IMMUNOLOGY AND SEROLOGY LAB

LECTURE 2: ANTI-STREPTOLYSIN O
KYLE VINCI P. SOLANO, RMT, MD
AUGUST 20, 2021
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Anti-Streptolysin O ● Both are beta hemolytic

Anti-streptolysin O ○ They have enough enzymes to digest RBC in
● Anti = its an antibody against the rest of the words. blood agar plate
● Strep = comes from the bacteria Streptococcus. ○ Clear hemolysis (they have consumed
● Lysin = lyses cell; quite a toxin that once it binds itself everything)
into the cell then facilitates the lysis or the explosion or ● Alpha-hemolytic (ex. Streptococcus pneumoniae)
the death of the cell ○ They don't have enough enzymes to digest the
● O = oxygen (?) entirety of RBC that's why naay mabilin na
Agglutination biliverdin
● More common principles of detection for a compound in ■ Makes the BAP have a greenish tint
immuno sero on alpha-hemolytic bacteria
● More larger components that are being bound together ● Streptococcus pyogenes
by your antibodies ○ Rheumatic fever, post streptococcal
Precipitation glomerulonephritis (Type III hypersensitivity
● Smaller components reaction), strep throat, scarlet fever,
Antigen and Antibody reaction endocarditis, pharyngitis, meningitis, toxic
● Most specific type of reaction in biology. shock syndrome, necrotizing fasciitis
● Used to detect certain compounds (organic compounds: ○ Occurs when streptococci has immune complex
lipids, carbohydrates, proteins) which are not easily deposition
detected via chemical means. ■ Antibodies are soluble biomolecules,
● Ex: red blobs as RBC and antibodies they can float around in water
○ Antibodies = plasma proteins: Gamma ■ If they float around in water and they
■ Gamma globulin- slowest to migrate have the antigen, they tend to flow
in electrophoresis. around the circulation
■ Produced by plasma cells; activated ■ If the antigen is like a toxin, part of the
by your B lymphocytes. cell wall of the bacteria, they tend to
○ Tips -fragment of antigen binding flow around = lots of immune
■ The one that attaches, not only complexes
bacteria but also anything that ■ Immune complexes happen when
stimulates the B cell. antibodies bind to a soluble antigen
● Immune system is very simple. (ex. Piece of bacterial cell wall)
○ If u are not with us then u are against us ○ They tend to deposit in high vascular area such
therefore you are foreign, then we will mount an as the glomeruli of the kidneys = immune
immune response against u. complex deposition in kidneys or heart
○ If B cell detects a foreign entity it will activate ● If there is an immune complex deposition, there will be
with the help of other cells, then it will produce inflammation in the area (glomerulonephritis) and will
an antibody. lead to fibrosis, which will decrease your glomerular
Streptococcus pyogenes filtration rate (GFR). Thus, causing kidney disease or
● Gram positive cocci in chains acute renal failure (ARF), which will eventually lead to
○ staining→ cell wall of the bacteria will adhere to End-Stage Renal Disease (ESRD) or dialysis
the gram stain (purple); if its negative then it will ● Purpose of ASO:
be stained by counterstain (red) ○ To control the infections
● Strep - in chains, greek ● If small amounts of immune complexes are deposited, it
● Cocci - spheres will eventually resolve, especially if taken with steroids or
● Pyogenes - type of infection such.
○ Pyo - pus-forming ● Antibiotics are taken to reduce the bacterial load
● Divides via binary fission ● Streptococcus pyogenes - normal flora of your mouth
○ Different from eukaryotes (usually divide by (oropharyngeal area)
mitosis) ○ If your immune system is weak, it could cause
● Prokaryotes divide in a single plane leading to be sore throat
attached from one to another ● If you have experienced a lot of sore throat every year, it
● catalase negative might be an indication to have tonsillectomy
● taxo-A/Bacitracin Sensitive ○ If sige kag sore throat, sige ka produce ug
antibodies with soluble antigens, sige ka
STREPTOCOCCAL INFECTIONS produce ng immune complex.
● Lancefield Group A (beta-hemolytic) ○ Once magdaghan na kaayo, it will go to your
○ Another type of classification kidney
○ Based on the carbohydrate in the cell wall of ○ Thus, you have to be observant if you always
bacteria have a sore throat to avoid post streptococcal
○ Lancefield A, B, C, D glomerulonephritis etc.
○ NO lancefield classification ● Cut off: 7 sore throats na proven na streptococcus
■ Streptococcus pneumoniae pyogenes pwede ka iadvice na mag tonsillectomy
■ Streptococcus viridans ● Toxic shock syndrome - caused by toxic shock like
○ Lancefield Group A - Streptococcus pyogenes toxin
○ Lancefield Group B - Streptococcus agalactiae ○ Most common bacteria: Staphylococcus aureus
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STREPTOCOCCAL ANTIGENS AND ANTIBODIES Aso Agglutination Test
● PRINCIPLE
Antigens Antibodies ○ Agglutination
Streptolysin O Anti-streptolysin O (ASO) ● REAGENT
Streptolysin S Anti-streptolysin S (ASS) ○ Polystyrene latex particles coated with
Hyaluronidase Anti-hyaluronidase (AHL) streptolysin O
● ANTIBODIES
Streptokinase Anti-streptokinase (ASK)
○ ASO antibodies (patient’s serum)
Deoxyribonuclease Anti-deoxyribonuclease (AND) ● REACTION
Erythrogenic toxin ○ Visible agglutination
Anti-nicotinamide adenine
dinucleotidase (ANAD)

● If you have infections in your soft tissues, always

remember that your hyaluronic acid is a part of your
connective tissue, which acts to hinder certain infections
from going deeper
○ Ex. If naa kay samad, this hyaluronic acid tries PASSIVE AGGLUTINATION
to block of further progression of the infection ● Antigen is enlarged: HOW?
● Streptococcus pyogenes has hyaluronidase, an enzyme ○ Carrier:
that breaks your hyaluronic acid. ■ Latex
● Thus, It has the ability to go deeper (flesh-eating ■ Rbc
infection) ■ Charcoal

● No special preparation is
required
● SERUM
● Storage:
○ 2-8C (<24hrs)
○ -20C (>24hrs)
○ Thawed at 37C
● Contraindications:
○ Lipemic
○ Hemolyzed
○ Bacterial contamination

RHEUMATOID FACTOR/RHEUMATOID ARTHRITIS:

● Body produces antibodies against the synovial tissue
● Immune complex deposition (Glomerulus) ● Chronic, multisystemic, autoimmune disorder
● Endothelia is the lining of your glomerulus which is a ● Progressive inflammatory disorder of the joints
simple stratified epithelium ● Factors:
● Y-shaped structures are your immune complexes which ○ Genetics
are the antibodies and your soluble antigen ○ Age
● Even though you don’t have an infection or the ○ Obesity
streptococcus bacteria in your kidney but because it ○ Physical activity
there is high blood flow in the kidney and it filters, so ● Most common in women 40-70 yrs old
there are areas na the movement of certain component
like your proteins are hindered.
● So instead na mag move along siya, it stays there and
deposits in the basement membrane of your glomeruli
leading to further infection
● This is your FAB portion or the Fragment of
Antigen Binding where your antigen is

● This is the FC portion or the Fragment of

Crystallization
○ A lot of neutrophils and macrophages
have receptors for it.
○ If they see an FC portion they will
automatically bind to it and produce
inflammation
● It is important to measure Streptolysin O because it is in
the blood and it is oxygen labile and when exposed to the
atmosphere, it will be destroyed.
● Since it is a toxin in the blood, the plasma cell will
recognize it as foreign and it will produce antibodies
against it
● Streptolysin O will only be produced by live or viable
Streptococcus pyogenes bacteria
● So if there is still the presence of Streptolysin O in the
blood, there is still bacterial infection
● With the increase of Streptolysin O there will be a
subsequent increase in your Anti-Streptolysin O because
there may be an ongoing infection by the Streptococcus
pyogenes
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 LABORATORY TESTING
● PRINCIPLE:
○ Agglutination
● REAGENT:
○ Latex suspension coated with albumin and
chemically bonded denatures of human IgG
○ Instead of binding the antigen to latex beads,
it’s actually the FC portion of the IgG that the
rheumatoid factor binds
● REACTION:
o Macroscopic agglutination
o Passive agglutination
● Antigen is enlarged: HOW?
○ CARRIER (also known as latex beads)
■ Latex
■ RB
■ charcoal
Osteoarthritis - cartilage is thin
Rheumatoid Arthritis - entire synovial fluid is inflamed
● 3 stages:
○ Synovitis
○ Subsequent immunologic events
○ Proliferative destruction of synovium - replaced
by fibrosis
Increase of autoantibodies such as:
● Rheumatoid Factor (RF)
○ Not specific
○ An autoantibody that binds to fc portion of IgG
● Anti-fillagrin antibodies (anti-citrullinated protein
antibodies - ACPA)
○ Anti-keratin antibodies (AKA)
○ Anti-periinuclear factor (APF)
● How does it work?
○ Antibodies to citrullinated peptides (ACCP)
○ the rheumatoid factor IgM binds to the FC
○ Anti-Sa antibodies
portion (fragment of crystallization) and of the
● Anti-RA33
IgG
○ In the fragment of antigen binding, you will see
hypervariable loops which is specific in
attracting the antigen

SPECIMEN PREPARATION
Specimen Preparation
● same results with the ASO
● No special preparation is required
● SERUM
● Storage:
○ 2° - 8° C (<24 hours)
○ -20° C (>24 hours)
○ Thawed at 37°C
● Contraindications
Rheumatoid Factors ○ Lipemic
● Immunoglobulins against the antigenic sites of the Fc ○ Hemolyzed
portion of IgG ○ Bacterial contamination
● Immunoglobulins G,M,and A - The process is quite simple and straightforward, once you add
● Correlates to: the serum in the reagent (which contains latex beads in the carrier)
○ Severity of the disease and agitate it slowly, within 15 minutes you will already see the
○ Nodules reaction.
○ Other organ involvement
○ Other infections VIDEO DEMO - ASO LATEX AGGLUTINATION TEST
(QUALITATIVE)
Material Needed:
● Serum sample
● Reagents
○ Red Cap = positive control
○ Blue Cap = negative control
○ Whtie Cap = ASO Latex reagent
● Black glass slide (or disposable slides) with 6 test cells
● Disposable mixing sticks
● Sample container for saline solution
● Glass marker (white)
● Pipette tips (yellow)

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QUALITATIVE TEST: 3. Mix using separate applicator sticks, and spread the mixtures
1. Place 1 drop of positive control on the test cell labeled over the entire area of the particular cell.
with PC (positive control)
2. Place 1 drop of negative control on the test cell labelled 4. Tilt the slide around slowly and evenly. You can do this in two
with NC (negative control) ways:
3. PLace 1 drop (40uL) of patient serum on the test cell
labelled with T ● Manual: Using your hands, rock back and forth at 8
4. Place 1 drop of ASO latex reagent on all test cells. Be to 10 times per minute for around 2 minutes.
careful no to contaminate the dropper with the previously ● Automated: Using a Laboratory Rotator, set the
dispensed fluids rotation at 100 rpm, and leave the glass slide to
5. Using the disposable stirrer, mix the fluid together within rotate for 2 minutes.
the circle. Use different mixing sticks on each test circle
6. Perform manual rotation for 2 minutes, Or subject the 5. Read the results under bright light.
slide to mechanical agitator at 100rpm for 2mins
7. Observe for clumping or agglutination If NEGATIVE, report this immediately.
8. It is expected that in the test cell for PC, agglutination is
observed. In NC, no agglutination should be seen If POSITIVE, proceed with SEMI-QUANTITATIVE
DETERMINATION.
VIDEO DEMO - ASO LATEX AGGLUTINATION TEST
B. SEMI-QUANTITATIVE DETERMINATION (for
(SEMI-QUANTITATIVE)
POSITIVE Qualitative Determination Results)
Saline buffer as diluent
1. Perform a two-fold serial dilution using NSS (refer to package
1. Add a volume of saline buffer in all the test cells. Refer to
insert) on the patient sample.
the package insert how much volume will be used
2. On the first cell, add a volume of serum sample. Refer to
2. Test each dilution using the procedure for QUALITATIVE
the package insert how much volume should be used.
DETERMINATION.
3. The same volume of fluid is used in transferring from one
test cell to another. Do not forget to mix after transferring 3. Read the TITER in the last dilution step with visible agglutination
the fluid to another test cell. Continue the serial dilution in (POSITIVE result).
all the remaining tubes
4. Add a drop of the ASO latex reagent in all the dilutions
you just made. Make sure to avoid contamination of the
reagent dropper
5. Using a disposable stirrer, mix the fluids in each test cel C. INTERPRETATION OF RESULTS
starting from the last cell with the highest dilution (circle
6) Multiply the TITER with the CONVERSION FACTOR of the RF
6. You may use one disposable stirrer in mixing the fluid in Latex Agglutination Slide Test: 12 IU/mL
all test cells PROVIDED THAT you will start on the test
cell with the highest dilution (last circle). In this video, it is NOTE: The conversion factor may vary among RF Latex
6 Agglutination Slide Test Kits manufactured. ALWAYS READ THE
7. Manual rotation for 2mins or mechanical rotation set at PACKAGE INSERT.
100rpm for 2mins
8. Observe which test cell displayed agglutination EXAMPLE:
9. The dilution of the LAST circle that yielded the positive
Titer after dilution is 1:16
reaction will be used to compute for the antibody titer
Computation: 16 x 12 [IU/mL] = 192 IU/mL
NOTE:

● The RF Latex Agglutination Slide Test is similar to

the ASO Latex Agglutination Slide Test. The RESULT TO BE RELEASED:
difference lies in the CONVERSION FACTORS
used. RHEUMATOID FACTOR (RF) LATEX AGGLUTINATION
● Bring all materials and samples to ROOM SLIDE TEST:
TEMPERATURE!
● Mix the latex reagent carefully prior to testing. This *Qualitative Determination: POSITIVE
is to suspend the latex particles completely.
● Make sure that the six-celled black glass slides are *Semi-Quantitative Determination: 192 IU/mL
clean and dry.
● Procedures may vary among test kits due to
differences in manufacturing. Thus, it is ESSENTIAL
to read the PACKAGE INSERTS prior to testing.

A. QUALITATIVE DETERMINATION
1. Pipette or drop onto separate cells of the glass slide the
following:

a. Positive Control = 1 drop

b. Negative Control = 1 drop

c. Serum Sample = 1 drop

2. Add each a drop of latex reagent onto the sample and control
cells.