Immunology and Serology Lab No.

1
Screening for Phagocytic Engulfment
Phagocytosis
- process by which cells ingest and degrade the following: microorganisms
Dead, senescent, damaged cells
Insoluble particles
- body’s defense mechanism
- takes into action when our body’s first line of defense is breached
 anatomical barriers
 skin
 mucous membranes
Phagocytic Cells
- cells capable of phagocytosis

1. Monocyte-Macrophage System
 Monocyte - cells in the circulation
 Macrophage - monocytes residing in tissues
2. Neutrophils
 Most predominant phagocytes because it is:
 The most abundant WBC in the circulation
 Actively motile
 Arrive first during injury except when the site of injury already has resident macrophage
Chemokines
- Chemoattractant substances
- Direct the phagocytic cells to the site of injury

7 STEPS OF PHAGOCYTOSIS

1. Chemotaxis
o Process of migration of phagocytic cells to the site of injury through the secretion of chemokines
2. Adherence
o Phagocytic cells will adhere/attach to offending particle
3. Engulfment
4. Phagosome Formation
o Phagosome - internal vesicle found inside phagocytic cells
5. Fusion
o Phagosome + Lysosome → Phagolysosome
o Phagosome fuses with lysosome to form a phagolysosome
o Lysosome - organelle with hydrolytic or degradative enzymes
o Phagolysosome - where degradation takes place
o Lysosomes will release degradative/hydrolytic enzymes and degrade engulfed material
6. Digestion and Destruction
7. Exocytosis
o Phagocytic cell expels the remnants of the phagocytosed material
 Buffy coat
- Depends on the amount of WBC present
- Separates plasma from packed RBCs
- Composed of:
o WBC
o Platelets
Bacterial Broth Culture
- Can either be:
o Bacillus subtilis
o Escherichia coli
o Coagulase Negative Staphylococcus spp. like Staphylococcus epidermidis

ANTICOAGULATED BLOOD →CENTRIFUGE

BUFFY COAT (4-8 drops) + BACTERIAL BROTH CULTURE (2 drops) → INCUBATE AT 37°C FOR: ideally, 30 mins
In the lab, 15 mins
MAKE BLOOD SMEARS (2) → AIR DRY → STAIN W/ WRIGHT STAIN → VIEW UNDER OIO
POSITIVE: Intracellular Bacteria

**If smear is predominantly LYMPHOCYTES:

- Too heavy bacterial load
- Neutrophils destroyed themselves in trying to engulf too much bacteria

FALSE NEGATIVE:
1. Not fresh specimen
2. Use of encapsulated bacteria
o Cannot be engulfed because phagocytic cells have no receptors for polysaccharide in the
capsule
3. Use of coagulase positive staphylococcus aureus
o Coagulase will bind with prothrombin in blood and form STAPHYLOTHROMBIN and will cause
the conversion of FIBRINOGEN to FIBRIN and results to CLOT FORMATION. The clot will coat the
organism and make it resistant to phagocytosis
o Coagulase + Prothrombin → Staphylothrombin
↓
Fibrinogen → Fibrin
4. Neutrophil disorders/dysfunction
o Not capable of phagocytosis

NOTE: This is only a screening test! It merely indicates that bacteria is engulfed, not destroyed.
 Immunology and Serology Lab No. 2
Agglutination Test: Blood Typing
Precipitation Test: Single Radial Immunodiffusion
Agglutination
- Visible aggregation of particles caused by a specific antibody and particulate antigens
*As seen on the surface of
particles
Precipitation
- Visible aggregation of particle caused by a soluble antibody and soluble antigen
Agglutinins
- Specific antibodies that cause agglutination

TYPES OF PARTICLES

1. Red Blood Cell

o Most commonly used
o Advantage:
 Many antigens specially polysaccharides are adsorbed spontaneously
*Most common form of antigen but not the best
o Disadvantage:
 Cause cross reactions
2. Bacteria
3. Inert Carriers
o Ex: latex particle
o Advantage:
 Inexpensive, relatively stable, not subject to cross reactions
 Large number of antibodies that can be bound
 Large amount of antigens detected; thus, reading the reaction is easy
4. Synthetic Beads
o Advantage:
 Consistency, uniformity, stability
 Reaction can be read easily

AGGLUTINATION IS A TWO-STEP PROCESS:

1. Sensitization
o Involves the antigen-antibody combination through single antigenic determinant
o Only one side of the is attached
o Follows the Law of Mass Action
o Ag + Ab ↔ Ag-Ab (reversible)
o Affected by the nature of antibody (IgG, IgM)
 IgM
 700x more efficient than IgG.
 Immunoglobulin of choice because of large size
 May be monomeric or pentameric
 2. Lattice Formation
o Formation of cross-links that form the vsible aggregates
o Stabilization of antigen-antibody complex with the binding of multiple antigenic determinants
o Represents interaction between antibody and multiple antigenic determinants

ENHANCEMENT OF LATTICE FORMATION

RBC and bacteria - slightly negative in charge

RBC
o sialoglycoprotein/ sialic acid on its membrane
o sialoglycoprotein/ sialic acid: imparts the slightly negative charge of RBC
o 25 nm apart → zeta potential

3 ways to enhance:

1. Decrease the buffer’s ionic strength

 LISS (Low Ionic Strength Saline/ Low Ionic Strength Solution)

2. Addition of 5-30% albumin

 Neutralizes the negative charge of RBC

3. Increase the viscosity of RBCs

 Enzymes:
o Cleave the chemical agent (Sialic Acid/ Siaoglycoprotein) on the RBC
surface. Thus, reducing the charge and decreasing the hydration
 Bromelin
 Papain
 Ficin
 Trypsin
 Agitation and centrifugation
 Altering the temperature and pH
o IgM
 Reacts best at 4°C and 27°C [Cold-reacting Antibody]
o IgG
 Reacts best at 30-37°C [Warm-reacting antibody]
o Optimal pH: 6.5-7.5
 Lower pH
 Anti-M (MNS blood group)
 Anti-P (P blood group)

TYPES OF AGGLUTINATION

1. Direct Agglutination
o Occurs when antigen is naturally on a particle
a. Hemagglutination: Blood typing
 Anti-A Anti-D Anti-B
(Blue) (colorless) (Yellow)

i. Slide Method: Whole Blood

ii. Tube Method: 3-5% blood cell suspension

o 1 drop of anti-sera + 3-5% red cell suspension

o Add anti-sera first before red cell in order to prevent false negative
results
o Grade agglutination to know the strength and relative concentration

Type Anti-A Anti-B A Antigen B Antigen

A - + + -
B + - - +
AB - - + +
O + + - -

 Antibody will be present only if the antigen is absent

 Type O: universal donor
 Type AB: universal recipient because no antigen against blood types

2. Passive Agglutination/ Indirect Agglutination

o Particles are coated with antigen not normally found
o Principle for detection of:
 Rheumatoid Factor
 Antinuclear Antibody (ANA)

3. Reverse Passive Agglutination

o Not naturally present
o Antibody is coated on particle
o Antibody, rather than antigen, is attached to the corner particles
o Principle for detection of:
 C-Reactive Protein
 Cryptococcal Antigen Latex Agglutination System (CALAS)

4. Agglutination Inhibitor
o Based on the competition between particles and soluble antigens for limited combining sites
o (+): lack of agglutination

5. Coagglutination
o Use bacteria as inert particles which the antibody is attached
o Most commonly used bacteria: Staphylococcus aureus
  Protein A will naturally absorb the portion of
antibody
o Disadvantage:
 Hard to read because bacteria are colorless

PRECIPITATION TEST

Radial Immuno Diffusion (RID)

- Passive immunodiffusion
o No electrical current used to enhance diffusion
- Single immunodiffusion
o Only one is allowed to diffuse

(+) precipitin ring

- C3
o Quantitated
o Part of the complement system
- Endpoint method of measurement by Mancini
o Anti-C3 is attached on the gel and is diffused in walls
o 5-10 uL serum sample and incubate for 24-72 hrs
- Antigens are diffused until it reach the Zone of Equivalence  (+) PRECIPITIN RING
 Immunology and Serology Lab No. 3
Qualitative Detection of Hepatitis B Surface Antigen
Qualitative Detection of Antibody to Hepatitis C Virus
Qualitative Detection of Dengue NS1 Antigen and IgG/IgM Antibodies

Hepatitis B Surface Antigen (HBsAg)

- Also known as Australia Antigen
- 1st marker to appear
o HBsAg  HBeAg anti-HBcanti-HBeanti-HBs
o Anti-HBsB: marker for recovery or recent immunization
- Marker of active infection
o Acute Hepatitis (2-6 months)
o Chronic Hepatitis (>6 months)
`
Transmission can occur via:

1. Parentheral
2. Sexual route
3. Vertical transmission (mother to child)

3 Ways to Acquire Hepatitis in Babies:

- In uterus
- Infected birth canal
- Breastfeeding

Hepatitis B Virus and Hepatitis C Virus

- Leading causes of viral hepatitis worldwide

- Hepatitis B Virus
o More infectious
- Chronicity may occur
- ↑ chances of recovery of HepB
- 85% of patients with HCV may lead to Chronic Liver Disease

HEPATITIS B VIRUS

- A.k.a Dane Particle (whole particles)

- Caused by Hepadna virus, DNA virus
- 3 antigens:
 o HBcAg
 Core
 Cannot be detected by any test because it is surrounded by HBeAg
o HBeAg
 envelope
o HBsAg
 Surface
- Antibodies:
o Anti-HBc
o Anti-HBe
o Anti-HBs

HEPATITIS C VIRUS

- RNA virus under flavivirus

- Causes Hepatitis C
- May be asymptomatic
- Most common cause of post-transfusion hepatitis
- Tests do not detect the antigen but the antibody [anti-HCV]
- Formerly known as: Non-A Non-B Hepatitis (NANBH)
- Capable of mutating and can escape immune system

**If qualitative test for HBsAg and Anti-HCV is positive, proceed to quantitative test

Confirmatory Test: MOLECULAR DIAGNOSTIC TEST

- Example: PCR
o HBV-DNA
o HCV-RNA

HUMAN IMMNODEFICIENCY VIRUS

- Formerly:
o Human T-Lymphotrophic Virus type III (HTLV-III)
o AIDS-Related Virus (ARV)
- Retrovirus (RNA)
- Subdivided into:
 o HIV-1
 Most common in US
o HIV-2
 Most common in Africa
- Screening test: ELISA
- Confirmatory test: Western Blot
o At least 2 antigens are required
o P24, gp120, gp41
 P24: should always be positive
p24 gp120 gp41
  x
 x 
- Principle: Lateral Flow Immunochromatographic Assay

TEST CONJUGATE PAD TEST PAD CONTROL PAD

HBsAg Mouse anti-HBsAG antibody Nonconjugated HBsAg Goat anti-mouse IgG antibody
conjugated with colloid gold antibody
Anti-HCV Recombinant HCV antigens Recombinant HCV antigens Goat anti-rabbit IgG
conjugated with colloid gold
HIV HIV 1: HIV 1: Goat anti-rabbit IgG
Recombinant HIV-1 antigen HIV-1 antigen
conjugated with colloid gold
HIV 2:
HIV-2: HIV-2 antigen
Recombinant HIV-2 antigen
conjugated with colloid golds

Rabbit IgG-gold conjugates

Conjugate Pad: chromogen that gives burgundy red color

Test Pad: uses nitrocellulose membrane

Test kits move by capillary action

Traditional Confirmatory Test for Hepatitis C: Radioimmunoblot Assay (RIBA)

Reporting:

- HBsAg: Nonreactive/Reactive
- Anti-HIV/anti-HCV: Positive/Negative
 Immunology and Serology Lab No. 4
Qualitative detection of Dengue NS1 and IgG/IgM Antibodies
Dengue Fever
- Viral hemorrhagic fever
o Ex: Yellow fever, EBOLA
- Mosquito borne
o Bites early in the morning and early dusk
- Caused by Dengue virus (DENV)
- Under Flavivirus (RNA)
o Positive sense RNA virus
 RNA can be classified:
 (+) Sense
 (-) Sense
- Vector: mosquito
o Once infected, they become lifetime carriers
 Aedes aegypti
 Only in the tropics
 Aedes albopictus
 Survive cold temperatures
 Leading cause of dengue in Western countries
- No vaccine yet because in order to produce a vaccine, all strains must be present
- Cross immunity to all serotypes upon recovery
o For short-term only (around 1 month)
- More severe when infected for the 2nd time
- ↑ hematocrit, ↓ platelet
o Bore holes on blood vessels, fluid leaks out; thus, decreased platelets
o Hematocrit
 Aka Packed Cell Volume (PCV)
 Increased due to fluid leakage

Stages
1. Classic Dengue Fever
2. Dengue Hemorrhagic Fever
3. Dengue Shock Fever

Ischemia
- Prolonged absence of blood supply
4 Serotypes:
1. DENV 1
2. DENV 2
3. DENV 3
4. DENV 4

Principle: IMMUNOCHROMATOGRAPHY

1. NS1 (Nonstructural Protein 1)

 o Antigen in dengue fever
o 1-9 days after onset of illness

2. IgM
o 3-5 days (primary infection)
o 20th day(secondary infection)
o If (+), indicates current infection/ primary infection

3. IgG
o 14th day (primary infection)
o 1-2 days (secondary infection)
o If (+), indicates past or recurring infection/ secondary infection

Infection: NS1 IgM IgG

Primary 1-9 days 3-5 days 14th day
Secondary 20th day 1-2 days

COMBO KIT

NS1 IgG/IgM
100 uL (3 drops) serum 10 uL serum
+
4 drops diluents
↓
Read within 15 minutes
 Immunology and Serology Lab No. 5
Flocculation Test: Rapid Plasma Reagin (RPR)
Venereal Disease Research Laboratory (VDRL)
Qualitative Detection of Antibodies to Treponema pallidum (Syphilis Test)

SYPHILIS
- Caused by Treponema pallidum subspecie pallidum
o Most commonly found in blood units (killed after 3 days refrigeration)
o Formerly: Spirochaeta pallid
o A spirochete
o Discovered by Christopher Columbus
 “the great traveler”
 Old World → New World: contracted smallpox
 New World → Old World: contracted syphilis
- Spanish call it the French Disease
- French call it Italian Disease
- Italians call it Spanish Disease

Mode of Transmission:
- Sexual contact
- Parenteral
- Mother to Fetus (Congenital Transmission
o Hutchinsonian Triad
 Notched teeth
 Keratitis
 Deafness

Treatment:
1. Heavy Metals
a. Arsenic
b. Arsphenamine
c. Salvarsan
2. DOC: Penicillin

Laboratory Diagnosis:

1. Direct observation of organism by Darkfield (DF) Microscopy

o True in 1° and 2° syphilis
o Coiled organism with corkscrew motility

2. Serologic Tests for Syphilis (STS)

a. 1st: WASSERMAN TEST
 Principle: Complement Fixation

3. Non-Treponemal Tests
o Most commonly used in manual laboratories
o Detects Reagin
  Antibody to cardiolipin
o Nonspecific
o Subject to false (+) results
 Examples:
 SLE
 IM
 Malaria
 Leprosy
 Viral Pneumonia

4. Treponemal Tests
o Specific
o Detection of Treponemal antibodies

a. Treponema pallidum Immobilization Test

 Mixing of patient’s serum with live, actively motile T. pallidum
 Reporting of results:
 POSITIVE: 50% or more
 NEGATIVE: 20% or less
 20-50% is doubtful and requires monitoring

b. Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)

 Patient’s serum is heat-inactivated and made wit sorbent consisting of nonpathogenic
treponemes (Reiter Strain) which removes cross reactivity with treponemes other than
T. pallidum

NICHOL’S STRAIN (Virulent Antigen)

+
Patient’s Serum containing Anti-Treponema
Antibody
+
Fluorescein Isothiocyanate (FITC)-labeled Anti-
human globulin (AHG)
↓
(+) FLUORESCENCE

OTHER TREPONEMA-ASSOCIATED DISEASE IN HUMANS

1. Treponema pallidum subsp. pertenue

o via wounded skin comes in contact with lesion
o Yaws
 Chronic nonvenereal lesions of skin and bones

2. Treponema pallidum subsp.endemicum

o Via mouth to mouth or utensil sharing
o Bejel
 Lesions in oral cavity
 3. Treponema carateum
o MOT same with T. pallidum subsp. pertenue
o Pinta
 Ulcerative skin disease

VENEREAL DISEASE RESEARCH LABORATORY (VDRL)

- Both qualitative and quantitative flocculation
- reagin combines to colloidal suspension of liids and cause formation of visible masses (flocculation)

1. VDRL slide with 14mm rings

2. VDRL stock antigen
a. 0.03% cardiolipin
o Main reacting component of VDRL stock antigen
b. 0.9% cholesterol
o Enhances reacting surface of cardiolipin
c. 0.21% lecithin
o Removes the anti-complement activity of cardiolipin

Procedure:
1. Inactivate patient’s serum
o Place in water bath at 56°C for 30 minutes to 1 hour
2. Allow to cool at RT
3. Place 0.5 mL of the inactivated serum into one of the 14 mm rings on the slide
4. Place 0.5 mL of (+) control
5. Place 0.5 mL of (-) control
6. Add one drop of antigen suspension
7. Rotate for 8 minutes

Reporting of Results:
NONREACTIVE: no clumps
WEAKLY REACTIVE: small clumps
REACTIVE: medium to large clumps

SYPHILIS RPR TEST

- Qualitative and quantitative flocculation test
- Modifies the VDRL stock antigen
o Added microparticulate charcoal to VDRL stock antigen
 WHITE
 Enhance reaction and help differentiate (-) and (+) result in white background
 Makes RPR easier to read

Green Cap

Red Cap
 - For POSITIVE patients - Add 1 drop of reagent and 50 uL of
o Proceed to serial dilution for A titer determination st
patient’s serum on the 1 dilution. Intepret
after 8 minutes.
- No the preceding dilutions, add 1 drop of
the previous dilution + 50 uL NSS
- Proceed until you arrive to a tube with no
flocculation
- Titer is the highest dilution that exhibits a
(+) result

ANTI-TP (Treponema pallidum) Test

- Sandwich method
- Principle: lateral flow immunochromatography
- Specimen:
o Plasma
 Green
 Blue
 Lavender
o Serum
- Test Kit:
o Conjugate Pad: Treponema pallidum antigen with colloid gold and rabbit IgG-gold conjugate
o Test Pad: non-conjugated recombinant Treponema pallidum antigens
o Control Pad: goat anti-rabbit IgG antibody

10 uL SAMPLE + 3 DROPS DILUENT + 1 DROP NSS

READ AFTER 15 MINUTES
 Immunology and Serology Lab No. 6
Latex Agglutination Test for C-Reactive Protein (CRP)
Latex Agglutination Test for Anti-Streptolysin O (ASO)

C-REACTIVE PROTEIN
- Sensitive for:
o Rheumatic Fever
o Rheumatic Arthritis
- C Polysaccharide of Pneumococcus
o Opsonin in microbes
- Acute Phase Reactant (↑ during inflammation)
o Serum Amyloid A
 chemotaxis
o Serum Amyloid D
 opsonin
o Alpha1-antitrypsin
 Serpin
 Serine protease
 Has the ability to down regulate inflammation
o Mannose-binding Protein
o Fibrinogen
 Coagulation factor
o Haptoglobin
 Carries hemoglobin
o Ceruloplasmin
- Signs of inflammation:
o Rubor (redness)
o Dolor (pain)
o Calor (heat)
o Tumor (swelling)
o Functio laesa (loss of function)

CRP Latex Agglutination Slide Test

- Detects presence of CRP antigen (antibody is coated)
- Principle: Reverse Passive Agglutination
- CRP- carrier articles coated with nonspecific anti-human CRP antibody
- Blue suspension of polystyrene coated with anti-human CRP antibody
- Contents:
o LR: CRP Latex Reagent
o PC: Control Serum (+)
o NC: Control Serum (-)
o GBS: Glycine-NaCl Buffer
 pH 8.2 0.2
- Specimen
o Serum
 Stability:
 24 hours at 2-8°C
  4 weeks at -20°C

- Pipetting Scheme
Sample 40 uL
PC 1 drop
NC 1 drop
LR 1 drop each
- Mix
- Rotate

- Interpretation of results
o (+) distinctly visible agglutination
 >6 mg/L
- Concentration of CRP

- Absence of agglutination= <6 mg/L

- ↑ conc. of Rheumatoid Factor = FALSE ↑ CRP TITER
- Prozone Phenomena (excess Ab)= FALSE ↓ CRP TITER

SIMILARITY DIFFERENCE
CRP Detects inflammation disorders Differ in specificity
ESR Rise/ increase in acute inflammation - CRP: specific (detects viral and bacterial
infections)
- ESR: non-specific

ANTI-STREPTOLYSIN O (ASO)
- Antibody against Streptolysin O
- Streptococcus pyogenes

Hemolysins

Streptolysin O Streptolysin S
Oxygen labile Oxygen stable
Antigenic Non-antigenic
B hemolysis on blood agar B hemolysis on blood agar

5 Properties of SLO:
1. Enzyme
2. Exoantigen
3. Antigenic (MW: 10,000 Da)
4. Oxygen labile, heat labile
5. Hemolytic factor
LATEX AGGLUTINATION SLIDE TEST
- Detecting antibody against carrier particles coated with antigen
- Principle: Passive Agglutination
- Carrier particles (latex)
o Polystyrene
o Bentonite
 Absorptive colloidal clay
o Charcoal
o RBC
- Yellow suspension with polystyrene latex particle coated with stabilized SLO
- Contents:
o LR: ASO Latex reagent
o PC: Control Serum (+)
o NC: Control Serum (-)
o GBS: Glycins-NaCl Buffer
 pH 8.2 0.2
- Specimen
o Serum
 Stability:
 7 days at 2-8°C
 3 months at -20°C
- Pipetting Scheme
Sample 40 uL
PC 1 drop
NC 1 drop
LR 1 drop each
- Mix
- Rotate ( minutes)
- Interpretation of results
o (+) distinctly visible agglutination
 >200 IU/mL
 Normal upper limit because <15-20% of healthy individuals demonstrate this
titer
- Concentration of (+) result
o Proceed to semi-quantitative test → dilution with GBS
- Concentration of ASO

Dilution ASO (IU/mL)

1:2 400
1:3 600
1:4 800
1:5 1,000

- Diagnostic value/ significance

 o Increased ASO titers:
 Rheumatoid fever
 Glomerulonephritis
o Elevated ASO titer (>200 IU/mL)
 Acute Streptococcal infection
- Titer of ASO monitored every 2 weeks over a period of 4-6 weeks