SEROLOGY DOCKIE NOTES

RED BLOOD CELL SUSPENSION

-Acts as indicator cells in the demonstration of antigen and antibody reactions in vitro
-Between 2-5% cell suspension provides optimum antigen concentration for the tube method for red
blood cell typing

1. PURPOSE OF WASHING RED CELLS

-To remove the unwanted plasma proteins including antibodies that might interfere in the
reactions
-Washing is done by repeatedly suspending the whole blood with normal saline solution (NSS),
mixing, centrifuging, and decanting completely the supernatant fluid

SIDENOTE:
➢ The following maybe in the plasma which may interfere with testing:
✓ Soluble antigens such as A and B (may neutralize the reagent)
✓ Wharton’s jelly in cord blood, cold-reacting autoimmune antibodies, and
increased levels of immunoglubulins that may cause either agglutination or
roleaux
✓ Hemolysed red blood cells due to a difficult draw will interfere in
grading/interpretation
✓ Fibrinogen can result in the formation of fibrin strands that will make grading
reactions difficult

2. APPLICATION/INDICATION
-Blood typing
-Crossmatching
-In transfusion medicine, washed red blood cells are indicated for:
• Patients with history of severe allergic reactions (anaphylactic or urticarial)
• Patients with IgA deficiency with documented anti-IgA antibodies
• Patients with recurrent febrile transfusion reactions not prevented by leukocyte
reduction
• Neonates or fetus patients with renal failure, before transfusion of potassium depleted
units

3. PREPARATION/PROCEDURE
a. Pipette 3mL of whole blood (from EDTA tube) into a 10mL capcity conical/centrifuge tube.
b. Fill the tube with normal saline solution until it reaches the 10mL mark
c. Cover the tube with its screw cap (or nesco film) and mix the suspension by gentle
inversion.
d. Centrifuge for 5 minutes at 3,400rpm (other reference: 3 000 to 5 000 rpm).
e. Decant by aspirating the supernatant using a pasteur pipette (or by simple pouring the
supernatant off the sink). Be sure that the packed red cells are not disturbed.
f. Resuspend the cells with another volume of NSS until it reaches again the 10mL mark.
g. Repeat steps b-e
***This process is called washing and is preferably done three times.

4. PREPARING RED CELL SUSPENSION OF DESIRED CONCENTRATION

a. Formula: % Red cell suspension = Amount of packed red blood cell (PRBC) X 100
Total Volume
Total Volume = Amount of washed PRBC + Amount of NSS to be added
DILUTION
-Indication of relative concentration
-Involves two entities:
1 = Amount of solute
• Solute
-The material being diluted
Dilution Total Volume
• Diluent
-The medium making up the rest of the solution

-Importance:
• For detection of the amount of antibodies; Serological tests require a visible end point in
order to establish the concentration of antibodies and this is achieved by dilution

1 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
1. SIMPLE DILUTIONS
-Involves SINGLE dilution

Table X.Computation for a Simple/Single Dilution

PROBLEM SOLUTION
1. Two mL of a 1:20 dilution a. Problem:
is needed to run a Amount of solute (Serum) –represented as x
specific serological Amount of diluent - ?
test. How much serum and b. Given:
how much diluent are 2mL-Total Volume
needed to make this 1:20 – Dilution
dilution? c. Formula:
1/Dilution = Amount of solute/Total Volume
1/20 = x/2
20x = 2
x = 2/20
x = 0.1 (Amount of solute/serum)
***To compute for the amount of diluent, subtract the
amount of solute from the total volume. That is,

Total Volume – Amount of solute = Amount of diluent

2mL – x = ?
2mL – 0.1mL = 1.9mL (Amount of diluent)
2. A 1:5 dilution of patient a. Problem:
serum is necessary to run Amount of diluent - ?
a serological test. There b. Given:
is 0.1mL of serum that 1:5 – Dilution
can be used. What amount 0.1mL – Amount of solute
of diluent is necessary ***The total volume will be represented by x in the
to make this dilution following formula.
using all of the serum? c. Formula:
1/Dilution = Amount of solute/Total Volume
1/5 = 0.1/x
x = 0.5 (Total Volume)
***To compute for the amount of diluent, subtract the
amount of solute from the total volume. That is,

Total Volume – Amount of solute = Amount of diluent

x – 0.1mL = ?
0.5mL – 0.1mL = 0.4mL

SIDENOTE:
➢ To calculate the concentration of a single dilution, multiply the original concentration by
the dilution expressed as a fraction.
o A specimen contains 500mg of substance of substance per deciliter of blood. A 1:5
dilution of this specimen is prepared by volumetrically measuring 1mL of the specimen
and adding 4mL of diluent. The concentration of substance in the dilution is:
500mg/dL x 1/5 = 100mg/dL

2. COMPOUND DILUTIONS
-Process of making a very large dilution from a small sample

a. Example
✓ A 1:500 dilution is to be prepared
✓ It is not normal to use 1mL of a sample and 499mL of diluent!
✓ This is wasteful especially when there is only a small amount of solution needed
to run the test. Furthermore, it will also result to inaccuracy because of improper
mixing.
✓ To calculate compound dilution, the first step is to plan the number and sizes of
simple dilutions to reach the desired endpoint.
✓ A 1:500 dilution can be achieved by making a 1:5 dilution of the original serum, a
1:10 dilution from the first dilution, and another 1:10 dilution.

Table X.Compound Dilution

1:5 DILUTION 1:10 DILUTION 1:10 DILUTION
0.1mL serum 0.1mL of 1:5 dilution 0.1mL of 1:10 dilution
O.4mL diluent 0.9mL diluent 0.9mL diluent
➢ RATIONALE:

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SEROLOGY DOCKIE NOTES
Multiplying 5 x 10 x 10 equals 500, or the total dilution. Each of the
simple dilutions is calculated individually by doing mental arithmetic or
by using the formula given for simple dilutions. The 1:500 dilution was
made using very little diluent in a series of test tubes, rather than
having to use larger volume in a flask. The volumes were kept small enough
so that mixing could take place easily, and the final volume of 1.0mL is
all that is necessary to perform a test.

3. SERIAL DILUTIONS
-A dilution series in which all dilutions, including or following the first one, are the same
-A general rule for calculating the concentrations of solutions obtained by dilution in series
is to multiply the original concentration by the first dilution (expressed as a fraction),
this by the second dilution, and so on until the desired concentration is known.

a. Example
✓ A five-tube twofold dilution is to be prepared
✓ A serum specimen is diluted 1:2
✓ A series of five tubes is prepared in which each succeeding tube is rediluted 1:2
✓ This is accomplished by placing 1mL of diluent into each four tubes (tubes 2 to 5)
✓ Tube 1 contains 1mL of undiluted serum
✓ Tube 2 contains 1mL of undiluted serum plus 1mL of diluent, resulting in a 1:2
dilution of serum
✓ Tube 3 contains 1mL portion of the 1:2 dilution of serum, resulting in a 1:4
dilution of serum (1/2 x 1/2 = 1/4)
✓ Tube 4 contains 1mL portion of 1:4 dilution from tube 3 resulting in a 1:8 dilution
(1/4 x1/2 = 1/8)
✓ Tube 5 contains 1mL portion of 1:8 dilution from tube 4 resulting in a 1:16 dilution
(1/8 x 1/2 = 1/16)
✓ The concentration of serum in terms of milliliters in each tube is calculated by
multiplying the previous concentration (mL) by the succeeding dilution.

Figure 10.Five-tube twofold

dilution. (Figure captured from
Turgeon’s)

SIDENOTE:
➢ Other example of Serial Dilution process:
o Each tube contains 0.2 mL of diluent. Patient serum (0.2 mL) is added to tube one.
This is carefully mixed, and then 0.2 mL is withdrawn and added to tube two. The
process is continued until the last tube is reached. The sample is mixed, and 0.2 mL
is discarded. Note that in this dilution, the amount of antibody is cut in half in
each successive tube.

Figure X.Serial dilution. (Figure

3 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible.
snipped off “ –Akihisa Yoshi
from Stevens)
SEROLOGY DOCKIE NOTES
BASIC IMMUNOLOGICAL PROCEDURES

PRECIPITATION
-Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are
visible

1. Terminologies
a. AFINITY
-Initital force of attraction that exists between a single Fab site on an antibody molecule
and a single epitope or determinant site on the corresponding antigen
-Noncovalent bonds binding the epitope and the binding site include Ionic bonds, Hydrogen
bonds, Hydrophobic bonds, Vander Waals forces, Electrostatic bond

b. AVIDITY
-Sum of all the attractive forces between an antigen and an antibody
-Measure of the overall stability of an antigen-antibody complex

c. CROSS-REACTIVITY
-Antibodies react with antigens that are structurally similar to the original antigen that
induced antibody production

2. Precipitation Curve
a. PROZONE
-Antibody excess
b. ZONE OF EQUIVALENCE
-Optimum precipitation occurs in
which the number of multivalent sites
of antigen and antibody are
approximately equal
c. POSTZONE
-Antigen excess

3. Measurement of Precipitation by Light

Scattering
a. TURBIDIMETRY
-Measure of turbidity or cloudiness of a solution
b. NEPHELOMETRY
-Measure of light scatter at a particular angle (usually 10-90 degrees)

4. Methods of Precipitation
a. PRECIPITATION IN A GEL MEDIUM
-Soluble antigen and antibody diffuse through the pores of the gel until their
concentration reaches the optimum ratio, forming a stable immunoprecipitate

Table X.Techniques of Immunological Precipitation in Gel

SINGLE DIFFUSION SINGLE DIFFUSION DOUBLE DIFFUSION DOUBLE DIFFUSION
SINGLE DIMENSION DOUBLE DIMENSION SINGLE DIMENSION DOUBLE DIMENSION
No. of reactants
(Ag or Ab)
No. of effective
dimension
Method

• OUDIN (SINGLE DIFFUSION-SINGLE DIMENSION)

1. Antibody (Anti-serum) is mixed in agarose
2. Antigen diluton is overlaid
*The concentration of the antigen must always be greater than the equivalence
concentration
3. The mobile antigen diffuses through the gel containing immobilized antibody,
forming Ag-Ab complexes until the size of the omplex becomes too large to pass
through the pores of the gel
4. At equivalence concentration at the point of slight Ag excess, a stable
precipitin band will form

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SEROLOGY DOCKIE NOTES
• RID (SINGLE DIFFUSION-DOUBLE DIMENSION)
Table X.Two Principal Radioimmunodiffusion Method
MANCINI FAHEY MCKELVEY
-Endpoint Method -Kinetic Methodd
-Antigen is allowed to diffuse fully to achieve -Maximum precipitation is not achieved because
maximum precipitation the measurement of the precipitation ring is
-This occurs between 24 and 72 hours taken before the point of equivalence, usually
at about 18 hours
-Square of the ring diameter is directly -The diameter of the precipitation ring is
proportional to the concentration of the proportional to the logarithm of the
antigen concentration of the standards
-Concentration of the standard is plotted on -Concentration of the standard is plotted on a
linear graph on the x-axis and the diameter semi-log paper on the log axis and the diameter
squared of the precipitation ring is plotted on of the precipitation ring on the arithmetic axis
the y-axis

-DISADAVANTAGE: It takes time to obtain results -ADVANTAGE: Results are obtained faster
-ADVANTAGE: Produces more reliable results
because it detects small amount of antigen

• OAKLEY AND FULTHROPE (DOUBLE DIFFUSION-SINGLE DIMENSION)

1. Overlay antibody (anti-serum) with agarose (neutral agar)
2. Overlay the gel with antigen
*Both the reactants diffuse so that the initial concentration of the antigen
does not need to be greater than the equivalence point with reference to the
original concentration of the antibody
3. Both antigen and antibody diffuse through the gel and form bands/layers of
precipitates near their zones of equivalence concentrations

• OUCHTERLONY (DOUBLE DIFFUSION-DOUBLE DIMENSION)

1. A pattern of wells is cut in an agarose gel (Petri dish or Slide)
2. Load with reactants
3. Incubate until lines of precipitate have fully developed
4. Interpret serological patterns

b. PRECIPITATION BY ELECTROPHORESIS
-Technique in which molecules with a net charge are precipitated when an electric field
is applied

Table X.Techniques of Immunological Precipitation by Electrophoresis

SINGLE DIFFUSION SINGLE DIFFUSION DOUBLE DIFFUSION DOUBLE DIFFUSION
SINGLE DIMENSION DOUBLE DIMENSION SINGLE DIMENSION DOUBLE DIMENSION
No. of reactants
(Ag or Ab)
No. of effective
dimension
Method

• ROCKET IMMUNOELECTROPHORESIS
1. Antibody is distributed in the gel and the antigen is placed in wells cut in
the gel, just as in RID
2. When the antigen diffuses out of the well, precipitation begins
3. As the concentration of antigen changes, , there is dissolution and reformation
of the precipitate at ever-increasing distances from the well
4. The end result is concical in shape, resembling a rocket

• CROSSED IMMUNOELECTROPHORESIS
1. Proteins are separated via electrophoresis
2. The separated proteins are subjected to a 2nd electrophoresis at a 90 degree
angle from the first electrophoresis wherein they will move through agarose gel
containing antibodies leading to formation of a precipitin arc (rocket/bell-
shaped)when Ag and Ab reach equivalence

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SEROLOGY DOCKIE NOTES
• COUNTER CURRENT IMMUNOELECTROPHORESIS
1. Antigen and antibody are added to opposite wells of an agar gel.
2. Electric field is applied
3. Antigen will migrate toward the anode and antibody wil midrate to the cathode
4. Precipitin band will form when the Ag and Ab meet in correct proportion

• CLASSIC IMMUNOELECTROPHORESIS
1. Antigen is introduced into a well and an electric field is applied resulting in
separation of proteins
2. Antibody is introduced in a trough located parallel to the separated proteins
3. Both serum proteins (Antigen) and the antibodies (in the trough) diffuse in the
gel resulting in a characteristic arc when Ag and Ab complexes form
SIDENOTE:
➢ Immunofixation electrophoresis as first described by ALper and Johnson,
is similar to classis immunoelectrophoresis except that after
electrophoresis takes place, antiserum is applied directly to the gel’s
surface rather than placed in a trough(depression).

AGGLUTINATION
-Process by which antigens such as cells aggregate to form larger complexes that are visible

1. Steps in Agglutination
a. SENSITIZATION
-Represents the physical attachment of antibody molecules to antigens on the erythrocyte
membrane
-The combination of antigen and antibody is a reversible chemical reaction. Altering the
physical conditions can result in the release of antibody from the antigen-binding site.
When physical conditions are purposely manipulated to break the antigen-antibody complex,
with subsequent release of the antibody into the surrounding medium, the procedure is
referred to as an elution.

b. LATTICE FORMATION
-Establishment of cross-links between sensitized particles (e.g., erythrocytes) and
antibodies, resulting in aggregation
-A much slower process than the sensitization phase

2. Types of Agglutination Reaction

a. DIRECT AGGLUTINATION
-Antigens are found naturally on the surface of the particle
• HEMAGGLUTINATION

• VIRAL HEMAGGLUTINATION

b. INDIRECT/PASSIVE AGGLUTINATION
-Antigens have been affixed or attached to a carrier/inert particle
-Agglutination occurs if patient antibody is present

Figure X.PASSIVE AGGLUTINATION (Figure snipped off from Stevens’)

• REVERSE PASSIVE AGGLUTINATION

-Antibodies have been affixed or attached to a carrier/inert particle
-Agglutination occurs if patient antigen is present
-Example: Reverse Passive Agglutination test for CRP

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SEROLOGY DOCKIE NOTES

Figure X. REVERSE PASSIVE AGGLUTINATION (Figure snipped off from Stevens’)

Table X.Applications of Direct and Indirect Agglutination

DIRECT
Blood typing
Cold Hemagglutination Test for M. pneumonia
Heterophile Agglutination test for EbV
(Paul bunnel, Davidsohn’s Monospot test)
Widal Test for S. typhi
Weil Felix Test for Rickecttsia
Viral Hemagglutination Test
INDIRECT
Hemagglutination test for syphilis
Passive Hemagglutination test for Rubella,
Rheumatoid Factor, Group A strep, T. spiralis,
T. pallidum
Reverse Passive Agglutination for CRP

c. COAGGLUTINATION
-Uses bacteria (usually S. aureus) as the inert particles to which antibody is attached

Figure X.COAGGLUTINATION (Figure snipped off from Stevens’)

d. AGGLUTINATION INHIBITION
-Based on competition between particulate and soluble antigens for limited antibody-
combining sites, and a lack of agglutination is a positive reaction
-Uses: hCG test, Detection of illicit drugs

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SEROLOGY DOCKIE NOTES

Figure X.AGGLUTINATION INHIBITION (Figure snipped off from Stevens’)

e. ANTIGEN-MEDIATED AGGLUTINATION
• ANTIHUMAN GLOBULIN (COOMB’S TEST)
-Detects non-agglutinating antibody by means of coupling with a second antibody
Table X.Types of Antihuman Globulin Test
DIRECT INDIRECT
-In Vivo agglutination -In Vitro Agglutination
-Detects IgG antibodies bound to the antigen on -Detects presence of antibodies in the serum
the red cells that is still to be attached to an analyte
(reagent red cells)
-APPLICATION: -APPLICATION:
HDN, HTR, AIHA, DIHA Cross-matching, Antibody detection and
identification, RBC antigen (Du) phenotyping

Figure X.DIRECT ANTOGLOBULIN TEST (Figure snipped off from Stevens’)

8 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES

Figure X.INDIRECT ANTOGLOBULIN TEST (Figure snipped off from Stevens’)

3. Grading of Agglutination
Table X.Widal Test
0 No agglutination
1+ 25% agglutination
2+ 50% agglutination
3+ 75% agglutination
4+ 100% agglutination
*A single rapid slide test is not considered positive unless a value of at least 2+ is assigned
in a sample taken in the first 2-3 weeks of infection.

Table X.Hemagglutination Test (Stevens)

TUBE METHOD SLIDE METHOD
Negative Smooth suspension Negative Even suspension, cloudy background
1+ Barely discernable clumps Weak
2+ Numerous smaller clumps Strong Small clumps, cloudy background
3+ Several large clumps
4+ One solid clump Large clumps, clear background

Table X.Hemagglutination Test (Harmening)

0 No agglutination or hemolysis
W Tiny agglutinates, Turbid background
1+ Small agglutinates,Turbid background
2+ Medium sized agglutinates, Clear background
3+ Several large agglutinates, Clear background
4+ One solid agglutinate

9 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
Table X.Reading Agglutination

***This table was snipped off from Turgeon’s

4. Quantitative Agglutination Reaction

Principle: Indirect/Passive Agglutination

Table X.Quantitative Agglutination Reaction

IMMUNOASSAY INERT PARTICLE
SPIA (Solid Particle Immunoassay)
DIA (Disperese Dye Immunoassay)
IMPACT (Immunoassay by Particle Counting)

5. Instrumentation
a. Particle Counting Immunoassay (PACIA) involves measuring the number of residual non-
agglutinating particles in a specimen.These are counted by means of a laser beam in an
optical particle counting similar to one that is designed to count blood cells.

10 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
6. Errors in Agglutination Testing
Table X.Causes of False-Positive and False-Negative Reactions in Agglutination Testing

***This table was snipped off from Stevens’

Continuation…

***This table was snipped off from Stevens’

LABELED IMMUNOASSAYS
-These are immunoassays where Antigen-Antibody complexes do not go beyond the first phase (Ag-Ab
union)

1. Fluorescence Immunoassay

a. DFA
-Antigen from patient sample is fixed to a slide
-Fluorescent labeled antibody is added
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SEROLOGY DOCKIE NOTES
-Antibody binds to antigen and become fixed to slide too
-Wash to remove unattached antibody
-Examine for fluorescence

b. IFA
-Antigen is fixed on slide and patient serum antibody is added, incubated then washed
-Fluorescent antihuman globulin is added, incubated and washed
-If the patient serum contains the corresponding antibody, it would be fixed to the antigen
on the slide
-Tagged or labeled reagent reacts with the Antibody fixed to the antigen

Figure X.DIRECT FLUORESCENT ASSAY (A) and INDIRECT FLUORESCENT ASSAY (B)
(Figure snipped off from Stevens’)

c. INHIBITION IMMUNOFLUORESCENCE
-The antigen is fixed to the slide and then flooded with patient serum
-If corresponding antibody is present in the serum, it will attach (fix) to the antigen
-When a specific fluorescent antibody reagent is added, it cannot become fixed because
all of the antigenic slides are already occupied by the unlabeled Px Ab
-Wash (Labeled reagent is washed off)
-Examine under fluorescence microscope

SIDENOTE:
➢ The inhibition assay is a blocking test in which an antigen is first exposed to unlabeled
antibody and then to labeled antibody, and is finally washed and examined. If the unlabeled
antibodies are both homologous to the antigen, there should be no fluorescence. This result
confirms the specificity of the FA technique. Antibody in an unknown serum can be detected
and identified by the inhibition test.

2. Radioimmunoassay
-Makes use of radioactive labels
Table X.Radioactive Labels
IODINE ISOTOPE TRITIUM
(125I and 131I) (3H)
Emitted Radiation
Measurement

a. COMPETITIVE
-Antigen in patient serum compete for antibody binding with a known quantity of same
antigen to which a radioactive isotope is covalently bond
-During incubation, the labeled and unlabeled antigen compete for limited number of
antibody combining sites
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SEROLOGY DOCKIE NOTES
-If unlabeled antigen is higher in concentration than the labeled antigen, more Ag-Ab
complexes will not be radioactive (decreased radioactivity)

-Free antigens are separated and the radioactivity of the bound phase and free phase is
quantified

Figure X.RADIOIMMUNOMETRIC ASSAY

b. NON COMPETITIVE (IMMUNORADIOMETRIC ASSAY/SANDWICH ASSAY)

-Antibody is bound to a solid phase
-The test analyte (Ag) is added
-After the interaction between the solid phase bound antibody and the reactant(Ag), washing
step follows in order to separate the bound and free phase
-A second radiolabeled antibody is added which reacts with the antigen(analyte) fixed on
the solid phase
-After the incubation and washing step stage, the radioactivity of the system is measured

3. Enzyme Immunoassay
-Uses enzymes as labels

a. COMPETITIVE IMMUNOASSAY
• HOMOGENOUS ASSAY (ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE)
-No separation step is necessary because enzyme activity diminishes when
binding of antibody and antigen occurs
-Reagent antigen is labeled with an enzyme tag. When antibody binds to
specific determinant site of the enzyme –tagged antigen, the active site on
the enzyme is blocked, hence decreased or loss of enzyme activity.
-Patient antigen competes with the enzyme labeled antigen for a limited
number of Ab-binding sites (hence “competitive” assay)
-If concentration of patient antigen is more than the reagent antigen,
enzyme activity is high since more patient antigen is bound to the antibody
and less active sites are blocked
-Enzyme activity is directly proportional to the concentration of patient
antigen or hapten present in the test solution
-A physical separation of bound and free analyte is thus not necessary

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SEROLOGY DOCKIE NOTES
• HETEROGENOUS ASSAY (ELISA)
-Requires separation step (e.g.,washing) of free and bound analyte
-Based on the principle of RIA
-Enzyme-labeled antigen competes with unlabeled patient antigen for a
limited number of binding sites on antibody molecules that are attached to
a solid phase.
-After carefully washing to remove any nonspecifically bound antigen, enzyme
activity is determined.
-Enzyme activity is inversely proportional to the concentration of the test
substance, meaning that the more patient antigen is bound, the less enzyme-
labeled antigen can attach.

b. NON COMPETITIVE IMMUNOASSAY

• INDIRECT ELISA
-Either Ag or Ab is bound to a solid phase support
• SANDWICH OR CAPTURE ASSAY
-Solid phase antibody is more commonly used
-Requires two washing steps

Figure X.NONCOMPETITIVE ELISA (Figure snipped from Stevens’)

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SEROLOGY DOCKIE NOTES

Figure X.NONCOMPETITIVE ELISA: Sandwich or Capture Assay (Figure snipped from Stevens’Stevens’)

COMPLEMENT FIXATION TEST

-Detects complement consumption in any
cellular or non-cellular antigen-antibody
reaction to which complement is bound
-Used in the detection of viral, fungal,
and rickettsial antibody
-When antigen-antibody occurs, complement
is ‘inactivated’ or fixed such that it
will be unavailable to cause lysis upon
addition of indicator cells (SRBCs coated
with amboceptor/hemolysin)

Figure X.Complement fixation Test

(Figure snipped off from Stevens’)

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES

SPIROCHETE DISEASES

1. Syphilis
-Caused by Treponema pallidum subspecies pallidum (originally called Spirochaeta pallida)
-Transmitted by sexual contact, direct blood transfusion or transplacental route
-The first diagnostic blood test for syphilis was the Wassermann test, a complement
fixation test developed in 1906

a. STAGES
Table X.Overview of Stages of Syphilis
STAGE MOST IMPORTANT FINDINGS/MANIFESTATIONS CORRELATION WITH SEROLOGY
Primary -Hard Chancre that is transient, painless and firm Darkfield Microscopy
-Lasts from 1-6 weeks, during which time the lesion
heals spontaneously
Secondary -Most contagious stage Darkfield Microscopy
-Generalized lymphadenopathy, malaise, fever, Serological test
pharyngitis and a rash often involving the mucous
membranes and the skin
-Condylomata lata (wart-like lesion)
*Condylomata acuminta (genital wart; HPV)
Latent -Lack of clinical symptoms Serological test

Tertiary -Gummas, Neurosyphilis, Cardiovascular disease Serological test

b. Antigens and Antibodies Related to Syphilis

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SEROLOGY
Table X.Antigen and Antibodies used in Syphilis Serology
Wasserman Antigen
Treponemal Antigen
✓ Reiter strain
✓ Nichol Strain
Treponemal Antibody
Non-treponemal Antibody
DOCKIE NOTES
ANTIGEN
-Also known as cardiolipin
-Identified as a phospholipid (Diphosphatidyl Gylcerol)
-Derived from the host
-Extract from beef heart
-Acts as a hapten
***The Wassermann antigen was derived from extracts of liver from
newborns who had died of congenital syphilis. Later Landsteiner
demonstrated that tissues from animals without syphilis,
particularly beef heart extracted in alcohol, could be used equally
well as antigens.
-Non-virulent variant of T. pallidum
-Virulent variant of T. pallidum
ANTIBODY
-Produced against the antigen of the organisms themselves
-Specific against outer membrane protein and endoflagellar protein
-Also known as Reagin/Anti-cardiolipin/Antilipoidal antibody
-Produced by infected patients against components of their own or
other mammalian bodies
-Almost always produced by persons with syphilis but also produced
in Malaria, Leprosy, IM, DPT immunization, SLE, Lymphogranuloma
Venereum, Hepatitis, Periarteritis Nodosa, Rehumatic Fever,
Measles, Chicken Pox, Pregnancy, Old Age

c. LABORATORY DETERMINATION
• Darkfield Microscopy (Phase Contrast Microscopy)
• Serological Test

Table X.Serological Tests for Syphilis

DESCRIPTION EXAMPLES
Non-Treponemal Tests -Screening tests VDRL
-Monitor efficacy of treatment RPR
-Based on Flocculation which is a special type of USR
precipitation TRUST
-Determine the presence of Reagin, an antibody that forms ART
against cardiolipin, a lipid material from damged cells
-An antigen that is a combination of cardiolipin,
Lecithin and cholesterol is used in the reaction
Treponemal Tests -Confirmatory tests TPI
-Uses either live or dead T. pallidum to demonstrate the FTA-ABS
presence of antibodies to the organism Treponemal
Agglutination
Tests

• NONTREPONEMAL TESTS

✓ VDRL
Table X.VDRL
RING/WELL DIAMETER NEEDLE GAUGE # OF DROPS ROTATION
DELIVERED/mL
Qualitative
Serum VDRL
Quantitative
Serum VDRL

CSF VDRL

-Examine flocculation MICROSCOPICALLY under 100x total magnification

-Syringe used for delivery is Hamilton syringe (Stevens)
-Specimen: 50uL (0.05mL) Serum/CSF

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SEROLOGY DOCKIE NOTES
✓ RPR
Table X.RPR
RING/WELL NEEDLE GAUGE # OF DROPS ROTATION
DIAMETER DELIVERED/mL
RPR

-Examine flocculation MACROSCOPICALLY

-Specimen: 50uL (0.05mL) unheated serum/plasma

• TREPNONEMAL TEST

✓ TREPONEMA PALLIDUM IMMOBILIZATION TEST

-Standard to which other treponemal tests are evaluated/Treponemal test of reference
-Involves mixing of patient serum with live actively motile T. pallidum extract from
testicular chancre of a rabbit and complement
-Test is considered positive if ≥50% Treponemes are immobilized

✓ FTA-ABS
-Indirect IF test
-Indicator label is FITC tagged with AHG

✓ HEMAGGLUTINATION TEST FOR SYPHILIS

-Employs blood cell coated with T. pallidum antigen

Table X.Interpretation of Results in Hemagglutination Tests for Syphilis

INTERPRETATION DESCRIPTION
Reactive Smooth matte at the bottom of the reaction well
Non-Reactive Compact button of cells with or without a hole in the center

Table X.Hemagglutination Tests

TYPE OF TEST ANTIGEN
TPHA Turkey Erythrocytes
MHA-TP Tanned Sheep Erythrocytes or Gel particles sensitized with T.
pallidum sonicate
HATTS Glutaraldehyde stabilized turkey erythrocytes

BACTERIAL INFECTIONS
1. Group A Streptococci
a. ANTISTREPTOLYSIN O
-Based on the principle of Neutralization
-Streptolysin O in its reduced form when added to red blood cells cause hemolysis. If a
patient’s serum containing antistreptolysin O antibody is added to streptolysin, an
antigen-antibody reaction occurs causing the neutralization of the toxin so that it cannot
react with red cells.
-The endpoint ASO titer is expressed in Todd units which is the reciprocal of the highest
serum dilution that shows no hemolysis of the indicator cells
-Titer of 166 Todd units or below is considered normal; moderately elevated if the titer
is at least 240 Todd units in an adult and 320 in child.

b. ANTI-DNASE B TESTING
-Measurement is based on Neutralization methodology. If anti-DNAse B antibodies are
present, they will neutralize reagent DNASE B preventing it from depolymerizing DNA (Hence,
No change in color)
-Presence of DNAse is measured by its effect on DNA-Methyl Green conjugate. This complex
is green in its intact form but when hydrolyzed by DNAse, the methyl green is reduced and
becomes colorless.
-An overnight incubation at 37degC is required in some testing methodologies to permit
anti-DNAse antibodies to inactivate the enzyme (DNAse B). Tubes are graded for color, with
a 4+ indicating that the intensity of color is unchanged and a 0 indicating a total loss
of color
-Nephelometry provides an automated means of testing that can be used for rapid
quantitation of anti-DNAse B

c. STREPTOZYME (MULTIENZYME) TEST

-Slide agglutination screening test for detection of antibodies to several Streptococcal
antigens
-Sheep RBCs are coated with Streptolysi, Streptokinase, Hyaluronidase, DNAse and NADase
so that antibodies to any of the streptococcal antigens can be detected

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SEROLOGY DOCKIE NOTES
2. Streptococcus MG
-A non-hemolytic gram + coccus that is isolated from the throats of patients with PAP
-It is seen in normal individual at a 1:10 titer

3. Mycoplasma pneumonia
-An extracellular bacteria with a single tripled layer membrane instead of cell wall that
causes PAP or Walking Pneumonia
-Serology testing for M. pneumonia involves testing for cold agglutinins, because these are
present in about 50% of patients with infection
-Cold agglutinins are autoantibodies that react with the I/I antigens on red blood cells and
cause their agglutination at temperatures below 37degC. Agglutination can be eliminated by
warming and reversed by recooling the specimen.
-Development of these antibodies is thought to result from cross-reaction of the I antigen
on human red cells or from alteration of the red cells by the organism

a. Cold Agglutinin Test

-No longer recommended for the detection of M. pneumonia infections since cold agglutinins
are also seen in patients with IM, Black Water Fever, Cirrhosis, Chronic sepsis,
Leishmaniasis, African Trypanosomiasis, Liver Disease, Relapsing Fever, Carcinoma,
Pernicious Anemia

b. EIA
-Most widely used method of detection

4. Rickettsia
-Short rods or coccobacilli, that are obligate, intracellular, gram-negative bacteria

a. WEIL-FELIX TEST
-Based on cross-reactivity of the patient’s antibodies with polysaccharide antigens
present on the OX-19 and OX-2 strains of P. vulgaris and the OX-K strain of P. mirabilis

Table X.Weil Felix Test Reactions

RICKETTSIAL DISEASE ORGANISM OX-19 OX-2 OX-K

RMSF

(Spotted Fever Group)

Murine/Endemic Typhus

Epidemic Typhus

Scrub Typhus

Q fever

Rickettsial Pox

Trench Fever

5. Salmonella typhi
-Etiologic agent of Typhoid fever

a. WIDAL TEST (FEBRILE AGGLUTINATION TEST)

Table X.Widal Test Interpretation
0 No agglutination
1+ 25% agglutination
2+ 50% agglutination
3+ 75% agglutination
4+ 100% agglutination
*A single rapid slide test is not considered positive unless a value of at least 2+ is assigned
in a sample taken in the first 2-3 weeks of infection.

b. TYPHI DOT
-Dot EIA which detects serum antibody to antigen dotted on a nitrocellulose membrane

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SEROLOGY DOCKIE NOTES
Table X.Clinicial Interpretation of Typhi Dot
IgM (O antigen) IgG (H antigen) INTERPRETATION
- - No typhoid fever
+ - Acute typhoid fever
+ + Middle stage of infection
- + Previous infection, Reinfection, Carrier state

AUTOIMMUNE DISEASES
1. Systemic Lupus Erythematosis
a. DEMEONSTRATION OF LE CELL

b. DETECTION OF ANA STAINING PATTERNS

Table X.ANA Staining Patterns

Homogenous -Characterizes anti-deoxyribonucleoprotein antibodies
(Solid, Diffuse) (i.e.,antibodies to nDNA, DsDNA, ssDNA, DNP or histones
-This pattern is typically seen in rheumatoid disorders
-High titers of homogenous ANA are suggestive of SLE
-Low titers may be found in SLE, RA, Sjogren’s syndrome and MCTD
Peripheral -The pattern results from antibodies to DNA
(Ring, Membranous, Shaggy or -Associated with SLE in the active stage of the disease and in
Thread) Sjogren’s syndrome
Speckled -Pattern occurs in the presence of antibody to any extractable
(Mottled/Pepper dot) nuclear antigen devoid of DNA or histone
-Anti-Sm antibodies have been shown to be specific with SLE
-Anti-RNP has been found in patients with wide variety of
rheumatic diseases including SLE, RA, Sjogren’s Syndrome,
Progressive Systemic Sclerosis, MCTD and Dermatomyositis
Nucleolar -Reflects an antibody to nucleolar RNA
-Present in about 50% patients with scleroderma (Progressive
Systemic Sclerosis), Sjogren’s Syndrome and in SLE
Anti-centromere Antibody -Antibody reacts with the centromeric chromatin of metaphase and
interphase cells
-Appears to be highly selective for the CREST variant of
progressive systemic sclerosis

2. Rheumatoid Arthritis
-Most common autoimmune disease
-Felty’s Syndrome is a variant of RA with splenomegaly, leukopenia and possibly
thrombocytopenia

a. RHEUMATOID FACTOR DETECTION

-Autoantibody present not only in RA but also in other diseases
-Most often of the IgM class that is directed against the Fc portion of IgG
• LATEX AGGLUTINATION
-Principle: Passive Agglutination
-When using RF latex tests, a titer of 1:80 or greater is generally considered a
positive reaction, a titer of 20-40 is considered a weakly positive reaction and if
there is no agglutination at 1:20, the specimen should be considered negative for RF,
even if subsequent dilution shows agglutination.
• ROSE WAALER TEST
-Agglutination test using tanned sheep red cells coated with small amounts of rabbit-
anti-SRBC immunoglobulin of the IgG class. These cell agglutinate when exposed to Rh
factor because of a cross-reaction between human and rabbit IgG
• SINGER AND PLOTZ TEST
-Latex Fixation Test
• SENSITIZED ALLIGATOR ERYTHROCYTE TEST (Cohen et.al.)
• BENTONITE FLOCCULATION TEST (Bloch and Bunim)

b. ANTI-CCP (Cyclic Citrullinated Peptides)

-Lead marker for detection of RA, because it is much more specific than RF

VIRAL INFECTIONS

1. Hepatitis
-General term that means inflammation of the liver
-It can be caused by several viruses (EBV, HSV, CMV) and by noninfectious agents, including
ionizing radiation, chemicals and autoimmune processes
19 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
Table X.Summary of HAV and its Associated Serological and Molecular Markers

***This table was snipped off from Stevens’

Table X.Summary of HBV and its Associated Serological and Molecular Markers

***This table was snipped off from Stevens’

Table X.Summary of HCV and its Associated Serological and Molecular Markers

***This table was snipped off from Stevens’

20 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
Table X.Summary of HDV and its Associated Serological and Molecular Markers

***This table was snipped off from Stevens’

Table X.Summary of HEV and its Associated Serological and Molecular Markers

***This table was snipped off from Stevens’

2. Infectious Mononucleosis
-Caused by Epstein Barr Virus
-The virus selectively infects host cells that are positive for the CD21 molecule, which
serves as a receptor for both the virus and the C3d protein of complement
-There are two types of antibodies formed in EBV infection

a. ANTIBODIES SPECIFIC FOR EBV

Table 60.Serological Responses of Patients with EBV-Associated Diseases

***This table was snipped off from Stevens

21 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
Table X.Characteristic Ab formation in IM Table X.Diagnostic Profile of EBV

*This table was snipped off from Turgeon’s *This table was snipped off from Turgeon’s

b. HETEROPHILE ANTIBODIES
-IgM antibodies that are capable of reacting with similar antigens from two or more
unrelated species
-Capable of reacting with horse red blood cells, sheep red blood cells, and bovine red
blood cells but not guinea pig kidney cells
-Titer does not correlate with the severity of disease

• PAUL-BUNNEL TEST
-Sheep red cells agglutinate in the presence of heterophile antibodies
-Only indicative of the presence or absence of heterophile antibodies
-A titer of 1:56 is considered to be presumptive test in the presence of clinical
and/or cytologic findings suggestive of IM

• DAVIDSOHN DIFFERENTIAL TEST

Table X.Absorption Patterns
HETEROPHILE ANTIBODY ABSORBED BY GUINE PIG KIDNEY ABSORBED BY BEEF (OX/BOVINE)
CELLS ERYTHROCYTES
Abs to Forsmann Ag
(Serum)
Abs in IM
Abs in Serum Sickness
This table was adapted from Navarro’s

Table X.Agglutination with Sheep Erythrocyte after Absorption

HETEROPHILE ANTIBODY AGGLUTINATION WITH SRBCs AFTER AGGLUTINATION WITH SRBCs AFTER
GUINEA PIG ABSORPTION BEEF ABSORPTION
Abs to Forsmann Ag
(Serum)
Abs in IM
Abs in Serum Sickness
This table was adapted from Navarro’s

• MONOSPOT TEST
-A rapid slide agglutination method which tests the ability of serum absorbed with
Guinea pig kidney or beef erythrocyte antigens to agglutinate horse red blood cells.
-The antibody could then be tittered by incubating serial dilutions of the patient’s
serum with sheep red blood cells in the Paul-Bunnel test

HUMAN IMMUNODEFICIENCY VIRUS

-A retrovirus under the genus Lentiviridae that causes AIDS

Table X.HIV—1 versus HIV-2

HIV-1
-Responsible for causing AIDS
-Identified independently by the laboratories of
Luc Montagnier of France and Robert Gallo and
Jay Levy of US in 1983 and 1984 respectively
HIV-2
-Majority of cases occurred in Africa
-May also cause AIDS but is less pathogenic and
has a lower rate of transmission

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SEROLOGY DOCKIE NOTES
-Formerly called Human T-cell Lymphotrophic
Virus-Type III (HTLV-III), Lymphadenopathy-
Associated Virus (LAV), and AID-associated Virus
(ARV)

1. Main Structural Genes

a. Gag
-codes for p55, precursor of p5,p9,p17
and p24; all four are located in the
nucleocapsid of the virus

b. Env
-Codes for glycoproteins gp160, gp120,
and gp41, which are found in the viral
envelope
-gp160 is a precursor protein that is
cleaved to form gp120 and gp41
-gp120 forms the numerous knobs or
spikes that protrude from the outer
envelope, while gp41 is a transmembrane
glycoprotein that spans the inner and
outer membrane and attaches to gp120
-both gp120 and gp41 are involved with
FUSING
and ATTACHING HIV to receptors on host
cells Figure X.Structure of HIV
(Figure snipped off from Turgeon’s)
c. Pol
-Codes for enzymes necessary for HIV replication namely p66 and p51. These are subunits
of reverse transcriptase p31 or integrase which mediates integration of the viral DNA
into the genome of infected host cells and of p10, a protease that cleaves protein
precursors into smaller active units. The p66 protein is also involved in degradation of
the original HIV RNA. These proteins are located in the core, in association with the
HIV RNA

Table X.Important Enzymes in HIV

Reverse Transcriptase Enables the virus to convert RNA to DNA
Integrase Inserts viral DNA into host DNA (genome)
Protease Cleaves other enzymes and structural proteins from their polyproteins
Important in formation of new virion

Table X.Enocding genes and antigen of HIV Virus

***This table was adapted from Turgeon’s

23 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES
2. HIV Replication
-Steps in the HIV replication cycle:
a. Fusion of the HIV cell to the host
cell surface
b. HIV RNA, reverse transcriptase,
integrase, and other viral proteins
enter the host cell
c. Viral DNA is formed by reverse
transcription
d. Viral DNA is transported across the
nucleus and integrates into the host
DNA
e. New viral RNA is used as genomic
RNA and to make viral proteins
f. New viral RNA and proteins move to
the cell surface and a new, immature,
HIV virus forms
g. The virus matures by protease
releasing individual HIV proteins.

Figure X.HIV Replication Cycle

(Figure snipped off from Turgeon)

Summary of HIV-1 Life Cycle

3. HIV Serology
a. CD4 T CELL ENNUMERATION
-The gold standard for CD4-T cell enumeration is Flow Cytometry

b. DETECTION OF HIV ANTIBODY

• ELISA
-Standard Screening Test
-When a sample screened for HIV Ab by ELISA yields a positive result, it should be
retested in duplicate by the same ELISA test. If 2 out of 3 specimens are reactive,
then the results must be confirmed (usually by Western blot)

• WESTERN BLOT (Immunoblot)

-Standard Confirmatory Test
-Result should be reported as positive if at least 2 of the following 3 bands are
present: p24, gp41, and gp120/160

24 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi
SEROLOGY DOCKIE NOTES

Figure 26.western Blot Immunoassay

(Figure snipped off from Turgeon’s)

SIDENOTE:
➢ Indeterminate results are from specimen that have some of the characteristic bands present
but do not meet the criteria for a positive result. Serum maybe collected in the early phase
of seroconversion or serum contains antibodies that cross-react with some of the immunoblot
antigens. The test must be repeated with same specimen; if result is still indeterminate,
testing may be performed with a new specimen obtained few weeks later; if pattern converts
to a positive result, the 1st specimen was obtained during the early phase of seroconversion;
if pattern is still indeterminate, it suggests that the pattern is due to a false positive
test rather than HIV infection

c. DETECTION OF HIV ANTIGEN

-p24 antigen testing

SIDENOTE:
➢ The most specific indicators of infection and disease progression include serodiagnostic
tests for HIV p24 antigen and antibody to p24 and other viral proteins.
➢ The first antibodies to be detected are directed against the gag proteins such as p24,
followed by the production of Abs to the env, pol and regulatory proteins

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SEROLOGY DOCKIE NOTES

d. NUCLEIC ACID TESTING/VIRAL LOAD ASSAY

• PCR (RT-PCR)
• Branched-Chain DNA Assay
• Nucleic Acid Sequence

Believe that you can become what you aspire for...,

if it’s for your dream, everything is worth risking and

trying.”
-MARCJAYGAGARIN, RMT, MD :3

REFERENCES:

Clinical Immunology and Serology – a Laboratory Perspective by Christine Dorrestyne Stevens

Immunology and Serology in Laboratory Medicine by Mary Louise Turgeon
Henry’s Clinical Diagnosis and Management by Laboratory Methods by McPherson
Laboratory Immunology and Serology by Neville Bryant
Essentials of Immunology and Serology by Jacqueline Stanley
Hematology: Clinicl Principles and Applications by Bernadette Rodak
Immunology and Serology Notes by Dean Arlene Lumbang
Immunology and Serology Notes by Miss Anna Leah Navarro
Immunology and Serology Notes by Dr. Turqueza
Internet URLs/Links

You must be prepared to face the worst possible scenarios…

Because harsh reality strikes without warning.
-Hunter x Hunter

26 MARCJAYGAGARIN, RMT, MD :3 “A human’s belief can make the impossible, possible. “ –Akihisa Yoshi