Visual Observations

of Serum Tubes
and Serum
Separator Tubes

This document provides illustrations of certain performance issues seen in serum and serum separator (gel) tubes.
These phenomena are not specific to any particular tube manufacturer. Their causes are multi-faceted and influenced
by patient factors, pre-analytical processing and handling, and analytical factors. The guide below explains the impact
of these phenomena and the steps to minimize occurrences.

Fibrin
What is it?
Fibrin consists of a three dimensional
network of protein strands which can entrap
red blood cells, white blood cells, and
platelets. Fibrin mass can range from a small
spot on the gel to a fully gelatinous serum
sample depending on the extent of fibrin
formation during or post centrifugation.
Presence of a fibrin strand or mass in the
serum indicates that the sample was not
completely clotted prior to centrifugation;
therefore, fibrin formation continued during
or post centrifugation. Fibrin can be present
due to poor mixing, short clot time, or
patient condition.
Aspiration of fibrin by sampling probes can
lead to physical obstruction of the probe Fibrin Strand Fibrin Mass
and/or insufficient sampled volume. This
can lead to instrument downtime, failure to Prevention
provide test results, or incorrect test results.1
A large fibrin mass can also reduce serum • Properly mix tubes to facilitate dispersion of the silica coating into the blood which assists
yield and/or interfere with the gel barrier the clotting process.
formation. • Allow adequate clotting time. Samples from patients with an intact clotting process
1
(“normal”) require 30-60 minutes to clot. Certain patient conditions may require longer
Data on file.
to clot. Refer to product insert for recommended clotting times by tube type.

Gel Globules/Oil Droplets

What is it?
Oil droplets are small spherical drops of
fluid resembling a fat droplet and may
sometimes be seen floating in the serum
or on the surface of the serum collected
in gel separator tubes. In some instances,
these droplets may appear as a uniform film
on the surface of the serum. Oil droplets
or an “oily film” may arise from material
expressed from the gel resin. It may also
be seen in certain patient conditions or as
a result of some therapies in specimens
collected in both gel and non-gel tubes.
Gel globules are similar in nature to oil
droplets but possess the properties of
the gel material. It is not possible to Gel Globules
differentiate between gel globules or oil
droplets by visual observation alone. They
are a potential problem with all types of Prevention
gel separator materials. Gel globules or oil
droplets can coat or occlude instrument • Use product within the shelf life.
probes, leading to erroneous results or • Store tubes between 4-25oC. Temperature fluctuations may impact gel performance.
instrument down time. Exposure to high temperatures prior to use and during centrifugation may lead to the
formation of gel globules or oil droplets in the serum.
Red Blood Cells on the Tube Wall
(Red Blood Cell Hangup)

What is it?
Presence of red blood cells on the tube
wall is due to the adhesion of red blood
cells to the inner tube wall in or above
the serum/plasma compartment after
centrifugation. This is referred to as
“red blood cell hangup”. The degree of
“hangup” is measured by the approximate
percentage of wall coverage as well as the
thickness of the coverage. The coverage
Red Blood Cells on the Tube Wall (L-R)
may range from a thin film or spot of red
cells to a thick coating which may also Acceptable Trace Moderate Gross Failure to
contain fibrin. Specimen Separate
A thin film or spot of red blood cells on
the tube wall is generally considered a
cosmetic issue.2 Prevention
• Properly mix tubes to facilitate dispersion of the silica coating.
2
• Use tubes within shelf life.
Data on file.
• Store tubes between 4-25oC.

Gel Smearing
What is it?
Gel smearing occurs during centrifugation
when the gel initially moves to a position
slightly higher in the tube than its final position
at the interface between the serum and clot.
As the separation continues and the gel moves
downward from this “high” position to its
“final” position, a thin “trail” of gel (or smear)
may be deposited on the wall of the tube.
Gel smearing is a normal observation in serum
gel tubes and can occur in any serum gel
product. It is generally a cosmetic issue only.
Gel Smearing Gel Smearing
However, if instrument analyzer probes enter
the tube at a point very close to the wall, there
is potential for the probe to aspirate the gel Prevention
smear and become partially or totally occluded. Use recommended centrifugation conditions for the tube and follow recommended
This can also occur if the tubes or probes are centrifuge maintenance schedules.
not properly aligned.

The following factors may contribute to increased gel smearing.

• Centrifugation temperatures exceeding 25°C.
• Very high rcf (relative centrifugal force or g-force) values.
• Excessive centrifuge “ramp-up” (rotor acceleration) or braking.
• Improper alignment of the tube and sample probes. Refer to instrument
manufacturer’s recommendations for tube rack and probe maintenance.

To learn about BD Vacutainer® specimen collection

products, educational materials, or services offered by
BD Diagnostics – Preanalytical Systems, please
contact your local BD Sales Consultant today.

e-mail: vacutainer_techservices@bd.com
Or visit us anytime online at www.bd.com/vacutainer
BD Global Technical Services at 1.800.631.0174 (USA only)

BD Diagnostics
Preanalytical Systems
1 Becton Drive
Franklin Lakes, NJ 07417
BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2007 BD www.bd.com/vacutainer
08/07 VS7864-G
TechTalk Volume 4, No. 2
Author: Lena Arzoumanian
November 2005

BD Global Technical Services receives many questions about BD products.

To address these questions, we have developed a periodic news bulletin called “Tech Talk.”

Q. What is the importance of A. The preparation of blood samples is a critical first step in the testing process. By understanding the
components of the product and adhering to the following processing instructions, the facility may
properly processing a
dramatically IMPROVE SPECIMEN INTEGRITY, resulting in a QUALITY SPECIMEN, a QUALITY
BD Vacutainer® SST™ Tube? RESULT, and ultimately assisting the doctor in providing QUALITY TREATMENT to patients.

Components of the BD Vacutainer ® SST ™ Tube

TUBE TYPES In general, glass is a natural clotting agent. The blood will clot due to contact activation causing
Plus Plastic – Polyethylene Terephthalate (PET); Glass – Soda Lime Glass the initiation of the clotting mechanism.
Plastic tubes require the clot activator, which helps accelerate the blood clotting mechanism.
Silicone and clot activator are applied to the interior surface of the tube.
CLOSURE TYPES • A silicone coating on the walls of most serum tubes reduces the adherence of red cells
BD Hemogard™ Closure; Conventional Stopper to the tube wall.1, 2
• The clot activator helps accelerate the blood clotting mechanism.1
ADDITIVES • The density of the polymer gel causes it to move upward during centrifugation to the
Clot Activator – Micronized Silica Particles; Barrier – Polymer Gel; Silicone Coating serum-clot interface, where it forms a barrier separating serum from the clot.1

The Importance of Proper Handling and Processing of the BD Vacutainer ® SST ™ Tube
PRECENTRIFUGATION
FILL tubes to the stated draw volume to ensure the proper TUBE STORAGE
4
5
blood-to-additive ratio. 6
7
8 • Tubes should be stored at 4-25°C (39-77°F).
9
10

11
Allow the tube to fill until the vacuum is exhausted and • Tubes should not be used beyond the designated expiration date.
12

13
14

15

blood flow ceases.3 • The expiration date is assigned to ensure adequate vacuum, barrier,
16

17
18

19

Most BD SST™ tubes have a 12-month shelf life. or additive performance.

20

21
22

23
24 °C

25

MIX the tube CLOT for 30 minutes in a vertical position in a tube rack. Observe a dense clot.
by 5 complete
• The recommended 30-minute minimum clotting time for the BD SST™ tube is based upon an intact clotting process.
inversions.
• Insufficient clotting (short clotting time) can result in the formation of fibrin. This fibrin formation may interfere
• Mixing is critical with barrier formation.
x5 to achieving
• Samples from certain populations of patients with impaired coagulation may require longer than 30 minutes to
appropriate
clot in a BD SST™ tube:
clotting times
and clot – Blood from patients on anticoagulant therapy (e.g., Coumadin®) may require longer clotting time.
formation. – Blood from patients on high doses of heparin may not clot at all.
• Mixing facilitates dispersion of the
– Certain diseases may require longer blood clotting times (e.g., liver disease).
silica into the blood, assisting the
clotting process. – Multiple Myeloma—the isolated myeloma globulin inhibits all three stages of fibrin formation: the proteolytic
• Inadequate mixing may result in action of thrombin on fibrinogen, the aggregation of fibrin monomers, and the stabilization of fibrin by
incomplete clotting. 30 minutes cross-linkages in the gamma and alpha chains.4
It is recommended that serum be physically separated from contact with cells as soon as possible with a maximum time limit of 2 hours
from the time of collection, unless conclusive evidence indicates that longer contact times do not contribute to error of the results.1, 8

CENTRIFUGATION Centrifugation recommendations:

The gel exhibits thixotropic properties (such that it is semi-solid under static conditions Product RCF (g force)
Time (min) Time (min)
and becomes less viscous when a force is applied), enabling it to flow during centrifugation. Swing-Bucket Fixed-Angle Bucket
Separator gels are designed with a specific density that falls between those of the serum and BD SST™ Glass Tube 1000-1300 10 15
cells, thus determining the location of the interface.5
BD SST Plus - 13 mm
™
1100-1300 10 15
Complete and adequate barrier formation is time, temperature and g-force dependent. BD SST™ Plus - 16 mm 1000-1300 10 15
Uniformity of the barrier is time dependent. BD SST™ Transport Tubes 1100-1300 15 15

• An incomplete barrier could result from shortened centrifugation times.

Conversion of RCF to RPM (radius in inches)
• For a horizontal (swing-bucket) centrifuge, the recommended spin time is 10 minutes.
Radius Speed–RPM Speed–RPM Radius Speed–RPM Speed–RPM
• For a fixed-angle centrifuge, the recommended spin time is 15 minutes. (Inches) Min. Max. (Inches) Min. Max.
A minimum g-force is required to get the gel moving, thus the recommendation is 1000 g.1, 5 3 3500 4000 7 2300 2600
• During the centrifugation process, centrifugal forces are applied to the gel in the tube. 4 3000 3500 8 2100 2400
At comparable g-force settings, the horizontal centrifuge is more efficient at gel barrier 5 2700 3100 9 2000 2300
formation than a fixed angle centrifuge, due to a higher axial force setting on the gel.
6 2500 2800 10 1900 2200
• The quality of the barrier formed from fixed-angle centrifugation also
depends upon the angle of the centrifuge head. Barriers formed in Conversion of RCF to RPM (radius in centimeters)
fixed-angle centrifuges contain a bias angle relative to the angle of
the head. These barriers are typically thinner than horizontal barriers, Radius Speed–RPM Speed–RPM Radius Speed–RPM Speed–RPM
(cm) Min. Max. (cm) Min. Max.
because the gel must cover a greater cross-sectional area in the tube.
8 3300 4000 16 2300 2600
The flow properties of the barrier material are temperature dependent.1
10 3000 3400 18 2200 2500
• Gel flow may be impeded if chilled before or during centrifugation.
12 2700 3100 20 2100 2400
To optimize flow and prevent heating during centrifugation,
set refrigerated centrifuges to 25°C (77°F).1 14 2500 2900 25 1900 2100
10 or 15 minutes
Rotor Axis Radius is the rotational radius of the
centrifuge as measured from the center of
Rotating Radius Rotating Radius the rotor head to the outside bottom of the
Angle Rotors Horizontal Rotors
rotor bucket (when the rotor bucket is held
at 180 degrees).1, 8

POSTCENTRIFUGATION Analytes from cellular leakage/exchange, accentuated by clot retraction, will then be
The specimen in the original tube should be centrifuged one time. Tubes should not be centrifuged into the serum being used for testing. If recentrifugation is required for
recentrifuged once the barrier is formed. A potential for inaccurate test results is possible. improved serum quality, then aspirate serum into a properly labeled clean test tube.

References:
1. BD Vacutainer® Evacuated Blood Collection System product insert available from www.bd.com/vacutainer/productinserts
2. Bush V, Skobe C, Dubrowny N, Cohen R, Pando S, Mohammad F. Assessment of Cellular Contamination of Serum for Routine Chemistry Analytes [abstract]. Clin Chem 1997
3. CLSI, Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard – 5th ed. H3-A5, Vol. 23 No. 32.
4. Soria J, Soria C, Samama M, Fine JM, Bousser J. Analysis of a fibrin formation abnormality in a case of multiple myeloma. Scand J Haematol. 1975; 15(3): 207-218
5. Fu CL, Cohen R, Losado R, Bush V. Cellular sedimentaion and barrier formation under centrifugal force in blood collection tubes. Lab Med. 2001; 32: 588-592
6. Burtis CA, Ashwood ER. Tietz Fundamentals of Clinical Chemistry. 5th ed. Bordez BG, editor. Philadelphia (PA): W.B. Saunders; 2001: 496-497
7. Hira K, Ohtani Y, Rahman M, Noguchi Y, Shimbo T, Fukui T. Pseudohypokalaemia caused by recentrifugation of blood samples after storage in gel separator tubes. Ann Clin Biochem.
2001 July; 38 (Pt 4): 386-390
8. CLSI, Procedures for the handling and processing of blood specimens; approved guideline. 3rd ed. H18-A3, Vol. 24 No. 38

Please call BD Global Technical Services for clinical support material. BD Diagnostics
Preanalytical Systems
BD Global Technical Services: 1.800.631.0174 BD Customer Service: 1.888.237.2762 1 Becton Drive
Coumadin is a registered trademark of Bristol-Myers Squibb Pharma Company. Franklin Lakes, NJ 07417
BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2006 BD. 11/05 VS7436-1 www.bd.com/vacutainer