IMMUNOSERO_AMTRMT

IMMUNOLOGY
- study of the molecules, cells, organs, and systems responsible for the recognition and disposal of foreign material.

SEROLOGY
- refer to the use of serologic tests to measure antibodies or antigens in infectious diseases or other conditions.

PRINCIPLES AND TESTS USED IN USHH-LAB

AGGLUTINATION

- the clumping and sedimentation of particulate or insoluble antigen/antibody complexes

STEPS IN AGGLUTINATION
1. SENSITIZATION
2. LATTICE FORMATION
3. ENHANCEMENT OF LATTICE FORMATION

DIRECT AGGLUTINATION
- insoluble particle is reacted with an antibody which results to aggregation of particles
- Ex: HEMAGGLUTINATION
- reacting antigen is native to the red blood cell
- Ex: ABO BLOOD GROUPING

ANTIGLOBULIN TEST
- detects the presence of non-agglutinating antibodies on red blood cells by adding a second antibody
a. DIRECT ANTIGLOBULIN TEST/DIRECT COOMB’S TEST
- used to demonstrate in vivo attachment of antibody or complement to an individual's red blood cells
- indicator of autoimmune hemolytic anemia, hemolytic disease of the newborn, sensitization of red blood cells caused by
the presence of drugs, or a transfusion reaction
b. INDIRECT ANTIGLOBULIN TEST/INDIRECT COOMB’S TEST
- determine the presence of a particular antibody in a patient, or it can be used to type patient red blood cells for specific
blood group antigens.
- Crossmatching, antibody identification and detection, RBC phenotyping (Weak D test)

IMMUNOCHROMATOGRAPHY

- Immunochromatography assay (ICA), namely lateral flow test, is a simple device intended to detect the presence or absence of the target
analyte. The concept of immune-chromatography is a combination of chromatography (separation of components of a sample based on
differences in their movement through a sorbent) and immunochemical reactions.

COMPONENTS OF IMMUNOCHROMATOGRAPHY ASSAY

1. SAMPLE APPLICATION PAD
2. CONJUGATE PAD
3. SUBSTRATE (NITROCELLULOSE) MEMBRANE
4. ADSORBENT PAD

TESTS IN USHH-LAB THAT USES IMMUNOCHROMATOGRAPHY ASSAY

1. DENGUE DUO TEST
2. H. PYLORI TEST
3. TYPHIDOT
4. WONDFO nCOV-2019
5. MALARIA TEST
6. LEPTOSPIRA TEST
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DENGUE

- Dengue virus is an enveloped positive-sense RNA virus

- Flavivirus
- 4 serotypes
- carried by Aedes aegypti mosquitoes

SEROLOGICAL TEST USED IN USHH-LAB

1) SD BIOLINE Dengue Duo rapid test contains two test devices (left side; Dengue NS1 Ag test,
right side; Dengue IgG/IgM test)
a) Dengue NS1 Ag TEST
- qualitative determination of dengue virus NS1 antigen in human serum,
plasma, or whole blood for the diagnosis of early acute dengue infection
b) Dengue IgG/IgM TEST
- qualitative, and differential detection of IgG and IgM antibodies to dengue
virus in human serum, plasma, or whole blood.

TEST INTERPRETATION OF DENGUE DUO

SD BIOLINE Dengue NS1 Ag

● Negative Result: The presence of only one color line in the “C” band within the result window indicates a negative result.
● Positive Result: The presence of two color lines (“T” band and “C” band) within the result window, no matter which line appears first,
indicates a positive result.
● Invalid Result: The control line (C) fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely
reasons for control line failure. Repeat the test using a new test device.

SD BIOLINE Dengue IgG/IgM

● Negative Result: The control line (C) is only visible on the test device. No IgG and IgM antibodies were detected. Retest in 3-5 days if
dengue infection is suspected.
● IgM Positive Result: The control line (C) and IgM line (M) are visible on the test device. This is positive for IgM antibodies to dengue virus.
This is indicative of primary dengue infection.
● IgG Positive Result: The control line (C) and IgG line (G) are visible on the test device. This is positive for IgG antibodies. This is indicative
of secondary or past infection.
● IgG and IgM Positive Result: The control line (C), IgM (M) and IgG line (G) are visible on the test device. This is positive for both IgM
and IgG antibodies. This is indicative of a late primary or early secondary dengue infection.
● Invalid Result: The control line (C) fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely
reasons for control line failure. Repeat the test using a new test device.

Dengue NS1 Ag (+) only Early acute dengue infection

Dengue IgM (+) only Primary dengue infection
Dengue IgG (+) only Secondary dengue infection
Dengue IgM/IgG (+/+) Late primary or Early secondary dengue infection

Read results within: 15-20 minutes

Specimen used: whole blood/serum/plasma

Helicobacter pylori INFECTION

- causes stomach inflammation, gastritis, peptic ulcers

- can be transmitted from person-to-person
- H. pylori bacteria grow in the protective mucus layer of the stomach lining, where they are less
exposed to the highly acidic juices produced by the stomach.
- H. pylori produces ammonia, which helps protect it from stomach acid and enables it to disrupt
and penetrate the mucus layer.
- H. pylori contributes to ulcer formation by increasing acid production, interfering with the
stomach's normal defenses against stomach acid, and producing toxins.

SEROLOGIC TEST USED IN USHH-LAB

1) SD BIOLINE H. pylori test
- qualitative detection of antibodies of all isotypes (IgG, IgM, IgA, etc)
specific to Helicobacter pylori in human serum or plasma.
- contains a membrane strip, which is pre-coated with H. pylori capture
antigen on test band region. The H. pylori antigen colloid gold conjugate
and serum sample moves along the membrane chromatographically to the
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test region (T) and forms a visible line as the antigen-antibody-antigen gold particle complex forms with high degree of
sensitivity and specificity.

TEST INTERPRETATION OF H. pylori TEST

1. As the test kit begins to work, a color band will appear at the left section of the result window to show that the test is working properly. This
band is the “Control Line”.
2. The right section of the result window indicates the test results. If another color band appears at the right section of the result window, this
band is the “Test Line”.
● Negative Result: The presence of only one purple color band (“C” band) within the result window indicates a negative result.
● Positive Result: The presence of two color bands (“T” band and “C” band) within the result window, no matter which band appears
first, indicates a positive result.
● Invalid Result: If the purple color band (“C” band) is not visible within the result window after performing the test, the result is
considered invalid. The directions may not have been followed correctly or the test may have deteriorated. It is recommended that
the specimen is re-tested.

Read results within: 10 minutes

Specimen used: serum/plasma
14C-Urea
2) Breath Test

PRINCIPLE

This product uses the method of ionization counting to measure 14C. The patient swallows urea capsule labelled with 13C/14C. If the patient
is infected with H. pylori, the test urea will be broken down and CO2 labelled with 13C/14C will be produced. CO2 moves from the blood to the lungs
and the exhaled breath will be collected and then detected by the analyzer.
In the analyzer, ß-ray emitted by 14C enters into GM tube and causes inert gas ionization. The anions of the gas ionized are speeded up to
move to the anode of the GM tube under strong field. More gases are ionized by colliding with other gas molecules, consequently lots of charge ions
are generated instantly. A detectable negative impulse is the generated and fed into the single-chip computer system and counted after it is amplified,
handled and discriminated.

TYPHOID FEVER

- Causative agent: Salmonella typhi

- SALMONELLOSIS: Infection caused by Salmonella species
- Salmonella enteritidis: most common cause of Salmonellosis in North America
- Salmonella typhi: most severe form of Salmonellosis
- Mode of transmission: Ingestion

THREE MAIN TYPES OF ANTIGENS OF SALMONELLA TYPHI

O ANTIGEN (THERMOSTABLE SOMATIC ANTIGEN) Polysaccharide in nature and is an endotoxin

H ANTIGEN (FLAGELLAR ANTIGEN) Protein in nature
Associated with virulent strains
Vi ANTIGEN (CAPSULAR ANTIGEN)
Indicative of carrier state

SEROLOGICAL TEST USED IN USHH-LAB

1) SD BIOLINE Salmonella typhi IgG/IgM Fast
- When a specimen is added to the test, anti-Salmonella typhi IgGs
and IgMs in the specimen sample react with Salmonella typhi
proteins of colloidal gold conjugates and forms a complex of
antibodies and colloidal gold conjugates. As this mixture migrates
along the length of the test strip by capillary action, the anti-
Salmonella typhi IgG or IgM complex is captured by the relevant
anti-human IgG and/or anti-human IgM immobilized in the two
lines across the test strip and generate a colored line.
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INTERPRETATION OF TYPHIDOT/Salmonella typhi IgG/IgM Fast

S. typhi IgM (+) and S. typhi IgM/IgG (+/-) Definite typhoid fever
Acute typhoid fever (middle stage of S. typhi infection)
S. typhi IgG (+) Previously successfully treated case of typhoid fever Reinfection of
typhoid fever
Typhoid carrier
S. typhi IgM and IgG (-/-) Not typhoid fever

Read results within: 15-30 minutes

Specimen used: whole blood/serum/plasma

COVID-19

- infectious disease caused by the SARS-CoV-2 virus

- Mode of transmission: Person-to-person contact via droplet transmission

SEROLOGICAL TEST USED IN USHH-LAB

1) WONDFO 2019-nCOV Antigen Test (Lateral Flow Method)
- based on the principle of immunochromatography sandwich for determination of 2019-nCoV antigen extracted from the
nasopharyngeal or oropharyngeal swab specimen. When the extracted specimen is added into the test device, the
specimen is absorbed into the device by capillary action, mixes with the 2019-nCoV antibody-dye conjugate and flows
across the pre-coated membrane.

TEST INTERPRETATION OF WONDFO 2019-nCOV Antigen Test

Positive Result: Colored bands appear at both test line (T) and control line (C). It indicates
a positive result for the 2019-nCoV antigen in the specimen.
Negative Result : Colored band appear at control line (C) only. It indicates that the
concentration of the 2019-nCoV antigen is zero or below the detection limit of the test.
Invalid Result: No visible colored band appears at control line after performing the test.
The directions may not have been followed correctly or the test may have deteriorated. It is
recommended to re-sampling and test.

Read results within: 15-20 minutes

Specimen used: nasopharyngeal swab/oropharyngeal swab

2) RT-PCR (Real-Time Reverse Transcription Polymerase Chain

Reaction)
- considered by the World Health Organization (WHO) as the standard
method for laboratory diagnosis of SARS-CoV-2.

MALARIA

- Causative agent: Blood parasites of the genus Plasmodium (P. falciparum, P. vivax, P. ovale and P. malariae)

HRP2 (Histidine-rich protein II): specific to P. falciparum

pLDH (Plasmodium lactate dehydrogenase): specific to Plasmodium species (Pan)

SEROLOGICAL TEST USED IN USHH-LAB

1) Bioline™ Malaria Ag P.f/Pan
- contains a membrane strip, which is precoated with mouse monoclonal antibodies specific to HRP2 of P. falciparum on
the test line P.f region and with mouse monoclonal antibodies specific to lactate dehydrogenase of Plasmodium species
Pan (P.falciparum, P.vivax, P. malariae and P. ovale) on the test line Pan region.
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TEST INTERPRETATION of Malaria Ag P.f/Pan

Negative result: The presence of control line (C) within the result window indicates a negative result.

Positive result
● P. falciparum positive:
The presence of two colored lines (test line “P.f” amd control line “C”) of three colored lines (test lines
“P.f”, “Pan” and control line “C”) within the result window, regardless of which line appears first,
indicate P.f positive result.
● Other Plasmodium species (P.v or P.m or P.o) positive:
The presence of two colored line (test line “Pan” and control line “C”) within the result window,
regardless of which line appears first, indicate Pan (P.v or P.m or P.o) positive result.
● P.f positive or mixed infection of P.f and P.v or P.m or P.o:
The presence of the three colored lines (test lines “P.f”, “Pan” and control line “C”) within the result
window, regardless of which line appears first, indicate P.f positive or mixed infection of P.f and P.v
or P.m or P.o

Invalid result: If the control line (C) is not visible within the result window after performing the test, the result is
considered invalid. Instructions may not have been followed correctly or the test may have deteriorated beyond the
expiration date. It is recommended that the specimen be retested using a new test device.

P.f. (+) and Pan (-) P. falciparum positive

P.f (-) and Pan (+) P. ovale, P. vivax, and P. malariae positive
P.f (+) and Pan (+) P. falciparum, P. ovale, P. vivax, P. malariae positive

Read results within: 15 minutes

Specimen used: whole blood

LEPTOSPIROSIS

- Leptospirosis is a disease that is caused by spirochete bacteria in the genus Leptospira.

- Spread by the urine of infected animals (rodents, dogs, livestock, pigs, horses, wildlife).
- Mode of transmission:
- Direct contact with the urine or reproductive fluids from infected animals
- Contact with urine-contaminated water (floodwater, rivers, streams, sewage) and wet soil
- Ingestion of food or water contaminated by urine or urine-contaminated water

SEROLOGICAL TEST USED IN USHH-LAB

1) LEPTOSPIRA IgG/IgM
- simultaneous detection and differentiation of IgG and IgM antibodies to Leptospira interrogans in human serum, plasma
or whole blood.

INTERPRETATION OF RESULTS OF LEPTOSPIRA IgG/IgM

IgG POSITIVE:
THE CONTROL AND IgG ARE VISIBLE ON THE TEST STRIP. THE TEST INDICATES THE PRESENCE OF ANTI-L. interrogans IgG.
THE RESULT IS ANTI-L. interrogans IgG POSITIVE OR REACTIVE

IgM POSITIVE:
THE CONTROL AND IgM ARE VISIBLE ON THE TEST STRIP. THE TEST INDICATES THE PRESENCE OF ANTI-L. interrogans IgM.
THE RESULT IS ANTI-L. interrogans IgM POSITIVE OR REACTIVE

IgM AND IgG POSITIVE:

THE CONTROL LINE, IgM AND IgG LINES ARE VISIBLE ON
THE TEST STRIP.
THE TEST IS INDICATES THE PRESENCE OF BOTH ANTI-L.
interrogans IgG AND IgM ANTIBODIES.
THE RESULT IS BOTH ANTI-l. interrogans IgG and IgM
POSITIVE OR REACTIVE.

IgM AND IgG NEGATIVE:

THE CONTROL LINE IS ONLY VISIBLE ON THE TEST
STRIP.
NO DETECTABLE ANTI-L. interrogans ANTIBODY IS
PRESENT IN THE SPECIMEN.
THE RESULT IS NEGATIVE OR NON-REACTIVE.
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FLUORESCENCE IMMUNOASSAY (FIA)

- uses antibodies labeled with fluorescent molecules that bind to the target protein or antigen of interest.

PRINCIPLE OF AFIAS AND ICHROMA machines: Fluorescence Immunoassay (FIA)

The tests uses a sandwich immunodetection method; the detector antibodies in buffer bind to antigens in the sample, forming antigen-antibody
complexes, and migrate onto nitrocellulose matrix to be captured by the other immobilized-antibodies on test strip. More antigens in the sample will
form more antigen- antibody complexes which lead to stronger fluorescence signal by detector antibodies.

C-Reactive Protein (CRP)

- acute phase reactant and marker of inflammation.

- measurement of CRP concentration has been used as a clinical tool for monitoring autoimmune diseases and infectious processes, such as
rheumatoid arthritis and systemic lupus erythematosus (SLE).

The sample type for AFIAS CRP is human whole blood/serum/plasma.

The cut-off (reference value): 10 mg/L

Human Chorionic Gonadotrophin (Total Βhcg)

- a glycoprotein hormone secreted by the developing placenta shortly after implantation

- can be detected in the urine and serum of pregnant women as early as 6 to 15 days after conception.

The sample type for AFIAS Total βhCG is human serum

The cut-off (Reference value): 20 mIU/mL

Anti-Streptolysin O (ASO)

- antibody produced in human blood against streptolysin O made from an infection of Streptococcus bacteria.
- An elevated or rising ASO titer may demonstrate recent streptococcal infections.
- aid in management and monitoring of scarlet fever, rheumatic fever and post infectious glomerulonephritis along with several other conditions

The sample type for ichroma™ ASO is human serum/plasma.

The cut-off (reference range): 160 IU/mL

Rheumatoid factor

- protein produced in the immune system that can attack healthy tissue in the body.
- commonly associated with rheumatoid arthritis because when the antibody was discovered, it was initially mostly used to diagnose
rheumatoid arthritis.

The sample type for ichroma™ RF IgM is human whole blood/serum /plasma.
The cut-off (reference value): 20 IU/mL

RADIAL IMMUNODIFFUSION (C3 EASY RID)

- The antigen (protein) inoculated in the well of the plate, diffuses radially in the agarose gel, reacting with specific antibodies incorporated in
agarose gel and forming immune complexes visible as precipitin rings.
- Diameter of precipitin ring is DIRECTLY PROPORTIONAL to the concentration of the relevant protein in the sample.

Specimen used in C3 EASY RID: serum

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C3 COMPLEMENT

- A protein that plays a vital role in the body’s innate immune system, providing the first line of defense against diseases
- One of the diseases that often causes abnormal C3 is systemic lupus erythematosus, an autoimmune disorder.

CHEMILUMINESCENCE IMMUNOASSAY (CLIA)

- an immunological analysis technique that combines highly specific immune reactions with highly sensitive chemiluminescent detection
- measure the serum concentrations of drugs, tumor markers, and antibodies.
- Its main advantages include high sensitivity, high signal intensity, wide dynamic range, high specificity, low reagent consumption, and high
automation.

PRINCIPLE OF VITROS 5600 AND ABBOTT ARCHITECT SYSTEM (IMMUNOASSAY TESTS): CLIA
The sample which contains antigen is mixed with a reagent containing labelled antibody to create a chemical complex. When a highly sensitive substrate
is mixed with this chemical complex, it results in the production of light (Luminescence) which is further amplified and detected by the photomultiplier
tube (PMT). The amount of light detected by the photomultiplier tube is directly proportional to the concentration of the analyte based on the assay
type.

Specimen used in blood screening tests in VITROS 5600 and ABBOTT ARCHITECT SYSTEM: serum and plasma

VIRAL HEPATITIS
- Hepatitis means inflammation of the liver.
- Cases can range from mild to severe, with many people not even realizing they are infected. The most common types of viral hepatitis are
hepatitis A, hepatitis B, and hepatitis C.

HEPATITIS A (HAV) HEPATITIS B (HBV) HEPATITIS C (HCV)

OTHER NAME Infectious hepatitis Serum hepatitis Post Transfusion hepatitis

Non-A, Non-B Hepatitis

MODE OF Fecal-oral Sexual contact Transplacental

TRANSMISSION Ingestion of contaminated Blood transfusion Sexual contact
food/water Sharing of needles Blood transfusion
Sharing of needles

FAMILY Picornaviridae (RNA) Hepadnaviridae (DNA) Flaviviridae (RNA)

SEROLOGIC MARKERS OF HEPATITIS A

1. HAV ANTIBODIES
a. ANTI-HAV IgM
- marker of acute hepatitis
- indicates recent infection
b. ANTI-HAV IgG
- indicate immunity to HAV, results from infection or immunization

SEROLOGIC MARKERS OF HEPATITIS B

1. HBsAg
- first marker to appear, indicates active infection either acute or chronic
- indicates acute infection or infectivity
2. Anti-HBs
- indicates past infection with immunity or immune response to HBV vaccines
- marker for immunity and recovery
3. Anti-HBc
- antibody produced against the core of HBV
- important marker during the window period
a. Anti-HBc IgM: indicates acute infection
b. Anti-HBc IgG: indicates past or chronic infection.
4. HBeAg
- marker for active viral replication and high infectivity of serum
5. Anti-HBe
- suggests low titer of HBV
- first serologic evidence of convalescence phase
6. HBV-DNA
- detected by PCR
- most sensitive test for detection of HBV
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SEROLOGIC MARKERS OF HEPATITIS C

1. Anti-HCV
- generally used to diagnose HCV infections
2. HCV-RNA
- used in monitoring response to antiviral therapy
- viral load

HUMAN IMMUNODEFICIENCY VIRUS (HIV)

- Causes AIDS
- Retrovirus
a. HIV-1: more pathogenic strain’
b. HIV-2: less pathogenic, lower transmission rate

CONFIRMATORY TEST FOR HIV

1. WESTERN BLOT ASSAY
- most sensitive and specific test for HIV-1
- detects IgG antibodies specific to HIV antigens
- The separated HIV-1 proteins are transferred to a nitrocellulose membrane
- if antibodiess to any HIV-1 antigens are presnt in the sample, bands corresponding to HIV-1 proteins will be seen.
- CRITERIA FOR POSITIVE INTERPRETATION (CDC CRITERIA):
- Presence of at least 2 out of possible 3 bands
- p24
- gp41
- gp120/160

SYPHILIS

- Causative Agent: Treponema pallidum ssp. pallidum

- Transmission: sexual contact, blood transfusion, transplacental route

SYPHILIS STAGES
1. PRIMARY STAGE
- lasts for 1-6 weeks
- highly contagious
- presence of HARD CHANCRE
- painless firm lesion at the site of entry
2. SECONDARY STAGE
- observed 1-2 months after the primary chancre appears
- most contagious stage
- all serologic test detect the infection
- CONDYLOMATA LATA
- wart like lesions in most area of the body
3. LATENT STAGE
- asymptomatic
- diagnosis can only be made by serologic methods
4. TERTIARY STAGE
- NEUROSYPHILIS
- most common complication in tertiary stage but can occur after primary stage
- TABES DORSALIS
- degeneration of lower spinal cord, and general paresis, or chronic progressive dementia
- GUMMAS (GRANULOMAS)
- areas of granulomatous inflammation that are most often found on bones, skin, or subcutaneous tissue

NON-TREPONEMAL SEROLOGICAL TESTS

- determine the presence of reagin
1. VENEREAL DISEASE RESEARCH LABORATORY (VDRL)
- serum or csf as sample
- principle: rapid slide microflocculation
2. RAPID PLASMA REAGIN (RPR)
- recommended for screening
- principle: flocculation/charcoal agglutination