Heparin Plasma Testing

in Clinical Chemistry

This technical article provides an in-depth discussion of the

following topics, including a review of the scientific literature:
• What is Heparin?
• Serum vs. Plasma
• Factors Influencing Plasma Specimen Quality
• Analyte Stability
• Instrument/Assay Considerations
 Introduction
Heparin plasma is the specimen of choice in clinical
chemistry testing for an increasing number of
laboratories. However, heparin plasma represents a
more complex specimen than conventional serum,
with unique considerations that may impact specimen
quality and test results. In recognition of these
considerations, the following article has been
created to provide laboratorians with an in-depth
discussion and review of heparin plasma. This article
discusses the technical factors that influence the
performance of heparin-plasma blood collection tubes
from collection through storage, with reference to the
scientific literature on heparin-plasma specimens.
A good practice guide is also included.
➤ Refer to page 12 for a list of specimen handling and 
processing steps to ensure high-quality heparin plasma.
What is Heparin?
Heparin is a heterogeneous mixture of straight-chain
anionic mucopolysaccharides with a molecular weight
distribution of roughly 3000 to 30000 Da. It belongs
to the glycosaminoglycan family naturally present in
several mammalian organs and tissues. Heparin is
strongly acidic due to the presence of covalently
linked sulfate and carboxylic acid groups. A variety
of heparin salts are available in which the acidic
protons are partially replaced by another positive ion.
CH2OSO3̄ COO¯ CH2OR
O O O O
COO¯
O O
OH O OH OSO3̄ O OH
NHR OH NHSO3̄ OSO3̄
R=H or SO3̄ R =SO3̄ or COCH3
Figure 1. Chemical structure of heparin1
Heparin is commonly used in several clinical
applications due to its properties as an anticoagulant.
It acts primarily through a complex that it forms
with antithrombin III. This complex inhibits
thrombin, which in turn prevents the formation
of fibrin from fibrinogen.
Heparin salts are used as anticoagulants in blood
collection devices to inhibit clotting of whole blood.
The most common salts for this application are
lithium heparin and sodium heparin, which are dry,
white hygroscopic powders. Electrolyte-balanced
lithium heparin is also used as an anticoagulant in
arterial blood gas syringes. Recommended heparin
activities for venous blood collection tubes of
10–30 USP units/mL blood or 12–30 IU/mL have
been published. 2,3
For plasma-based testing in clinical chemistry, heparin
salts are the preferred anticoagulants for most tests.
EDTA, citrate, and oxalate cannot be used for routine
chemistry testing since they bind calcium and have
commonly measured counterions (potassium, sodium).
Lithium heparin is used more often than sodium heparin
in chemistry testing to avoid interference with sodium
determinations. Sodium heparin is used more commonly
in immune cell function and other cellular testing.
This article will focus on the use of blood collection
tubes containing lithium heparin in plasma-based
clinical chemistry testing.
Serum vs. Plasma
Serum is the most commonly used type of specimen
in both routine and special chemistry testing. The wide
preference for serum can be attributed in large part to
the essentially cell-free nature of serum produced from
properly clotted blood specimens. This characteristic
allows serum that has been separated from the clot to
remain stable for extended periods of time, which is
CH2OSO3̄
especially desirable in settings where specimens are not
O analyzed soon after centrifugation.
O
OH O An additional reason for the wide use of serum may
NHSO3̄ be the historical development of assays in clinical
chemistry. Development and validation of assays
has long focused on serum-based methodologies due
to the ease of handling, manipulation, and storage
associated with the relatively clean matrix of serum.
Validation of methods for plasma specimens was
less common and limited to certain instrument
manufacturers and tests. However, trends in clinical
laboratory requirements and expectations over the
past decade have created increased interest in and
demand for the unique attributes of plasma specimens.
As a result, development and validation of assays for
both serum and plasma are now standard among
many instrument manufacturers. Two important
requirements—turnaround time and specimen
quality—are discussed in the following sections.

Turnaround Time Specimen Quality
Reducing test result turnaround time (TAT) is an Specimen quality has been another important factor
important performance improvement initiative for prompting some laboratories to switch to plasma.
many laboratories. Numerous approaches have been As noted above, serum specimens are subject to latent
taken to reduce TAT by shortening the time interval fibrin formation when clotting is inadequate. This can
between blood collection and testing. These include occur not only when insufficient time is allowed for
the use of pneumatic tube systems, the placement of clotting, but also with specimens from patients receiving
satellite/STAT laboratories, and/or the implementation anticoagulant or thrombolytic therapy. In both cases,
of point-of-care testing. Due to the clotting time coagulation may continue after centrifugation, with
necessary to produce a serum sample, many formation of fibrin in the serum ranging from thin
laboratories have also switched from serum to strands to large cloud-like masses. This in turn can lead
plasma to reduce TAT. Recommended clotting times to a variety of undesirable outcomes when serum is
for serum blood collection tubes generally range from sampled for testing. Aspiration of fibrin by sampling
30–60 minutes. The impact of this time requirement probes can lead to physical obstruction of the probe
depends on the specimen transport conditions in and/or insufficient sampled volume. Aspiration of
use. Where the time interval between collection and smaller fibrin “microclots” that do not cause probe
centrifugation is within this timeframe, transport blockage can result in gradual deposition of fibrin in
and clotting can occur in parallel, and use of plasma reaction pathways or chambers, and/or interference
tubes does not help improve TAT. However, when the with measurement systems or reagents. Latent fibrin
time interval between collection and centrifugation formation may also continue within the analyzer.
is shorter than the recommended clotting time, These various effects of fibrin can lead to instrument
laboratories are faced with the decision of whether to downtime, failure to provide test results, or erroneous
hold specimens to ensure complete clotting, or process test results.4-6 Instrument fibrin issues are often
and test specimens that may continue to clot and form incorrectly attributed to gel contamination.
fibrin in the serum (discussed further below).
Various approaches have been used to minimize the
Use of plasma allows laboratories to process and test
impact of fibrin formation in serum specimens.
specimens upon receipt, while avoiding latent fibrin
These include holding specimens to allow additional
formation due to incomplete clotting. A time-based
time for clotting, addition of agents to promote
flowchart for selection of serum vs. plasma is shown
clotting (e.g., thrombin) or neutralize heparin
in Figure 2.
(e.g., protamine), centrifugation at higher g-force/spin
time or recentrifugation, and/or visual inspection
and rimming to remove fibrin prior to analysis.
However, these approaches require user intervention,
increase TAT, and may not be recommended.
Time from Serum
Another approach is the use of analyzers with
YES
phlebotomy to or clot detection modules to help reduce time spent in
centrifugation Plasma the identification and resolution of fibrin clots.
> 30 min
While issues associated with fibrin formation can
be reduced, they can be difficult to eliminate,
especially in settings with patients on anticoagulant
NO
therapy and/or where specimens are ready for
processing well before recommended clotting
Are serum
times have elapsed. To help reduce these issues,
specimens held YES Use of some laboratories have switched to plasma
to ensure plasma can specimens. However, plasma specimens also have
minimum clot improve TAT
unique characteristics concerning specimen quality
time?
and integrity. These are discussed in more detail in
the next section.
NO
Figure 2. Serum vs. plasma
flowchart. NOTE – actual
Use of
recommended minimum
plasma can
clot times may differ from
reduce fibrin
the 30-minute example used


Clot Formation and Gel Movement
The formation of a physical clot in serum blood
collection tubes also leads to other differences between
serum and plasma specimens. Clot formation and
retraction typically result in a solid clot that does not
conform to the interior tube dimensions. As a result,
some serum is trapped between the clot and the tube
wall and is not readily accessible (Figure 3). When
blood is anticoagulated, the cellular components
compact downward in a relatively uniform manner
during centrifugation, and the yield of plasma
above the cells is generally greater than the yield of
serum above the clot in serum tubes. Plasma yield is
generally near 50% of blood draw but depends on
patient factors such as hematocrit.7,8 The clot also
has a significant influence on the movement of gel in
gel-containing blood collection tubes. When a clot is
present, the gel is forced to move upward around the
clot, against the tube wall, during centrifugation.
With anticoagulated blood, however, there is no
obstruction to upward gel movement, and the gel
moves up in pieces similar to a ‘lava lamp’ (Figure 4).
Hence, the time required for the gel to complete its
upward movement to form a thick barrier is generally
shorter with plasma tubes than with serum tubes Figure 4. Gel movement (simulated) in serum (top) and
(under the same centrifugation conditions).9 Depending plasma (bottom) tube during centrifugation
on the centrifugation conditions used, this can
result in more reproducible gel barrier formation in
plasma tubes. Rapid gel movement in plasma tubes
has an adverse effect on plasma purity, however, by
trapping cells and platelets in the plasma compartment
Rapid gel movement in plasma
before their separation is complete. The ‘lava lamp’ tubes traps some cells and platelets
mechanism of gel movement in plasma tubes also in the plasma compartment before
entraps more cells in the formed gel barrier after
centrifugation compared with serum gel tubes. their separation is complete.

serum plasma
Table 1 summarizes general advantages and
disadvantages with both serum and plasma specimens.
Some of these have already been discussed while
others are discussed in the following sections.

Figure 3. Blood specimens after centrifugation
Test Results
An important consideration when comparing serum
vs. plasma specimens is assay/test result equivalence.
In general, most assays in clinical chemistry are
compatible with both serum and heparin plasma,
and test results are sufficiently equivalent that the
same reference ranges can be used. However, for
certain assays or test methods, plasma specimens
may be unacceptable, or differences in results may
be sufficient to warrant a change in reference range.



Table 1. Serum vs. Plasma
Advantages and Disadvantages
Advantages
Serum – nearly cell-free
– good storage stability for most analytes
– wide range of assays available
Plasma – shorter TAT: can be centrifuged immediately
– faster gel movement in gel tubes/more reproducible
gel barrier formation
– more representative of in vivo state
– available plasma yield 15-20% higher than serum10
Disadvantages
Serum – may cause pseudohyperkalemia, especially in patients
with thrombocytosis
– longer TAT
– instrument or test interference from fibrin, especially with
anticoagulation therapy
Plasma – higher cell/platelet counts; increased potential for testing
errors with certain tests/instruments
– reduced storage stability for certain analytes (especially in
gel tubes); fibrin formation during storage
– interference from anticoagulant
– interference from fibrinogen (e.g., protein electrophoresis)
– some tests may not be supported
Analytical differences between serum and plasma
specimens can be grouped into two broad categories:
inherent differences due to the nature of the
sample/specimen, and differences associated with
the tube additives.
The absence of a clotting process in anticoagulated
blood leads to differences in several analytes between
serum and plasma specimens. The clot formed during
coagulation consumes fibrinogen and entraps cells
and platelets within a network of fibrin. Due to
the presence of fibrinogen, total protein levels are
generally slightly increased in plasma.10-13 Interference
from fibrinogen may also make plasma an unsuitable
specimen for certain protein analysis methods
(e.g., protein electrophoresis).13 Conversely, generation
of ammonia/ammonium ion from deamination of
amino acids during clotting precludes the use of serum
specimens for ammonia testing (the ammonium salt of
heparin should also not be used).13 Activities of certain
enzymes (e.g., LD, ALKP) may exhibit serum-plasma
differences, however, not all literature reports are in
agreement in this area.10-15 The number of cells and
platelets in the plasma, combined with differences
in analytical methodology, may be a factor in these
serum-plasma discrepancies.16
Concentrations of potassium and inorganic phosphate
are higher in serum than in plasma due to release

from platelets during coagulation.10-14,17,18 Potassium
concentrations in serum have been shown to be
0.2–0.7 mmol/L higher than those in plasma.17
Mean serum-plasma potassium differences of
0.32–0.37 mmol/L at time 0 hours were reported
to increase to 0.41–0.47 when tubes were left to
stand for 3 hours at room temperature before
centrifugation and analysis.18 The authors also noted
significant variability in these differences, attributed
to variation in release of potassium during clotting
or during delays in the separation of serum from
cells. A linear correlation has been shown between
platelet count and the increase in serum potassium.13
Patients with essential thrombocythaemia and
significant thrombocytosis were found to have serum
pseudohyperkalaemia (> 5.5 mmol/L) that corrected
when measured on plasma.19 Other studies have
also demonstrated a significant correlation between
thrombocytosis and pseudohyperkalaemia, 20,21 with
an increase of 0.07 to 0.15 mmol/L of potassium
estimated for every 100 x 109/L of platelets. 21 Since
hyperkalaemia is a potentially life-threatening event
requiring accurate determination of its etiology, it
has been suggested that when there is no obvious
explanation for an elevated serum potassium,
the plasma potassium level should be measured,
particularly in patients with elevated platelet counts.22
Patients with essential
thrombocythaemia and significant
thrombocytosis were found to have
serum pseudohyperkalaemia 
(>5.5 mmol/L) that corrected when
measured on plasma.
Certain tests may also be affected by the heparin
counterion or heparin itself. The lithium salt of
heparin should not be used for lithium determination
due to interference from lithium. 23 Sodium will
be increased when sodium heparin is used as the
anticoagulant, especially when tubes are underfilled
with blood. To avoid interference with sodium
determinations, lithium heparin is used much more
commonly than sodium heparin for plasma testing
in clinical chemistry. Samples with IgM paraproteins
have been reported to produce interference with
certain glucose and GGT methods when collected as
lithium-heparin plasma specimens. 24 Heparin has also
been reported to affect results with certain troponin Standardized procedures for
immunoassays, 25-28 although instrument companies
continue to work to make assays compatible with both specimen handling, processing, and
serum and heparin plasma samples. testing are especially important
Tests which may exhibit serum-plasma differences are for plasma specimens to minimize
summarized in Table 2. It is important to note that variation in test results.
the occurrence and magnitude of such differences
can depend on specimen handling and processing
procedures and/or the specific instrument/assay
methodology used. Plasma specimens may also exhibit
an increased frequency of duplicate errors with certain
Factors Influencing
instrument/test combinations, due to platelets, cell Plasma Specimen Quality
aggregates, or microclots. 29,30 Plasma specimens with
Specimen quality is an important factor that influences
increased cellular and platelet concentrations also
both instrument operation and test results. Guidelines
exhibit reduced stability of certain common analytes
have been published for the handling and processing
that are involved in cell/platelet-mediated metabolic
of blood specimens to help ensure acceptable specimen
processes and/or are present in higher concentrations
quality.10,32 To avoid inaccurate test results, it has
in cells or platelets. Analytes that can be affected
been recommended that serum/plasma be physically
by these factors include glucose, potassium, total
separated from contact with cells within a maximum
carbon dioxide, inorganic phosphate, aspartate amino
of two hours from the time of collection. 32 For serum
transferase (AST), and lactate dehydrogenase (LD).
and plasma specimens, specimen quality can be
Serum-plasma differences may be evident with
equated with specimen ‘purity’ – i.e., the extent to
these analytes depending on plasma cell/platelet
which the serum or plasma is free from undesirable
content and the time interval between centrifugation
constituents such as fibrin, cells, and platelets.
and testing. Standardized procedures for specimen
The ideal plasma specimen would thus be one that
handling, processing, and testing are therefore
is cell/platelet-free and in which the anticoagulant
especially important for plasma specimens to
functions to inhibit clotting and fibrin formation
minimize variation in test results. Plasma specimen
during collection and continuing over extended
quality and analyte stability are discussed in more
periods of time. In practice, this ideal is often not
detail in the next two sections.
attained with heparin-plasma specimens.

Table 2. Potential Serum vs. Heparin Plasma
Test Result Differences*
serum not recommended due to generation of
Ammonia ammonia during clotting
differences in certain enzymes (e.g., LD, ALKP, AST)
Enzymes may be seen
Lithium increased with use of lithium heparin
increased in serum due to release from cells/platelets
Phosphorus during clotting
increased in serum due to release from cells/platelets
Potassium during clotting
Sodium increased with use of sodium heparin
Total Protein increased in plasma due to presence of fibrinogen
Other heparin binds divalent cations (e.g., ionized calcium) 31
and may interfere with certain immunoassays;
analytes that are involved in cell/platelet-mediated
metabolic processes and/or are present in higher
concentrations in cells or platelets may show
differences depending on plasma cell/platelet
content and time of testing
* Listing is intended to be illustrative and is not necessarily comprehensive.
Differences may depend on specimen handling/processing conditions and assay
platform/manufacturer.
The separation of plasma from other blood components
during centrifugation is based upon a density gradient.
The relative presence of these components in the
plasma after centrifugation should therefore decrease
with increasing density: platelets (least dense) > white
blood cells > red blood cells (most dense). Platelets are
typically the most abundant of these components in
heparin plasma, followed by white blood cells (mainly
lymphocytes and monocytes). 33 As described in this
section, several factors determine the actual cell and
platelet counts obtained. Heparin plasma can also
contain fibrin which is formed due to incomplete
anticoagulation. This fibrin is generally present in the
form of thin fibrin strands. Incomplete dispersion of
heparin (inadequate specimen mixing/tube inversions
immediately after collection) and patient factors such
as disease state and medication can both diminish
the efficacy of heparin activity and lead to increased
fibrin formation and ‘microclots’. In addition, platelet
aggregates can form which may also contain fibrin
and/or white blood cells. These aggregates can be large


enough to be visible to the unaided eye and have been
termed ‘white particulate matter’ (WPM) due to their
typical white color. 34 WPM has also been observed
in blood bags used in transfusion medicine, where it
was determined to consist predominantly of platelets,
with white blood cells and cellular debris including
fragments and granules from white blood cells, and
occasional fibrin strands. 35 As a result of the potential
for variable amounts of cells, platelets, fibrin, and
WPM, heparin plasma is generally a more complicated
matrix to manage than serum. A proper understanding
of the factors that influence the amount of these
components in the plasma is therefore essential to
ensure that specimen quality is acceptable.
Specimen Handling After Centrifugation
An important factor that influences plasma specimen
quality is how the specimen is handled after
centrifugation. In practice, this is often a function of
whether the blood collection tube used contains a gel
barrier or not. Before discussing specimen handling,
it is informative to examine how gel influences the
situation of white blood cells and platelets following
centrifugation. In both heparin-plasma tubes with
and without gel, packing of cells with concurrent
expression of plasma above the cells continues during
centrifugation. In the tube with gel, however, the
gel begins to form a barrier between the cells and the
plasma within one minute, and barrier formation is
almost complete within 2–3 minutes.9 The quick
formation of the gel barrier traps some cells (mostly
white blood cells) and platelets in the plasma
compartment before their sedimentation is complete.
After centrifugation, these cells and platelets are
concentrated on or near the gel surface, versus being
concentrated at the red blood cell-plasma interface
in a tube without gel. These cells in plasma tubes
are sometimes referred to as the ‘buffy coat’ (visible
in Figure 3).
When tubes are kept upright and not mixed/agitated
after centrifugation, plasma sampled or aliquoted
from the top of the plasma column will be relatively
‘clean’ with only small amounts of cells and platelets.
This applies to both tubes with and without gel,
although the actual amounts of cells and platelets
will depend on the centrifugation conditions used
and the depth of sampling. Plasma obtained from
tubes without gel is generally of high quality for the
simple reason that the plasma is usually aliquoted
from the top of the tube after centrifugation. The use
of aliquoting must be weighed against the additional
cost of labor/time and supplies required for aliquoting,

as well as the potential for mislabelling of secondary
tubes. Gel tubes offer the benefit of not having to
aliquot the plasma. However, gel tubes may be held
in a variety of orientations after centrifugation,
especially when transported to reference laboratories
or other off-site testing facilities. This can lead to
mixing and resuspension of components that were
previously on or near the gel surface. Table 3 shows
cell and platelet counts in plasma obtained from
lithium-heparin plasma tubes with gel. 33 Two aliquots
(1 mL) were sampled and tested from each tube.
The first 1 mL aliquot was taken from the top of
the plasma column after centrifugation with the tube
kept still and upright (‘unmixed’). Each tube was
then gently inverted 180° and back three times.
The second 1 mL aliquot was then taken from the
top of the remaining plasma column (‘mixed’).
As shown in Table 3, cell and platelet counts increased
substantially after mixing the plasma. While the
mixed plasma counts are also increased due to
the concentration effect from the reduced plasma
volume (volume lower by 1 mL due to first aliquot),
the substantial effect of mixing is still evident.
Any fibrin and WPM present on or above the gel
after centrifugation will also be resuspended.
Hence, plasma specimens in gel tubes which have
been mixed after centrifugation may appear slightly
darker or cloudy due to the resuspension of these
components. This difference in the visual appearance
between mixed and unmixed plasma is evident in
the photographs in Figure 5.
Table 3. Cell Counts in Mixed vs. Unmixed
Plasma Obtained from Gel Tubes (mean ± sd)*
Plasma RBC WBC PLT
Aliquot x 106/mm3 x 103/mm3 x 103/mm3
Unmixed 0.000 ± 0.000 0.03 ± 0.05 11 ± 8
Mixed 0.013 ± 0.006 0.54 ± 0.22 125 ± 71
* 13 x 100 mm plastic lithium-heparin gel tubes (n= 40) were centrifuged for
10 minutes at 1300g in a swing-bucket centrifuge. The mean plasma volume
obtained after centrifugation was 2.24 mL. Two 1-mL aliquots were removed
from each tube before and after mixing as described in text. Data from
reference 33.
Adequate sedimentation of cells
and platelets during centrifugation
depends on centrifuge g-force
(RCF), spin time, and brake time.
 unmixed mixed 160

140

120

Platelets x 103/mm3
100

80

60

40

20

0
6000 9000 12000 15000
G-min

Figure 5. Effect of mixing plasma separator tube after Figure 6. Effect of g-min (RCF x spin time) on plasma platelet
centrifugation counts in lithium-heparin tubes with ( ■ ) and without (◆) gel.
From data in Table 4

Centrifugation Conditions
The centrifugation conditions used also influence gel drawn from the same subject population were
specimen quality by determining the sedimentation centrifuged at four conditions consisting of two RCF
of cells and platelets in the plasma. In general, settings (1800, 3000g) and two time settings (4 min,
sedimentation increases with increasing relative 5 min). The same centrifuge and ramp-up/down
centrifugal force (RCF) and spin time. Plasma platelet settings were used for all tubes. After centrifugation,
counts in lithium-heparin tubes with gel decreased an aliquot was removed from the top of the plasma
from 321 to 5 (both x 109/L) when centrifugation was column in each tube and tested for cell and platelet
increased from 500g/5 min to 3000g/10 min.15 counts. As shown in Table 4, platelet counts decreased
The results of a more detailed study are shown in with both increased RCF and spin time in both tube
Table 4. 33 Lithium-heparin tubes with and without types. These platelet counts as a function of g-min
(g-force multiplied by spin time in minutes) are shown
in Figure 6. Red and white blood cell counts were
Table 4. Effect of Centrifuge RCF relatively unaffected by the conditions used in this
and Spin Time on Cell Counts (mean ± sd)* study. Of interest, for all four centrifuge conditions,
RCF (g) RBC WBC PLT plasma platelet counts were higher in the tubes
/minutes x 106/mm3 x 103/mm3 x 103/mm3 without gel. This may be an artifact of the centrifuge
Lithium-Heparin Tubes Without Gel
braking process, during which cells/platelets are
subject to free upward movement in tubes without
1800/4 min 0.004 ± 0.013 0.05 ± 0.07 149 ± 113
gel, compared with gel tubes where the immobile
1800/5 min 0.001 ± 0.003 0.05 ± 0.06 107 ± 81 gel barrier may reduce the momentum of upward
movement. A higher frequency of duplicate errors
3000/4 min 0.000 ± 0.002 0.05 ± 0.05 73 ± 40
in LD measurements has been reported when direct
3000/5 min 0.004 ± 0.027 0.04 ± 0.05 47 ± 27 sampling from heparin-plasma tubes without gel
Lithium-Heparin Tubes With Gel separator compared with heparin-plasma tubes with
gel separator. 29 These errors may be due to LD release
1800/4 min 0.000 ± 0.000 0.06 ± 0.05 43 ± 20
from platelets that are sampled and then lysed.16
1800/5 min 0.000 ± 0.000 0.04 ± 0.05 37 ± 17 Rapid centrifuge braking (deceleration) may be used in
3000/4 min 0.000 ± 0.000 0.04 ± 0.05 24 ± 11
front-end automation systems to increase throughput
and reduce TAT; the potential for cell/platelet
3000/5 min 0.010 ± 0.060 0.05 ± 0.05 18 ± 9 resuspension in these systems should be considered
* 13 x 100 mm plastic lithium-heparin tubes with and without gel (n= 40) were when assessing plasma specimen quality.
centrifuged together in a swing-bucket centrifuge. One aliquot was removed
from the top of the (unmixed) plasma column in each tube and analyzed for
cell counts and platelets. Counts reflect concentration in the sampled aliquot
only, not the entire volume of plasma obtained from centrifugation. Data from
reference 33.


Other Factors
The cellular content of plasma can lead to fibrin
formation through a process of ‘heparin neutralization’.
This occurs due to the fact that heparin binds to
certain proteins that compete with antithrombin III
(AT III) for heparin binding, in particular platelet
factor 4 (PF4) which is stored in platelet alpha
granules and released when platelets are activated. 36-38
This binding reduces the availability of heparin for
anticoagulation. Conversion of fibrinogen to fibrin in
heparin anticoagulated blood varies widely, dependant
on the number of cells and platelets and on the
concentration of AT III and other plasma proteins.
Poor phlebotomy technique may also contribute to
heparin neutralization and subsequent fibrin formation
by causing platelet activation and release of PF4.
Similarly, heparin neutralization may also be increased
in specimens from patients with clinical states
associated with activation of platelets.
Temperature can also influence the formation of
fibrin in heparin plasma. The contact activation
of Factor XII and other plasma proteins is accelerated
at low temperatures (< 37° C) irrespective of the
presence of heparin. 38,39 Cold-promoted Factor VII
activation is the result of activation by both activated
contact proteins and the trace amounts of thrombin
they generate. Since this activation is accelerated
at reduced temperatures, refrigeration may drive
the reaction towards clotting and thus may be
antagonistic to the anticoagulant action of heparin.38
These mechanisms can thus lead to increased fibrin
formation when heparin plasma is refrigerated.
When needed, recentrifugation of stored plasma
aliquots can help clean up the sample prior to testing.
As described in this section, a wide variety of factors
can influence heparin-plasma specimen quality.
Careful attention to all aspects of specimen handling
from collection to storage is required to ensure
specimen quality is acceptable. Depending on the
plasma content of cells, platelets, fibrin, and/or
aggregates such as WPM after centrifugation, several
undesirable outcomes can occur, including issues
with instrument operation, analyte instability, and
erroneous test results. These effects are discussed in
the next two sections.

Table 5. Factors Impacting Heparin-Plasma
Specimen Quality and Overall Performance
Disease state
Patient
Medication
Handling Pre- Tube type (gel/non-gel)
Laboratory Phlebotomy technique
Inadequate mixing
Transport time and conditions
Handling in Centrifugation conditions
Laboratory Handling after centrifugation (aliquoting, mixing)
Storage time and temperature
Instrument Probe depth/diameter
Predilution
Reagent pH
Automation, transport belts (agitation)
Analyte Stability in Plasma
Literature reports on analyte stability in heparin-
plasma specimens are few. Studies on the overnight
stability of plasma specimens40 and the effect of
anticoagulants and storage temperatures on plasma
and serum hormones41 have been published.
A detailed study was also published on the stability
of analytes in serum and plasma specimens both
uncentrifuged, and centrifuged and aliquoted.42
Storage of uncentrifuged specimens beyond 24 hours
caused significant changes in most analytes
investigated, with analyte instability higher in the
plasma specimens.
As noted earlier, heparin-plasma specimens with
increased cellular and platelet concentrations exhibit
reduced stability of certain common analytes that
are involved in cell/platelet-mediated metabolic
processes and/or are present in higher concentrations
in cells or platelets. Table 6 shows data from one
study where lithium heparin plasma specimens were
tested at initial time and again after 24 hours storage
at room temperature.43,44 Both tubes with and without
gel were included. After centrifugation, plasma
from tubes without gel was carefully aliquoted into
secondary plastic containers, whereas plasma in tubes
with gel remained on the gel in the primary tube.
 Table 6. Storage Stability of Selected Analytes in Lithium Heparin Plasma (mean ± sd)*
TIME 0 TIME 24h
ANALYTE UNITS Aliquot from Aliquot from
Gel Tube Gel Tube
Non-Gel Tube Non-Gel Tube
Glucose mg/dL 100.8 ± 50.8 98.3 ± 50.8 101.2 ± 51.0 62.0 ± 52.4

Potassium mmol/L 3.87 ± 0.26 3.96 ± 0.27 3.88 ± 0.25 4.25 ± 0.29

Carbon Dioxide† mmol/L 20.6 ± 2.2 19.6 ± 2.0 20.7 ± 2.1 16.0 ± 1.8

Phosphorus mg/dL 3.06 ± 0.56 3.11 ± 0.55 3.09 ± 0.55 3.46 ± 0.53

Triglycerides mg/dL 134.4 ± 91.1 131.8 ± 88.7 126.2 ± 86.8 125.9 ± 85.9

GGT U/L 32.9 ± 51.2 33.1 ± 49.9 32.6 ± 51.4 34.7 ± 49.8

AST U/L 21.6 ± 7.4 22.5 ± 7.1 21.0 ± 6.9 23.9 ± 6.8

LD U/L 139.1 ± 16.6 154.8 ± 16.5 138.1 ± 16.9 184.9 ± 25.7

* 13 x 100 mm plastic lithium heparin tubes with and without gel (n= 40) were centrifuged at 1300g for 10 minutes in a swing-bucket centrifuge.
Testing was performed on an Olympus® AU5400 analyzer.
†
n=38 for CO2 in gel tubes, due to values below limit of detection after 24 hours storage.

As shown in Table 6, most analytes in aliquoted
plasma (from the ‘non–gel’ tube) remained stable
over the 24-hour storage interval. This stability is
comparable to that seen with serum specimens and is
attributed to the relatively low cell and platelet counts
in the plasma aliquots. In contrast, these analytes
were less stable in plasma stored in the gel tubes—
in some cases significantly. Glucose and total
carbon dioxide exhibited large decreases in plasma
stored in gel tubes, due to cell/platelet-mediated
metabolic processes over time. Potassium,
inorganic phosphate and certain enzymes (GGT,
AST, LD) exhibited moderate to large increases,
attributed to release from (lysis of) cells and
platelets. The reduced stability of these analytes in
plasma gel tubes is attributed to the higher counts
of cells and platelets, most of which remain on or
near the gel surface after centrifugation when tubes
are stored upright and not agitated. Decreases in
triglycerides were observed in both tube types
and may be due to the activation of lipase activity
by heparin.45,46 Other routine chemistry analytes
evaluated in this study were stable in both tube types
or demonstrated small changes over 24 hours that
were considered clinically acceptable: sodium,
chloride, BUN, creatinine, uric acid, phosphorus,
calcium, cholesterol, triglycerides, total protein,
albumin, total bilirubin, direct bilirubin, ALKP,
ALT, iron, and magnesium.
The instability of certain analytes in heparin plasma
will be proportional to the amount of cells and
platelets in contact with the plasma. These changes
are due to the mechanisms described above involving
cells and platelets, and are not due to the presence
or absence of gel itself. When plasma from lithium-
heparin gel tubes was aliquoted and further processed
to be cell-free, routine analytes were considered to be
stable up to 56 hours.42 Depending on the elapsed time
between collection and testing, and whether plasma
is aliquoted or not after centrifugation, changes in
these analytes may be clinically significant. While
large changes in most settings will only be encountered
after storage of specimens (i.e., after initial time
testing), these mechanisms may also influence test
results at initial time if aliquoting and/or testing is
not performed soon after centrifugation. As some
mechanisms are temperature-dependant, storage
temperature can also influence analyte stability.
Increases in plasma potassium were reported to
be higher in refrigerated blood samples compared
to samples stored at room temperature; this was
attributed to inhibition of the cellular Na+,K+-
ATPase activity brought about by cold.47,48 Thus, it
is important that laboratories evaluate the need for
delayed or add-on testing when considering the use
of heparin-plasma specimens. Stability studies and
implementation of procedures such as aliquoting
may be needed, depending on each institution’s
specific needs and requirements.
The stability of certain analytes 
in heparin plasma depends 
on plasma cell/platelet counts 
and storage temperature.


Instrument/Assay Considerations
The use of heparin-plasma specimens for clinical
chemistry testing involves certain important
considerations involving the manufacturer of the
instrument(s) to be used for plasma testing. First and
foremost is whether the required assays are validated
and supported for plasma by the manufacturer.
Today, use of heparin-plasma specimens is supported
by many clinical chemistry instrument companies,
however assay coverage is variable and some tests may
not be compatible with heparin plasma on specific
platforms. As assays are updated and improved, earlier
literature reports discussing specimen compatibility
on specific instruments may be outdated. Instrument
manufacturers should therefore be consulted for the
most current information on this topic.
The presence of cells, platelets, and other components
in heparin plasma can also lead to issues with
instrument operation. Whether issues are experienced
will depend on the amount and location of these
components in the plasma after centrifugation, as
well as the specific instrument platform. One report
has described the potential for instrument sampling
probes that descend only slightly below the plasma
meniscus to aspirate cell aggregates or microclots at
or near the meniscus. 30 The authors noted that this
can lead to sampling problems (e.g., blockage or
short-sampling) and/or release of cellular contents
due to cell rupture as a result of reagent pH changes.
Additional potential mechanisms of interference
were also described, such as optical interference in
reaction cuvettes due to floating microclots. As such
issues are highly dependant on the specific instrument
platform, it is again recommended that instrument
manufacturers be consulted to identify potential issues
and their possible resolution.
10
Summary
Heparin-plasma specimens are an attractive alternative
to serum specimens for many laboratories, allowing
them to improve test result TAT while minimizing
issues with ongoing fibrin formation due to incomplete
clotting. However, as demonstrated in this article,
heparin plasma is a more complex specimen than
serum. Factors involved in the collection, handling
and processing of heparin-plasma specimens can
influence plasma specimen quality, with corresponding
potential effects on both instrument operation and
analyte stability. The benefits and limitations of both
serum and plasma should therefore be examined when
determining which specimen is most suitable in a
specific institution or setting. Specimen management
protocols are of particular importance when using
heparin plasma, to ensure plasma specimen quality
and overall performance are acceptable. It is hoped the
information in this article will assist laboratories in
developing such protocols and maximizing the benefits
of heparin plasma in clinical chemistry testing.
The data presented in this article are intended to be
illustrative; actual results will depend on numerous
variables, including but not limited to the blood
collection tube, the handling and processing conditions
used, and the instrument platform and test method.
Whenever changing any manufacturer’s blood
collection tube type, size, handling, processing,
or storage condition for a particular laboratory
assay, the laboratory personnel should review
the tube manufacturer’s data and their own
data to establish/verify the reference range for
a specific instrument/reagent system. Based on
such information, the laboratory can then decide
if a change is appropriate.
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11
 Steps to Ensuring High-Quality Heparin Plasma
Adhering to the following specimen handling and processing steps will help
to ensure acceptable plasma specimen quality and overall performance.

➤ E nsure correct phlebotomy technique to

minimize hemolysis and platelet activation.
➤ Fill evacuated blood collection tubes to the
stated draw volume. This will ensure the proper
blood-to-additive ratio in the tube.
➤ I nvert tubes 8 to 10 times immediately after
collection to ensure that the blood and heparin
are mixed thoroughly.
➤ E nsure centrifuge g-force (RCF), spin time, and
brake time are sufficient to obtain adequate
sedimentation of cells, platelets, and other debris.
➤ C entrifuge tubes within 2 hours of collection
to separate plasma from cells.
➤ Carefully aliquot plasma from non-gel tubes
after centrifugation.
➤ Avoid mixing/agitation of plasma gel tubes
between centrifugation and testing.
➤ To help reduce fibrin formation and potassium
increases during storage, store heparin plasma at
room temperature.
➤ Evaluate and enforce “add-on testing” policies
that your facility has validated for analyte
stability in heparin plasma, including the effects
of specimens containing latent fibrin.

12
For technical assistance on BD Vacutainer® products:
U.S. customers please call BD Global Technical Services at 1-800-631-0174.
Customers outside the U.S. please contact your local BD sales consultant.
www.bd.com/vacutainer

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