Chapter Two

Serologic techniques
 Learning Objective

At the end of this chapter, the students should be

able to:

1. List material and equipment for serological tests

2. Collect, preserve and prepare serological specimens

3. Run complement inactivation procedure and state

its importance

4. Run serial dilution, determine end point and titer.

 Outline

2.1. Introduction

2.2. Materials necessary for basic serologic tests

2.3. Collection, preparation and preservation of

serologic al tests

2.4. Shipment of serological specimens

2.5. Complement inactivation

2.6. Dilution

2.6.1. Serial dilution

 2.1. Introduction
 Dilution is the act of making a weaker solution
from a strong solution.
 Serial dilution The systematic re-dilution of a
fluid number of times is called a Serial dilution
 Titer is the reciprocal of the highest dilution
showing a positive reaction
 Complement is a group of non-immunoglobulin
plasma proteins that are sequentially activated by
Ag–Ab complexes
2.2. Materials necessary for basic
serologic tests

Types of glassware include:

 Test tubes
 Glass slides
 Serological pipette with a size of 10ml, 5ml, 2ml
and 1ml.
2.2. Materials necessary for basic
serologic tests
Glassware
 Dirty glassware easily affects serological tests.
 After using all the glassware (test tube, beaker,
pipette, etc) they should be soaked in detergent
for several hours and rinsed several times in tap
water.

 Finally, allow drying by placing in a dry oven or

dust free place. Test tubes and pipettes should
not be scratched or broken, which will interfere
with the reading of a test.
Glassware's and plastic wares
2.2. Materials necessary for basic
serologic tests

Constant Temperature Device

 Incubators and water baths are used in
serological tests. These materials are
electrically operated and have thermostat that
hold the temperature within the required
limits. These devices should be checked prior
to use by a thermometer.
2.2. Materials necessary for basic
serologic tests

Rotating Machine
 Rotating machines are required to facilitate
antigen antibody reactions. Such machines have
a flat plate, which rotate at a prescribed rate of
speed. A knob located on the front of the
machine controls the number of revolutions per
minute.
 2.3. Collection, Preparation And
Preservation Of Specimens
For Serologic tests
 Specimens that are used for serologic test
include: serum, plasma and cerebrospinal fluid.
 Serum or plasma samples could be obtained from
venous blood, which can be collected by the
laboratory personnel.
 CSF should be collected by a physician or trained
nurse.
 2.3. Collection, Preparation And
Preservation Of Specimens
For Serologic tests

Serum or plasma sample collection

 Collect 2-3ml of venous blood from a patient
using a sterile syringe and needle.
 If serum is required, allow the whole blood to clot
at room temperature for at least one hour,
 Centrifuge the clotted blood for 10 minutes at
2000 rpm.
 2.3. Collection, Preparation And
Preservation Of Specimens
For Serologic tests

 Transfer the serum to a labeled tube with a

paster pipette and rubber bulb.
 Plasma samples are obtained by treating fresh
blood with anticoagulant,
 Centrifuge and separate the supernatant.
 2.3. Collection, Preparation And
Preservation Of Specimens
For Serologic tests
 The specimen should be free from hemolyzed blood.
 Finally, seal the specimen containing tube; the tube
should be labeled with full patient's identification
(age, sex, code number, etc).
 The test should be performed within hours after
sample collection, if this could not be done preserve
it at -20oc.
 2.4. Shipment of serological
specimens
 Most health center and clinic laboratories are
limited in the diagnostic procedures that can be
carried out and have to ship serologic specimens
to other laboratories.

 Before shipment, the following things should be

considered.
 Don't ship whole blood unless the tests to be
performed require whole blood.
 Don't inactivate serum or plasma.
 2.4. Shipment of serological
specimens

Serum, plasma, and CSF should be handled as follows:

 Collect and process specimens under sterile
conditions.
 Ship specimens by the fastest route as soon after
collection as possible.
 Don't ship whole blood unless the test to be performed
required whole blood.
 Remove cells from plasma and clot from serum before
shipment.
 2.4. Shipment of serological
specimens

 Don't inactivate serum or plasma before mailing.

 Keep the specimen and packing container in the
refrigerator until time of shipment.
 Shipment is requires several days preserve by
refrigeration in transit. First, freeze the specimen;
then pack and ship in a well-insulated container
with dry ice.
 2.5. Complement inactivation

 Complement is a group of non-immunoglobulin

plasma proteins that are sequentially activated by
Ag–Ab complexes (or directly by microbial
constituents) and cause irreversible damage to
membrane of cellular target
 2.5. Complement inactivation

 Complement molecules circulate in the blood in

an inactive form but activation of the first
complement component sets in motion a ripple
effect. As each component is activated, it acts in
turn on the next component in a precise
sequence called complement cascade.
 2.5. Complement inactivation

 Some tests need inactivated serum. Others do

not.
 Inactivation may be important since complement
promotes lysis of erythrocytes and can contribute
to false test results in tests using RBCs.
 Some complement components may also cause
false agglutination in some tests.
 2.5. Complement inactivation

 Complement components can be inactivated by of

three mechanism
 Spontaneous decay
 Enzymatic degradation of C4, C3 and C5
rapidly decay
 Stoichiometric inhibition
 2.5. Complement inactivation

 The complement in serum must be inactivated

usually by stoichiometric inhibition for most
serological testing.
 To inactivate complement, place tubes of serum
in hot water bath (56c) for 30min
 If the protein complement is not inactivated it will
promote lysis of the red cells and other types of
cells and can therefore produce invalid results
 2.5. Complement inactivation

 Complement is also known to interfere with

certain tests for syphilis.
 Serum samples to be tested more than 4 hours
after inactivation should be reheated at 560c for
10 minutes and allowed to cool to room
temperature
 2.6. Dilution

 Dilution is the act of making a weaker solution

from a strong solution.
 Adding a diluent such as water or saline, which
contains none of the material being diluted, is
used to do this.
 Dilutions are usually expressed as 1 unit of the
final solution.
 2.6. Dilution
Dilution techniques
 Dilutions can be used in the laboratory to change
the concentration of the body fluids, such as serum
so that it is consistent with the range of an assay.
 Making dilutions can also be necessary to prepare
reagents and standards.
 Dilution has two parts: diluents and solute.
 2.6. Dilution

 A dilution involves adding of a substance, the

diluent to other substances, the solute.
 Dilutions show the relative amount of the solute
in the dilute solution.
 It is an indicator of concentration, not volume.
 2.6. Dilution
 Expression of dilution
Dilution is usually expressed as:
a to b
a:b
a/b
Whereas;
a, is the volume of the original materials
that was diluted e. g. serum
(solute)
b, is the total volume to which it was
diluted.
It contains solute a and diluent b.
 2.6. Dilution
 The dilution factor is the inverse of the dilution
statement. For a 1:10 dilution, the dilution factor is
10. For a : b dilution the dilution factor is b.
 2.6. Dilution

Technique
 Two liquids of very different compositions (density,
or surface tension) is required
 An exact volume of concentrated solute is added to
a calibrated flask or container, and then diluent is
added to the required volume.
 Adequate mixing must take place to ensure
homogeneity
 2.6. Dilution
E.g.,
if you want to prepare 1:10 dilution
 Take 1 ml solute
1st

 Take 9 ml solvent
2nd

 Then mix
 2.6. Dilution

Method
 Add 1-ml solute into10 ml graduated volumetric
flask and then add water up to the 10-ml mark or
graduation of the flask.
 2.6. Dilution
 When a solution is diluted with water, its concentration is
decreased and its volume is increased. But the total
amount of solute remains constant.

 Mathematical expressions of the dilutions are;

CiVi = CfVf Where, Ci is initial concentration
Vi is initial volume.
Cf final concentration
Vf is final volume.
 2.6. Dilution

2.6.1. Serial dilutions

 The systematic re-dilution of a fluid number of
times is called a "serial dilution".

 Serial dilutions are most commonly employed in

serological procedure to obtain quantitative
estimations of antigen or antibody content.
 2.6. Dilution

 Serial dilutions are a unique type of dilution

techniques.

 In serial dilution, all dilutions, except the 1st are

prepared from the previous dilution and all
dilutions made after the initial dilution are the
same.
 2.6. Dilution

 Serial dilutions are used to prepare sets of

standard solutions and are also used to prepare
patient's samples to analyze components that can
exist over a wide concentration range, such as
antibody titers.
 Serial dilutions must be prepared with care as
errors can be compounded during the serial
technique.
 2.6. Dilution

0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5 0.5

0.5 0.5 0.5 0.5

0.9 0.5 0.5 0.5 0.5 0.5

initial
Tube 1 2 3 4 5 6 7 8 9 10

Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120

Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120
 2.6. Dilution
An example of the serial dilution is as follows: -

 Into each of ten test tubes is measured 0.5 ml of

saline 1/2 ml of serum is placed in the 1st tube and
mixed.
 Since there is 0.5 ml of serum in a total volume of
1.0 ml; a 0.5:1 or a 1:2 dilution exists in the first
tube.
 2.6. Dilution
 Now, 0.5 ml of this solution is removed and mixed
with the 0.5 ml of saline in the 2nd tube; this gives
another 1:2 dilution, but since the 0.5 ml of solution
put into the 2nd tube is already a 1:2 dilution of the
serum, the dilution of serum in the 2nd tube is one
half that of the 1st tube or 1/2 of ½ =1/4 or 1:4.

 This and, by applying the above reasoning, the

dilutions of serum are found to be (1/2)10 = 1/1024
or 1: 1024 in the 10th tube.
 2.6. Dilution

Class work

Q. Calculate the volume of serum

in 2nd tube and next respective
tubes?
 2.6. Dilution
The titer
 The titer (French; Titer = standard) may be defined
as the quantity of a substance required to produce a
reaction with a given volume of another substances
or the amount of one substances required to
correspond with a given amount of another
substances.
 It is also defined as the reciprocal of the highest
dilution showing a positive reaction (agglutination,
hemolysis, etc,).
 2.6. Dilution

 In clinical serology titer is usually referred to as a

measure of the number of antibody molecules per
unit volume of the original serum and gives and
indication of the antibody concentration in the
patient’s serum.
 2.6. Dilution
 An antibody titer of serum is the highest dilution
of serum that will give a reaction with antigen.
 For example, if the last tube showing a ratio
contains 1ml. Volume, and the serum in this
tube is 1 part in a total of 640 parts, the titer
is 640 units/ml of serum, or 1:640.

 Generally a maximum dilution of a specific

antibody that gives a measurable reaction with a
specific antigen; usually expressed as the respect
of that dilution is called titer
Review questions

Try the following problems

 For ASO titer, tube 1 contains 0.8ml 0f saline,
tubes 2 to 5 contain 0.5ml of saline; 0.2ml of
serum is added to tube 1, and serial dilutions
using 0.5ml are carried out in the remaining
tubes. What is the dilution in each tube?
 Explain the shipment of specimen and
complement inactivation.
Reference

1. Tizard. Immunology an introduction,4th

edition ,Saunders publishing,1994

2. Naville J. Bryant Laboratory Immunology and

Serology 3rd edition. Serological services
Ltd.Toronto,Ontario,Canada,1992

3. Mary Louise .Immunology and Serology in

Laboratory medicine 3rd edition