IMMUNOLOGY AND SEROLOGY LAB

LECTURE 1: DILUTION
MIRALYN MADEL QUIRIT, RMT
AUGUST 17, 2021
For updates and corrections → @mar4rii on Twitter

DILUTION 5
● Weakend solutions = 20
● Involves a particular solution with a definite concentration =
1
● Process of adding additional solvent to a solution to 4

decrease its concentration

● A mixture having 2 components:
○ Substance to be diluted/solute (serum, red EXAMPLE NO. 2
cells, urine, acids, etc)
○ Diluent/solvent (distilled water, saline solution, Dilute 3 mL of a serum with 25 mL of saline. What is the
buffer, or indicated chemical reagent) dilution?
● Mixing a solute with the diluent results to the reduction of
a particular solution
● Common solute in Serology: Serum or other body fluids

● Total volume of solution = solute + solvent

● When performing dilution, get the volume of solute and - answer can be expressed as a fraction or with the use of a
add it with the volume of your diluent colon
● RATIO: the relationship of the solute and the solvent
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒 What is the resulting dilution when 0.5 mL of a serum is
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 added with 1.5 mL saline?
● DILUTION FACTOR

SINGLE DILUTION
● Stock solution + distilled water = single dilution
● One step process of adding diluent to the stock solution
● Example: Dilute 1mL of serum with 9mL of saline

0.5/2.0 is an acceptable dilution, but it is recommended

to expressed it as a whole number
1𝑚𝑙
= 1+9𝑚𝐿
1
= 10
● If 1/10 is the dilution what is the ratio?
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
1
𝑅𝑎𝑡𝑖𝑜 = 9

EXAMPLE NO 3
EXAMPLE NO 1

Make 250 mL of a 1/10 dilution of serum in saline.

Five milliliters of serum is diluted up to 25mL with saline. What is
the serum dissolution? What is the serum to saline ratio?
given: TV - 250 mL, Dilution – 1/10
𝑠𝑜𝑙𝑢𝑡𝑒 problem: how much volume of serum is used?
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
5𝑚𝐿
= 25𝑚𝐿
1
= 5
If the total volume of the solution is 25, what is the total volume
of the solvent?
Total volume = solute + solvent
25mL = 5mL + x
25mL - 5mL = x
20mL = x 25 mL serum is added with saline to bring the total volume
up to 250 mL/
What is the serum to saline ratio?
𝑣𝑜𝑙. 𝑠𝑜𝑙𝑢𝑡𝑒 Problem: How much saline (solvent) is needed to add to 25 mL
𝑅𝑎𝑡𝑖𝑜 = 𝑣𝑜𝑙. 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
(serum) in order to make a 250 mL volume of a solution having
a 1:10 dilution?

1
TV = 25 + y
y = 250 – 25
ysolvent = 225 mL

to check:
serum = 8 mL
8 mL + 32 mL solvent = ⅕ dilution
TV = 40 mL

SERIAL DILUTION
● We do series of the steps
● We repeat the procedure more than once.
- by using 25 mL serum, added with 225 mL saline, we will ● Production of solutions having different concentrations of
yield a 1:10 dilution with a total volume of 250 mL. the same substance.
● The concentration of the solution being diluted decreases
● Make 1/10 dilution of serum in saline. with increased dilution.

EXAMPLE NO. 7

- You can choose to use other values but just reduce it to its Ex: When is the dilution if you add 1 mL aliquot of a specimen to
simplest form. 9 mL of diluent?

● If you want to have a total volume of 70 mL:

Step 1 : D= 1/10
All successive tubes would have 9 mL of diluent. You
7 mL – serum would then transfer 1 mL of the initial diluted sample into the
63 mL - solvent next tube, mix, transfer 1 mL to the next, mix and so on.
● Prepare a series of tubes (ex. 3 tubes)
Given : 1/10 dilution ● In the successive tubes, you are going to add the
● No definite volume of solvent same volume of the solvent used in the first step
○ Serum = 2 mL ● In series of tubes, you have to use the same volume
of diluent that you used in the first step
● Transfer 1ml of the initially diluted sample to the next
tube, so on and so forth
10= 2 + y
10 - 2 = y
Y= 8 mL (solvent)
CAN BE USE THIS FORMULA FOR BIGGER VALUES
● Solute = 20
● TV= 1/10

X = 10 (20)
X= 200 (TV)

solvent= 180 mL
● You prepared 5 tubes
● Stock solution (solute)
● Mix 1 ml of your solute and 9ml of diluent
= 1/10 ○ Dapat same ang volume ng diluent in all of the
tubes
○ Place 9ml of diluent in all of the tubes prepared
● In step 1: you will get a volume from your stock solution,
EXAMPLE NO. 4
from the problem, it needs 1 ml. Transfer to Tube 1 that
contains 9 ml of the diluent
○ Total volume of the solution now becomes 10
Determine the amount of serum in 40 mL of a ⅕ dilution of
ml
serum saline.
○ Having a dilution of 1/10
● Get from tube 1 and transfer it to tube 2
○ Use the same volume of solute that you’re
going to transfer from tube 1 to tube 2 = 1 ml
● Tube 2 total volume becomes 10ml
○ Dilution changes (increases) = 1/100
2
 ○ The color also changes; becomes lighter ○ Discarded vol: 1ml
○ The concentration of substance decreases, but ○ Get 1ml from the last tube and discard
the value for dilution increases ● Tubes will be subjected for testing
● Repeat the same process to the succeeding tubes ● As we transfer 1 ml from the previous tube to the next,
○ Tube 3 total volume becomes 10mL with a the volume of the previous tubes will decrease. (from 10
dilution of 1/1000 (increases) ml to 9ml)
○ Concentration decreases (lighter blue) ● If you had 5 tubes, what would be the final dilution of
● In serial dilution, the concentration of the substance tube 4?
decreases in the successive tubes whereas the value ○ 1/10000
for dilution increases. ○ How did we come up with that value?
● Final step: Discard volume
○ Volume of solute we set

SERIAL DILUTION (ex no. 7)

1 2 3 4 5

Volume of
1ml 1ml 1ml 1ml 1ml
solute

Volume of
0ml 9ml 9ml 9ml 9ml
diluent

Total volume 10ml 10ml 10ml 10ml 10ml

Tube dilution 1/10 1/10 1/10 1/10 1/10

Final dilution 1/10 1/100 1/1000 1/10000 1/100000

● Tube dilution - focuses on the dilution of the tube itself without considering the initially diluted sample
● Final dilution - must determine first the tube dilution
○ Multiply the tube dilution with the previous final dilution

MIXED DILUTION
● Pag mixed dilution, iba ang volume ng diluent sa Tube 1, iba din ang volume ng diluent sa Tubes 2 and onwards

● Transfer 1mL of serum in the first tube ● The same principle still applied na as the tube
● Sa first tube, naglagay ka ng 9mL of the diluent for the progresses,
first tube and 4mL of diluent on Tubes 2,3,4, and 5. The concentration decreases but the value of the dilution
● Form tube 1, you get 4mL and transfer it to the increases
succeeding tubes ● We are still going to use the same table in the
● Get 4 mL from the last tube then discard computation
● Problem: ● So how do we do it in the computation?
○ A serial dilution is made by placing 9mL ○ Place the corresponding values in the table
diluent in the first tube of a series and 4mL in ○ Then compute for the tube dilution and the final
each of the remaining four tubes. 1mL of dilution the same thing we did in the basic serial
serum is added to the first tube and 4mL from solution
the first tube is transferred to the second tube
and then to each succeeding tube. The last
4mL transferred and discarded.
○ Process ng pag transfer is still the same

3
 MIXED DILUTION

1 2 3 4 5

Volume of 1mL 4mL 4mL 4mL 4mL

solute

Volume of 9mL 4mL 4mL 4mL 4mL

diluent

Total 10mL 8mL 8mL 8mL 8mL

volume

Tube 1/10 1/2 1/2 1/2 1/2

dilution

Final 1/10 1/20 1/40 1/80 1/160

dilution

● Then we get the dilution of Tube 1

D = 1/10

● For Tube 2

D = 4/8 converted to ½
(the same for the succeeding tubes)

● For the computation of the final volume

○ The final dilution of tube 1 is the tube itself so it will be 1/10
○ The final dilution of Tubes 2 to 5 will be the same kung ano ginawa sa serial dilution kanina.
■ Multiply the tube dilution of the next tube to the Final dilution of the previous tube
■ 1/10 * ½ = 1/20

EXAMPLE NO. 8

● You are given a series of 5 tubes, each of which contains 2 mL of diluent. 0.5 mL is added to the first tube and 0.5 mL is carried out in
the remaining tubes. What is the dilution of the mixture in tube 3 and tube 5?

SERIAL DILUTION

1 2 3 4 5

Volume of 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL

solute

Volume of 2mL 2mL 2mL 2mL 2mL

diluent

Total 2.5mL 2.5mL 2.5mL 2.5mL 2.5mL

volume

Tube 1/5 1/5 1/5 1/5 1/5

dilution

Final 1/5 1/25 1/125 1/625 1/3125

dilution

4
 EXAMPLE NO. 9

● You are given a series of 10 tubes, each of which contains 4mL of diluent. 1 mL of fluid is added to the first tube and dilution using
0.5mL is carried out in the remaining tubes. What is the serum concentration in tubes 4 and 8?

SERIAL DILUTION

1 2 3 4 5 6 7 8 9 10

Volume of 1mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL
solute

Volume of 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL 4mL
diluent

Total volume 5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL 4.5mL

Tube dilution 1/5 1/9 1/9 1/9 1/9 1/9 1/9 1/9 1/9 1/9

Final dilution 1/5 1/45 1/405 1/3645 1/32805 1/295245 1/2657205 1/2391484 1/4.65x10^ 1/5.16x10
5 -9 ^-10

DILUTION as TEST ENDPOINTS

● TITERS
○ To determine the value of the titer
○ Endpoint reaction can be: (most common)
■ Agglutination
■ Precipitation
○ Perform serial dilution

● Serially diluted sample = becuase the dilution increases DILUTION FACTOR

in the succeeding tubes and the concentration decreases
● 4 tubes with reactions = 1,2,3 ,4
○ 5 & 6 = no reaction; disregard 1
● Which among the 4 has the smallest amount of Titer dilution =
8
concentration that produces a particular endpoint
reaction? 1
○ Tube 4 = has the least amount of Dilution factor = or 8
8
concentration; masyadong diluted kaya mahina
na ang reaction; this tube is still counted Dilution factor = reciprocal of the dilution
because it still has observable reaction
○ Titer = 1/160 Ex. ASO test
■ Used for the computation of the - when performing semi-quantitative, u will use a dilution factor of
concentration of the antigen ot the 200 iu/L (concentration of the reagent in the kit)
antibody
● RED CELLS = most common indicator
200 x 8 = 1,600 iu/L (reported concentration of ASO)
○ It is necessary to use indicator for us to
visualize the reaction of the antigen and
antibody - Reporting is in the form of numbers
○ The common observation is agglutination - Semi-quantitative because we did not exactly measure the
antibodies, we just did the titer determination and multiply it with
the factor of the test

5
 IMMUNOLOGY AND SEROLOGY LAB

LECTURE 2: ANTI-STREPTOLYSIN O
KYLE VINCI P. SOLANO, RMT, MD
AUGUST 20, 2021
For updates and corrections → @mar4rii on Twitter

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
1
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
2
 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)

3
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.

4
 IMMUNOLOGY AND SEROLOGY LAB

LECTURE 3: C REACTIVE PROTEIN

PROF. CHALEE SIENES-REYES, RMT, MSMT
AUGUST 30, 2021
For updates and corrections → @mar4rii on Twitter

WHAT IS C-REACTIVE PROTEIN? ● Binding free chromatin

● C- Reactive Protein (CRP) ● Inducing cytokine release
○ Studied by William S. Tille and Thomas
Francis. They were able to detect form
pneumococcal infections
● CRP was first detected by Tille and Francis in 1930
● Detected in the blood of patients with Streptococcal
pneumoniae infection that formed a precipitate when
combined with polysaccharide of the cell wall of the
pneumococcus and hence called- C- reactive
substance – CRP.
● a pentameric protein
● Regulated by CRP gene (located in chromosome 1)
● synthesized by the liver in minimal amounts in normal
condition
● elevated in response to IL-6 which has a pleiotropic
● activity
○ note: IL-6 is a single glycoprotein produced by
monocytes, endothelial cells, and adipose
tissues
● is an acute phase reactant protein (APRP) that is
elevated in the plasma when there is inflammation
throughout the body
○ (tissue damage, necrosis, dead cells or Complement component: c3
apoptotic cells) release There are 5 endpoints when the compliment is activated:
“lysophosphatidylcholine” embedded in the 1. Chemotactic anaphylatoxin production
cellular membrane) which will be acted upon by - Capable of causing vasodilation
the CRP - Allows passage and leakage of some
● Belongs to the Pentraxin family : fluids thereby inflammation happens
○ Five symmetric circular identical non covalent - Accumulation of cells in the area =
sub units with a phosphocholine (PCh) swelling
binding site 2. Formation of immune complexes that will eventually
be taken out of the body
HOW IS CRP PRODUCED? - However, when these immune complexes
● Normal range: 0.8mg/L and 3.0 mg/L cannot be removed because it is bigger, they
will be deposited in some areas of the body and
will eventually cause inflammation. At some
point, there is the cause of an autoimmune
disease
3. activation of antigen received by the antibody as
immune complex
4. Activation of the complement directly having the
lysis of the foreign agent
5. Opsonization and bacterial phagocytosis

Binding of Free Chromatin

IL-6 produced by the monocytes, macrophages, and endothelial
cells
↓
IL-6 will stimulate the liver
↓
The liver will produce CRP
↓
CRP will be attracted to lysophosphatidylcholine on sites that are
undergoing tissue damage, injury, and necrosis
↓
Effector cells: monocytes, macrophages, and endothelial cells will
release again IL-6
↓
Elevation of CRP

FUNCTIONS OF C-REACTIVE PROTEIN ● Free chromatin is released by cells undergoing apoptosis

● Activate complement pathway ● LPC will be acted upon by the CRP
● Opsonizing bacteria, fungi, and parasites
● Agglutinating particles Inducing cytokine released
1
 the blood as to fibrinolysis
○ Tissue damage
● Result:
○ Atherosclerosis: deposit of LDL
○ Influx of the cell
○ Aggregation of platelets
○ Vasoconstriction
RECALL
● Facilitates infiltration of monocytes into the vessel wall
○ Monocytes = production of IL 6→ stimulate the
liver for the production of the CRP
○ CRP goes high.
● Induces complement activation
○ Can cause immune complexes to be deposited
in some other parts→ cause influx of cells
stimulated in the form of chemotactic factor
allowing cells fo to the area to combat those
cells (foreign agents)
● Increase of LDL uptake and oxidation
○ Cause of atherosclerosis
● Increase nitric oxide production
○ Preventing homeostasis
● Induce production of tissue factor
Pro-IL 1 (inactivated) will be converted to Activated IL -1 by the ● Upregulates the expression of adhesion molecules
caspase-1 (interleukin 1 activator enzyme) with inflammasome ○ Platelets are aggregated
↓ ● Inhibits fibrinolysis by increasing expression of PAI- 1
IL-1 ○ Leaving the tissue damaged
↓
IL-6 Importance of CRP detection
↓ A. Differential diagnosis and classification of inflammatory
Liver is stimulated disease
↓ B. Diagnosis and management of infection
The liver will produce CRP and fibrinogen C. Assessment of disease activity in inflammatory
↓ conditions
The endothelial lining will produce their substance in response to
the stimuli

SLE RA
When CRP is elevated
● No longer helpful C3 lower high
● Capable of inhibiting the activities of endothelial nitric
oxide synthase. CRP Increased CRP but high
○ Synthase - accumulation way lower than RA
○ NO - maintain vascular homeostasis:
modulation of vascular dilation, regulation of
cell growth, protection of vessel; prevent ● CRP higher is brought about by the RA
aggregation in the endothelial lining. ● C3 - the amplification of loop
● Increased ROS ○ Inflammation of joints is responsible for the
○ ROS - upon metabolism of oxygen, there is a increase in CRP level.
product called superoxide = responsible for
immune response in killing microbial invasion; A. Differential diagnosis and classification of inflammatory
serves as a messenger in normal cell which disease
signals transduction and cell cycling and
homeostasis.
● Triggers production of ET- 1 (endothelin)
○ Capable of vasoconstriction
■ Vasocontriction = not good;
contributes to vascular dysfunction.
● Opsonization
○ Uptake of cells to eliminate foreign agents.
● Croh’s Disease versus Ulcerative colitis
● Fibrosis
○ CD is associated with a strong CRP
○ When there’s injury, theres also dissolution of
response, UC has only a modest to
2
 absent CRP response Remember :
○ UC - inflammation in the large bowel
(confined in the mucosa)
○ CD - inflammation anywhere in the
GIT (transmural)
■ From the esophageal area
to the anus - greater area
for the manifestation of
inflammation

A. Classification of Inflammatory conditions

Acute Conditions Chronic Conditions
Bacterial Infections ● Arthritis
● Lungs ● Systemic Lupus
● Urinary Tract Erythematosus
● Digestive Tract ● Inflammatory Bowel
● Skin and other sites disease Crohn’s
with or without sepsis disease)
● Ulcerative colitis
● Make sure that the serum sample collected is free from
● Acute - infection right at the very onset
any red cell contamination or any hemolysis.
○ The possible increase of the CRP level in
○ How to know if it is not contaminated?
response to the removal of bacterial agents and
○ Remove and transfer the plasma or serum to
expresses LPC (ligand for the phosphocholine
another tube and spin again
of CRP)
○ When there is stippling of RBCs, it was just
● Chronic - you have it for a longer period
contaminated, thus must be transferred again
○ When there is a tinge of red and no
B. Diagnosis and Management of Infection
sedimentation, there is hemolysis
● Bacterial Endocarditis
■ Cannot be used and should
● Postoperative complications including infection
re-collect
and Thromboembolism
● If testing can not be Done
● Neonatal Septicemia and meningitis
● Samples can be refrigerated within 3 days at
● Intercurrent infection in Leukemia and its
2-8 degree C
treatment
● Frozen at -20oC if more than 3 days
● Intercurrent infection in SLE
● Bring all to Room Temperature before USE:
● Management on how the CRP level progresses
○ Reagents
● The moment the inflammation subsides, the
○ Frozen Samples
CRP goes down until to a normal level
○ Refrigerated sample
○ Note: Latex reagent should be mixed
C. Assessment of disease activity in inflammatory
thoroughly before use.
Conditions
■ Because particles tend to settle the
● The greater the intensity of the CRP level, the greater the
moment you drop
chance of the damage
→ if it is not latex reagent, there are also other test methods:
- Heart Disease - Acute Vasculitis
- Diabetes pancreatitis (inflammation in the Other Test Methods
- Allergic - Anklosing blood vessels) : ● Enzyme link Immunosorbent Assay (ELISA)
reaction spondylitis - Polyarteritis ● Radio Immunosorbent Assay (RIA)
- Malignancy - Crohn Disease nodosa ● Chemiluminescence immunoassay (CLI)
- Graft versus - Familial Fevers - Bechet ● Fluorescence immunoassay (FI)
Host (the graft including familial syndrome ● Immunoturbidimetry or Turbidemetry
will have - Mediterranean - Polymyalgia ● Nephelometry
greater power fever Psoriatic ● Radial immunodiffusion (RID)
than the host) arthropathy → these different methods would also have different techniques
- Rheumatoid
Arthritis Bring all to room temperature before USE:
(Juvenile ● Reagens
chronic) ● Frozen samples
- Rheumatoid ● Refrigerated samples
Arthritis
- Rheumatic Always read the package inserts or the manufacturer’s
Fever literature
- Reiter disease
Qualitative and Semi Quantitative CRP Test
How is the CRP Test done?
● There is No special instruction given to patients. Latex Agglutination Method
● No special preparation to be made by the patient. ● Materials in the KIT
● It can be collected anytime of the day, but make sure it is ○ CRP Latex Reagent: A suspension of uniform
proper polystyrene particles coated with monospecific
antihuman CRP (goat) in saline solution , pH
7.5 + 0.5. Reagent sensitivity is adjusted to
approximately 6(5-10) mg / L mix well before
using.
○ CRP Positive Control Serum: A stabilized
prediluted human serum containing >20mg/L
CRP
○ CRP Negative Control Serum: A stabilized

3
 prediluted human. B. Semi-Quantitative Test
○ Glycine Buffer (20x): add one part to nineteen 1. Set-up at least five test tubes: 1:2, 1:4, 1:8, 1:16, 1:32,
parts of distilled water before use. etc.
■ Used as the diluent when you 2. Dilute sample according to dilution factor on each test
continuo on for the semi-quantitative tube with saline solution
○ Reaction Slide 3. Place one drop of each of the positive and negative
○ Stirring Sticks controls onto the slide ring. Place one drop of each
○ Test Cards dilution in successive fields.
4. Gently resuspend the CRP Latex reagent and add one
drop to each test field
5. Mix well with provided stirring sticks. Gently rock the slide
for two (2) minutes (or by using a laboratory rotator
machine for 1 minute at 100 rpm) and read immediately
under direct light.
● Semi-quantitative is the same as quantitative, except that
specimen used in semi-quanti is a diluted sample.
● So long as you see agglutination occurring, you continue with
your test
● In the determination of titer, you will have the last positive
dilution that gives you the endpoint dilution
● Discard the test if there is any inconsistencies in the results
→ Different manufacturers may have different presentation or ● When preparing for a dilution, take note of how much you are
packaging going to discard
→ Take note of the tubes cap or vials cap: red, green. Black, white ● In the mixture, they will all have one drop of the reagents
- These are indicators only
- Ex. Green - negative control ● Most often could be dropped upside down, or it could be in an
- Red - positive control ampule or a vial with a built-in dropper with a curve
→ always read labels ○ Designed to create uniform drops
→ black slides with blue ring, black slides with red ring and white ● The specimen should not be in the laboratory rotator for 2
with black rings minutes.
- Those are test cards, may come in glass or in cards
- Whenever in cards, make sure when you mix, mix lightly, Limitations
to avoid scratching ● Reaction time is critical. If reaction time exceeds two (2)
- Scratches may have cause absorption of some particles minutes, drying of the reaction mixture may cause
of the simple = erroneous result false-positive results.
○ Drying could cause particle formations
● Materials required but not provided interpreting it as positive
○ Timer - to have a uniformed time depending on ○ There will also be possible contamination of
the test manufacturing literature any particles will be trapped in the surface area
○ Test Tubes ● Freezing CRP Latex Reagent will result in spontaneous
■ Microtubes agglutination
○ Test Tube Rack ○ It should be indicated in the CRP Latex
○ Serological pipettes Reagent, package insert, or te vial that
■ Laboratory rotator machine temperature must be in 2-3 degrees
■ Pasteur Pipette ● The intensity of agglutination is not necessarily indicative
■ Pipette tips - 1 for each patient of relative CRP concentration; therefore, screening
sample; disposable reactions should not be graded.
○ It is will be reported as positive.
● A false-negative can be attributed to a prozone
phenomenon (antigen excess). Therefore, it is
recommended to check all negative sera by retesting at a
1:10 dilution with Saline Solution.
○ When you have the pro-zone, you also have
the post-zone phenomenon that causes a false
negative.
○ True negative and true positive can be found in
the zone of equivalence
● If there is increased concentration in the solution, you
might not be able to appreciate the presence of the
antibody is overwhelmed by the process of antigen
A. Qualitative Test: ● There is a recommendation to have it in a ten-fold
1. Bring reagents and specimens to room temperature dilution or 1:10 with saline
before use. ● Test again in the same amount
2. Place one drop (40 µl) of CRP Positive Control on field ● If it gives you a negative reaction, then it is negative.
#1 of the reaction slide. Place one drop (40 µl) of the
CRP Negative Control on field #2. Using a serological Interpretation
pipette place (40µl) of undiluted test sample to field #3. - Both qualitative and semi-quantitative have the very
Continue likewise with additional unknowns. Use different same interpretation as to positive and negative
pipette tips for each sample. A. Qualitative Test:
3. Gently resuspend the CRP Latex Reagent and add one ● A negative reaction is indicated by a uniform milky
drop to each test field. suspension with no agglutination as observed with the
4. Mix well with the provided stirring sticks. CRP Negative Control.
5. Rotate the slide for 2 minutes and read immediately ● A positive reaction is indicated by any observable
under an oblique indirect light (or by using laboratory agglutination in the reaction mixture.
rotator machine 1 minute at100rpm} ● The specimen reaction should be compared to the CRP
Negative

4
 ○ Maximize the number of test sample in each
testing time rather than to test two patients

Sensitivity: 6 (5-10) mg/L

● Whenever you have a positive, 6 mg/L becomes your
value to be multiplied with whatever the factor would be
● If negative, the value is less than 6 mg/L

B. Semi-Quantitative Test:
● The approximate CRP concentration in the patient
sample is calculated as follow: 6×CRP titer = mg/L

Dilution CRP mg/L

1:2 12
1:4 24
1:8 48
1:16 96
1:32 192
1:64 384
● Ex. Patient 4 gives you the last reaction visible at 1:32
and negative on the 64. Thus, your titer is 32.
● Highest serum dilution that gives you the endpoint
reaction. So this is the least concentration of serum but it
has the highest dilution = Titer
● 6x32 = 192
● Report: 192mg/L
● How much was the normal range?
○ 0.8mg/L - 3mg/L = way above the normal level
○ Thus, the severity of the inflammation is there.

5
 IMMUNOLOGY AND SEROLOGY LAB

LECTURE 4: SEROLOGIC TESTS FOR

SYPHILIS
PROF. BENJO A. DALUPAN, RMT
AUGUST 30, 2021
For updates and corrections → @mar4rii on Twitter

SYPHILIS ○ Primary chancre appear on hands, mouth, skin

● Caused: Spirochete Treponema pallidum subsp. pallidum and other parts of the body.
● A sexually transmitted disease that affects the skin and ○ Typically heal on their own over a few months
mucous membranes of the external genitalia and also ● Some spirochetes go to nearby lymph nodes where they
sometimes the mouth. caused by lymphadenopathy (enlargement of lymph
● Gram negative bacteria that look like corkscrews. nodes)
● Possess 6 thin endoflagella called the axial filaments ● If it is acquired thru a blood transfusion, there may not be
which renders them move in a unique spinning motion. early localized stage at all or no primary chancre.
● It cannot be cultured in an ordinary media. ● Primary syphilis
● Too small to be seen in the light microscope that's why ○ Can usually be found in the chancre
special procedures and serologic tests are required to ○ Treponyms can usually be found in the
help screen the Treponema pallidum. chancres
■ We can view or observe them in dark
Mode of Transmissions: field microscope (appear white
1. Acquired Syphilis against a black background)
● Happens when T.pallidum enters the body through body ● People with primary syphilis tend to have a non reactive
fluids. result to serologic tests
○ Cuts/Breaks
○ Sexua lContact. 2. Secondary Syphilis
■ Including oral, anal and vaginal sex. ● Dissemination stage
○ Contaminated Needles ● Appears 6-8 weeks
○ Direct Contact’ ● Generalized rash and secondary lesions (eyes, joints or
■ With a skin lesion of an infected CNS)
person. ● Condylomata lata
■ Lesion - covered with rich of ○ smooth , white, painless wartlike lesions
spirochetes ○ Appear on most areas like the genitals, around
2. Congenital the anal region, and armpit
● When a pregnant person has syphilis and T.pallidum ● (+) dark field microscopy
enters a baby either in the uterus or while the baby exits ● Reactive to serologic tests
thru the vagina at birth.
● Also called as vertical transmission 3. Latent Syphilis (Hidden syphilis)
● Disease enters a dormant or asymptomatic phase
4 CLINICAL STAGE OF SYPHILIS ● 2nd year of infection
● No clinical symptoms
1. Syphilis Primary (early) ● Serologically reactive
2. Secondary ● Spirochetes can mostly be found in the tiny capillaries of
3. Latent various body organs and tissues
4. Tertiary (late) ● Therefore, the results will be serologically reactive

1. Primary/ Early Syphilis 4. Tertiary/Late Syphilis

● Appears 2-8 weeks ● Gumma/ Gummata
○ After it lands on the skin or mucous ● 80% - CNS involvement “neurosyphilis”
membranes. ● 10% - heart involvement “cardiovascular syphilis”
● HARD CHANCRE - painless (chancUred) ● Serologically reactive
● lasts for 1-5 weeks ● Type IV hypersensitivity reaction
● (+) Darkfield microscopy ○ Immune response lead by the t cells
○ Typically appear white against a dark ○ Recruit phagocytes and causes release of
background pro-inflammatory cytokines such as the tumor
● Nonreactive to Serologic tests necrosis factor, IL-1 and IL-6
● Destroy the soft tissue and skin→ formation of ulcers ○ All of these immune responses leads to local
called as syphilitic chancres swelling or edema, redness, warm and
○ Painless systemic symptoms like fever
○ Has a hard base ● In some cases, the immune cells start to huddle around
○ Usually covered by fluids rich in spirochetes. and form a granulomatous lesion called
○ This can spread to other parts of the body and Gumma/Gummata
other individuals. ○ Has lots of different types of immune cells that
● Soft chancre get surrounded by the outermost layer
○ Caused by another bacteria Haemophilus fibroblast
ducreyi. ○ There aren't any spirochetes in gumma/
● In individuals who acquire syphilis thru sexual contact, gummata, its just immune cells getting excited
primary chancre develops around external genitalia. for no apparent reason
● Physical touch, lesion and in some other way ○ The tissues at the center often ends up without

1
 oxygen leading to coagulative necrosis NONTREPONEMAL TEST
● in tertiary syphilis, various organs like the heart and ● Venereal disease research laboratory (VDRL) test
blood vessels are damaged --- Cardiovascular syphilis ● Rapid plasma reagin (RPR)
○ Around 10% of the patients develop ● These non-treponemal tests both use cardiolipin or the
cardiovascular problems which results in aortic wasserman antigen
aneurysm ● They detect the reagin or the anti-cardiolipin
● Brain and spinal cord can also get damaged, called as
Neurosyphilis
○ Around 80% patients experience CNS
involvement which can result in paralysis or
dementia
● Other organs may also get damaged, like the liver, joints
Venereal Disease Research Laboratory (VDRL) Test
and testes which haven't earned their unique names yet
● Patients with tertiary syphilis will be serologically reactive ● Principle: Flocculation
○ Flocculation occurs when the precipitate floats
4 Clinical Stages of Syphilis instead of sedimentation
1. Primary (early) - Hard chancre ● Equipment:
2. Secondary - Condylomata lata ○ Antigen Needles
3. Latent - Asymptomatic ■ Qualitative - 1/60 mL (60 drops/mL) of
4. Tertiary (late) - Gumma antigen
■ Quantitative - 1/75 mL (75 drops/mL)
Darkfield Microscopy of antigen
○ Saline Needles - Quantitative - 1/100 mL (100
drops/mL) of saline
○ Mechanical rotator - 180 rpm
○ Glass Slides
○ Serological Pipets

● Diagnosis of acquired syphilis starts with identifying the

spirochetes in the fluid from chanre which can be done
using darkfield microscopy or dark field microscope
● A darkfield microscope shines thin slivers of light on a
slide so that the background appears dark while the
extremely thin spirochetes lights up
● Spirochetes appears to be white against the black ● Ex. Patients with syphilis develop an antibody known as
background reagin to a tissue derived substance which is known as
cardiolipin or antigen
Nontreponemal Treponemal ● So when the cardiolipin, the nontreponemal antigen is
Antigens Wasserman antigen Nichols added to heated serum of patient containing the reagin or
(Cardiolipin) Reiter the antibody, there will be antigen and antibody reaction
Antibodies Reagin Anti-treponemal resulting in flocculation
(Anti-cardiolipin) antibodies ● Positive result of this test: Flocculation
● The diagnosis is confirmed with serological tests which ● Reagent:
look for antibodies against treponema pallidum antigens ○ Antigen’
● 2 type of antigens: Alcoholic solution of
○ Nontreponemal antigens “Cardiolipin-cholesterol-lecithin”
■ Wasserman (Cardiolipin) ■ Cardiolipin
● is a normal constituent of ● Serves as an antigen which
host tissue and it can be is a phospholipid in nature
released when there is isolated from the beef heart
damage in the cells ■ Cholesterol
● Not specific for it ● Center of absorption of
○ Treponemal antigens tissue lipids to increase the
■ Nichols strain size of the antigen
● Strain of treponema ■ Lecithin
pallidum ● Produce standard reactivity
● Pathogenic & virulent ○ 1% buffered saline, pH 6.0
■ Reiter strain
● Comes from the treponemes ● Sample Preparation:
but not from the treponema ○ INACTIVATION
pallidum ■ Serum- heated at 56 degrees Celsius
● Non pathogenic for 30 minutes
● For the antibodies, ■ Heating the serum at 56 degrees
○ Nontreponemal celsius for 30 minutes inactivate the
■ Anti-cardiolipin complements in the serum
● Anti-cardiolipin antibodies = ■ Compliments interfere with tests
called as reagin in the blood ○ RE-INACTIVATION
○ Anti- treponemal antibodies ■ Serum- heated at 56 degrees Celsius
■ Detects antibodies directed against 10 minutes
the treponema pallidum or detects the ■ Serum should be re-inactivated if
anti-treponemal antibody there will be an interval of 4 hours or
more between inactivation and testing

2
Qualitative Quantitative
● We need to inactivate the complement by heating the ● Recall the “dilution Lesson”
serum at 56 degrees Celsius for 30 minutes ● Perform on “reactive and Minimally Reactive Sera”
● Serum + 1 drop (1/60mL) antigen ● Twofold dilutions serum ranging in 0.9% saline
○ Serum:Antigen Ratio (3:1) ● Report the highest dilution giving reactive results
○ Antigen: Cadiolipin-Cholesterol-Antigen solution
● Rotate on a mechanical rotator for 4 minutes at 180 rpm
● Observe MICROSCOPICALLY for the presence of
flocculation IMPORTANT NOTES TO REMEMBER

False Positive
● VDRL - SLE, RF, IM, Malaria, and Pregnancy
● RPR - IM, Leprosy, RF

POSITIVE RESULTS (VDRL/RPR) ⟶ MORE SPECIFIC

TREPONEMAL TEST
● Interpretation ● All positive results in VDRL or RPR must be confirmed
○ Reactive when there are medium to large by a more specific treponemal test
clumps
○ Weakly reactive if there are small clumps TREPONEMAL TEST
○ Non-reactive- no clumps
● All sera with reactive or weakly reactive results in TREPONEMAL TEST
qualitative VDRL must be tested with quantitative VDRL 1. Treponema pallidum immobilixation (TPI) test
2. Fluorescent treponema pallidum antibody absorbed
Quantitative (FTA-ABS) test
● Recall the “Dilution Lesson” 3. Microhemagglutination-treponema pallidum (MHA-TP)
○ Do two-fold serum dilution ranging from 1:2 to test
1:32
● Perform on “Reactive and Weakly Reactive Sera” These treponemal tests use treponemal antigens like:
● Twofold dilutions serum ranging from 1:2 to 1:32 ● Nichols (pathogenic and virulent)
● Reporting: ● Reiter (Not T. pallidum and not pathogenic)
○ WR - Weakly Reactive ○ These stains are used to detect
○ Reactive Anti-treponemal antibodies present in the
● Highest dilution showing a positive result serum of the px
● TITER (reactive in (1:2 dilutions)
● Reciprocal of dilution (Reactive 2 dilution) TREPONEMA PALLIDUM IMMOBILIZATION (TPI) TEST
● Principle:
RAPID PLASMA REAGIN (RPR) ○ Antibodies against T. pallidum - immobilize live
● Nontreponemal treponemes
○ WASSERMAN Antigen (Cardiolipin) ● Antigen:
○ Detect nontreponemal antibody ○ MOTILE T. pallidum (NICHOLS STRAIN)
■ Reagin (Anti-Cardiolipin) ● Preparation:
● Principle: Flocculation ○ Diluted patient’s serum + antigen suspension
○ Advantage ⟶ DARKFIELD ILLUMINATION
■ No need to inactivate serum ● Result:
■ No heating ○ HIGHEST serum dilution - immobilizes the
○ Equipment treponemes ⟶ IMMOBILIZING ANTIBODY
■ Plastic coated slide TITER
○ Reagent
■ Cardiolipin with added charcoal
● Charcoal particles form FLUORESCENT TREPONEMA PALLIDUM ANTIBODY
larger particles that are ABSORPTION (FTA-ABS) TEST
visible as clumps when
● Preparation:
aggregated by an antibody
○ Pre-absorption of serum with Reiter strain (non
● Help the test to be read
pathogenic treponemes)
macroscopically
■ Enhances the specificity of FTA-ABS
test
○ Antigen - Nichols strain is fixed (slide)
■ The virulent strains came from the
rabbit testicles
○ Addition of serum
○ Addition of conjugate (fluorescent-labeled AHG)
○ POSITIVE - FLUORESCENCE observed
Qualitative
against a black background
● Spread 0.05mL unheated serum to fill the circle
● +1 drop (1/60mL) antigen without mixing
● Rotate on a mechanical rotator for 8 minutes at 100rpm
● Observe macroscopically for clumping
● Result:
○ Reactive - medium to large clumps
○ Minimally reactive - small clumps
○ Nonreactive - no clumps

3
MICROHEMAGGLUTINATION-TREPONEMA PALLIDUM
(MHA-TP) TEST
● PRINCIPLE: Simple Passive Hemagglutination

○ The sheep RBCs are coated with Treponemal

antigen, resulting to sensitized sheep RBCs
● ANTIGEN: Tanned formalin sheep RBC coated with
treponemal antigen
● POSITIVE RESULT: HEMAGGLUTINATION
● Use as substitute for FTA-ABS
● TPI, MHA-TP, FTA-ABS are highly sensitive and specific
tests but not routinely used
● However, currently there are many treponemal tests
available in the market
● RESULTS INTERPRETATION

RPR & FTA-ABS Reactive POSITIVE FOR SYPHILIS

RPR; Reactive; NEGATIVE FOR SYPHILIS
FTA-ABS Non Reactive
RPR; Non Reactive; LATE/LATENT/PREVIOUS
FTA-ABS Reactive

Recall:
● RPR is a non treponemal test and nonspecific test; there
are a lot of diseases that could give a false positive result
in RPR
● So, in order to confirm the positive RPR result, we need
to proceed to a more specific treponemal test (FTA-ABS)
● If the FTA-ABS is non reactive, we can conclude that the
RPR positive result is a false positive
● The RPR will eventually become non reactive if the
patient is treated
● The FTA-ABS test remains reactive since Treponemal
antibodies persists for life

4
 OUTLINE ● Antigens
→ Thermolabile flagellar
I. Bacterial Agglutination G. Typhidot
→ Thermostable somatic
II. Typhoid Fever H. Tubex Test
→ Thermolabile capsular
(Salmonellasis) III. Typhus Fever (Rickettsia
A. Flagellar Antigen Infection)
B. Somatic Antigen A. Weil-Felix Test
C. Capsular Antigen B. Indirect
D. Febrile Agglutinins Immunofluorescence
E. Specimen C. Laboratory Diagnosis
Preparation INDEX: REVIEW QUESTIONS
F. Widal Test INDEX: APPENDIX

I. BACTERIAL AGGLUTINATION TEST

● A group of serologic test which is usually requested to
determine whether the fever of the patient is caused by a
bacterial pathogen
● a.k.a. Febrile agglutination tests
● Detects the presence of particular antibodies against the
pathogen or the presence of that particular antigen
A. FLAGELLAR ANTIGENS
→ able to detect an intact antigen directly on the organism´s
● Hauch antigen or H antigen
surface
● Thermolabile flagellar
→ it is the organisms which is incorporated in the reagent
● Bacteria (with the H antigen) + saline with 2% formalin
used for BAT
→ 2% formalin: destroys O antigens so that only H antigens
● Principle
are present
→ DIRECT AGGLUTINATION
● Detects flagellar agglutinins
→ inactivated whole organism suspension
→ observance of visible aggregates B. SOMATIC ANTIGENS
● Detection of antibodies against pathogens ● Ohne antigen or O antigen
→ Typhoid fever (Salmonella typhi) ● Thermostable somatic
→ Typhus fever (Rickettsia rickettsi) ● Found on bacterial body
→ Brucellosis (Brucella abortus) ● Bacteria + phenol/alcohol
→ Tularemia (Franciscella tularensis) → destroys H antigens so that only O antigens are present
● Also utilized to detect the presence of: ● Detect somatic agglutinins against Salmonella
→ Tetanus (Clostridium tetani)
C. CAPSULAR ANTIGENS
→ Yersiniosis (Yersinia pestis)
● Vi antigen
→ Leptospirosis (Leptospira interrogans)
→ K antigen in other bacteria
→ Parasites (e.g. Malaria, Toxoplasma, Leishmaniasis)
● Thermolabile capsular
● Used for:
● Capsules or envelope of the bacterial body
→ detection of serum antibodies
→ serologic identification of bacteria D. FEBRILE AGGLUTININS
▪ antisera: prepared by hyperimmunizing rabbits with the ● Antibodies against bacterial febrile diseases starts to appear
respective bacteria → producing antibodies against it 7-10 days
→ diagnosing diseases in which the bacterial agent is difficult → varies from one type to another
to cultivate in vitro ▪ maximum by the 14th day (typhus fever)
● Seldom used today: ▪ 3-5 weeks (typhoid fever)
→ culture is the preferred method ● O agglutinins
→ there are new methods for identifying the pathogens in → rises earlier in the disease; good indicator for the actual
which there is increased specificity disease
→ Cross-reactivity issues → drops faster than H agglutinins
▪ lessen or lower the specificity of the test → not affected by immunization (IgM form)
→ Non-specific ● H agglutinins
→ Biosafety hazards → slow to rise
▪ some organisms can be possibly used as bioweapon → remain elevated for several years
→ Laboratory capability → indicator for past salmonella infection
▪ laboratory must be at biosafety level 2 to handle such → increases after immunization (IgG form)
samples; biosafety level 3 to perform culture
E. SPECIMEN PREPARATION
● Common cause of laboratory-acquired infections
● No special preparation is required
II. TYPHOID FEVER (SALMONELLASIS) ● Serum (yields more antibodies compared to plasma)
● Accompanied with headache, high fever, abdominal → 2° - 8° C (<24 hours)
cramping, diarrhea and confusion → -20° C (>24 hours)
● Transmission → thawed at 37°C
→ food and water contamination ● Contraindications
● Infection → lipemic
→ Typhoid fever (Salmonella typhi) → hemolysed
→ Paratyphoid fever (Salmonella paratyphi) → bacterial contamination

MLS 412 GGGL, AJDM, JAGT BSMLS 2022 1 of 6

 F. WIDAL TEST
● oldest method for detection of typhoid fever
● WIDAL AGGLUTINATION TEST
→ measures Anti-O and Anti-H
→ Anti-O (early infection)
→ Anti-H (late development)
Reagents and Materials
● O antigens of Salmonella typhi
● H antigens of Salmonella typhi
● H antigens of Salmonella paratyphi B
● H antigens of Salmonella paratyphi A
● Positive control
● NSS (diluent) G. TYPHIDOT
● Glass slide ● Most commonly used
● plastic stirrers → aid in diagnosis of infection with S. typhi and S. parathyphi
● incubator ● Lateral flow chromatographic immunoassay
● timer ● Detects IgM and IgG against Salmonella
● automated pipette w the tips ● Screening test
● test tubes Reagents and Materials
Slide Method ● Individually sealed foil pouches containing
● Procedure (refer to the video: Bacterial Agglutination (13:18)) → One cassette device
→ Place a drop of the positive control and a drop NSS → One desiccant: to absorb moisture
(serves as the negative control) ● Plastic droppers
→ Place a drop of the sample on each circles ● Sample diluent: aids in the capillary action of the serum action
→ For both controls, we make use of O antigens of towards the testing area
Salmonella typhi ● One package insert
→ For other circles, the designated reagents are added
according to its label
→ Mix using the plastic stirrers
→ Manual rotation (1-2 mins) or lab rotator
● Result interpretation (refer to the video)
→ test wells showing agglutination with O and AH antigen:
corresponding antibodies present
▪ Salmonella paratyphi A is present
Interpretation
● In the Widal Test, O antigen is present in all Salmonella Procedure
organisms. They are only differentiated by the H ● Aspirate a specified amount of the sample and place it in the
antigens sample well
● Add a drop of the diluent
Score Interpretation ● Set the time for 15 minutes before reading the results
100% ++++ (+4) strong reactions
75% +++ (+3) strong reactions
50% ++ (+2) weak reactions
25% + (+1) weak reactions
0% 0 (non-reactive)

Principle: Lateral Flow Chromatographic Immunoassay

● Parts
→ sample pad: where the sample is placed
→ conjugating pad: contains recombinant H and O antigens,
and control antibody; each are conjugated with colloidal
gold
→ 2 test lines: contains nitrocellulose membrane strips
Tube Method ▪ M LINE: first test line; precoated with monoclonal
● Serial dilution anti-human IgM for the detection of S. typhi and S.
● Suspension of fine aggregates paratyphi IgM antibodies
● Four-fold rise (acute infection) ▪ G LINE: precoated with reagents that can detect the
● Procedure (refer to the video: Bacterial Agglutination (16:30) ) presence of anti-S. typhi and anti-S. paratyphi IgG
→ Prepare the sample in 1/10 dilution and is serially diluted in antibodies
1/2 dilution → control line: contains immobilized anti-rabbit IgG
→ Add the reagents ● The dispensed patient sample migrates by capillary action
→ Shake and cover with cotton plugs across the test cassette
→ Incubate (24 hours) ● If IgM antibodies are present, it will bind to the H-O
→ Observe for any presence of agglutination reactions conjugates
▪ last tube which has given the agglutination reaction is → Immunocomplex is captured in the membrane by the
considered as the titer (concentration of an antibody, precoated anti-human IgM antibody as it reaches the M
determined by finding the highest dilution of serum at line → burgundy colored M line is formed → indicates the
which it is still able to cause agglutination of the detection of anti-S.typhi or anti-S. paratyphi IgM antibodies
antigen)

MLS 412 Bacterial Agglutination 2 of 6

● If IgG antibodies are present, it will bind to the H-O → the conjugated control antibody will also travel until it
conjugates reaches the control line; test is valid → color band
→ Immunocomplex is captured in the membrane by the formation
precoated reagents as it reaches the G line → burgundy
colored G line is formed → indicates the detection of
anti-S.typhi or anti-S. paratyphi IgG antibodies
● Since the conjugated pad also contains control antibody
conjugated with colloidal gold, it will also travel along the test
cassette → control line will also form a burgundy colored line
since it contains an immobilized anti-rabbit IgG which is
against the IgG from the conjugate pad
● A color in the control line indicates that the test is valid
● Any faint line is still indicative of the presence of antibodies

● If the patient contain both IgG and IgM antibodies against S.

typhi, it would react with the immobilized anti-human IgM and
IgG
→ formation of colored band in both G line and M line
→ the conjugated control antibody will also travel until it
reaches the control line; test is valid → color band
formation

Result Interpretation
● If the patient does not contain any IgG or IgM antibodies
against S. typhi, antigens in the conjugated pad will not be
carried
→ no formation of colored band in both M line and G line
→ the control antibody will be the one to flow along the test
cassette until it reaches the control line
→ the test is valid because there is a color production in the
control area ● Test is considered invalid if there is no presence of color band
in the control area
→ test cassette may be defective
→ problem in the flowing of the sample
→ absence of control antibodies in the conjugate pad

● If the patient contain IgM antibodies against S. typhi, it would

react with the immobilized anti-human IgM
→ formation of colored band in M line but none in G line
→ the conjugated control antibody will also travel until it
reaches the control line; test is valid → color band
formation ● No color band formation in the control line →still considered
as invalid regardless of the color formation in M line and/or G
line
→ there could be a possibility that there is another antibody
that cross-reacted with the reagents and antibodies
present in the pad

● If the patient contain IgG antibodies against S. typhi, it would

react with the immobilized anti-human IgG
→ formation of colored band in G line but none in M line

MLS 412 Bacterial Agglutination 3 of 6

 Result Interpretation
● The more antibodies coated in the indicator particle is
suspended in the supernatant →the darker the color (BLUE)
● If the result is in borderline, repeat test
→ if it still has the same result after repeating the test, repeat
H. TUBEX TEST the test in some other day
● Inhibition magnetic binding Score Interpretation
● Semiquantitative colorimetric assay <2 Negative
● Detects Salmonella typhi IgM anti-O9 antibodies 3 Borderline
→ much more specific for Salmonella 4 Weak Positive
Principle 6-10 Positive
● MAGNETIC INHIBITION REACTION
● Brown reagent mixed with the serum is a magnetic particle
coated with the Salmonella O9 antigen
● Blue reagent contains an indicator particle coated with an
antibody against Salmonella antigen
● If the serum contains the antibody against Salmonella O9
antigen, it will bind with the antigen
→ the blue reagent will no longer bind with the antigen and
will remain in the solution
● Antibodies present in the serum has inhibited the reaction of
the indicator particle coated with antibody to the magnetic
particle coated with the O9 antigen

I. LABORATORY DIAGNOSIS
● CULTURE METHOD
→ standard technique for diagnosis of typhoid fever
→ blood (early – 1-2 weeks)
→ urine (late – 3-4 weeks)
→ stool (indefinite)
● COUNTER IMMUNOELECTROPHORESIS
→ more sensitive

Procedure
● Both sample and Tubex brown reagent should have the same
amount (45microliter)
● Mix for 5-10 times
● Incubate for 2 minutes
● Add another Tubex blue reagent (90microliter)
● Shake for 2 minutes;
● Allow separation in room temperature (5 mins)
● Read results

MLS 412 Bacterial Agglutination 4 of 6

 II. TYPHUS FEVER (RICKETTSIAL INFECTION)
● RICKETTSIA
→ Small, gram-negative obligate intracellular bacteria
▪ cannot survive outside the host cell
→ Arthropod bite or fecal contamination
→ Endothelial cells
▪ vasculitis
▪ cell necrosis
▪ thrombosis
▪ skin rashes
▪ organ dysfunction

A. WEIL-FELIX TEST
● AGGLUTINATION TEST
→ non-specific (found in Proteus organisms)
→ based on cross-reacting antibodies with
POLYSACCHARIDE O (Proteus vulgaris)
● Antigens
→ Proteus OX2 (Proteus vulgaris)
→ Proteus OX19 (Proteus vulgaris)
→ Proteus OXK (Proteus mirabilis)
● Rickettsia antigens cannot be utilized as reagents for
bacterial agglutination tests
→ difficult to cultivate
→ they are intracellular
● Four-fold rise of serum antibodies
B. INDIRECT IMMUNOFLUORESCENCE
● Most preferred method in determining the presence of
rickettsia
● Utilizes two types of antibodies (primary and secondary
antibodies)
● The antibody present in the patient serum will attach to the
known antigen
● The secondary antibody is specific to the antibody of interest
● Once complex is formed between the antibody present in the
patient serum and the secondary antibody, it gives off
fluorescence

C. LABORATORY DIAGNOSIS
● Polymerase chain reaction
● Immunohistochemistry
→ get skin scrapings from patients with rickettsial infections
● Bacterial culture

MLS 412 Bacterial Agglutination 5 of 6

 INDEX: APPENDIX

MLS 412 Bacterial Agglutination 6 of 6