Activity NO. 3 - COLLECTION AND PREPARATION OF SEROLOGICAL SPECIMENS

Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.
: (083) 228-2019, Fax: (083) 228-4015, Email: st.alexiuscollege@yahoo.com
ACTIVITY NO. 3
COLLECTION AND PREPARATION OF
SEROLOGICAL SPECIMENS
I. INTRODUCTION
Blood specimens should be collected before meal to avoid the presence of chyle, an emulsion of
fat globules that often appears in serum during digestion. Blood should be collected by venipuncture. If
syringes and needles are used care must be taken to allow the blood to run gently into the clean collecting
tube to avoid rupturing of cells. The vacutainer system consists of a needle holder and glass vacuum tube
instead of the syringe barrel and plunger. Once the vein is punctured, and the vacutainer tube is
appropriately in contact with the needle, the required quantity of blood flows automatically into the
vacutainers tube so that the need to pull the plunger out is obviated. It offers leak proof tubes and allows
standardization of specimen quality. It is simple, quicker, cleaner and safer to use. Many procedures done
in the laboratory require demonstration of in vitro antigen and antibody reactions. Thus, preparing plasma
and serum as antibody source is needed.
II. OBJECTIVES
At the end of this experiment, the students will be able to:

1. Identify the steps of preparing samples used for serological procedures
2. Manipulate appropriately the equipment used for preparation of samples used for serology
3. Describe the characteristics of samples that are accepted for serological tests
III. MATERIALS
Centrifuge
Aspirator bulb
Pasteur pipette
Venipuncture set
Test tubes
Test tube rack
Parafilm
Evacuated tubes (red and lavender top)
IV. PROCEDURE
1. Whole Blood Preparation
a. Draw a sufficient amount of blood corresponding to the volume of the additive/anticoagulant
b. To achieve an optimum ratio of blood to additive/anticoagulant, the volume of blood should
(1) fill the tube to the line indicated on the evacuated tube label, or
(2) be according to the appropriate ratio of the sample to the additive/anticoagulant
c. Mix evacuated tube immediately after collection, before clotting can occur, gently by inversion
according to the type of additive/anticoagulant
d. Label the specimen container with patient information including the date and time of collection
MT 309 Immunology and Serology Laboratory Manual Page 1 of 10

Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.: (083) 228-2019, Fax: (083) 228-4015, Email: st.alexiuscollege@yahoo.com
2. Plasma Preparation
a. Draw a sufficient amount of blood into an evacuated tube with the required anticoagulant to
yield the plasma volume required by the test.
b. Gently mix immediately after collection by appropriate number of inversions according to the
type of anticoagulant used. Avoid hemolysis of the specimen during collection and mixing.
c. Place the specimen in a rack at room temperature and centrifuge within 2 hours of collection. Do
not refrigerate the sample until the plasma is separated from the cells.
d. Samples should undergo centrifugation immediately. Centrifuge anticoagulated blood for 5 – 10
minutes at 2,500 rpm or refer to speed and duration recommended by manufacturer of the
evacuated tubes. Do not use brake to stop centrifuge.
e. After centrifugation, three layers are formed: (from top to bottom) plasma, buffy coat, red blood
cells. The plasma should appear clear and no pink to red tinge is manifested
f. Carefully aspirate or collect the supernatant (plasma) with a Pasteur pipette and transfer to a test
tube. Take care not to disrupt the packed red blood cell layer or transfer any cells.
g. Label the sample container
3. Serum Preparation
a. Draw a sufficient amount of blood to yield the serum volume required by the test.
b. Allow the tube containing venous blood to stand in an upright position at room temperature for
20 – 30 minutes (no longer than 60 min) to allow complete clotting to occur. Centrifuging
specimens before coagulation is complete causes fibrin clots to form in the serum.
(1) If using a clot activator tube, invert carefully 5 – 6 times to mix clot activator and blood.
(2) If using a serum separator tube (SST), mix tubes well by carefully inverting the collection
tube 8 – 10 times for clot formation to occur.
Avoid hemolysis of the specimen during collection and mixing.
c. After 20 – 30 minutes, check for complete clotting by inclining the tube gently and when blood
does not ooze from the clot, coagulation time is reached.
d. Rim or ring the sides of the clot by thrusting the applicator stick to a depth of 1 cm and perform
full turn at the sides of the clot gently. Do this once.
Note: If the clot clings to the stick, dislodge the clot from the stick carefully and
include the clot for centrifugation. Do not discard the clot.
e. Centrifuge clotted blood for 5 – 10 minutes at 2,500 rpm or refer to speed and duration
recommended by manufacturer of the evacuated tubes. Do not use brake to stop centrifuge.
Note: Centrifuge within 2 hours of collection. Do not refrigerate the sample until
the serum is separated from the cells.
f. After centrifugation, check the supernatant (serum) and carefully aspirate it with a Pasteur
pipette and transfer to a test tube. Take care not to disrupt the packed red blood cell layer or
transfer any cells.
g. Inspect serum for turbidity. Turbid samples should be re-centrifuged to remove remaining
insoluble matter. Serum should appear clear and no pink to red tinge is manifested
h. Label the sample container

PERFORMANCE ASSESSMENT SHEET

ACTIVITY NO. 3
SAMPLE PREPARATION FOR SEROLOGIC PROCEDURES
Name:__________________________________________ Date: __________________
PROCEDURE RATING COMMENTS

You must:
1. Wash hands
2. Wear proper Personal Protective Equipment (PPE).
3. Prepare all the materials and reagents needed.
Whole Blood Preparation
1. Draw a sufficient amount of blood corresponding to the
volume of the additive/anticoagulant
2. To achieve an optimum ratio of blood to
additive/anticoagulant, the volume of blood should
(a) fill the tube to the line indicated on the evacuated
tube label, or
(b) be according to the appropriate ratio of the
sample to the additive/anticoagulant
3. Mix evacuated tube immediately after collection, before
clotting can occur, gently by inversion according to the
type of additive/anticoagulant
4. Label the specimen container with patient information
including the date and time of collection
Plasma Preparation
1. Draw a sufficient amount of blood into an evacuated
tube with the required anticoagulant to yield the plasma
volume required by the test.
2. Gently mix immediately after collection by appropriate
number of inversions according to the type of
anticoagulant used. Avoid hemolysis of the specimen
during collection and mixing.
3. Place the specimen in a rack at room temperature and
centrifuge within 2 hours of collection. Do not
refrigerate the sample until the plasma is separated from
the cells.
4. Samples should undergo centrifugation immediately.
Centrifuge anticoagulated blood for 5 – 10 minutes at
2,500 rpm or refer to speed and duration recommended
by manufacturer of the evacuated tubes. Do not use
brake to stop centrifuge.

5. After centrifugation, three layers are formed: (from top

to bottom) plasma, buffy coat, red blood cells. The
plasma should appear clear and no pink to red tinge is
manifested
6. Carefully aspirate or collect the supernatant (plasma)
with a Pasteur pipette and transfer to a test tube. Take
care not to disrupt the packed red blood cell layer or
transfer any cells.
7. Label the sample container
Serum Preparation
1. Draw a sufficient amount of blood to yield the serum
volume required by the test.
2. Allow the tube containing venous blood to stand in an
upright position at room temperature for 20 – 30
minutes (no longer than 60 min) to allow complete
clotting to occur. Centrifuging specimens before
coagulation is complete causes fibrin clots to form in
the serum.
(a) If using a clot activator tube, invert carefully 5 –
6 times to mix clot activator and blood.
(b) If using a serum separator tube (SST), mix tubes
well by carefully inverting the collection tube 8 –
10 times for clot formation to occur.
Avoid hemolysis of the specimen during
collection and mixing.
3. After 20 – 30 minutes, check for complete clotting by
inclining the tube gently and when blood does not ooze
from the clot, coagulation time is reached.
4. Rim or ring the sides of the clot by thrusting the
applicator stick to a depth of 1 cm and perform full turn
at the sides of the clot gently. Do this once.
Note: If the clot clings to the stick, dislodge the
clot from the stick carefully and include the clot
for centrifugation. Do not discard the clot.
5. Centrifuge clotted blood for 5 – 10 minutes at 2,500
rpm or refer to speed and duration recommended by
manufacturer of the evacuated tubes. Do not use brake
to stop centrifuge.
Note: Centrifuge within 2 hours of collection.
Do not refrigerate the sample until the serum is
separated from the cells.

6. After centrifugation, check the supernatant (serum) and

carefully aspirate it with a Pasteur pipette and transfer
to a test tube. Take care not to disrupt the packed red
blood cell layer or transfer any cells.
7. Inspect serum for turbidity. Turbid samples should be
re-centrifuged to remove remaining insoluble matter.
Serum should appear clear and no pink to red tinge is
manifested
8. Label the sample container
RATING:
4- Excellent
3-Very Satisfactory
2- Satisfactory
1-Poor
0-Not Done

REPORT SHEET
ACTIVITY NO. 3
COLLECTION AND PREPARATION OF
SEROLOGICAL SPECIMENS
Name: ______________________________________________Rating: _____________________

Date Performed: ______________________________________Date Submitted: ______________
--------------------------------------------------------------------------------------------------------------------------
ILLUSTRATIONS:
1. Illustrate and label accordingly the important steps in whole blood preparation

2. Illustrate and label accordingly the important steps in plasma preparation.

3. Illustrate and label accordingly the important steps in serum preparation

REVIEW QUESTIONS:
1. List the possible serological specimens.
2. Explain how serum and plasma are collected, prepared and preserved for serologic tests
4. Between plasma and serum, which is the preferred sample for serologic testing and why?

5. Why is complete clotting necessary during serum preparation?
6. Describe and discuss why the following samples are NOT used in serologic testing
a. Hemolyzed sample
b. Lipemic sample
c. Icteric sample

Activity NO. 3 - COLLECTION AND PREPARATION OF SEROLOGICAL SPECIMENS

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Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.

: (083) 228-2019, Fax: (083) 228-4015, Email: st.alexiuscollege@yahoo.com

At the end of this experiment, the students will be able to:

MT 309 Immunology and Serology Laboratory Manual Page 1 of 10

MT 309 Immunology and Serology Laboratory Manual Page 2 of 10

PERFORMANCE ASSESSMENT SHEET

Name:________________________ Date:

PROCEDURE RATING COMMENTS

MT 309 Immunology and Serology Laboratory Manual Page 3 of 10

5. After centrifugation, three layers are formed: (from top

MT 309 Immunology and Serology Laboratory Manual Page 4 of 10

6. After centrifugation, check the supernatant (serum) and

MT 309 Immunology and Serology Laboratory Manual Page 5 of 10

Name: __________________________Rating: _

MT 309 Immunology and Serology Laboratory Manual Page 6 of 10

2. Illustrate and label accordingly the important steps in plasma preparation.

MT 309 Immunology and Serology Laboratory Manual Page 7 of 10

3. Illustrate and label accordingly the important steps in serum preparation

MT 309 Immunology and Serology Laboratory Manual Page 8 of 10

MT 309 Immunology and Serology Laboratory Manual Page 9 of 10

5. Why is complete clotting necessary during serum preparation?

MT 309 Immunology and Serology Laboratory Manual Page 10 of 10

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