Chapter 16

Specimen collection and preparation

for serological tests

7/6/2023 By Dejene G (Medical Immunologist) 1

 Learning Objective
At the end of this chapter, the students should
be able to:
1. Collect, prepare , preserve and ship
serological specimens
2. Run complement inactivation procedure and
state its importance
3. Run serial dilution, determine end point and
titer

7/6/2023 By Dejene G (Medical Immunologist) 2

 Outlines
Safety, specimen collection, preparation,
preservation and Shipment of serological
specimens

Dilution (Serial dilutions and Determination of

end point and titer) and Complement
inactivation

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 Specimen collection
Specimens include:
Whole blood
Serum Can be collected by the laboratory
Plasma personnel

Urine
Peritonial fluid
Pericardial fluid Should be collected
by a physician or
Plurial fluid trained nurse

Cerebrospinal fluid

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 Guideline in blood collection
Blood represents a large percentage of the total
specimens used in laboratory determinations
There are three general sources of blood for
clinical laboratory tests:-
Venous blood
Peripheral, or capillary blood
Arterial blood ( rare cases like in blood gas
analysis)

7/6/2023 By Dejene G (Medical Immunologist) 5

 Guideline in blood collection …….
1. Collection sites:
a. Capillary blood
o Ring/middle finger - adults & children – half way b/n
centre & ball (vascular & fleshy)
o Heel - infant < 3 months – side of heel
o Big toe – older infants (> 3 months) – side of great toe
o Ear lobe – today is not routinely used as a blood
collection site Big toe
Heel

Earloabe

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 Guideline in blood…….
b. Collection of venous blood
– Venous blood is collected from a vein.
– Veins in the forearm are most commonly used for vein
puncture.
– Veins in the wrist or ankle may also be used for vein
puncture (if the forearm site is not available).
• The three main veins in the forearm are
– cephalic
– median
– basilic.
The median cubital vein is usually chosen for vein puncture. (Because it
is larger, closer to the surface, easier to enter).
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 Veins of fore arm

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 Guideline in blood…….
Equipment and Supplies
 Gloves
 Eye goggles
 Needle
 Holder
 Tubes
 Gauze
 Alcohol Pad
 Band Aid
 Sharps Container
 Tourniquet

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Procedure for serum/plasma separations
 Collect 2-3ml of venous blood from a patient using a
sterile syringe and needle or vacationer system
 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.
 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
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 Normal Plasma and hemolysed serum

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 Serum Vs plasma

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Procedure for serum/plasma separations…
 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
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 Preservation of serum/plasma
Serum should be stored at 4–8°C until
shipment takes place, or for max. 7 days

When kept for longer periods, serum samples

should be frozen at −20°C or lower and
transported to the testing laboratory on frozen
ice packs

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 Preservation of serum/plasma…
If there is a delay in centrifuging the sample
for any reason, keep the sample at room temp.
and don’t refrigerate, which may lead to
hemolysis
If there is a delay in the test, then keep serum
or plasma at 4 °C
If the temperature of 4 °C is not suitable for
the special test, keep the serum at -20 °C

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 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

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 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
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Shipment of serological specimens…

Keep the specimen and packing container in

the refrigerator until time of shipment
Shipment requires several days preserve by
refrigeration in transit
First, freeze the specimen; then pack and ship
in a well-insulated container with dry ice

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 Complement inactivations
 Some tests need inactivated serum, others do not

 May be important since complement promotes lysis

of erythrocytes and can contribute to false test results
in tests using RBCs

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 Complement in activations…
Complement components can be inactivated
by of three mechanism
–Spontaneous decay

–Enzymatic degradation of C4, C3 and

C5 rapidly decay

–Stoichiometric inhibition

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 Complement in activations…
 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

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 Complement in activations…
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

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 Dilutions
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

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 Dilutions…
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.
 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
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 Dilutions…
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 a and diluent b.
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 Dilutions…

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.

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 Dilutions…
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

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 Di lDilutions… …..cont
E.g.
If you want to prepare 1:10 dilution
 Take 1 ml solute
1st

 Take 9 ml solvent
2nd

 Then mix

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 DilutdiDilutions…ons …..cont
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

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 Dilutions…
 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.
 Dilution increases volume but does not change the solute

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 Dilutions…
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

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 Dilutions…

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

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 Dilutions…
 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

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 Dilutions…
…..cont
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

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 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.
 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.

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 Dilutions…
Procedure of serial dilution determining the dilutions:
1. Add 0.9 ml of saline to the first tube using a 1ml pipette.
2. Add 0.5 ml of saline to the remaining 6 tubes.
3. Add 0.1 ml serum to the first tube; wipe of the pipette tip.
4. Draw up 0.5ml and allow to drain into tube 2
5. Add 0.5 ml from the first tube to the second tube and Mix it
6. Transfer 0.5 ml from second tube to third tube
7. Continue transferring 0.5 ml until the last tube is reached.
8. Remove 0.5 ml from the last tube and discard it.
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Exercise for simple and serial dilutions
 2 mL of a 1:20 dilution is needed to run a specific serological test. How
much serum and how much diluent are needed to make this dilution?

 A 1:5 dilution of patient serum is necessary to run a serological test. There is 0.1
mL of serum that can be used. What amount of diluents is necessary to make this
dilution using all of the serum?

 How much diluent needs to be added to 0.2 ml of serum

to make a 1:20 dilution?

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 The titer
 The titer (Fr. 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,).
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 The titer…
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.

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 The titer…
 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

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Review question
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.

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