latex, silica, or RBC)
- CRP acts somewhat like an antibody, as it is
- Reagent: 1% suspension of polystyrene latex
capable of opsonization, agglutination,
particles coated with anti-human CRP (antibody)
precipitation, and activation of complement by the
produced in goats or rabbits
classical pathway.
o Usually, the color of the reagent is white
** opsonin sila yung mga substances that will bind to
** we will be using latex particles in order to
foreign microorganisms to make them more susceptible
enhance the agglutination
to phagocytosis
- Preservative: Sodium Azide
- Normal levels range from 0.1 mg/dL in newborns to
- Specimen: SERUM
0.5 mg/dL in adults.
** meaning u’re going to extract blood and u’re
going to place it on the red top tube and stand it for
Clinical Uses of CRP 10-15mins, once na clotted na yung specimen u’re
• Most widely used indicator of acute inflammation going to centrifuge it and after centrifugation u’re
o CRP Elevations can be found on bacterial going to get the serum
infections, if patient has rheumatic fever,
viral infections, tuberculosis, and after Note:
heart attack • In serology, always add clear solution first
• Used to monitor patient’s response in antibiotic • CRP, ASO, and RF tests procedures are the same
therapy (just change the positive and negative control
• Used to monitor patient’s response to reagents)
chemotherapy
• Used to monitor patient’s response to organ Procedures
transplantation
1. Wipe the black test card with tissue paper
o The level of CRP rises after tissue injury or
2. Label
surgery
o During uncomplicated cases, they will • 1st circle: CRP positive control
reach the peak level about 2 days after • 2nd circle: patient’s name, age, gender,
surgery, and return to normal level within date and time of collection
7 to 10 days • 3rd circle: CRP negative control
• Used to detect whether the patient has a risk of 3. Check the test kits
developing heart attack or stroke in the future. • Should be stored at room temperature
o Both CRP and LDL (Low Density 4. Check the materials
Lipoprotein in chemistry for cholesterol 5. Add 1 drop of positive control on the 1st circle using
levels) can be elevated for persons at risk a pipette
for cardiovascular diseases 6. Add 1 drop of serum on the 2nd circle (Patient’s
sample) using a pipette
7. Add 1 drop of negative control on the 3rdcircle
using a pipette
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

** dapat yung pipette is in perpendicular position or ** u can also perform the other test for CRP such as
nakatayo lang sya and press it and allow it to drop in a yung magpeperform na tayo ng dilution
falling drop (1drop)
8. Add 1 drop of CRP reagent on all of the circles
9. Using an applicator stick, mix the added drops until
it covers the whole circle (use different stick on
each circle)
10. Rotate the slide for 2 minutes
11. Check the results
• Positive Reaction: agglutination
• Negative Reaction: no agglutination
(presence of opaque fluid)

** ito yung black test card amd black ang ginagamit natin
bcuz yung reagent natin is color white, in order to easily
observe the agglutination we will be using the BLACK TEST
CARD

Sources of error/interference
➢ False negative reaction
o due to high levels of CRP in undiluted
specimens
** to have visible agglutination we must have
an equal amount of antigen and antibody)
➢ False positive reactions
o Read the reaction time longer that 2
minutes
o specimens are lipemic, hemolyzed, or
contaminated with bacteria

Limitations
- for screening test only
- This 2-minute slide latex agglutination test has a
detection level of 1 mg CRP/ dL ; therefore, patients
with CRP values less than 1 mg/ dL may go
undetected.
- The sensitivity of the procedure has been assessed
at 93%.
- Because the latex slide agglutination test is a
qualitative and semiquantitative procedure, other
methods such as nephelometry should be used for
the quantitative determination of the CRP level
when indicated.
- The strength of the agglutination reaction is not
always indicative of the CRP concentration.
- Weak reactions may be produced in samples
with elevated or low CRP values.
- Results may vary, depending on the patient’s
condition.
** so again we have to consider the false negative and
false positive reactions
** this test, latex slide agglutination will serve only as
SCREENING TEST
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Rheumatic Factor Test, Antinuclear Antibody, and Febrile Antigen

o Add FICT conjugate (fluorescein
isothiocyanate – conjugated goat anti-
human globulin)
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) ▪ This will bind to the antigen-
- It is an autoimmune disease antibody complex
**Autoimmune disease – the immune system is o Incubate the slide, then wash it to
attacking your own cells. remove the excess dyes.
**Autoantibodies are the ones that attack our cells. o After washing, view the slide under
Sa SLE, nagdedevelop siya ng autoantibodies. fluorescent microscope.
- Usually affects people within 20-40 years old **Positive with the patient’s antibody =
- a chronic systemic inflammatory disease that presence of fluorescence under
affects between 40 and more than 200 persons microscope
per 100,000, depending on the population **Negative = without presence of
- Women are more likely to be affected that fluorescence kasi wala naman naform na
antigen-antibody complex; so wala rin
men (9:1)
pagbabindan si FICT conjugate.
**Determination of anti-nuclear antibodies is used for ✓ SLE Latex Agglutination Test
diagnosis of Systemic Lupus Erythematosus (SLE) o Detects autoantibody (Anti-DNP)
which is present in the serum
Laboratory Diagnosis o Reagent: SLE Latex Reagent
▪ Suspended Inert latex
particles coated with DNP
o Principle: Passive or Indirect
Agglutination
**Because we used latex particles which
coats the antigen.
o Positive Reaction: agglutination
(nagbind anti-DNP kay DNP)

Screening test for Anti-Nuclear Antibodies: (not specific

- Principle: Passive/ Indirect Agglutination
for SLE)
✓ Fluorescent Antinuclear Antibody (FANA) Test - RF is an autoantibody that can be seen on
– Most widely used and accepted test because patients with rheumatoid arthritis.
it is highly sensitive, detects a wide range of - Materials and Reagents:
antibodies, and is inexpensive and easy to o RF Latex Reagent: A suspension of
perform uniform polystyrene particles coated
**Dedetect natin dito yung mismong antibodies. with IgG (human) in glycine buffer, pH
o Principle: indirect 8.2; reagent sensitivity is standardized
immunofluorescence with the World Health Organization RF
o The glass slide is incorporated with a Standard. MIX WELL BEFORE USING.
known antigen (mouse kidney cells or o RF Positive Control Serum: A
Hep2 cells), then the patient’s sample stabilized, prediluted human serum
is added to detect the presence of containing at least 8 IU/mL of RF.
antibody o RF Negative Control Serum: A
**If the antibody is present, it will react or stabilized, prediluted human serum
bind to the known antigen. containing less than 8 IU/mL of RF.
o Incubate the slide, then wash it to o Glycine-Saline Buffer (20x): pH 8.2 ±
remove the excess antibodies 0.1M glycine and 0.15M NaCl
**Incubate para magkaroon ng time to o Reaction Slide. (black test cards –
bind si antigen kay antibody. commonly used kasi mas Madali makita
agglutination)
o Pipette / Stir Sticks
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Procedure that can also affect multiple organs such as

Qualitative Method heart and the lungs.
1. Allow each components of the test kit - Caused by inflammatory process that results in
(reagents, controls) to reach room the destruction of bone and cartilage
**Nag-aaccumulate sa bone yung mga napproduce
temperature.
na antibody.
2. Gently shake the latex reagent to disperse the
**RF – most common antibody that can be seen
particles. with patients that have rheumatoid arthritis.
3. Place a drop of undiluted serum into a circle of - Anti-CCP: second major type of antibody
a test slide. associated with RA.
4. Add one drop of Rf Latex reagent next to the
drop of serum.
5. Spread the reagent and serum sample over the
entire area of the test circle using a separate
stirrer for each sample.
6. Gently tilt the test slide backwards and
forwards for two minutes.
7. Observe and interpret the results
• Note: Positive and Negative Controls Clinical Sign and Symptoms
should be run at regular intervals • Initial symptoms involve the joints, tendons,
and bursae
**Steps based sa video: • Nonspecific symptoms such as malaise,
1. Add 1 drop of clear solution first to each circle fatigue, fever, weight loss, and transient joint
(Positive control, negative control, and serum) pain that begin in the small joints of hands and
to prevent incorrect procedures.
feet
**Nakakalimutan na kasi ng iba mag-add ng clear
solution/s kapag nauna yung reagent since colored
• Joint pain can lead into muscle spasms and
siya. limitation of motion
**In all serological tests, always add the clear • If left untreated, may result in permanent joint
solution first. dysfunction and deformity
2. Add 1 drop of reagent • A small percent may develop Felty's Syndrome
3. Mix the components in the circle using a mixing (a combination of chronic, nodular RA coupled
stick (use a new mixing stick for each circle, with neutropenia and splenomegaly)
bawal yung same gagamitin). • Most common cause of death: Cardiovascular
4. Incubate for 2 mins (according sa vid) disease
5. Tilt/round the slides gently.
6. Observe for the presence of agglutination. Laboratory Diagnosis
**Positive = agglutination ✓ Manual Agglutination tests using charcoal or
**Negative = no agglutination
latex particles coated with IgG
✓ ELISA
Results ✓ Chemiluminescence Immunoassay
Qualitative Test ✓ Nephelometric methods
• Negative Result: A negative reaction is
indicated by a uniform milky suspension with
no agglutination observed with the RF
Negative Control. - a standard panel of serologic antigens used in
• Positive Result: A positive reaction is indicated screening patients with unexplained fever.
by any observable agglutination in the
reaction mixture. Febrile Diseases
Note: The specimen reaction should be compared to - group of microbial infections characterized by
the RF Negative and Positive Controls fever and the production of antibodies known
as febrile agglutinins.
RHEUMATOID ARTHRITIS - These diseases include:
o Brucellosis, which is caused by the
- can be characterized as a chronic, symmetric,
bacteria Brucella abortus
and erosive arthritis of the peripheral joints
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

o Paratyphoid fever, which is caused by significant levels of the corresponding

Salmonella paratyphi antibody in the patient serum.
o Rocky Mountain spotted fever, which - No agglutination is a negative test result and
is caused by rickettsiae indicates absence of clinically significant levels
o Tularemia, which is caused by of the corresponding antibody in the patient
Francisella tularensis serum.
o Q fever, which is caused by rickettsiae

WIDAL TEST
- an agglutination test which detects the
presence of serum agglutinins (H and O) in
patients serum with typhoid and paratyphoid
fever
- Developed by Ferdinand Widal in 1896.
- Principle: Direct agglutination Rapid Slide Titration Technique
- Antigens used: - With the use of a pipettor, dispense 0.08ml(or
o Somatic Antigen or “O” Antigen 80µl), 40µl, 20µl, 10µl, and 5µl of undiluted
▪ Present serum on the circles.
▪ OA, OB, OC: paratyphoid - Shake the reagent bottle, then add one drop of
▪ OD: typhoid undiluted antigen suspension to each of the
o Flagellar Antigen or “H” Antigen serum aliquot.
▪ Past/Previous - Mix it well using a stirring stick and rotate it for
▪ HA, HB, HC: paratyphoid 1 min.
▪ HD: typhoid - Observe for the presence of agglutination.
o Capsular Antigen or “K” Antigen or “Vi”
Antigen
- Salmonella antibody starts appearing in serum
at the end of first week and rise sharply during
the 3rd week of endemic fever.
- In acute typhoid fever, O Agglutinins can
usually be detected 6-8 days after the onset of
fever and H Agglutinins after 10 12 days.

Tube Agglutination Test

**All positive results obtained on the slide test should

be confirmed using the tube agglutination test.
**Steps:
- Prepare 8 test tubes.
Slide Method - Using a pipette, dispense 1.9ml of 0.85% of
**Since this is a direct agglutination, we will just add a drop saline into the first tube, and 1ml of saline into
of serum and a drop of the antigen. the remaining 7 test tubes.
- Agglutination is a positive test result and if the - Using another pipette, dispense 0.1ml of
positive reaction is observed with 20 uL of test patient’s undiluted serum on the first test
sample, it indicates presence of clinically
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

tube. Mix it well using a large pipette volume **Proteus and Rickettsia has the same glycolytic
and (eat?? what da heal ende q tlg magets) antigenic determinant and polysaccharide. Since
- Using another pipette, dispense 1ml from the they have almost the same structure, they have the
first tube to second tube. Then, mekus mekus ability for cross-reactivity.
mo nayan insan tapos ulitin lang sa mga o Same glycolipid antigenic determinant
remaining test tubes. o Same polysaccharide
**For the 7th test tube, discard 1ml because the 8th - Antigen used:
tube will serve as the negative control; kailangan o Proteus OX-2 (P. vulgaris)
wala siyang serum. o Proteus OX-19 (P. mirabilis)
- Shake the reagent bottle well. Add 1 drop of o Proteus OX-K (Kingsbury strain)
the antigen suspension to each of the test - Methods:
tubes. Mekus mekus mo nayan ulit insan. o Slide method
- Incubate all the tubes into water bath. o Rapid slide method
**For Salmonella O antigens and proteus – incubate o Tube method
at 50°C for 4 hours
**For Salmonella H antigens – incubate at 50°C for
2 hours Interpretation
- After incubation, check each test tube for the OX-2 OX-19 OX-K
Rickettsia rickettsi 1+ 4+ 0
presence of agglutination.
Rickettsia mooseri 1+ 4+ 0
**Compute for the titer since we used dilution.
Rickettsia prowazeki 1+ 4+ 0
Rickettsia tsutsugamushi 0 0 3+
Interpretation: Rickettsia akari 0 0 0
• INC titer on OD Ag: indicates acute or recent Rickettsia burnetti 0 0 0
typhoid fever **Rickettsia rickettsia, Rickettsia mooseri, and Rickettsia
• INC titer on OA, OB, and OC: indicates acute or prowazekii cross reacts on the antigens of P. vulgaris(OX-2),
and P. mirabilis (OX-19). While the Rickettsia tsutsugamushi
recent paratyphoid fever
tends to cross react with Kingsbury strain (OX-K).
• INC titer on HD Ag: indicates previous or past **1:160 = most significant titer in Weil-Felix test.
infection with typhoid fever
o Convalescent or vaccination against Additional infos from video (weil-felix):
typhoid fever Requirements for the test:
• INC titer on HA, HC, HB: indicates past 1. Patient serum sample
infection on S. paratyphi 2. OX2, OX19, and OXK antigens (thaw all reagents to
• INC titer on O, and H Ag: indicates mixed the room temperature)
infection 3. Plastic cards with circles
**For Widal test, we have two types of agglutination test 4. Disposable sticks for mixing
that we can observe: 5. Micropipette with tips
➢ Somatic agglutination – compact aggregate that 6. Gloves
are not easily dispersed; stronger agglutination 7. Discard jar
than H antigen
➢ Flagellar agglutination – fluffy; easily dispersed
- Transfer 20 µl of serum sample on to the rings. This is
**Significant titer = 1:80 or 80
<80 = normal equivalent to 1 in 80 dilution.
>80 = indicative of infection - First & second rows shown in this video are of positive
& negative controls, respectively.
- Next, transfer one drop of each of the three antigens
WEIL-FELIX TEST
for positive control, negative control and test
- allows the diagnosis of Rickettsiosis specimen.
- principle: direct agglutination - The serum-antigen mixture is initially mixed using
- based on the fact that three strains of Proteus plastic sticks. It can be followed by using a rotator or
which share somatic antigenic components rotating the card manually.
with Rickettsia, are agglutinated by antibodies - As seen in this video, appearance of clumps indicates
present in the serum of infected patients agglutination, which is seen only in the positive control
- Several rickettsiae, such as Rickettsia specimen.
prowazekii, Rickettsia mooseri, and R. rickettsii - No agglutination is seen in▸ the test serum specimen
posses antigens that cross react with antigens for any of the three Proteus antigens.
of OX strains of Proteus vulgaris
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

- For completion sake, we will perform "dilution" on the • Discard used reagents and sample as per BMW
positive control sample to determine its titre. disposal guidelines followed in your healthcare setting
- Transfer 10 μl to obtain a "dilution" of 1 in 160.
- Similarly, transfer 5 µl to obtain a "dilution" of 1 in Potential Sources of Variability
320. - Shake the antigenic suspensions well before use
- Add a drop of antigen, against which the titre has to - Ensure suspension: Homogenous
be determined. In this case, we are testing against - Moderate rise in titer of all three 'H' agglutinins
OX19 antigen. occurring simultaneously
- We made another "dilution" of 1 in 1280 by taking - Against all three H antigens: Recent TAB vaccination
1.25 µl of serum. Agglutination was seen only in the - Antibiotic treatment: Prevents rise in titer
dilution of 1 in 320, which is the titre. - Negative result cannot rule out Typhoid fever:
- In this test, the serum had antibodies of 1 in 320 o Sample taken early in course of illness (earlier
dilution only towards OX19 antigen, suggestive of the than 7 days from the onset of infection
Typhus group. because antibodies have not reached the
detectable limit within this period.
Additional infos from video (widal test): o Diagnosis of typhoid fever can be more
Antibodies specific → Flagellar Antigen 'H' Somatic Antigen 'O' definitively established from increasing titers
**Detectable in blood after 7 days from onset of of H and O antibodies and successive tests
infection rather than from single test results of H and O
titers.
Test facilitates quantitative estimation of antibodies
- Salmonella Antigens in human serum by slide
- Tube agglutination test

• Titers of TO, TH AH, BH above 1:80 & above:

Diagnostically significant
• Diagnosis should be confirmed through:
- Positive cultures
- Rapid test
- Clinical presentation
- Laboratory tests

Principle:
- When the coloured, smooth, attenuated
antigen suspensions are mixed and incubated
with patient serum
Anti-salmonella antibodies + Antigen suspensions → agglutination

Primary Sample
• No special preparation required prior to sample
collection
• Collect 2 ml of venous blood in a plain red topped
vacutainer
• Do not use haemolyzed and turbid samples
• Freshly collected serum : Preferable
• Samples: Stored at 2-8°C up to 72 hours in delay
testing

**di ko na sinama steps ng tests na pinakita sa vid kasi same lang

naman ng andito.

Safety Precautions
• Handle all samples as potentially infectious
• Handle all reagents with care and avoid contact with
eye, mouth and skin
• Do not perform mouth pipetting
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Slide Agglutination, Febrile Antigens, ASO

AGGLUTINATION
- process by which particulate antigens such as cells
aggregate to form larger complexes when a specific
antibody is present.
** we have the present of particulate antigens and
these antigens will bind when the specific antibody is
present
** partuculate antigen plus antibody will tend to
aggregate to form a larger complex TYPES OF AGGLUTINATION REACTIONS
** larger complex is yung nakikita natin macroscopically ➢ Direct Agglutination – occurs when antigens are
and even microscopically found naturally on a particle
o Reagent used: true antigen
o Examples:
▪ Widal test: true salmonella Ag
▪ Weil-Felix test: proteus Ag
➢ Passive or Indirect Agglutination – employs
particles that are coated with antigens not normally
found on their surfaces.
o A variety of particles, including
erythrocytes, latex, gelatin, and silicates,
are used for this purpose

2 STEPS IN AGGLUTINATION
Sensitization
- involves antigen-antibody combination through
single antigenic determinants on the particle
surface
** this is a reversible reaction
** FACTORS THAT AFFECT THE SENSITIZATION:
incubation time and temperature
** we have to take note the antibody that were going to ➢ Reverse Passive Agglutination – antibody rather
use and remember we have the cold antibodies (IgM) than antigen is attached to a carrier particle
and the warm antibodies (IgG) o Antibody is coated with a carrier particle
** once the antigen binds to the antibody, it will be o Example: CRP latex test
followed by a lattice formation

Lattice Formation
- representing the sum of interactions between
antibody and multiple antigenic determinants on a
particle
** in this step magkakaroon na po ng networks to have
a visible agglutination
- ENHANCEMENT OF LATTICE FORMATION:
o agitation or centrifugation
** this is why we need to centrifuge the rbc
para magsettle sila sa bottom bcuz remember
the rbc has slightly negative charge and like ➢ Agglutination Inhibition – based on competition
charges tend to repel to each other between particulate and soluble antigens for
o use of enzyme (e.g bromelin, papain, limited antibody combining sites, and a lack of
ficin) agglutination is an indicator of a positive reaction.
o increasing the viscosity o Presence of agglutination indicates a
** this can be done using dextran and albumin negative reaction
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Hemagglutination Red blood cells Lack of agglutination

Inhibition spontaneously is a positive test,
agglutinate if indicating the
viral particles are presence of patient
present. antibody.
** both agglutination inhibition and hemagglutination- for
that type of reaction so there is a competition between the
particles and a patient’s antigen
** so with that lack of agglutination will be the positive result

GRADING OF AGGLUTINATION REACTION

0 no agglutination
1+ Very small clumping with an opaque fluid background
➢ Coagglutination – name given to systems using 2+ Small clumping with a slightly opaque fluid
background
bacteria as the inert particles to which antibody is
3+ Moderate clumping with a fairly clear fluid background
attached.
(medium clump)
o Staphylococcus aureus is most frequently 4+ Large clumping with a clear fluid background (one
used because of the presence of Protein A solid clump)
** here ure going to obeserve the presence of agglutination
under the direct light source

Type of Reaction Principle Results

Direct Antigen is Agglutination
Agglutination naturally found indicates the
on a particle presence of patient
antibody
Ex: we have the
weil felix test and
the widal test, by
that we will be
using a true
bacterial antigen DIRECT BLOOD TYPING
as our reagent - Principle: direct agglutination
** direct po sya kase here ang gagamitin natin is red
Indirect (Passive) Particles coated Agglutination
cells and remember sa red cells andon po yung mga
Agglutination with antigens indicates the
not normally presence of patient antigens (A, B, AB, and O) and as for reagent, we will be
found on their antibody using a commercially prepared A antigen or B antigen
surfaces - The ABO blood groups (A, B, AB, and O) represent
** antigen is the antigens expressed on the erythrocytes (red
coated w/ a blood cells, RBCs) of each group. Reagent typing
carrier particles sera contains specific antibodies to A antigen and B
(such as latex, antigen.
silica, - When an unknown patient’s RBCs are mixed with
erythrocytes and
known antibody A or antibody B, agglutination of
so on)
Reverse Passive Carrier Particles Agglutination the RBCs will occur if a specific antigen antibody
are coated with indicates the reaction occurs.
reagent antibody presence of patient ** for our activity we will be performing a slide abo
antibody agglutination test or ABO blood typing
Agglutination Haptens Lack of agglutination ** ang ginagamit natin here is si color yellow bcuz Anti
Inhibition attached to is a positive test, B is for Rh reactions, that’s why meron tayong A positive
carrier particles. indicating the B negative and Ab negative and so on
Particles presence of patient ** so, the positive and negative pertains to the Rh
compete with antigen
reactions
patient antigens
** may pinanood si maam na vid about BLOOD
for a limited
number of GROUPING EXPERIMENT ang time stamp is from 6:34 –
antibody sites. 9:00
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

** for the result para malaman natin kung ano po yung

blood group ni patient we have to look for the presence of
agglutination
** so tatandaan natin guys, that the antigen-antibody
binding must be specific so kung merong presence mg
antigen plus specific antibody, so what happened is that
they will form agglutination
** meaning koag nagkaroon ng agglutination whether on
anti a or anti b, so there is a presence of antigen
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

LABORATORY DIAGNOSIS
AGGLUTINATION TEST A. CULTURE
o plated the specimen on sheep blood agar and
GROUP A STREPTOCOCCAL INFECTION incubated
- Streptococcus pyogenes (Group A β haemolytic ** adter incubation we’re going to observe the-
Streptococci) o Small translucent colonies surrounded by a clear
- Frequent infections can lead to sequelae infections zone of β hemolysis will be visible
such as Rheumatic Heart Disease and Acute
Glomerulonephritis B. IDENTIFICATION
** ASO LATEX AGGLUTINATION TEST is a test used to o susceptibility to bacitracin
check group a streptococcal infection ** identification can be done by BACITRACIN
** yung Acute Rheumatic Fever usually it develops a SUSCEPTIBILITY TESTING
sequelae infection to pharyngitis or tonsillitis o testing for L-pyrrolidonyl-β-naphthylamide (PYR)
** usually 2-3% of infected individuals can develop activity
acute rheumatic fever o Lancefield typing
** take note also that it does not occur as a result of skin
infection
DETECTION OF GROUP A STREPTOCOCCAL ANTIGENS
** usually features of acute rheumatic fever includes
➢ LATERAL FLOW IMMUNOCHROMATOGRAPHIC ASSAYS
fever, pain, inflammation in the joints and even
(LFA)
inflammation of the heart
o used for the detection of bacterial, viral, fungal,
** Acute Glomerulonephritis – a damage in glomeruli
of the kidney and parasitic antigens in clinical sample
** usually this condition may follow infection of either o strep A antigen extracted from a throat swab
the skin or the pharynx whereas again yung rheumatic reacts with an enzyme-labeled antibody on a test
membrane
fever is follows after upper respiratory tract infection
** usually yung mga group a antigens na yan can be
** this glomerulonephritis caused by streptococcal
extracted by either enzymatic or chemical means
infections is usually common in the children between
the ages of 2 and 12 years old ** the process can take anywhere for 2-30 mins
** and usually it is prevalent during the winter season depending on the particular technique
** so now what happened in this test is that the antigen-
antibody complex is captured by amother antibody at a
specific location on the membrane ibig sabihin when a
color line is produced
** ibig sabihin the sample is positive for the antigen
becuz nagkaroon ng antigen – antibody complex
** many of these test? Require with no more than 2-5
minutes of hands on time when we talk about the LFA
** this assay is usedd to detect the group a
streptococcal ANTIGEN

DETECTION OF STREPTOCOCCAL ANTIBODIES

✓ Antistreptolysin O (ASO) Testing
✓ Anti-DNase B Testing
✓ Streptozyme Testing
BACTERIAL TOXINS
** we all know that the streptococcus pyogenes are able to ** THE MOST DIGNOSTICALLY IMPORTANT ANTIBODIES OF
produce the bacterial toxins STREPTOCOCCUS: ASO, ANTI- DNase, ANTI-NADase, ANTI-
➢ Streptolysin O (SLO) HYLAURONIDASE
o is an oxygen-labile enzyme that causes ** the assay for the detection of the antubody can be
hemolysis by binding to cholesterol in the performed individually or with the use of streotozyme test
RBC membrane. ** streptozyme test can detect all antibodies of these
o It is antigenic, and the presence of product
antibodies to SLO is an indicator of recent ** the two most commonly test is ASO and anti dnase for the
streptococcal infection. detection of streptococcal antibodies and also these are
➢ Streptolysin S individual tests
o a nonantigenic, oxygen stable enzyme.
o It causes hemolysis by disrupting the Antistreptolysin O (ASO) Testing
selective permeability of the RBC
- detect antibodies to the streptolysin O enzyme
membrane.
produced by Group A streptococcus.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

** meaning if u are infected with Group A ** we have here 14 test tubes and in the ASO test tube we
streptococcus, so ure immune system will be able to will be performing COMPOUND DILUTION meaning every
detect antibody against the streptolysin O and that’s test tube magkakaiba sila ng composition ng diluent and
what we called as ANTISTREPTOLYSIN O serum
- we can check for the ASO titer – tube method na ** sa test tube 1&2 may 1:10 dilution and we can have it as
ginagamit natin 0.5ml serum and 4.5 ml of buffer
** what we are going to do is to check for the presence ** test tube 4,5,6&7 – 1:100 dilution (1ml coming from the
of the antibody on the patient’s serum 1:10 dilution plus the 9ml of diluent)
- based on the ability of antibodies in the patient’s ** test tube 8,9,10,11&12 – 1:500 dilution (2ml coming from
serum to neutralize the hemolytic activity of 1:100 dilution plus the 8ml diluent)
streptolysin O. ** every test tube were going to add serum and the diluent
- involved preparing dilutions of patient serum to ** nasa picture yung values nung serum and diluent for
which a measured amount of streptolysin O reagent every tubes
was added. ** sa tube 13, diluent lang meron and walang serum bcuz
- These were allowed to combine during an red cell control natin syan
incubation period after which reagent RBCs were - Add 0.5 ml strep O Ag on all tubes except tube 13
added as an indicator. (red cell control)
- If enough antibodies were present, the streptolysin ** ofc if nag add tayo ng streptolysin o antigen,
O was neutralized and no hemolysis occurred. maneuneutralize si red cell
- The titer was reported as the reciprocal of the - Tube 14 (streptolysin O control tube)
highest dilution demonstrating no hemolysis. - Shakes the tube and incubate for 15min, 37°C
- This titer could be expressed in either Todd units o Add 0.5 ml Type O human red cells to all
** hindi nabanggit yan lahat tubes
o Incubate again for 30 mins at 37°C
SERODIAGNOSTIC TESTS o Centrifuge, check for presence of
hemolysis
ASO Tube Method / Rantz & Randall Procedure ▪ Positive: absence of hemolysis
- THE TEST ESTIMATES THE AMOUNT OF ANTIBODY ▪ Negative: hemolysis (red
(ASO) THAT IN THE PRESENCE OF CONSTANT DOSE supernatant)
OF SLO CAN COMPLETELY INHIBIT HEMOLYSIS OF A - Titer: last tube with no hemolysis (clear
CONSTANT GIVEN NUMBER OF RED CELLS supernatant)
** used to detect for the presence of the - Tube 13: no hemolysis
Antistreptolysin O - Tube 14: with hemolysis (sa trans pero sabi ni
- Principle: Neutralization / Enzyme Inhibition Test maam without hemolysis kasi streptolysin O control
- TODD Unit = Titer daw sya)
** titer is yung last tube showing visible agglutination - Significant/diagnosis titer: 166 todd units
but here sa ASO tube, titer is the last tube showing the ** refer to the picture above sa paghahanap ng todd unit and
neutralization (last tube with no hemolysis) the formula mentioned above

ASO Slide Method

- Principle: Passive/Indirect Agglutination
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 ** the antigen is coated with a latex carrier particle
𝑇𝑂𝐷𝐷 𝑈𝑛𝑖𝑡 = 𝑥 𝑑𝑖𝑙
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 - Sample: serum
- Positive Result: No Hemolysis - Reagent:
- Negative Result: With Hemolysis o ASO Latex Reagent – Streptolysin O Ag
- Normal Values: attached to Latex Ag; suspension of
o Children: <125 Todd units polystyrene latex particles sensitized with
o Adults: <166 Todd units streptococcal exoenzyme (Streptolysin O)
▪ ASO positive control – stabilized
COMPOUND DILUTION human serum containing ASO
reactive with ASO latex reagent.
▪ ASO negative control – a
stabilized human serum
containing ASO non-reactive
with ASO latex reagent.
- All reagents contain 0.1% Sodium azide as a
preservative
- Analytical sensitivity:
o 200 IU/mL
o Positive: if titer is > 200 IU/mL
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

o Negative: if titer is < 200 IU/mL ** in this test we will be needing an overnight
incubation at 37 celcius (ginagawa natin to in order to
Materials permit the antibodies to inactivate the enzyme)
• ASO latex reagent (white) ** and after that the tubes will be graded by the color:
• Pipette 4+ = intensity ng color (green color so the color is
• Control sera (Positive and Negative Controls) unchanged) // 0 = colorless so total change of color
** NORMAL TITER for children: 240-640 TODD units
• Applicator stick (for mixing)
• Black test cards
• Mechanical rotator
Streptozyme Test
** it measures all antibodies against the streptococcal
PROCEDURES antigen
** take note yung procedure almost the same lang sa CRP **ASO, ANTI- Dnase B, ANTI-HYALURONIDASE, ANTI-
determination yung paggamit ng black test card and pag STREPTOKINASE, ANTI-NICOTINAMIDE-
label nung three circles also yung idrodrop sa every circle ADENINEDINUCLEOTIDE (ANTI-NAD)
lahat yun sinabi ni maam and yang sa baba hindi nabanggit
basta YUNG PROCEDURE NI CRP DETERMINATION SAME - A slide agglutination screening test for the
LANG HERE detection of antibodies against streptococcal
1. Allow each components of the test kit (reagents, antigens
controls) to reach room temperature. - positive in 95% of patients with acute
2. Gently shake the latex reagent to disperse the poststreptococcal glomerulonephritis because of
particles. GAS pharyngitis
3. Place a drop of undiluted serum into a circle of a - Sheep RBCs are coated with streptolysin,
test slide. streptokinase, hyaluronidase, DNase, and NADase
4. Add one drop of ASO Latex reagent next to the drop ** now we are going to do is that the reagent RBC wil be
of serum. mixed with 1:100 dilution of patient’s serum
5. Spread the reagent and serum sample over the ** (+) result – w/ hemagglutination
entire area of the test circle using a separate stirrer ** streptozyme test can be used in conjunction with anti
for each sample. streptolysin Obor anti-dnase b testing when sequelae of
6. Gently tilt the test slide backwards and forwards for group a streptococcal infections are suspected
two minutes. - Multi enzyme test, multiple antibody test
7. Observe and interpret the results for 2 minutes. - Principle: Passive or Indirect Agglutination
- Reagent: Sheep RBC
Note: o Preserved with Formalin, Formaldehyde or
• Positive and Negative Controls should be run at Aldehyde
regular intervals

Anti-Dnase B TestIng
- useful in patients suspected of having
glomerulonephritis preceded by streptococcal skin
infections
** bcuz usually guys, yung mga ASO antibodies are not
stimulated/ stimulated?? by these type of diseases yung
mga skin infections
** Dnase B- produced by group a streptococci kaya
ginagamit sya for testing of group a streptococci
sequalae
- Antibodies to DNase B: may be detected in
patients with acute rheumatic fever who have a
negative ASO test result
- based on a neutralization methodology
** meaning to say if the anti-dnase b antibody is present
so they will neutralize the reagent dnase b so they will
prevent it from depolimerizing dna
** and then the result will be measured by its effect on
the DNA-methyl green conjugate
** thi complex is green and intact in form however
kapag na neutralize sya ni Dnase the methyl green will
be reduced and they will become COLORLESS
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Syphilis
** In some cases, people affected with
syphilis fails to notice the lesions/sore
- most commonly acquired spirochete disease in because it is painless. Pwedeng natago rin
the US siya sa vagina tsaka sa rectum.
- it is a bacterial infection caused by spirochetes
- Causative agent: Treponema pallidum subs. ➢ Secondary Stage
pallidum o 6 to 8 weeks after chancres first appear
- Mode of Transmission: o Rashes will start to appear on the trunk,
o Sexual transmission (most common) then it will eventually cover the whole
o Transmission to the fetus is possible in body
mothers with clinically latent disease ▪ Rashes are not itchy
(mother to baby) ▪ Some people may experience
o Parenteral exposure hair loss, fever, sore throat,
and swollen lymph nodes
** Once infected with the bacteria, the disease will start as o generalized lymphadenopathy,
painless sore. May sore or lesion whether on the genital area, malaise, fever, pharyngitis, and rash on
rectum, or mouth – kung saan nagkaroon contact with the skin and mucous membranes, and
bacteria.
Involvement of the CNS.
** After those lesions, the bacteria can remain inactive for a
decade, then pwede siya maging active ulit. o lesions are highly contagious, but heal
** It can be cured using penicillin (an antibiotic) spontaneously within 2 to 6 weeks.
** If syphilis is not treated, it can lead to heart, brain, or other o Serologic tests are positive in
organs’ damage (life-threatening in some instances). secondary syphilis.
o Antibodies are mostly IgG.
- Detect antibodies against treponemal antigens
and nontreponemal cardiolipin antigens ➢ Latent Stage (hidden stage)
(Wasser-mann antigens) develop and elicit a o contagious and is generally considered
cell-mediated and humoral Immune response, to begin after the second year of
which results in the formation of immune infection.
complexes. ** very contagious sabi ni ma’am
** If hindi ka na-treat from syphilis, the
** Aside from the Treponema pallidum subs. pallidum that disease may progress from secondary
causes syphilis, we also have other treponemes: stage up to the latent stage.
o Characterized by lack of clinical
symptoms.
** There is no appearance of signs and
symptoms and it can last for years
** Wala nang lesions/chancre
o Serologic tests are still positive.
** Meaning, kahit wala na yung mga
- Tropic trepanematoses: lesions/chancre, present parin yung
o Pinta: caused by T. carateum bacteria sa katawan mo. So ‘pag
o Bejel: caused by T. endemicum nagperform ng serologic test, it will appear
(endemic syphilis) positive.
o Yaws: caused by T. pertenue (tropic ** Dark field microscopy is not advisable
granuloma) because of lack of clinical symptoms (nasa
transes, wala sa ppt)
- Incubation period: T. pallidum enters the body, ** When performing dark field microscopy
reaches the bloodstream, and is disseminated for syphilis, the site for specimen collection
to all organs. This early asymptomatic phase is the area of infection, wherein the clinical
lasts 10 days to 10 weeks symptoms (lesions/chancre) are present.
However, dark field microscopy is not
Stages of Syphilis advisable because the clinical symptoms
➢ Primary (Early) Stage are not present at this stage. (summary ng
explanation ni ma’am why ‘di advisable dark
o Initial lesion: chancre (small
field microscopy sa latent stage)
sore/lesion)
o Patients are non-infectious except for
▪ Lesions are painless
pregnant women who can pass the
o appear 2 to 8 weeks after infection and disease to the fetus
last for 1 to 5 weeks
o Serum tests for syphilis are positive in
➢ Tertiary Stage
90% of patients after 3 weeks. (IgM)
Genital area of male Genital area of female
o Occurs between 10-30 years after
secondary stage
o 3 Manifestations: (binanggit lang ni
maam ‘tong tatlong naka-bold)
** The sore will appear at the spot where the
bacteria entered your body.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

Gummatous syphilis (Gummas) – localized areas o can be found on patients with syphilis
of granulomatous inflammation that are most often - Principle: Flocculation – precipitation of very
found in bones, skin, or subcutaneous tissues fine particles that clump together
Cardiovascular disease – usually involve the
ascending aorta, symptoms are due to destruction
of elastic tissue, especially in the ascending and
transverse segment of the aortic arch.
Neurosyphilis – complication most often associated
- Positive within 1-4 weeks after the appearance
with the tertiary stage, but it can actually occur
of primary chancre.
anytime after the primary stage and can span all
- Components of Non-treponemal methods:
stages of the disease.
Cardiolipin, Cholesterol, and Lecithin
** affected brain (“neuro”)
Types of Non-treponemal Serologic Test
Venereal Disease Research Laboratory (VDRL)
- both qualitative and quantitative slide flocculation
test for serum
- Serum is heated at 56°C for 30 minutes to
inactivate complement
- Composition of VDRL Ag Reagent:
o 0.03% cardiolipin: main reacting
component
o 0.9% cholesterol: used to enhance the
** Inoculation→ Primary Syphilis (may lesions sa infected
area), if not treated→ Secondary Syphilis (may rashes)→ reacting surface of cardiolipin
Latent Syphilis (no appearance of signs and symptoms→ o 0.21% lecithin: removes the anti-
Tertiary Syphilis complement activity of cardiolipin

CONGENITAL SYPHILIS Procedures:

1. Sera and patient samples are spread out to
- Infection of the fetus causes late abortion,
fill the entire ring.
stillbirth, neonatal death, neonatal disease, or
2. One drop (1/60mL) of the VDRL antigen is
latent infection.
then added to each ring using Hamilton
- Outcome: depends on the stage of the mother's
syringe
disease
3. The slide is rotated for 4 minutes on a rotator
- Most newborns with congenital syphilis have no
at 180 rpm.
symptoms, although some babies can have
4. It is read microscopically to determine the
rashes on their hands and feet
presence of flocculation
- Diffuse maculopapular desquamatous rash Interpretation of results:
(particularly around the mouth and on the palms
• Reactive: medium to large clumps
and soles)
• Weakly reactive: small clumps
- Hemolytic anemia, jaundice,
hepatosplenomegaly, abnormal cartilage and • Non-reactive: no clumps
bone involvement, and mental retardation. Specimens used:
➢ Serum VDRL
o Size of ring: 14mm diameter
Laboratory Diagnosis o Ag delivery needle: 18 gauge needle
➢ CSF VDRL: used for testing neurosyphilis
Direct Detection o Size of ring: 16mm diameter
- Dark field microscopy or Fluorescent o Ag delivery needle: 21 or 22 gauge needle
Antibody testing o Rotate for 8 mins at 180 rpm
- Direct detection of spirochetes
Rapid Plasma Reagin (RPR)
- Primary and secondary syphilis can be
- modified VDRL test involving macroscopic
diagnosed
agglutination
- Not applicable in the latent stage
- RPR Antigen Reagent: similar to the VDRL
** kasi wala nang active lesion sa latent
antigen with the addition of the following:
- Requirement for direct detection: ACTIVE
o Charcoal for easier visualization
LESION
** Sa specimen collection requirement daw ‘to. o EDTA to prevent oxidation of lipid
** Active lesion kasi directly sa lesion magcocollect o Thimerosal which acts as a preservative
o Choline chloride which inactivates the
Serological Tests complement
** used in latent stage - Specimen: 0.05 mL of serum
1. Non-treponemal Serologic Test
- determine the presence of an antibody that Procedures:
forms against cardiolipin (Reagin) – lipid 1. Patient serum is placed in a test circle (18 mm)
material released from damaged cells on a plastic-coated disposable card.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)

2. Antigen is dispensed from a small plastic o 1+: minimally reactive, test must be
dispensing bottle with a calibrated 20-gauge repeated with a second specimen drawn in
needle. 1-2 weeks
3. The card is mechanically rotated for 8 mins at o 2-4+: REACTIVE
100rpm. Hemagglutination Treponemal Test for Syphilis
4. Cards are read under a high-intensity light - Rgt antigen: glutaraldehyde stabilized turkey
source. RBC coated with treponemal antigen
- Microtiter well is used
Interpretation of results: ** May gelatin sa mga microtiter wells
• Reactive: with clumps ** Then we’re going to add glutaraldehyde stabilized
• Non-reactive: no clumps turkey RBC coated with treponemal antigen
- Principle: indirect / passive hemagglutination
** Indirect/passive because the treponemal antigen
2. Treponemal Serologic Test
will be coated with RBC.
- detects antibody directed against the T.
pallidum organism or against specific
Treponema pallidum Immobilization Test
treponemal antigens
- Standard test
- 100% reactive in secondary and latent syphilis
- Principle: the antibody produced vs T. pallidum +
- 2 Sources of Ag:
complement can immobilized the live
o Non-pathogenic Reiter strain
treponemes
o Pathogenic Reiter strain
- Rgt antigen: live actively motile T. pallidum
organisms
Types of Treponemal Serologic Test
- Positive Result: >50% immobilized
Fluorescent Treponemal Antibody Absorption treponemes
Test (FTA-ABS)
- Principle: Indirect Immunofluorescence
Treponema pallidum Particle Agglutination
- one of the earliest confirmatory test
(TP-PA) Test
- Highly sensitive and specific, but it is time
- use colored gelatin particles coated with
consuming to perform
treponemal antigens
o Rgt antigen: Nichol’s strain dried and
- More sensitive in detecting primary syphilis
fixed on slide
o Absorbent: Reiter treponemes
Traditional testing algorithm for syphilis
Procedures:
1. Slides: contains Nichol’s strain (Ag) of T.
pallidum
2. Specimen: 1:5 heat inactivated serum (Ab)
reacted with sorbent containing non-pathogenic
treponemes (Reiter strain)
3. Incubated at 37°C for 30 minutes
** During incubation nagkakaroon ng time to bind
yung antibody kay antigen. ** For the diagnosis of syphilis, ang first na ginagawa ay initial
4. Washed with deionized water then rinse with screen using the non-treponemal test (either RPR or VDRL).
phosphate-buffer saline for 5 minutes Nowadays, RPR is commonly performed.
** Wash para matanggal yung excess antigen ** Even sa pregnant women, may lab tests na ginagawa aside
5. Add (antihuman immunoglobulin conjugated with from the lipid profile, urinalysis, CBC, etc. Nagpapatest din sila
fluorescein I isothiocyanate label) for different diseases, like STD, Hepatitis B, HIV, and even the
** Add antibody conjugate (sabi ma’am) RPR.
6. Incubated at 37°C for 30 minutes ** Kapag sa RPR ang result is non-reactive it means negative,
7. Wash negative na rin sa syphilis. Wala na additional testing na
gagawin.
8. Examine under fluorescent microscope
** Kapag nag-reactive ka for the RPR, possible na may
syphilis. We are going to perform treponemal test for
Interpretation of results: confirmation.
• Positive result: green fluorescence ** If negative sa treponemal test, possible na mayroon lang
** Presence of antibody = fluorescence false-positive reactions na nagyari during the RPR test. If
** Kapag wala antibody, walang magbabind sa reactive naman, that’s the time na ittreat ka na from syphilis
antigen, and wala rin pagbabindan yung fluorescent (like penicillin).
dye or antibody conjugate. So, under the microscope
ay walang makikita. ** Video (33:06-40:27) of RPR procedure (same lang sa
procedure rito mas detailed lang).

• REPORTED on a scale of 0-4+

o 0: no fluorescence (non-reactive)
 Dengue, Hepatitis, and HIV
IMMUNOLOGY AND SEROLOGY (LABORATORY)

- mosquito borne viral infection.

- Caused by Flavivirus (Flaviviridae family)
- causes flu like illness, and occasionally develops
into a potentially lethal complication called
severe dengue.
o Dengue hemorrhagic fever
o Dengue shock syndrome - During the onset of symptoms, there will be a
- Transmission: transmitted to humans through high level of virus inside the body
the bites of infected female mosquitoes - NS1 antigen is also detected during the first to
- Vectors: Aedes aegypti and Aedes albopictus ninth day of infection
o Aside from dengue, these vectors can o Positive in NS1 dengue
also transmit chikungunya, yellow fever, - Little by little, antibodies will be developed
and zika virus. (IgM)(red curve in the illustration above)
- Once we recover, IgG will then increase (green
Aedes aegypti curve in the illustration above)
- primary vector of dengue LABORATORY DIAGNOSIS
- After virus incubation for 4-10 days, an infected  Dengue IgG/IgM
mosquito is capable of transmitting the virus for o Antibody Capture Test (Solid Phase
the rest of its life. Immunochromatography)
- a day-time feeder, its peak biting periods are o Dengue IgM antibodies indicates
early in the morning and in the evening before primary infection, which appears 5-10
dusk. days after infection
- Can also cause Chikungunya, Yellow fever, and o Dengue IgG antibodies indicates past
Zika infection infection, which appears 4-5 days after
infection
Aedes albopictus Notes:
- secondary dengue vector IgG- past dengue infection
- Its spread is due to its tolerance to IgM- present/acute dengue infection
temperatures below freezing, hibernation, and
ability to shelter in microhabitats.

CHARACTERISTICS
 a severe, flu like illness that affects infants,
young children, and adults, but seldom causes
death (severe cases)
 Symptoms usually last for 2-7 days, after an
incubation period of 4-10 days after the bite ** This is a Solid phase Immunochromatography
from an infected mosquito. ** Inside it is a nitrocellulose membrane and coated
 Suspected Dengue: when a high fever antibodies
(40°C/104°F) is accompanied by two of the Square: sample well; where we drop the sample
following symptoms: Circle: where we add dengue diluent
o severe headache -Upon addition of sample and dengue diluent, it
o pain behind the eyes will migrate up to the end.
o muscle and joint pains -Refer to the illustration above for the specific
o Nausea antibodies present in each red line
o Vomiting - If the antigen is present, it’ll react to
o swollen glands antibodies present in nitrocellulose membrane
o rash forming a pink/purple solid line

SEROTYPES IgM: can appear 5-10 days after infection

 DENV-1 IgG: can appear during 4-5 days after infection
 DENV-2
 DENV-3
 DENV-4
**were not elaborated

DENGUE INFECTIONS
 Primary Dengue Fever Infection
 Secondary Dengue Fever Infection
 Dengue Hemorrhagic Fever
 Dengue Shock Fever
o Dengue hemorrhagic and dengue shock
fever are the severe cases of dengue
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
Test procedure as seen in the video:  Primary hepatitis viruses: A, B,
1. Draw the blood specimen using the provided disposable C, D, E, and GB virus C
dropper.  Secondary hepatitis viruses:
2. Add 1 drop into the sample well (square) of the NS1 Epstein Barr virus (EBV),
device. cytomegalovirus (CMV),
3. Load the specimen into the sample well (square) of the herpesvirus
IgG/IgM device.
4. Add 2 drops of NS1 assay buffer into the sample well
(square) of NS1 device
5. Add 3-4 drops of IgG/IgM assay buffer into the buffer
well (circle) of IgG/IgM device
6. Read result w/in 20 mins

● Whether negative or positive, there should be a

line in your control region. If none, this is invalid.
● Presence of line in G region: IgG positive (past
dengue infection) HEPATITIS A
● Presence of line in M region: IgM positive (acute - “Infectious Hepatitis”
dengue infection) - Non-enveloped, single stranded RNA virus
● Presence of line in G,M, and control region: - Belongs to the genus Hepatovirus of the
convalescent phase (recovery phase) Picornaviridae family
● Absence of line anywhere but control region: no - Transmission: Fecal Oral (contaminated foods,
history of dengue water), Blood Transfusion (Rare)
- Average Incubation Period: 28 days
- They can produce symptoms of acute hepatitis
in the majority of infected adults
- Most infections in children are asymptomatic
- Does not progress to a chronic state
- It is usually self-limiting
- Symptoms typically resolving within 2 months
- Treatment: supportive (bed rest, nutritional
support, medication for fever, nausea)
 Dengue NS1 Ag Test - Vaccination: formalin killed HAV
o Antibody Capture Test (Solid Phase o Administered within 2 weeks of
Immunochromatography) exposure
o Same test kit but different antibodies
attached in nitrocellulose membrane Serological and
Clinical Significance
o Specimen used: serum, whole blood Molecular Markers
o If antigen is present, it’ll react on NS1 IgM anti-HAV Acute HAV
Total anti-HAV Immunity to Hepatitis A
antibody HAV RNA Detection of HAV in clinical food or
water samples (molecular assays)

HEPATITIS B
- The virus responsible is a DNA virus, belonging
to the Hepadnaviridae family
- Transmission: parenteral (contact with
contaminated blood), sexual contact, blood
transfusion, and perinatal (from infected
mother to infant)
- Vaccines: Recombinant Hepatitis B Surface
Antigen (HBsAg) – administered during 0, 1,
- Inflammation of the liver and 6 months
- Early and acute stages of hepatitis can be o Has booster dose after 5 years
characterized as flu-like symptoms, usually mild - Incubation period: 40-180 days (according to
to moderate pain, in the upper right quadrant Turgeon) and 30-180 days (according to Stevens)
of the abdomen - Chronic infection results in inflammation and
o Upper right abdomen: where liver is damage to the liver , increased risk of
located developing cirrhosis or hepatocellular
- It can lead to hepatomegaly (liver enlargement), carcinoma (cancer)
jaundice (yellowing of the skin and eyes), dark
urine, and light feces Hepatitis B Markers
o because ofelevation of bilirubi n and  HBsAg – active hepatitis B infection
other liver enzymes. o Hepatitis B surface antigen
- Etiology o first marker that appears at the end of
o Viral hepatitis is the most common liver the incubation period
disease worldwide. o indicator of acute infection or chronic
o Divided into two major groups: infection with unresolved antigenemia
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
 HBeAg – active hepatitis B with high degree of
infectivity
o Hepatitis B envelop antigen
o indicates active viral replication
o HBV is most infectious when the Be
8antigen is detectable. Test for Hepatitis B Surface Antigen (HBsAg)
REMEMBER:
 Antibody must be specific to the antigen
 If antigen produces HbsAg, body will produce Anti-
Hbs antibody

 Anti-HBc
o "core window“
o peaks at the end of the acute infection
stage after HBsAg is no longer
detectable and before antibody to anti-
HBs can be detected.
 Anti-Hbe – indicates recovery from hepatitis B
o appear shortly after the antigen
disappears.
o Their presence in patients with acute
hepatitis B suggests that the infection is Specimen used: plasma, serum, or whole blood
resolving. Test procedure as seen on the video:
o indicates that the infection is resolving 1. Place the test device on the clean and level surface
or that there is no complicating liver 2. Add 2 drops of serum/plasma (50uL) into sample well
disease. may be seen in chronic 3. Read result w/in 20 mins.
asymptomatic carriers. Interpretation:
 Anti-HBs – indicates immunity to hepatitis B Positive: 2 purple color band at position C and T
o It appears at the end of the acute stage Negative: 1 purple color band at position C
and the beginning of the recovery stage.
o Its concentration peaks, then plateaus
during recovery and never disappears. HEPATITIS C
o Can be detected if a person has vaccine - Previously classified as “nonA-nonB” Hepatitis
 Marker that appears as positive - Belongs to the family Flaviviridae and the genus
when we have vaccine against Hepacivirus
Hepa B. - Transmission: contaminated blood, parenteral,
sexual, perinatal
- Also known as posttransfusion hepatitis
o patients that undergo blood transfusion
- Incubation period: 7 weeks (range is 2 to 30
weeks)
- 85% of patient can develop chronic infection
- Majority of the infections are asymptomatic,
which may develop to cirrhosis
- Patients may also develop hepatocellular
carcinoma.

Serological and
Clinical Significance
Molecular Markers
Anti-HCV Current or past hepatitis C infection
HCV RNA Current hepatitis C infection

HEPATITIS D
- Also known as delta hepatitis
- The only member within the Deltavirus genus
- It is a defective virus
- Transmision Mostly parenteral, but also sexual,
perinatal; HBV infection required (for
replication)
- Coinfection: occurs when patients acquire HBV
and HDV infections simultaneously.
(magkasabay)
- Superinfection: occurs in patients with an
established HBV infection who acquire HDV
*Table: diniscuss lang ni ma’am as is. infection (HBV first then HDV)
o superinfections can occur and progress
to chronic HBV/HDV infection.
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
- Treatment: Combination of interferon alpha
and antiviral drugs

Serological and
Clinical Significance
Molecular Markers
IgM anti-HDV Acute or chronic hepatitis D
IgG anti-HDV Recovery from hepatitis D or chronic
hepatitis D
HDV RNA Active HDV infection
Characteristics of HIV
HEPATITIS E  Composition of the virus:
- Belongs to genus Hepevirus in the family o Belongs to the genus Lentivirinae of the
Hepeviridae virus family Retroviridae
- Transmission: consumption of infected pork and o Classified as Retrovirus
possibly by direct contact with infected animals  Contains ribonucleic acid (RNA)
or fecally contaminated water, and blood as its nucleic acid and a unique
transfusion enzyme called reverse
o Fecal-oral just like Hepa A transcriptase, which transcribes
- Incubation period: 2 to 6 weeks RNA into DNA
- It can lead to complications:  Three Main Structural Genes:
o Fulminant hepatitis – liver failure may o Gag: codes for p55
occur. It is common among pregnant o Env
women o Pol

Serological and
Clinical Significance
Molecular Markers
IgM anti-HEV Current hepatitis E infection
IgG anti-HEV Current or past hepatitis E infection
HEV RNA Current hepatitis E infection

- Etiologic agent of acquired immunodeficiency

syndrome (AIDS)
o The virus causing AIDS (HIV 1) was
identified independently by the
laboratories of Luc Montagnier , Robert
Gallo and Jay Levy
o Two serogroups:
 HIV-1 is the predominant strain,
and it is found worldwide.
 HIV-2 is limited primarily to
West Africa.
- HIV targets T cells, specifically CD4
- Robert Gallo called HIV as Human T-
lymphotropic Virus (Type III) during their time
- Luc Montagnier named HIV as *Table: diniscuss lang ni ma’am as is.
Lymphadenopathy Associated Virus
- HIV target T-cells specifically CD4 Viral Replication
- HIV 1 Subtypes:
o Group M: main or major group
o Group O: outlier group
o Group N
- Transmission:
o Intimate sexual contact
o Parenteral: Contact with blood or other
body fluids (wounds/abrasion)
o Perinatally: from infected mother to
infant
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
- The virus will attach to the susceptible host cell, Clinical Symptoms of HIV
which targets the CD4 antigen present in T cells,  Acute or Early Stage – usually appear 3-6 weeks
as well as in monocyte and macrophage o Many patients are asymptomatic,
o HIV viruses that infect T cells = T-tropic showing mild chronic lymphadenopathy
or X4 strains o Can last from many month to years
o Both macrophages and T cells = M- after the viral exposure
tropic or R5 strains o Viremia – presence of virus in the blood
- HIV surface has gp120 glycoprotein which  detectable
initiate the attachment to the host cell o Acute retroviral syndrome may develop
- Co receptors: o Flu like or infectious mononucleosis like
o CXCR4 = required for HIV to enter T symptoms
lymphocytes  Period of clinical latency
o CCR5 = required for entry into o Decrease in viremia (decrease virus in
macrophages blood)
- Once they bind to the coreceptors, the viral o Patient will become asymptomatic, but
particle will be taken into the cell  uncoating the virus is still present
of the particle  Final Stage
- Reverse transcriptase produces complementary o Profound immunosuppression with very
DNA and will convert the RNA into a proviral low numbers of CD4+ T cells
DNA o Results to AIDS
- The proviral DNA will be integrated into the
genomic RNA, and they will be transported into
ACQUIRED IMMUNODEFICIENCY
the cytoplasm
- New virus particles will be produced
SYNDROME (AIDS)
- New viral RNA will be used as the genomic RNA - a disease, atleast moderately predictive of a
to make the viral proteins and replication starts. defect in cell mediated immunity, occurring in a
person with no known cause for diminished
SUMMARY: resistance to that disease
1. The virus will attach to the CD4 membrane - can have opportunistic infections
receptor and will shed its protein coat to expose
RNA core. GP120 will bind to CD4 and to co-
receptors (CXCR4 and/or CCR5)
2. Enzyme reverse transcriptase will convert RNA
into proviral DNA.
3. Proviral DNA will be integrated into the genome
and new virus particles will be produced
4. Replication starts

Immune Response and HIV Immunologic Manifestations

 Serologic effects - Increased level of p24 antigen will be the
o Antibodies to HIV generally appear indication of the initial viral replication
about 12 weeks after infection. - Antibodies to be detected:
o Neutralizing antibodies: appear about 1 o gp41 transmembrane glycoprotein
year after infection. o gag proteins
 These neutralizing antibodies o Env
can interfere with the infection o Pol
of the host cell o regulatory proteins
o HIV is able to escape the immune LABORATORY TESTING FOR HIV INFECTIONS
response by undergoing antigenic
Serological Tests
variation
 Effect on T cells  ELISA – standard screening method and easy to
o As the disease progresses, there is a perform, can be adapted to test a large number
depletion of CD4+ T helper cells. of samples, and are highly sensitive and specific
 AIDS: CD4 count is < 200/mm3 o Confirmatory test: Western Blot or
Immunoblot – At least two of the
 Because of this, you can
following three bands are present: p24,
acquire opportunistic
gp41 and gp120 / gp160
infections like
pnuemonia, TB, retinitis.
 Normal: CD4 count is
1000/mm3
o HIV compromises the immune response
by destroying T helper cells.
o The ratio of CD4 to CD8 cells is reduced
from 2:1 (normal)
 IMMUNOLOGY AND SEROLOGY (LABORATORY)
First generation of ELISAs
- based on a solid phase, indirect assay system that detected Qualitative Nucleic Acid Tests (NATs)
antibodies to only HIV-1 - used to determine whether or not a detectable
- HIV antibodies in patient serum were detected after binding
to a solid support coated with viral lysate antigens from HIV-
level of HIV nucleic acid is present in human
1 cultured in human T cell lines, followed by adding an plasma
enzyme labeled conjugate and substrate.
- Prone to false positive results
TREATMENT AND PREVENTION
Second generation ELISAs
- indirect binding assays that used highly purified recombinant  Supportive care
or synthetic antigens from both HIV-1 and HIV-2, rather than  Antiretroviral therapy: nucleoside analogue
crude cell lysates. reverse transcriptase inhibitors, nonnucleoside
- improved specificity and sensitivity overall and were able to
detect antibodies to both HIV-1 and HIV-2. reverse transcriptase inhibitors, protease
Third generation assays inhibitors, integrase inhibitors, fusion inhibitors,
- use the sandwich technique, based on the ability of antibody entry inhibitors
to bind with more than one antigen.  Combination antiretroviral therapy (CART) or
- This format improved sensitivity by simultaneously detecting
HIV antibodies of different immunoglobulin classes, including
Highly active antiretroviral therapy (HAART)
IgM combination of drugs and at least 2
Fourth generation assays antiretroviral drug classes
- can simultaneously detect HIV-1 antibodies, HIV-2
antibodies, and p24 antigen(active viral replication)
“Detuned” assays
TESTING OF INFANTS AND YOUNG CHILDREN
- ELISAs with reduced sensitivity serological tests are not reliable
- used to differentiate a recent HIV infection from a more  Qualitative HIV-1 DNA PCR
established infection.
o preferred method
- Individuals with more recent infection (< 4 months) will give
a positive result only with the standard highly sensitive ELISA o detects proviral DNA within the infants’
- These individuals can have a negative results in detuned peripheral blood mononuclear cells
assays  Quantitative HIV RNA assays
 CD4 T-Cell Enumeration – played a central role o used to diagnose HIV infection in infants
in evaluating the degree of immune suppression and young children and are more likely
in HIV infected patients for several years. to detect infections with strains other
o Determine the number of CD4+ T cells than subtype B.
o Flow cytometry – gold standard for o Testing for p24 antigen is not
enumerating CD4 T cells recommended, as it has a low
 IFA (indirect immunofluorescence assay) – sensitivity in infants
antibodies are detected to HIV antigens  Nucleic acid tests for HIV
expressed on the surface of infected T cell lines o performed in infants with known
fixed onto a glass slide. perinatal exposure at the ages of 14 to
o Advantage: able to detect antibodies to 21 days, 1 to 2 months, 4 to 6 months,
many HIV antigens but requires a well and possibly at birth.
maintained fluorescent microscope and o A positive test result should be
subjective interpretation of the results. confirmed by repeat testing.
o T-cell line incorporated onto a slide, add o Negative test results provide
antibodies (patient’s serum), then add presumptive evidence for the absence
antibody conjugate. Antibody of HIV infection and should be
fluorescent conjugate then binds if confirmed by serological tests at 12 to
there is a presence of antigen-antibody 18 months of age
binding
 RIPA (radioimmunoprecipitation assay) – uses
minimally denatured antigens but requires
radioactive materials
o its use is limited to research
laboratories that can maintain HIV in
cell cultures
 Line immunoassays – are similar to Western
blots (uses test strips) except that recombinant
or synthetic HIV proteins are applied to the test
strips, rather than being electrophoresed from
cultured virus preparations.
o These assays have allowed for easier
interpretation because of fewer bands
and false positive results.
 p24 Antigen Testing
o detects p24 antigen from the core of
the HIV-1 virion
o high in the initial weeks of infection
during the early burst of viral replication
o used to achieve earlier diagnosis, detect
HIV infection in newborns, and monitor
patients on antiretroviral therapy.
 PRECIPITATION  Avidity

 Involves combining soluble antigen with soluble antibody o Represents the overall strength of antigen-antibody
to produce insoluble complexes that are visible (precipitate) binding and is the sum of the affinities of all the individual
 First noted in 1897 by Kraus, who found that culture filtrates antibody-antigen combining sites
of enteric bacteria would precipitate when they are mixed with o Strength in which a multivalent antibody binds a
specific antibodies multivalent antigen and is a measure of overall stability
of an antigen-antibody complex
o The more bonds that form between antigen and
antibody, the higher the avidity is.
o Once the binding occurs, there is force that keeps the
molecules together.

LAW OF MASS ACTION

 This law states that free reactants are in equilibrium with

bound reactants

 In order to observe visible reactions, it must have specific

antigen and antibody, so they must fit together.
 Both soluble antigen and soluble antibody must have binding
sites and their concentrations must be equal.
 The equilibrium constant is thus:
Antigen-Antibody Binding
K= K1/K2 = [AgAb]/[Ab][Ag]
 AFFINITY
o Initial force of attraction that exists between a single K = stands for equilibrium
Fab site on an antibody molecule and a single epitope
K1 = forward reaction (antigen-antibody complex)
(found on the antigen like bacterial cell) or determinant
site on the corresponding antigen K2 = reverse reaction (free antigen and free
o The more the cross-reacting antigen resembles the antibody)
original antigen, the stronger the bond will be between
the antigen and the binding site Forward reaction – Antigen will bind to the antibody forming antigen
 Cross-reactivity antibody complex
o Antibodies are capable of reacting with antigens that are
structurally similar to the original antigen that induced Reverse reaction – antigen antibody will dissociate to form a single
antibody production (not the true antigen) antigen and a single antibody (antigen-antibody complex will dissociate
o When the affinity is higher, the assay reaction is more forming FREE antigen and FREE antibody)
sensitive because more antigen-antibody complexes
will be formed and visualized more easily Where [AgAb] = concentration of the antigen-antibody complex (mol/L)
o Binding between the structurally similar antigen is not
[Ab] = concentration of antigen (mol/L)
that strong compared to the original antigen
[Ag] = concentration of antigen (mol/L)

 PRECIPITATION CURVE
o Equal amount of antigen and antibody can be found
on the zone of equivalence (where optimum
precipitation occur)
 Will have visible reactions or stable
network or lattice
 Precipitation was formed because of
equal amount of antigen and antibody
o Prozone reaction
 Excess in Ab compared to antigen
  No precipitation observed
 Can lead to false negative
reaction due to high A
  Remedy: dilute the antibody PASSIVE IMMUNODIFFUSION TECHNIQUES
and perform the test again
because in dilution, there can
be decreasing amount of the
antibody to meet the zone of
equivalence
o Postzone reaction
 Excess in antigen compared to Ab
 No precipitation observed
 Remedy: do not dilute since dilution is
only performed on antibody, repeat the
test after a week instead so the patient
can produce equal amount of antigen
and antibody
 Agar and agarose (serve as supporting medium) help stabilize
PRECIPITATION CURVE
the diffusion process and allow visualization of the precipitin
bands
 Antigen and antibody are added to wells in the gel and
antigen-antibody combination occurs by means of diffusion
 No electrical current is used to speed up the process
 The rate of diffusion is affected by:
o Size of the particles
o Temperature
o Gel viscosity
o Amount of hydration

1. RADIAL IMMUNODIFFUSION

 Single diffusion technique (Ag is only added)

o Antibody is uniformly distributed in the support gel
MEASUREMENT OF PRECIPITATION BY LIGHT and antigen is applied to a well cut into the gel
o As the antigen diffuses out from the well, antigen-
SCATTERING
antibody combination occurs in changing
 Turbidimetry proportions until the zone of equivalence is reached
o a measure of the turbidity or cloudiness of a and stable lattice is formed in the gel
solution o The area of the ring obtained is a measure of
o The more turbid, more analytes are present in antigen concentration, and this can be compared to
the solution a standard curve obtained by using antigens of
o measures the reduction of light intensity caused by known concentration
reflection, absorption, or scatter
o The amount of scatter is proportional to the size,
shape, and concentration of molecules present in
solution
o Measurements are made using a
spectrophotocometer or an automated clinical
chemistry analyzer
 Nephelometry
o Measures the light that is scattered at a
particular angle from the incident beam as it passes
through a suspension
o End point nephelometry – the reaction is allowed
to run essentially to completion, but large particles
tend to fall out of solution and decrease the amount
of scatter
o Kinetic or rate nephelometry – the rate of
scattering increase is measured immediately after
the reagent is added

o Technique was developed by James Oudin

o (+): precipitin ring
 End-point method
 o Antigen is allowed to diffuse to completion, and (C) fusion of two lines with a spur indicates partial identity.
when equivalence is reached, there is no further
change in the ring diameter ◦ two antigens share a common epitope, but some
o This occurs between 24 and 72 hours antibody molecules are not captured by antigen and
o The square of the diameter is then directly
proportional to the concentration of the antigen
o Developed by Mancini
 Kinetic Method
o Uses measurements taken before the point of
equivalence is reached
o The diameter is proportional to the log of the
concentration
o Readings are taken at about 18 hours
o Developed by Fahey and mcKelvey

Sources of Errors:
1. Overfilling and underfilling of wells
2. Nicking the side of the wells when filling
3. Spilling sample outside the wells
4. Improper incubation time and temperature
Incorrect measurement

2. OUCHTERLONY DOUBLE DIFFUSION

 Both antigen and antibody diffuse independently through a

semisolid medium in two dimensions, horizontally and travel through the initial precipitin line to combine
vertically. with additional epitopes found in the more complex
 Wells are cut in a gel, and reactants are added to the wells. antigen.
 After an incubation period of between 12 and 48 hours in a
moist chamber, precipitin lines form where the moving front of ◦ Therefore, the spur always points to the simpler
antigen meets that of antibody. antigen

 It is called double diffusion because both antigen and antibody

are added
 (+): precipitin lines
 Several patterns are possible:

(A) Fusion of the lines at their junction to form an arc represents

serological identity or the presence of a common epitope,

(B) a pattern of crossed lines demonstrates two separate

reactions and indicates that the compared antigens share
no common epitopes
 ELECTROPHORESIS TECHNIQUES

 Electrophoresis
o Separates molecules according to differences in
their electric
o charge when they are placed in an electric field.
o A direct current is forced through the gel, causing
antigen, antibody, or both to migrate.
o migration of charged solutes or particles in an
electrical field
o As diffusion takes place, distinct precipitin bands
are formed.
o Electric current is used in order to speed up the
reaction

1. ROCKET IMMUNOELECTROPHORESIS  Positive result: Lines or arcs

 One-dimension electroimmunodiffusion – developed by

Laurell
 Electrophoresis is used to facilitate migration of the antigen
into the agar. When the antigen diffuses out of the well,
precipitation begins.
 End result: precipitin line

3. IMMUNOFIXATION ELECTROPHORESIS

 Similar to immunoelectrophoresis except that after

electrophoresis takes place, antiserum is applied directly to
the gel’s surface rather than placed in a trough.
 Developed by Alper and Johnson
 Uses agarose or cellulose
 The unknown antigen is placed on the gel, electrophoretic
separation takes place, and then the reagent antibody is
applied.

2. IMMUNOELECTROPHORESIS Sources of Error in Electrophoresis

 Application of current in the wrong direction
 Incorrect pH of the buffer
 Double-diffusion technique that incorporates electrophoresis  Incorrect electrophoresis time
current to enhance results.  Concentrations of antigen and antibody
 Developed by Grabar and Williams  Amount of current applied
 Source of the antigens: SERUM
 Antiserum is placed in the trough, and the gel is incubated for
18 to 24 hours.
TABLE: SUMMARY OF TECHNIQUES
  a much slower process than the sensitization phase.
 Methods of Enhancing Agglutination
AGGLUTINATION o Centrifugation
o Treatment with proteolytic enzymes
 process by which particulate antigens such as cells o Use of colloids
aggregate to form larger complexes (agglutination) when a o AHG testing
specific antibody is present.
 Gruber and Durham published the first report about the TYPES OF AGGLUTINATION REACTIONS
ability of antibody to clump cells, based on observations of
agglutination of bacterial cells by serum. 1. Direct Agglutination
 Widal and Sicard developed one of the earliest diagnostic
◦ occurs when antigens are found naturally on a particle
tests in 1896 for the detection of antibodies occurring in
typhoid fever, brucellosis, and tularemia. ◦ true antigen is used

STEPS IN AGGLUTINATION ◦ Widal Test (used to diagnose Salmonella typhi so we use True
Salmonella Ag)
 Sensitization
o Involves antigen–antibody combination through ◦ Weil-Felix Test (Proteus Ag)
single antigenic determinants on the particle and is
rapid and reversible 2. Passive Agglutination/Indirect Agglutination
o Antibody will bind to the antigen
o No reaction observed or no agglutination ◦ employs particles that are coated with antigens not
 Lattice Formation normally found on their surfaces.
o formation of cross-links that form the visible
◦ A variety of particles, including erythrocytes, latex,
aggregates.
gelatin, and silicates, are used for this purpose
o represents the stabilization of antigen– antibody
complexes with the binding together of multiple 3. Reverse Passive Agglutination
antigenic determinants
o Visible agglutination ◦ antibody rather than antigen is attached to a carrier
particle. (like latex, erythrocytes, silicates)

◦ CRP Latex Test

4. Agglutination Inhibition

◦ based on competition between particulate and soluble

antigens for limited antibody- combining sites, and a lack of
agglutination is an indicator of a positive reaction.

◦ On the patient’s sample, we are able to detect the antigen and

upon the presence of reagent antibody and they will bind to
each other
SENSITIZATION
◦ We will add antigen coated particles and since the antigen and
 represents the physical attachment of antibody molecules to reagent antibody are binded, antigen coated particles cannot
antigens on the erythrocyte membrane bind (walang pagbabind-an) so no agglutination happens,
 FACTORS THAT INFLUENCE SENSITIZATION: there is competition
o Particle Charge
 RBCs have slight negative charge (zeta ◦ If there is no antigen, nothing will bind on the reagent
potential) antibody; then the presence of antigen coated particles will
 Centrifugation: cells will aggregate to the bind to the reagent antibody (since there is no antigen of the
bottom patient); no competition; will form lattice formation
o Antibody type
5. Coagglutination
 IgM antibodies are pentamer; they have larger
size, they will have strong agglutination ◦ name given to systems using bacteria as the inert particles to
o Antigen-Antibody ratio which antibody is attached.
o pH (must have an optimum ph of 6.5 – 7.5)
o Temperature and length of incubation. ◦ Staphylococcus aureus is most frequently used because of
 IgM for cold temperature the presence of Protein A
 IgG for warm temperature

LATTICE FORMATION

 the establishment of cross-links between sensitized particles

and antibodies, resulting in aggregation
COMPARISON OF AGGLUTINATION REACTIONS
 Labeled Immunoassay
IMMUNOLOGY AND SEROLOGY (LECTURE)

- (Recall) Unlabeled immunoassays: precipitation DETECTION OF THE LABEL

and agglutination - For Radioimmunoassays: involves a system for
o Simple techniques to be performed counting radioactivity,
o Uses different equipment - For other labels such as enzymes, fluorescence,
o Needs to have enough concentration of or chemiluminescence: typically a change in
unknown to visualize the reaction absorbance in a substrate is measured by
** we have learned that precipitation and agglutination spectrophotometry and fluorescence microscope
are very simple techniques to perform. We have to use (for fluorescence)
different equipments and enough concentration of
unknown in order to visualize the reaction (precipitation, QUALITY CONTROL
clumping, agglutination) - Run a blank tube, usually phosphate buffered
- Labeled immunoassay is designed for antigens saline, with every test. (To check the quality of
and antibodies that may be small in size or reagents)
present in very low concentrations. - A negative control and a high and a low positive
** we only have a small size/concentration of either control should be run in addition.
antigen or antibody - This serves as a check on the quality of the
** labelled coz we use labels reagents to make sure the label is readily
- The presence of such antigens and antibodies is detectable under current testing conditions.
determined indirectly by using a labeled
reactant to detect whether or not specific
binding has taken place
o Label used in labeled immunoassays:
enzyme, radioisotopes, fluorochrome, COMPETITIVE IMMUNOASSAYS
and chemiluminescence probes
- All reactants are mixed together simultaneously;
- Analyte – substance to be measured
labelled antigen competes with unlabelled
o Example: bacterial antigens, tumor
patient antigen for a limited number of antibody-
markers, different immunoglobulins,
binding sites.
hormones, and drugs
** May competition, we’re going to add the reactants
** since we have the analyte, we have to use another
(labelled antigen and unlabelled patient antigen or
molecule that will react to the analyte. Kung ang analyte
unknown)
is antigen, gagamit ng antibody to have a reaction
** if we add them together, they will have an competition
** REMEMBER: the antigens and antibodies should be
- The amount of bound label is inversely
specific
proportional to the concentration of the labeled
ANTIBODIES antigen.
- high affinity for the antigen - If there are different antigens that bind to the
- the higher the affinity of antibody for antigen, antibody, there will be no reaction detected
the larger the amount of antigen bound to o Patient antigen is present
antibody and the more accurately specific
binding can be measured.
- The ultimate sensitivity of the immunoassay, in
fact, depends largely on the magnitude of affinity.

STANDARDS OR CALIBRATORS
- are unlabeled analytes that are made up in
known concentrations of the substance to be
measured.
- Used as controls
- used to establish a relationship between the
labeled analyte measured and any unlabeled
analyte that might be present in patient
specimens
** EX. Our solid phase vehicle, we have here the
SEPARATION METHODS antibody (solid phase bound antibody) nakaincorporate
- Solid phase vehicle: na sa solid phase vehicle yung antibody. We have two
o polystyrene test tubes type of antigen to be added (labeled and unlabeled
o microtiter plates antigen). For the competitive immunoassay, we’re going
o glass or polystyrene beads to add it simultaneously (sabay). Pag inadd, magkakaron
o magnetic beads ng competition at maguunahan sa pagbind kay limited
o cellulose membranes (nasa loob ng mga antibody, it’s either on one side, nandyan yung labeled
test kits) analyte and other side nandon yung unknown. Kapag
- The bound and unbound fractions are usually ganyan ang result, magkaiba yung antigen sa nagbind,
separated by physical means, including kaya walang madedetect na color change/reaction (dahil
decanting, centrifugation, or filtration. magkaiba)
- This is followed by a washing step to remove ** the signal strength is usually inversely
any remaining unbound analyte proportional/related (kapag walang nadetech na color
 IMMUNOLOGY AND SEROLOGY (LECTURE)
change/reaction/positive ibig sabihin present ang - rapid and sensitive method that can be used to
patient’s antigen dahil nagkaron ng competition) detect small amounts of antigen or antibody.
** sample A, let’s assume na walang patient’s antigen or - Valuable in measuring a number of substances
negative ang result ni patient, walang kacompetition si such as hormones, serum proteins, and vitamins
labeled analyte. Magkakaobserve tayo ng reaction. that either occur at very low levels in blood
plasma or are so small that they could not be
NONCOMPETITIVE IMMUNOASSAY detected otherwise.
- Antibody, often called capture antibody , is - Radioactive substances as a label
first passively absorbed to a solid phase. o They have a nucleus that will decay
Unknown patient antigen is then allowed to react spontaneously (will emit matter and
with and be captured by the antibody. After energy)
washing to remove unbound antigen, a second o 125-I (Iodine 125) – most commonly
antibody with a label is added to the reaction. used radioactive label
** yung antibody/solid phase/captured antibody, were  Most popular
going going to add first the patient antigen/unknown.  It is easily incorporated into the
Kapag nagbind na sa solid phase, we ’ re to wash it to protein molecule
remove the unbound antigen and the after that we ’ re  They emits gamma radiation
going to add the secondary label which can be detected with the
- The amount of label measured is directly use of gamma counter
proportional to the amount of patient antigen.  It can also measure even very
- If patient antigen is not present, the second low quantity of activity
antibody will not bind, therefore there will be no - Disadvantages:
reaction detected o Health hazard (dealing with radioactive
** kapag nagkaroon ng antigen and antibody substances)
combination/complex with the presence of capture o Disposal problems
antibody plus the patient ’s antigen, doon po mag bbind o Short shelf life
yung secondary label antibody. Kapag nagbind meron o Needs for expensive equipment
tayong makikitang reaction
** kapag negative, meron tayong capture antibody na
nakaattach sa solid phase, kapag inadd yung patient ’ s
sample since wala namang antigen, walanv magbbind sa
solid phase antigen. Upon adding the secondary, walang
pagbbindan since wala namang antigen/antibody
reaction

HETEROGENEOUS ENZYME
IMMUNOASSAYS
- require a step to physically separate free from
bound analyte.
** usually pineperform to the means of washing (para
maremove yung unbound analyte, ang maiiwan is yung ** kapag competitive binding, iadd simultaneously si
bound analyte) antigen at unlabeled antigen (magcocompete sila with
- This step provides a simple way to separate the limited antibody binding sites)
bound and free reactants. ** kapag negative/absent yung antigen kay patient
- Washing: the sample is then thoroughly washed sample, walang kakumpetensya (may reaction)
and the remaining activity is determined.
Two types of tests for radioimmunoassay
HOMOGENEOUS ENZYME  Radioallergosorbent test (RAST) – is a RIA
IMMUNOASSAYS method specifically designed to measure antigen
- do not need a separation step. specific IgE
- simpler to perform as there is no washing step.  Radioimmunosorbent test (RIST) – is a
** di na nagpperform ng washing in order to separate competitive binding technique used to quantitate
free from bound antibiodies IgE (total IgE)

ENZYME IMMUNOASSAYS
RADIOIMMUNOASSAYS - Using enzymes as labels, developed as
- Pioneered by Yalow and Berson (late 1950s) alternative to RIA.
- To determine the level of insulin and anti-insulin - Enzymes are naturally occurring molecules that
complexes in diabetic patients catalyze certain biochemical reactions.
** during that time ginagamit si radioimmunoassay in o They can react with the different
order to determine the level of insulin and anti-insulin substrates producing products that can
complex in diabetic patients be chromogenic (nagbabago color) ,
- Principle: Competitive binding (competition fluorogenic, or luminescent
between labeled and unlabeled) - Advantages:
- Result: inversely proportional o Cheap
o Readily available
 IMMUNOLOGY AND SEROLOGY (LECTURE)
o Have long shelf life (as compared to ** we have the solid phase antigen then iaadd first si
radioactive substances) patient’s antibody, magbbind sa solid phase antigen.
o Adapted to automation Wash to remove unbound or free antibody, then magadd
o Can cause changes that can be measured ng enzyme labeled antibody ay magbbind kay antigen-
using inexpensive equipment antibody complex. That’s why the color change is
- Enzymes that have been used as labels in directly proportional to the amount of antibody in the
colorimetric reaction: specimen

Horseradish Most commonly used because they ** pag negative. Pag absent yung patient’s antibody
peroxidase have the highest turnover rates upon adding the patient’s sample, walang magbbind sa
(highest conversion of substrate),

Alkaline
high sensitivity, and they are easy
solid phase, walang iwash din. Upon the addition of the
phosphatase enzyme label antibody, wala siyang pagbbindan since
to detect
 β-D- galactosidase wala namang naging antigen-antibody reaction na
nangyari. No color change

Capture Assays
- Sandwich immunoassays
- These are best suited to antigens that have
multiple determinants such as antibodies,
cytokines, proteins, tumor markers, and
microorganisms (especially viruses)
- If the antibody, rather the antigen, is bound to the
** 1. Antigen bound solid phase
solid phase.
** 2. Magaadd lang ng enzyme labeled antibody
- Enzymatic activity is directly proportional to
** 3. Upon the addition of the substrate and solution, it
the amount of antigen in the test sample
will result to color change
Heterogenous Enzyme Immunoassays
- Principle: Competitive Enzyme
Immunoassays
- Enzyme-labelled antigen competes with
unlabelled patient antigen for a limited number ** antibodies is attached into the solid phase vehicle,
of binding sites on antibody molecules that are ang hinahanap natin sa patient ’ s sample ay patient ’ s
attached to a solid phase antigen.
- Enzyme activity is inversely proportional to the ** solid phase antibody + patient’s antigen kung present
concentration of the test substance. si patient antigen magbbind sa solid phase antibody.
Wash in order to remove the excess antigen then add the
enzyme labeled antibody
** if there is an antigen-antibody binding, dyan
magbbind si enzyme label antibody resulting in change
in color
** kaya tinawag na sandwich immunoassay (antibody-
antigen-antibody) naka sandwich siya
** kapag absent ang patient’s antigen, walang magbbind.
Upon the addition of the enzyme labeled antibody,
walang pagbbindan since wala namang tayong antigen-
antibody complex na naform. No color change (inversely
Noncompetitive Enzyme Immunoassays
proportional)
- Indirect immunoassays or Enzyme-linked ** color change - presence of antigen
immunosorbent assays (ELISA) ** no color change - no presence of antigen
- The enzyme-labelled reagent only binds after the
initial antigen-antibody reaction has taken place Homogenous Enzyme Immunoassays
- The amount of color, fluorescence, or
- Principle: Change in enzyme activity as specific
luminescence detected is directly proportional
antigen-antibody combination occurs.
to the amount of antibody in the specimen.
- Reagent antigen is labelled with an enzyme tag
- Sensitivity is determined by the following:
o Detectability of enzymatic activity
o Change in that activity when antibody
binds to antigen
o Strength of the antibody’s binding
o Susceptibility of the assay to
interference from endogenous enzyme
activity
- Advantages:
o No washing required
o Less sensitive than heterogeneous
immunoassays
o Rapid and simple to perform
o Adapt easily to automation
 IMMUNOLOGY AND SEROLOGY (LECTURE)
Rapid Immunoassays cell suspensions with a high degree of sensitivity
- Membrane-based, easy to perform, and give and specificity.
reproducible results. - Fluorophores or fluorochromes – fluorescent
- Example: immunochromatography used in compounds that absorb energy from a light
serology (test kits) source
o They will convert energy into a light of
longer wavelength
- Commonly used compounds:
o Fluorescein – absorb light at 490-495
nm and will emit green light color at 520
nm
o Rhodamine or tetramethylrhodamine –
absorb light at 550 nm and will emit red
light color at 585 nm
** since meron difference in emission patterns and
absorbance pede natin gamitin pareho si Fluorescein
and Rhodamine
- Other fluorescent compounds: Phycobiliprotein,
Europium, and Lucifer yellow

Direct Immunofluorescent Assays

- Antibody that is conjugated with a fluorescent
tag is added directly to unknown antigen that is
fixed to a microscope slide.
- After incubation and a wash step, the slide is
read using a fluorescence microscope.
- Best used for antigen detection in tissue or body
fluids
- Antigens – Bright apple green or yellow-orange
objects against a dark background
** meron tayong glass slide and the patient’s antigen is
fixed there. We are going to incubate it directly with the
- The sample will be applied on one end of the fluorescent labeled antibody, after that we ’ re going to
strip, and it will migrate up to the distal end wash to remove the free/unbound antibody and then we’
(absorbed by the nitrocellulose membrane) re going to check for the presence of fluorescence
- The patient sample will be added to a cassette ** best used: for the antigen detection in tissue and body
containing the antibody labeled with colloidal fluids (dahil madaling ilagay sa glass slides)
gold particles
- The sample and the antibody will be combined, Indirect Immunofluorescent Assays
and they will move through the capillary flow, - Most commonly used
then the monoclonal antibody will capture the - Involve two steps, the first of which is incubation
patient’s antigen attached to the gold labeled of patient serum with a known antigen attached
antibody to a solid phase.
- Result: visible line - The slide is washed, and then an antihuman
** sa loob ng rapid immunoassay meron nitrocellulose immunoglobulin containing a fluorescent tag is
membrane at may mga nakaattach na antibody added.
** ex. Immuno chromatography na ginagamit natin in - This combines with the first antibody to form a
serology (test kits) may nitrocellulose membranes at may sandwich, which localizes the fluorescence.
nakaattach na either antigen or antibody (depende sa - Example: ANA test for Systemic Lupus
specific disease na dinedetect) Erythematosus (SLE)
** the sample will be applied on one end of the strip then ** we’re going to incubate the patients sample with the
magmigrate sa distal end (absorbed by nitrocellulose known antigen and add patients antibody. During the
membrane) incubation, kung present si patients antibody magbbind
** patient sample will be added to a cassette containing yan sa solid phase antigen. Wash to remove the
the antibody labeled with colloidal gold particles and free/unbound antibody. Add the anti-human labeled
then upon the addition of the patient ’ s sample, antibody. Kapag may presence of antigen-antibody
magcocombine yan with the antibody and then will be reaction, magbbind yung label antibody natin. Pag
moved to the capillary flow. The monoclonal antibody tinignan under fluorescence microscope, may makikita
magrereact/capture the patient’s antigen attached to the kang fluorescence.
gold labeled antibody (magkakaron ng line)
FLUORESCENT IMMUNOASSAYS
- Restricted to qualitative observations involving
the use of a fluorescence microscope.
** usually when it comes to the fluorescent immunoassay
pwede tayo gumamit ng fluorescence microscope
- In this manner, many types of antigens can be
detected either in fixed tissue sections or in live
 IMMUNOLOGY AND SEROLOGY (LECTURE)
Indirect Reagent antigen is If fluorescence is
Fluorescent attached to a slide. detected, patient
Patient antibody is antibody is present and
allowed to react. the test is positive
A second
fluorescent
labelled antibody
is added.
Fluorescent Fluorescent When patient antigen
Polarization labelled antigen binds, less reagent
competes with antigen binds and less
Fluorescence Polarization Immunoassays patient antigen for polarization will be
- Type of homogeneous fluorescent immunoassay a limited number detected.
of soluble Inverse ratio between
- Based on the change in polarization of antibody binding patient antigen and
fluorescent light emitted from a labelled sites. amount of polarization.
molecule when it is bound by antibody. Immuno Patient sample is If test is positive, a line
- When an antigen is bound to the antibody, the Chromatographi added to a test or plus sign will form
c strip and migrates on the test strip where
polarization of light increases
through the strip. patient antigen or
- Labeled antigens compete with unlabelled Labeled antigen antibody is captured.
antigen in the patient sample for a limited or antibody binds
number of antibody-binding sites. and is captured by
a second reagent
in the detection
zone.

CHEMILUMINESCENT IMMUNOASSAYS
- Chemiluminescence – emission of light caused
by chemical reaction, typically an oxidation
reaction, producing an excited molecule that
decays back to its original ground state.
- Most common substances used:
o Luminol
o Acridinium esters
o Ruthenium derivatives
o Nitrophenyl oxalates

Type of Assay Principle Results

Competitive Patient antigen Inverse ratio: The
competes with more patient antigen is
labelled antigen present, the less label
for limited detected
antibody binding
sites
Noncompetitive Excess solid All patient antibody is
or Indirect Elisa phase antigen allowed to bind.
binds patient Amount of label is
antibody and a directly proportional to
second labelled the amount of patient
antibody is added antibody present.
Capture or Excess solid All patient antibody is
Sandwich phase antibody allowed to bind.
binds patient Amount of label is
antigen and a directly proportional to
second labelled the amount of patient
antibody is added antibody present.
Homogenous Patient antigen No separation step.
and enzyme Antibody in solution.
labelled antigen Inverse ratio between
react with reagent patient antigen and
antibody in amount of labelled
solution. detected.
Enzyme label is
inactivated when
reagent antigen
binds to antibody
Direct Patient antigen is If fluorescence is
Fluorescent attached to a slide. detected, patient
Specific antigen is present and
fluorescent test is positive
labelled antibody
is added