SEROLOGY

Vital Health REVIEW CENTER

 Beginning of Serology
• Serology as a science began in 1901.
• Austrian American immunologist Karl
Landsteiner (1868-1943) identified groups of
red blood cells as A, B, and O.
• From that discovery came the recognition that
cells of all types, including blood cells, cells of
the body, and microorganisms carry proteins
and other molecules on their surface that are
recognized by cells of the immune system.
 Karl Landsteiner (1868-1943)
• An Austrian physician by
training, Landsteiner
played an integral part in
the identification of
blood groups.

• He demonstrated the
catastrophic effect of
transfusing with the
wrong type of blood,
Specific antibodies establishes Blood
grouping system
 Purpose of Serological Tests
• Serological tests may be performed for
diagnostic purposes
– when an infection is suspected
– in autoimmune diseases
– in many other situations, such as checking an
individual's blood type.
 Serology
• Serology is the scientific
study of blood serum.
• In practice, the term
usually refers to the
diagnostic identification
of antibodies and
antigens in the serum
 Serology
• The branch of laboratory
medicine that studies
blood serum for evidence
of infection and other
parameters by evaluating
antigen-antibody
reactions in vitro
 Terms used in Evaluating Serological
Methodologies

• Sensitivity
– Analytical Sensitivity – ability of a test to detect
very small amounts of a substance
– Clinical Sensitivity – ability of test to give
positive result if patient has the disease (no false
negative results)
 Specificity
• Analytical Specificity – ability of test to
detect substance without interference from
cross-reacting substances
• Clinical Specificity – ability of test to give
negative result if patient does not have disease
(no false positive results)
 Affinity
• Affinity refers to the strength
of binding between a single
antigenic determinant and
an individual antibody
combining site.

• Affinity is the equilibrium

constant that describes the
antigen-antibody reaction
• Antibody affinity is the strength of the reaction
between a single antigenic determinant and a
single combining site on the antibody.

• It is the sum of the attractive and repulsive

forces operating between the antigenic
determinant and the combining site .
Avidity
• Avidity is a measure of the
overall strength of binding
of an antigen with many
antigenic determinants and
multivalent antibodies

• Avidity is influenced by both

the valence of the antibody
and the valence of the
antigen.

• Avidity is more than the

sum of the individual
affinities.
 Dilution
• Estimating the antibody
by determining the
greatest degree to which
the serum may be diluted
without losing the power
to given an observable
effect in a mixture with
specific antigen
Titer
• Different dilutions of
serum are tested in
mixture with a constant
amount of antigen and
greatest reacting dilution
is taken as the measure or
Titer
 Expression of Titers
• Expressed in term of the was
in which they are made

• Dilution 1 in 8 is a dilution
made by mixing one volume
of serum with seven
volumes of diluents (Normal
Saline )

• Incorrect to express
dilution as 1/8
Common methods in creating dilutions
 Sero Conversion
• Seroconversion is the
development of
detectable specific
antibodies to
microorganisms in the
blood serum as a result of
infection or
immunization.
Sero reversion
• Seroreversion is the
opposite of
seroconversion. This is
when the tests can no
longer detect antibodies
or antigens in a patient’s
serum
 Testing paired Samples

• Testing for infectious

diseases is performed on
acute and convalescent
specimens (about 2 weeks
apart) Paired sample.

• Must see 4-fold or 2-tube

rise in titre to be clinically
significant
 Majority Diagnostic tests are Serological tests

• There are several

serology techniques that
can be used depending
on the antibodies being
studied. These include:
ELISA, agglutination,
precipitation,
complement-fixation,
and fluorescent
antibodies.
 DETECTION AND APPLICATION OF
ANTIGEN–ANTIBODY REACTIONS

• Precipitation
• Agglutination
• Complement fixation
• Immuno assay using labelled reagents
• Immuno histochemistry (Immunofluorescence)
• Cytokine immunoassays (ELISPOTR )
• DNA innunoassays
 Antigen-Antibody Combinations
• Primary Phenomenon
– Initial antigen antibody binding
• RIA, EIA, IFA
• Secondary Phenomenon
– Aggregates of immune complexes
– Agglutination and Precipitation reactions
• Tertiary Phenomenon
– Invivo/body’s reaction to immune complexes
– Inflammation, phagocytosis, chemotaxis
SOLUBLE ANTIGEN WITH ANTIBODY

PRECIPITATION
MECHANISM
– Interaction of a soluble antigen with antibody in
correct proportion.
– Soluble antigens
– Protein
– Carbohydrates
– Occurrence is due to divalent antibodies cross
linking with a multivalent antigen forming a
lattice.
• Prozone phenomenon is a term which is used to
describe suboptimal precipitation or incomplete
lattice formation which occurs in the region of
antibody excess.
– Due to use of fresh serum containing complement
– Maybe due to the presence of both IgM and IgG
antibodies
• Post zone phenomenon occurs in the region of
antigen excess wherein there is a suboptimal
precipitation or incomplete lattice formation.
Precipitation curve
TURBIDIMETRY
NEPHELOMETRY

PRECIPITATION BY LIGHT
SCATTERING METHODS
PRECIPITATION BY LIGHT SCATTERING

• TURBIDIMETRY

– Measures the turbidity or cloudiness of a solution.

– Measures reduction in light intensity due to
reflection, absorption scatter.
– Utilizes a spectrophotometer or an automated
analyzer
PRECIPITATION BY LIGHT SCATTERING

• NEPHELOMETRY
– Measures light that is scattered at a particular angle
from the incident beam as it passes trough a
suspension.

– Used for quantification of

• IgA, IgM, IgG, complement
• Alpha-antitrypsin
• Alpha 2 macroglobulin
• Fibrinogen, ceruloplasmin, RF
PRECIPITATION BY PASSIVE
IMMUNODIFFUSION
• Best demonstrated by immunodiffusion
technique.
• Involves random movement of antigen or
antibody to form complexes in a medium
• Agar
• Agarose
• Methods
• One dimensional ID
• Two dimensional ID
One-Dimensional ID
• Antigen or antibody remains fixed in a semi-
solid medium.
• Reactant moves and complexes with the
antigen or antibody
• Methods:
A. Single Linear Diffusion (Oudin Technique)
B. Single Radial Diffusion (Fahey or Mancini)
A. Single Linear Diffusion
• The Oudin Technique

– Known antibody is fixed in an agar tube

– Overlaid with an unknown antigen
– The antigen moves trough the gel forming
precipitin bands at point of slight antigen excess.
– Used to detect multiple antigen-antibody reactions
B. Single Radial Diffusion
• The specific known antibody is fixed in agar
plate
• Unknown antigen from the specimen is added
into a well cut in an agar plate
• Incubated for 24-48 hours
• Used in the detection and semi-quantification
of many antigens.
Methods of RID
A. Mancini
• End Point Method
• Antigen is allowed to
diffuse to completion
– IgG: 24 hrs
– IgM: 50-72 hours
• Concentration of the
Antigen = the square of the
diameter of the ring
B. Fahey-McKerley
• Kinetic Method
• Antigen is not allowed to
diffuse to completion
• 18 hours
• Log of standard concentration
is proportional to the diameter
of precipitin rings
• Y axis: analyte concentration
• X axis: diameter of the ring
Two Dimensional ID
• Ouchterlony Technique
• The antigen and antibody are both free to move
toward each other in a medium
– Methods:
A. Double Linear Diffusion
B. Double Radial Diffusion
A. Double Linear Diffusion
• Two wells are cut in a plate
• The antigen is placed in one well and the
antibody into the other.
• The two diffuse towards each other and at
optimal concentrations a precipitate line is
formed
• Sources of error
– Over or under filling of wells
– Spilling the patient’s serum on the gel
– Nicking the side of the well when filling
– Improper incubation time and temperature
– Specimen contamination
B. Double Radial Diffusion
• Two antigens and one Antibody wells are cut
at angles in an agar plate.
• Useful for establishing relationship between
various substances
• Sources of Error
– Overfilling of wells
– Irregular well punching
– Non level incubation
– Gel Drying
– Over heating
– Inadequate time for diffusion
– Antibody degradation
– Bacterial or Fungal contamination
• Double diffusion in agar can also be used for semi-
quantification of antigen or antibody reactants as shown in.
• It is still an important initial test for the detection of serologic
reactions to various human, animal and plant antigens.
Rocket Immunoelectrophoresis
Counter Immunoelectrophoresis
Immunoelectrophoresis
Immunofixation Electrophoresis

PRECIPITATION BY
ELECTROPHORESIS
• Uses the principle of separation of individual
proteins in an electrical field.
• The medium used in this purpose should not
impede or enhance the flow of molecules in an
electrical field.
– agarose
– cellulose acetate strips
Test principle:

• The biologic fluid is spotted at the origin and

subjected to an electrical current, allowing for
separation of individual constituents.
• Large molecular weight substances move slowly
and traverse a shorter distance in the given time
period.
• Smaller molecular weight fragments traverse
further
• Densitometer scanning converts the separated
bands into peaks.
A. Rocket Immunoelectrophoresis (Laurell)

• The antiserum to the particular antigen is

incorporated into an agarose medium and poured
over a glass slide.
– This ensures that antibody does not migrate.
• The specimen containing the unknown antigen is
placed in a small well.
• Electrophoresis of the antigen into the antibody is
then performed.
• The resultant pattern of immuno-precipitation
resembles a spike or rocket
– hence the term rocket electrophoresis
• The principal application of this technique is
to quantify antigens in biological fluids.
• The rocket pattern
– Is formed due to the precipitation occuring along
the lateral margins of the moving boundary of
antigen, as it is driven into the agar containing the
antibody.
– Gradually as the antigen is lost through
precipitation its concentration at the leading edge
diminishes and the lateral margins converge to
form a sharp point.
• The total distance of antigen migration(length
of spike) for a given antiserum concentration is
linearly proportionate to the antigen
concentration.
• This system cannot be used to quantify
immunoglobulins because their weak negative
charge prevents their electrophoretic mobility
in this system.
B. Counter Current Immunoelectrophoresis

• Gel is poured onto a plate

• Two columns of wells are cut, with antigen in one
well and antibody in the other.
• The plate is placed in an electric filed.
• At pH 8.6 antibodies migrates towards the cathode
and antigens towards the anode
• Can be semi quantitiative if serial dilutions are used
• Useful to the detection of autoantibodies, antibodies
to infectious antigens and certain microbial agents
• Sources of error:
– Reversal of wells so that the current applied is in
wrong direction
– Improper pH and buffer
– Insufficient electrophoresis time
– Prozone or Postzone
– Wells are not parallel
C. Immunoelectrophoresis

• Combination of electrophoresis and diffusion

techniques.
• Principle:
– Under an electrical current unknown antigen migrates
trough a gel medium
– The current is stopped when desired migration is reached.
– A through is cut in the agar and filled with unknown
antibody
– A precipitin arc is then formed.
– The arc closest to the trough at a point where antigen is in
highest concentration.
• Applications of immunoelectrophoresis
• diagnosis of paraproteinemias
• hypo or agammaglobulinemia
• identification of L chains in the urine of patients with
plasma cell dyscrasias or autoimmune disorders.
• identifying increased amounts of proteins present in the
cerebro spinal fluid in patients with various neurologic
diseases.
• Used to identify urine proteins
D. Immunofixation Electrophoresis

– Similar to immunoelectrophoresis
• Difference:
– After electrophoresis has taken place, antiserum is applied
directly to the surface of the gel rather then being placed in the
trough
• Application:
– Western blot Technique
Comparison of Precipitation
Techniques
Particulate antigen and antibody

AGGLUTINATION
Mechanisms:
• Reaction of antibody with multivalent antigen by
an antibody
– Antigen particulately cellular in nature
• Bacteria
• Yeasts
• Erythrocytes
• Latex
• Cross linking or lattice formation results to
clumping in vitro
• Antibodies agglutination bacteria in vivo is
opsonization.
Methods:
• Slide Tests
– Rapid tests requiring only 2-5 minutes of rotation at
room temperature
• Tube Tests:
– They usually require longer incubation
• 15 mins-24 hrs
• 4C, 20C, 37C
• Microtiter Techniques
– Adaptation of test tube procedure
• Less patient sample
• Less amount of reagents
DIRECT AGGLUTINATION
DIRECT BACTERIAL AGGLUTINATION
INDIRECT OR PASSIVE AGGLUTINATION
REVERSE PASSIVE AGGLUTINATION
CONGLUTINATION
ANTIGLOBULIN TECHNIQUE
AGGLUTINATION INHIBITION REACTION

TYPES OF AGGLUTINATION
• Whereas precipitation reactions involve
soluble antigens, agglutination is the visible
aggregation of particles caused by combination
with specific antibody.
• Antibodies that produce such reactions are
often called agglutinins.
 Steps in Agglutination
• Sensitization
– antigen–antibody
combination through
single antigenic
determinants on the
particle surface

– This initial reaction

follows the law of mass
action and is rapid and
reversible.
– The second step is the formation of cross-links that
form the visible aggregates.

– Represents the stabilization of antigen–antibody

complexes with the binding together of multiple
antigenic determinants
• Lattice Formation

– The second stage, representing the sum of

interactions between antibody and multiple
antigenic determinants on a particle, is dependent
on environmental conditions and the relative
concentrations of antigen and antibody
• Lattice formation is governed by
physicochemical factors such as the milieu’s
ionic strength, pH, and temperature.

• Antibody must be able to bridge the gap

between cells in such a way that one molecule
can bind to a site on each of two different
cells.
Direct Agglutination
•Employs antigens found naturally on the surface of cells
• Direct Hemagglutination
– Agglutination of RBC to
detect ABO and Rh antigens
– Detection of Cold
Agglutinins that react with I
antigens on patient’s own
RBC or Type O RBC
– Detection of Antibodies of
EBV (heterophile
antibodies) reacting with
horse, sheep or beef RBC
• Direct Bacterial
Agglutination Test
– Simple direct technique, a cell
or insoluble particle is
agglutinated directly by
antibody.
– Examples
• Slide Test: Kauffman and
White Test
• Tube Test: Widal, Weil
Felix Test
• Widal: 1:160
• Brucella: 1:132
• Tularemia: 1:40
Passive or Indirect Agglutination
• Soluble antigen is attached or coated to a
particulate carrier
• Charcoal
• Latex
• Gelatin
• RBC
• Uses to detect
• Reagin
• Rheumatoid Factor
• Rheumatoid Arthritis
• Rubella Antibody
• Thyroglobulin Antibody
Reverse Passive Agglutination
• The antibody is attached to the particulate
carrier to detect the unknown antigen
• Used to detect
• C Reactive protein
Conglutination
• Employs bacteria as the inert particle to which
antibody is attached
• Frequently used:
• Protein A of staphylococcus aureus
• Naturally absorbs the Fc portion of an antibody
molecule
Agglutination Inhibition:
• Two step procedure:
1. Soluble antigen in sample is incubated with known
antibody reagent. If antigen is present a reaction will
take place but not visible.
2. The particulate antigen is added
– If the antigen was present in the sample, no free
antibody will attach to the particulate
• No agglutination: Positive test
– If antigen is absent then free antibody will attach
to the particulate
• Agglutination: Negative test
• Used to detect
• HCG in serum and urine
• Presence of soluble A, B and H substances in body
fluids
Antiglobulin Mediated Agglutination Reaction

– The Coomb’s Test

– Note that IgG is a non agglutinating Antibody
• Incomplete or blocking antibodies
• Rh antigenic determinant on cells
– However can sensitize the cell without causing a
visible agglutination
– The addition of AHG binds the Fc portion of IgG
that acts a bridge to make agglutintation visible
a. Direct Coomb’s Test
• Detects invivo sensitization of red blood cells
– HDN, HTR
• Specimen is used is RBC
b. Indirect Coomb’s test
• Detects invitro sensitization of RBC
– X-matching, antibody detection, Antibody identification and
RBC phenotyping
• Sample to be used depends on the test, either serum or
RBC.

Known Ag + Unknown AB---------- wash (remove excess Ab)

+
AHG

(agglutination)
Grading of agglutination
reactions
Causes of False-Positive and False-Negative
Reactions in Agglutination Testing
 Neutralization reactions
• Principle:
– antibody neutralizes toxin
– After neutralization antibody is not available to react in indicator
system
• Results:
– NO agglutination or NO hemolysis = positive reaction
– Agglutination or hemolysis = negative reaction (antibody not
bound in origin

• Generally, positive control samples used should show no reaction

and negative control samples show a reaction

• Example
– Antistreptolysin O test (ASO)
 Complement fixation (CF)
• Use the ability of complement to lyse cells as an
indicator of whether an antigen-antibody took place
• Use of indicator system (Sheep’s cell)
• Can test for antigen or antibody
• Positive test:
– Ag + Ab + C + indicator cell = no hemolysis
• Negative test
– Ag or Ab + C + indicator cell = hemolysis
• CF is only applicable to IgM antibodies
• Application:
– RMSF
– Herpes simplex infection
– Infuenza
BINDER-LIGAND ASSAYS
LABELLED IMMUNOASSAY
Labelled Immunoassay
• Primarily used to detect or demonstrate
immune complexes
• Designed for antigens and antibodies that may
be small in size or present in very low
concentration
• Immune complexes is determined indirectly by
using a labelled reactant to detect whether or
not specific binding has taken place.
Radioimmunoassay
Enzyme-Linked Immunosorbent Assay
Flourescence Immunoassay
Chemiluniscence Immunoassay

LABELLED IMMUNOASSAY
 Labelled Immunoassay Format
Heterogenous Immunoassay Homogenous Immunoassay
• Involve a solid phase • Consist of only a liquid
– Microwell phase
– Bead • Do not require washing
• Require washing steps to steps
remove unbound antigens or • Faster and easier to
antibodies automate
• Format: • Format:
– Competitive – Uncompetitive
– Uncompetitive
Radioimmunoassay
• Has been used to quantify hormones in plasma.
• Hormones like insulin, GH, ACTH, T3, T4, and estrogen
• Serum proteins like CEA, anti-DNA
• Metabolites like folic acid
• Drugs like digoxin, digitoxin, morphine
• Microbial agents and antibodies such as HBsAg
• Makes use of radioactive substances as labels
• 3H triatiated hydrogen (beta emmiter)
• 125I most common labelling radioactive substance
• 131I
 Types of RIA
Non-competitive
Competitive Binding Assays Immunoradiometric Assay
• Analyte competes with a • This method uses labeled
radiolabled analyte for a
limited number of binding sites antibody that is present in
• The concentration of excess
radioactive analyte is in • The amount of bound
excess, so that all the binding labelled antibody is in direct
sites on the antibody will be
occupied.
proportion to the amount of
• If patient antigen is present,
patient analyte.
some of the binding sites will
be filled with unlabeled
analyte, decreasing the amount
radioactive label
 Methods of RIA
Liquid Phase
• Depends on the competition
between
– Labelled antigen
– Unlabelled antigen
• The Ag-Ab complexes formed
are separated by their
physiochemical or
immunologic means and their
radioactivity is determined
• Measurement of unknown
antigen is determined by
reference to a standard curve
Solid Phase
• Involves adsorption or
covalent linkage to antibody to
a solid matrix.
• Unlabelled antigen is added
followed by a labelled antigen.
• Determination of bound versus
free labelled antigen is then
made.
• The amount of antigen in the
known sample is calculated by
reference to a standard.
Radioallergo Sorbent Test (RAST)
Radioimmunosorbent Test (RIST)

APPLICATIONS OF RIA
• Radio Immuno
Sorbent Test
– Total IgE
concentration
– usually done in a
microtiter poly -
carbonate plate with
multiple wells
• Radio
Allergosorbent
Test
– measures the amount
of IgE to specific
antigens (allergens) in
the patient’s serum
 Radio Immunoassay
Advantage
• It is sensitive and precise
technic
• Capable of detecting and
measuring trace amounts of
analytes
Disadvantage
• Health hazard is involved in
working with radioactive
substances
• Disposal of radioactive
wasted is an environmental
problem
• RIA requires expensive
equipment
Enzyme-Linked Immunosorbent Assay
• Can be used to assay both antigens and antibodies
• Requirements:
– Either antigen or antibody that can be attaced to a solid
phase support
– Either antigen or antibody that can be linked to an
enzyme
• Horse raddish peroxidase
• Alkaline phosphatase
• G-6-PD
• Glucose oxidase
• Urease
• Beta-d-galactosidase
– Enzyme label is linked to an antibody or analyte
by several means
• Glutaraldehyde often used as a cross linker
– Join amino groups of enzymes and the molecule to be labelled
• Maleimide derivatives
NONCOMPETITIVE
COMPETITIVE
CAPTURE

TYPES OF ENZYME
IMMUNOASSAY
HETEROGENOUS ENZYME
IMMUNOASSAY
Competitive ELISA
• Involves a solid surface to
which a specific antigen is
attached.
• Patient serum potentially
containing antibody and
antibody specific to the test
antibody are mixed
• Chromogenic substrate is then
added.
• The amount of the color
produced is inversely
proportional to the antibody.
Non Competitive ELISA
• Specific antigen is attached
to a solid phase surface
• Patient serum that could
contain antibody is added to
the solid phase, followed by
an enzyme labelled antibody
specific to the test antibody.
• The added chromogenic
substrate changes its color,
and proportional to the
amount of antibody present
Non-Competitive Elisa
Capture Enzyme Immunoassay

– Designed to detect a specific type of antibody .

– Antibody specific for IgG and IgM is attached to a solid phase.

– The patient serum that could contain IgG or IgM is added.

– Specific antigen is added

– Finally chormogenic substrate is added which in the presence of

enzyme changes in color.

– The amount of color that develops is proportional to the amount of

antigen specific IgM or IgG in patient serum
Homogenous Enzyme Immunoassays

• Antibody-Antigen system in which no

separation step is necessary
• Based on the principle of change in enzyme
activity as specific antigen-antibody
combination occurs
• Rapid simple to perform and as adapt easily to
automation but generally less sensitive than
heterogenous assay.
Membrane-based Cassette Assays
– Primarily for POC
– Either antigen or antibody can be coupled to the
membrane, and the reaction is read by looking for
the presence of a colored reaction product.
– The membrane is usually nitrocellulose which is
able to easily immobilize proteins and nucleic
acids.
Membrane cassette assay
 ELISA
Advantage
• Sensitivity is similar to RIA
without creating health
hazard or causing disposal
problem
• No need for expensive
instrumentation
Disadvantage
• Some specimens contains
enzyme inhibitors
• Enzymes are sensitive to
temperature
Flourescence Immunoassay
• Principle:

– Makes us of fluorescent compounds known as flourophores or

flourochromes as labels or conjugates of known antibody
molecules to allow visualization of antigen-antibody reactions
via ultraviolet light and fluorescence microscope

– Such labelled antibody are used to identify unknown antigens.

– Unknown antigens can be detected either in fixed tissue sections

or live cell suspension with sensitivity and specificity
Principle of Fluorescent Method
Flourescent Dyes
– Flourescein isothiocyanate (FITC)
• Emits green color at 517 nm
• Absorption at 490-495
– Tetra methyl rhodamine isothiocyanate (TRITC)
• Emits bright red light at 580-585 nm
• Absorption at 580 nm
– Phycobiliproteins
• Derived from algae, porphyrins and chlorophylls
• Red fluorescence at 600 nm
Reading Devices
• The amount or the degree of flourescence
produced in FIA are measured by

– Spectroflourometer
– Fluorometer
– Flow cytometer
– Flourescence microscope
 Types of FIA
Direct
• An antibody is conjugated
with a flourescent label is
added directly to unknown
antigen that is fixed to a
microscopic slide
• Best for antigen detection
• Antigens are visualized as
bright apple green or yellow
objects against a dark
background
Indirect
• A known antigen is attached
to a solid phase is added
with the patient serum
• The resulting antigen-
antibody complex is then
added with AHG reagent
containing a fluorescent dye
• Used for both antigen and
antibody detection.
Direct Versus Indirect FIA
 Chemiluminescence
• Chemiluminescence is the
emission of light with
limited emission of heat
(luminescence), as the
result of a chemical
reaction.
 Chemiluminescent Immunoenzymatic
Assay
• Process for the quantitative and qualitative
determination of antigens, antibodies and their
complexes by means of a chemiluminescing labelling
substance activated or excited to chemiluminescence's
by an analytical reagent.
• By means of a serological reaction, initially an
antigen/antibody complex is formed which is treated
with a chemiluminescing conjugate containing
chemiluminescing triphenylmethane dyes and the
chemiluminescence of the chemiluminescing
complex formed is measured.
 Flow Cytometry
• Method of choice for T- and B-cell analysis (lymphocyte
phenotyping)
• Principle
– Incubate specimen with 1 or 2 monoclonal antibodies
tagged with fluorochrome
– Single cells pass through incident light of instrument (laser)
which excites fluorochrome and results in emitted light of
different wavelength
– Intensity of fluorescence measured to detect cells
possessing surface markers for the specific monoclonal
antibodies that were employed
– Forward light scatter indicates cell size or volume
– 90° side-scattered light indicates granula
 Common uses Flow Cytometry

• DNA analysis
• Reticulocyte counts
• Leukaemia/lymphoma
classification
• CD 4 cell estimations in
AIDS/HIV patients.