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Quality Standards for Sample Processing, Transportation, and Storage in

Hemostasis Testing

Article  in  Seminars in Thrombosis and Hemostasis · June 2012

DOI: 10.1055/s-0032-1319768 · Source: PubMed

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576

Quality Standards for Sample Processing,

Transportation, and Storage in
Hemostasis Testing
Dorothy M. Adcock Funk, M.D. 1 Giuseppe Lippi, M.D. 2
Emmanuel J. Favaloro, Ph.D., M.A.I.M.S., F.F.Sc. (RCPA) 3

1 Esoterix Inc., Englewood, Colorado Address for correspondence and reprint requests Dorothy M. Adcock
2 Laboratory of Clinical Chemistry and Hematology, Department of Funk, M.D., Laboratory and Medical Director, Coagulation, Esoterix
Pathology and Laboratory Medicine, Academic Hospital of Parma, Inc., 8490 Upland Drive Englewood, CO 80112
Parma, Italy (e-mail: [email protected]).
3 Department of Haematology, Institute of Clinical Pathology and
Medical Research (ICPMR), Westmead Hospital, New South Wales,
Australia

Semin Thromb Hemost 2012;38:576–585.

Abstract Samples for hemostasis testing drawn into sodium citrate anticoagulant are vulnerable
Keywords to the effects of preanalytical variables associated with sample processing, transporta-
► preanalytical tion, and storage. These variables include the temperature at which samples are
variability transported and stored; the stability of the samples once processed; whether main-
► coagulation testing tained at room temperature, refrigerated, or frozen; methods of centrifugation; as well
► prothrombin time as the potential impact of using an automated line. Acknowledgment of these variables,
► activated partial as well as understanding their potential impact on assay results, is imperative to the
thromboplastin time reporting of high quality and accurate results. This article discusses the preanalytical
► laboratory errors issues associated with sample processing, transportation, and storage and also presents
► quality standards the ideal conditions for sample handling.

To provide accurate and reliable laboratory test results, clini- patient care.4 Laboratory errors attributed to the preanalyt-
cal laboratories must employ a well-developed, implemented, ical phase of testing can be significant and exceed those that
and monitored quality management system.1 One of the occur in the analytical phase.5–8 Samples for routine or
fundamental elements of a quality management system are specialized hemostasis testing are especially vulnerable to
quality standards, such as those developed for sample proc- such errors compared with other types of laboratory samples.
essing, transportation, and storage of specimens for hemo- It is imperative, therefore, that these quality standards are
stasis testing. Quality standards must be clearly written and closely followed and any deviations validated. This article will
document not only proper procedures, but also what impact highlight standards that should be applied to sample proc-
may occur to the sample and ultimately patient care, if these essing, transportation, and storage for hemostasis assays, for
are not followed. Training and regular competency assess- those samples collected in sodium citrate anticoagulant.
ments surrounding these quality standards are also key Sample collection standards for coagulation testing are dis-
components to a quality system.2 Improper procedures asso- cussed elsewhere in this issue of Seminars in Thrombosis &
ciated with sample processing, transportation, and storage Hemostasis.9
may alter the sample such that the results do not reflect the Whole blood samples for plasma-based coagulation assays
true condition of the patient, although they may be accurate would, in ideal circumstances, be collected and processed to
for the test sample.3 Such errors may lead to mistaken or produce platelet-poor plasma (PPP) within 1 hour.6,10 Trans-
inappropriate diagnosis with resultant detrimental impact on portation and storage of sodium citrate whole blood samples

published online Issue Theme Quality in Hemostasis and Copyright © 2012 by Thieme Medical DOI http://dx.doi.org/
June 16, 2012 Thrombosis, Part I; Guest Editors, Publishers, Inc., 333 Seventh Avenue, 10.1055/s-0032-1319768.
Emmanuel J. Favaloro, Ph.D., M.A.I.M.S., New York, NY 10001, USA. ISSN 0094-6176.
F.F.Sc. (RCPA), Mario Plebani, M.D., and Tel: +1(212) 584-4662.
Giuseppe Lippi, M.D.
 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al. 577

would occur at ambient temperature (15 to 22°C) and whole Department of Transportation have regulations governing the
blood samples would not be placed on ice, in an iced water transport of clinical specimens.10
bath, or refrigerated. Sample analysis for routine assays would
be completed within 4 hours of collection, with the notable
Transportation and Storage of Unprocessed
exception that samples for prothrombin time (PT) testing are
or Processed Sodium Citrate Tubes for
stable for 24 hours. Sample analysis for more complex hemo-
Plasma-Based Coagulation Assays
stasis assays would also ideally be undertaken before delete-
rious effects of sample aging, within similar time frames Samples transported from outlying facilities to central coag-
(dependent on the stability of the test analyte, see ►Table 1). ulation laboratories are often maintained unprocessed during
Often, however, specimens for coagulation testing are transportation and storage. This has several advantages. For
collected at a site other than the testing facility. It is common, example, drawing sites do not require equipment (such as
for example, for patient samples to be drawn in physician centrifuges) to process samples and the receiving coagulation
offices or extended care facilities. Reference or hospital labo- laboratory can better evaluate primary collection tubes for
ratories also frequently utilize conveniently located patient certain preanalytical variables such as fill volume, presence of
draw stations. Each of these scenarios requires that samples clots, hematocrit, and correct collection tube type. However,
then be couriered from the sites of collection to regional sample integrity for certain specific assays, such as for
facilities for testing. Finally, laboratories may ship samples activated partial thromboplastin time (APTT) or anti-Xa assay
that require more esoteric coagulation assays to centralized in the presence of unfractionated heparin (UFH), may be
specialized laboratories. Depending on the testing ordered, as enhanced if samples are centrifuged immediately after blood
well as the distance and time required between collection and collection, rather than shipped unprocessed.
analysis, samples may be transported and stored in any of the Specimens should arrive in the testing facility allowing
following three conditions: (1) unprocessed as sodium citrate sufficient time for the samples to be processed (if necessary)
whole blood samples, (2) centrifuged, but maintained in and analyzed in agreement with the specified time frames
the primary sodium citrate tube, or (3) processed by centrifu- listed in the guidelines below. Unprocessed or processed
gation and plasma aliquoted into a secondary tube. samples collected in sodium citrate, should remain capped
Samples must always be transported in a manner that and maintained at ambient (room) temperature (18 to
avoids infectious potential and in accordance with institu- 25°C).10 Whole blood samples must not be refrigerated or
tional policies and procedures. Many regulations require that stored on ice or in an iced water bath. Whole blood samples
samples are transported inside of impermeable plastic bags should be transported in a vertical rather than horizontal
which are sealed. Facilities must also be aware of applicable position and if processed, should not be agitated as to remix
regulations regarding packaging and shipment of hazardous the plasma and cellular components.11 The sample may be
materials. For example, in the United States, the Centers for compromised and therefore develop the potential to generate
Disease Control, International Air Transport Association, and erroneous results, which may then be unwittingly reported to

Table 1 Stability of Whole Blood Sample for Common Hemostasis Assays

Assay Stability of Whole Blood Sample

CLSI H21 A510 Other30–35
APTT 4h 18–24 h
PT 24 h 24–72 h
APTT or anti-Xa assay for sample containing UFH 1h
APTT or Anti-Xa assay for sample containing LMWH 4h 24 h
Factors II, VII, IX, X, and XI activities 4h 48 h
Factors V and VIII 4h 24 h
VWF:Ag and VWF:RCo 4h 24–48 h
Fibrinogen 4h 48 h to 7 d
D-dimer 4h 48 h
Antithrombin activity 4h 48 h to 7 d
PC activity 4h 48 h
PS activity 4h 4–6 h
Free PS 4h 24 h

CLSI, Clinical and Laboratory Standards Institute; APTT, activated partial thromboplastin time; h, hours; PT, prothrombin time; UFH, unfractionated
heparin; LMWH, low-molecular weight heparin; VWF:Ag, von Willebrand factor antigen; VWF:RCo, von Willebrand factor: Ristocetin cofactor; d, days;
PC, protein C; PS, protein S.

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

578 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al.

the requesting clinician, because of any of the following variations of routine coagulation tests (i.e., PT, APTT, fibrino-
conditions: changes in sample pH; extremes of temperature; gen, and D-dimer) in whole blood samples left uncentrifuged
physical trauma to the specimen; excessive delay between and stored at 4°C for up to 6 hours. Significant variations were
collection and analysis; and inappropriate condition of sepa- instead observed at 24 hours for APTT, but not for PT,
ration. Each of these variables and their impact on assay fibrinogen, and D-dimer.24 Extremes of temperature may
results is discussed below. occur if whole blood samples are stored in laboratory collec-
tion boxes that are maintained out of doors depending on
Sample pH external temperatures, or if samples are transported without
The buffering capacity of citric acid, which is a key component adequate protection against the elements (e.g., stored in a
of commercial trisodium citrate collection tubes, maintains vehicle’s trunk without an insulated container).25
the sample pH between 7.30 and 7.45.12 Maintenance of
physiologic pH is critical to obtain accurate plasma-based Sample Transportation
hemostasis testing as well as platelet function studies. An Whole blood samples should be transported in a manner that
increase in specimen pH occurs if samples are stored un- avoids significant physical trauma. If samples are transported
capped for more than 30 minutes, as carbon dioxide diffuses after centrifugation but plasma is not aliquoted, centrifuga-
from the plasma into the ambient atmosphere.13 The use of tion should always be repeated before analysis. Samples
small caliber tubes, to minimize the surface area-to-volume should be transported in an upright position. High speed
ratio, will help maintain pH.13,14 Processed samples are more pneumatic tube systems are often employed to allow rapid
susceptible to change in pH than are whole blood samples due transport of patient samples. It has been historically reported
to the loss of the hemoglobin buffering capacity.13 Increase that, due to the rapid acceleration and deceleration forces that
in pH leads to clinically significant prolongations of the APTT occur in some of these systems (especially the older ones),
and PT, and also affects a variety of specialized coagulation samples may suffer trauma, resulting in platelet activation
assays including loss of platelet reactivity. For example, a pH and red-cell fragmentation and/or release of adenosine di-
change of as little as 0.8 units may prolong the APTT of a phosphate (ADP). Nevertheless, while use of pneumatic tube
normal sample by greater than 20 seconds, depending on the transport systems is generally not recommended for samples
buffering capacity of the reagent used in the test system.15 that will be subject to platelet function studies, no ill-effects
have been documented for samples for plasma-based coagu-
Temperature Effects lation assays.26,27 Specifically, no significant effects were
Hemostasis factors, including procoagulant factors, naturally demonstrated in the evaluation of paired samples (one
occurring anticoagulant factors, as well as platelets, become sample hand delivered and the other transported by pneu-
labile in an ex vivo environment. These components may matic tube) tested using PT and APTT assays.28 This study also
undergo either in vitro degradation or activation that is both included a comparison of paired fibrin monomer results, an
time and temperature dependent. For example, FVIII and extremely sensitive marker of activation of the coagulation
protein S (PS) are particularly labile factors that are prone cascade, and demonstrated no significant difference. In a
to degradation, such that loss of activity occurs within 4 to more recent study by Wallin et al, no preanalytical effect of
6 hours if blood is maintained at room temperature.16,17 pneumatic tube transport could be recorded for most coagu-
Degradation may be accelerated at very warm temperatures lation parameters and Platelet Function Analyzer (PFA-100®
and essentially all factors will lose activity if maintained at System, Siemens Healthcare Diagnostics Inc., Marburg,
58°C for a period of time.18 Conversely, platelets and FVII are Germany) analysis.29 Only for thromboelastographic analysis
activated by cold temperatures. Indeed, platelets may under- did the time to clot formation exhibit a significant shortening
go spontaneous aggregation in the cold.19 Cold activation of (i.e., 16%) in samples shipped by a pneumatic transport tube
FVII can result in elevation of FVII activity by 150% or more system.29 It is thereby conceivable that pneumatic transpor-
with a resultant decrease in PT.20 Cold activation of whole tation of specimens would only negligibly affect most coagu-
blood samples may also lead to clinically significant loss of lation tests, and it seems reasonable that only samples for
FVIII and von Willebrand factor (VWF) leading to a mistaken platelet aggregation studies need be manually transported to
diagnosis of hemophilia A or von Willebrand disease.21 In a the laboratory.
study published by Favaloro et al, 50% of whole blood samples
demonstrated to have FVIII activity and VWF levels in the
Guidelines for Storage and Transportation of
normal reference intervals when properly processed, fell
Unprocessed or Processed Whole Blood
below a normal reference interval of 50% when stored at
Samples
4°C for 3.5 hours. This effect was slightly greater using a
functional measure of VWF activity than the measure of The following guidelines for the storage and transportation of
antigen.22 Whole blood samples therefore should not be unprocessed or processed, whole blood samples in sodium
stored or transported on ice or in a refrigerator. If for some citrate evacuated tubes should generally be followed, unless
reason, whole blood samples destined for VWF and FVIII other conditions have been validated. Whole blood samples
analysis are stored in the cold, samples should be warmed should be maintained capped, in an upright position, and at
and thoroughly mixed before processing.23 In a further study, ambient temperatures and not be placed in refrigerated
however, Salvagno et al failed to observe clinically significant storage (from 2 to 8°C). The allowable times listed should

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al. 579

also encompass the time required for sample processing and rich plasma (PRP) or whole blood samples. Samples for
analyses. platelet function testing should not be transported using a
pneumatic tube system, especially if this has not been vali-
1. Specimens for APTT and most special coagulation assays
dated, as the potential physical trauma may lead to platelet
(such as factor assays, assays for the detection of lupus
activation and release of ADP from erythrocytes, which will
anticoagulants (LA), and VWF assays) that do not contain
blunt platelet responsiveness.25 Platelets should always be
UFH are stable for up to four hours from the time of
transported in an upright position and stored at room tem-
specimen collection, according to the CLSI H21 guideline.10
perature.13 Exposure to cold temperatures causes physical
Limiting samples to a four-hour stability is conservative
alterations to the platelets including shape changes and loss
and samples for APTT and most special coagulation assays
of their microtubular systems.13 Storage at refrigerated tem-
are likely stable for longer periods of time. A host of
peratures also causes spontaneous platelet activation result-
published studies have demonstrated 6 to 8 hours or
ing in aggregation, while storage at 37°C leads to impaired
longer stability of APTT-based assay samples and even
responsiveness.13 The extent of variation in the responsive-
24-hour stability for some special coagulation assays.30–33
ness of platelets also depends on the time interval between
Zürcher and colleagues demonstrated that with the ex-
venipuncture and testing, as whether the sample is tested as
ception of FV and FVIII activities and free PS antigen,
PRP or whole blood.
several hemostasis assays are stable for 24 to 48 hours
following collection (see ►Table 1).31 Samples for fibrino-
Platelet Rich Plasma
gen and antithrombin activity have been shown to be
Platelets demonstrate less responsiveness for the first 30
stable for up to 7 days.30 It has also been demonstrated that
minutes after processing and it has been suggested that
whole blood samples from patients on low-molecular
this initial refractoriness is likely a result of ADP released
weight heparin are stable for 24 hours after collection, if
from erythrocytes and platelets during centrifugation.13 The
samples will be subject to heparin monitoring using a
platelets in PRP maintain ideal responsiveness for only 3 to 4
chromogenic anti-Xa assay.33 Samples for most VWF-
hours following processing and lack of response beyond this is
based assays are also stable for up to 24 hours when stored
likely a reflection of the increase in sample pH and exhaustion
as whole blood at ambient temperature.34
of energy reserves.13,14 For this reason, testing is often
2. Samples suspected or known to contain UFH that will be
performed after 30 minutes but within 3 to 4 hours of PRP
subjected to APTT or anti-Xa analysis, should be main-
preparation. Efforts to help maintain pH of the PRP should be
tained at room temperature, and centrifuged within 1 hour
employed such as using small caliber tubes and limiting
of collection. If samples are left unprocessed for longer
mixing/agitation of the PRP.13,14
periods of time, platelet factor 4 released from platelets
For use in platelet aggregation studies, the PRP is often
will neutralize the UFH present in the sample leading to
adjusted using autologous PPP to achieve a standardized
clinically significant, factitiously low APTT and anti-Xa
platelet number (typically 200  109/L to 300  109/L).
results.35 If samples are centrifuged within 1 hour of
This practice is discouraged by Cattaneo et al who have
collection, samples are stable as long as testing is complet-
demonstrated that substances released by platelets and other
ed within 4 hours.35
blood cells during centrifugation necessary to obtain PPP has
3. Samples for PT or international normalized ratio (INR)
an inhibitor effect on platelet aggregation.39 In support of this,
assays are stable for at least 24 hours when maintained
Linnemann et al demonstrated that adjustment of the platelet
at room temperature, unprocessed or processed, according
count in PRP is time-consuming and of no advantage when
to CLSI H21 guideline.10 Indeed, up to 3-day stability of PT/
performing platelet function testing and is therefore unnec-
INR determinations in patients on warfarin therapy, using
essary.40 A comparative analysis performed by Favaloro and
a variety of thromboplastin reagents has been demon-
Mohammed also noted deleterious effects of platelet count
strated.36 Mechanical agitation of the whole blood sample
adjustment in a small case series.41
should be avoided during transportation or storage, as this
has been shown to lead to a spurious increase in PT/INR
Whole Blood
results due to an unknown mechanism.11 Measured activ-
Recommendations for handling of PRP samples should gen-
ities of the vitamin K-dependent factors (FII, FVII, FIX, and
erally be applied to whole blood samples for platelet function
FX) have been shown to be stable for up to 24 hours if
analysis. Use of whole blood samples avoids the artifacts that
unprocessed or processed samples are stored at room
may occur secondary to centrifugation, including platelet
temperature.37
activation and release of platelet inhibitor cellular products.42
According to manufacturer instructions, whole blood sam-
Transportation and Storage of Sodium
ples for testing in the PFA-100® system should sit for
Citrate Collection Tubes for Platelet
10 minutes following phlebotomy. If the sample has been
Function Studies
transported from another site, samples should sit for
Samples for platelet function testing require special han- 30 minutes so that the platelets return to their “resting” state.
dling.38 This includes determination of closure time using a Platelet function testing using whole blood samples should
or platelet aggregation studies, employing light transmit- be completed within 3 hours of phlebotomy.13 It may also be
tance or impedance methodologies using either platelet- worthwhile to verify (e.g., by systematic centrifugation of the

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

580 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al.

specimens after analysis has been completed) whether col- tube, receipt of the incorrect tube is easily determined. Once
lection and handling of whole blood samples has generated aliquoted into a secondary tube, however, serum and all types
spurious hemolysis, because platelet as well as leukocyte of plasma have essentially an identical appearance and it is
and erythrocyte injury dramatically affects test results of not obvious that the sample type is inappropriate. Collection
PFA-100.43 of blood for most plasma-based coagulation assays into a tube
other than sodium citrate is a cause for specimen rejection.
When testing is performed on the incorrect sample type, the
Transportation and Storage of Plasma
laboratory might issue a test result that reflects the true
Aliquoted from a Sodium Citrate Collection
status of the test samples provided (e.g., serum or EDTA
Tube into a Secondary Aliquot Tube for
plasma), but this would not reflect the true status of the
Plasma-Based Coagulation Studies
patient under investigation.47 The effects of these different
If samples cannot be transported as whole blood to a facility matrices on hemostasis assays are variable and depends on
for testing under ideal conditions or within their specified the sample type received as well as the test performed
time frames, they should be processed by centrifugation and (►Table 2).6 While a sample that results in “no clot detected”
aliquoted into secondary aliquot tubes. Samples that will for APTT and PT assays may be readily recognized to inappro-
ultimately be aliquoted should be processed promptly, ideally priately be serum or lithium heparin, the effects of the
within 1 hour of collection and certainly within 4 hours. different sample matrices on other test results may be
Aliquot tubes must be composed on an appropriate material, much more subtle.6,7 While patterns of test results may
consisting of a nonactivating substance such as polypropyl- suggest an incorrect sample type, the application of simple
ene. Polystyrene tubes are not acceptable for coagulation algorithms incorporating the measurement of sodium, potas-
studies. Once aliquoted, samples must be capped to avoid sium, and citrate can greatly assist in classifying a sample as
changes in pH. Aliquot tubes must be properly labeled with other than sodium citrate plasma.47 As specifically regards
the appropriate patient identification and labeled as to the citrated plasma, there is a significant decrease in potassium,
matrix of the sample (e.g., citrated plasma, ethylenediami- chloride, calcium, and magnesium, and a substantial increase
netetraacetic acid [EDTA] plasma, heparin plasma, and in sodium, when compared with EDTA, serum, and lithium-
serum).44 heparin plasma. Although two different algorithms have been
Most hemostasis tests require citrated plasma. Moreover, developed and validated to verify the nature of the sample
EDTA plasma, heparin plasma, and serum are generally matrices, the simplest requires performance of only potassi-
unsuitable for most hemostasis tests. Collection of samples um and possibly sodium (►Fig. 1).
into an incorrect type of evacuated tube accounts for 5 to Separated plasma can generally be maintained at room
13% of all unsuitable samples received by clinical laboratories, temperature or refrigerated for a few hours without adverse
and up to 2% of all samples received in the coagulation effect on coagulation. If samples are stored for greater than
laboratory.45,46 When transported in the primary collection 4 hours from collection, they should be maintained in an

Table 2 Effect of Sample Matrix on Common Hemostasis Assays

Tube Type 3.2% Citrate EDTA Sodium Heparin Serum

Assay Mean/Range
APTT (s) 29/25–33 68/45–92 NCD NCD
PT (s) 12.4/11.5–13.2 23/19–27 NCD NCD
dRVVT (s) 34.6/27–43 55/45–64 NCD NCD
FV activity (%) 113/84–142 71/39–103 81/59–103 23/13–33
FVII activity (%) 115/50–180 116/51–182 77/43–107 308/80–437
FVIII activity (%) 141/80–202 7.5/2–19 <1 4.5/1.3–7.7
FIX activity (%) 122/97–148 115/63–168 <1 350/135–565
VWF:Ag (%) 122/50–194 143/59–228 70/42–98 101/32–169
VWF:RCo (%) 114/41–188 131/46–215 37/13–60 74/25–124
PC activity (%) 111/66–155 152/100–205 <1 21.6/0–70
PS activity (%) 96/73–119 30/17–42 <1 15.3/0–39.5
Free PS Ag (%) 108/72–144 131/91–171 126/94–159 131/97–164
AT activity (%) 102/86–118 121/105–138 126/108–143 47/30–65

EDTA, ethylenediaminetetraacetic acid; APTT, activated partial thromboplastin time; s, second(s); NCD, no clot detected; PT, prothrombin time;
dRVVT, dilute Russell viper venom time: F, factor; VWF:Ag, von Willebrand factor antigen; WF:RCo, von Willebrand factor: Ristocetin cofactor; PC,
protein C; PS, protein S; Free PS Ag, free protein S antigen; AT, antithrombin.

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al. 581

Figure 1 A recommended algorithm to assess for potentially unsuitable sample matrices. A sample that is received in a secondary tube and
suspected to not be citrate plasma; can be tested for potassium level. A high level suggests the sample is an ethylenediaminetetraacetic acid
plasma sample. Testing by thrombin time (TT) should be normal, but prothrombin time (PT) and activated partial thromboplastin time (APTT)
would be prolonged. Mixing with normal plasma would only show partial correction. If potassium level is not high measure sodium; low sodium
would suggest either serum or lithium heparin. These will both show prolonged or no clot with TT, PT, and APTT, but can be differentiated with
mixing studies. High sodium would suggest a sodium citrate sample, the correct sample matrix. However, whether this sample has been
appropriately processed, and whether clot-based tests will be normal or not, may require alternate and additional investigation. Figure updated
and modified from reference.47 EDTA, ethylenediaminetetraacetic acid.

appropriate freezer.10 If a 70°C or colder freezer is not Sample Processing

available, plasma can be stored in a 20°C freezer that does
not undergo automatic freeze/thaw cycles. Frost-free freezers Before centrifugation, samples for plasma-based coagulation
that have automatic defrost cycles are generally unsuitable, as assays must be inspected visually to ensure that the specimen
they cycle freeze–thaw events to maintain the frost-free has been collected into a properly labeled sodium citrate tube
environment, and this can result in cold activation of FVII within its expiry date and that it is adequately filled (mini-
and degradation of other factors. However, the use of frost- mum 90% fill unless shorter collection volumes have been
free freezers for patient samples is acceptable when freezers validated for the test being performed).9,50,51 Specimens
are monitored by a continuous-monitoring temperature re- should always be rejected when these conditions are not
cording device, or a minimum–maximum thermometer, met.10 It has long been standard practice to check collection
which enables the laboratory to show that the acceptable tubes for clots before centrifugation by uncapping the sample
temperature range is never exceeded. Storage at 20°C is and inserting two wooden applicator sticks. This practice is
generally sufficient for tests that are performed within no longer recommended due to the infectious potential and
2 weeks or less.10 Storage for extended (greater than 2 weeks) as it has been difficult to maintain this practice in the era of
periods of time should occur in 70°C freezer or colder.48 automation. While not as effective in the detection of clots as
If plasma aliquots are to be shipped to another facility and inserting wooden applicator sticks, gentle inversion of the
the time between collection and projected analysis exceeds collection tube and visual inspection may detect the presence
acceptable standards, samples should be frozen and shipped of clots. Inspecting the sample in a back-lit setting (e.g., in
on dry ice or another means to maintain samples in a frozen front of a view box) may aid in clot detection. Small, even
state. Sufficient dry ice must be included to maintain the microclots, however, may be missed and might still interfere
sample frozen for the required transportation time. Samples with results. The presence of a clot, even a microclot may lead
stored in dry ice (solid carbon dioxide, or CO2) containers to consumption of or possibly activation of factors and
should be thawed uncapped at 37°C for at least 15 minutes, to platelets. The presence of a clot, regardless of size therefore,
allow equilibration with ambient air, and evaporation of is a cause of specimen rejection. While it may not be possible
plasma CO2, before testing.49 If CO2 is retained in the plasma, to check every sample for the presence of a clot before
it can cause spuriously prolonged clotting times due to analysis, this possibility should certainly be investigated if
increased sample pH.12 the result of testing suggests such a possibility, that is, a

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

582 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al.

greatly extended APTT and/or PT. This of course can only be Centrifugation times of 15 or more minutes can create a
accomplished if the sample has remained in the primary bottleneck in the laboratory and delay result turnaround
collection tube. A recommendation to perform routine in- time. Shorter centrifuge times at 1500 g are acceptable for
spection of samples destined for platelet function testing, routine coagulation tests if testing is performed on fresh
including that with the PFA-100®, can also be supported. samples immediately postcentrifugation, but only when
Once a sample has been processed, fibrin clots may be there are no subsequent test requirements, thereby ensuring
visible in the plasma. These may be seen in fresh samples, but that plasma will not be frozen or processed for additional
also may appear in aliquots after a freeze–thaw cycle. Fibrin assays.54 Another means to reduce the time needed for
clots have the appearance of an opaque or gelatinous globule centrifugation, but still achieve a PPP, is to increase the
or strands of variable size (see ►Fig. 2). If suspected, their relative centrifugal force (RCF) used. Using centrifugal forces
presence may be confirmed; for example, if present, they can greater than 1500 g are generally discouraged as this may
often be fished out of the sample with wooden applicator induce platelet activation and lysis of red blood cells.54 To the
sticks (see ►Fig. 2). These fibrin clots may affect results, and contrary, several studies have reported no adverse effect on
are a cause of specimen rejection of a sodium citrate plasma routine coagulation testing, such as APTT, PT, and fibrinogen,
sample. Fibrin clots may result from activation of the sample if centrifuged at high speed (i.e., 11,000 g) with short (i.e.,
before analysis. A shortened APTT result, below the reference 2 minutes) durations.55,56 It has been cautioned, however,
range, may serve as a flag for close inspection of the sample that samples spun in this manner should be tested within
tube for the presence of a fibrin clot. 10 minutes if sampled from the primary tube or promptly
Most coagulation-based tests, including PT, APTT, and aliquoted to a secondary tube, to avert the drift of platelets,
clotting factor assays, are performed on plasma derived which cling to the side of the tube at high RCF, back into the
from once-centrifuged samples. In general, samples should plasma.57 It can also be noted that the latest guidelines for LA
be centrifuged to achieve PPP, such that the postcentrifuga- testing recommend centrifugation speeds of 2000 and
tion plasma contains <10  109 (10,000/µL).10 The residual 2500 g for sample processing by double centrifugation
platelet count of processed samples can be easily verified (see also below).58
using an automated cell counter. Adequacy of centrifugation Centrifugation should ideally occur at ambient (room)
to achieve PPP should be documented and confirmed at least temperatures (15 to 22°C), but this is sometimes difficult to
annually as part of a quality assurance process. The genera- control in laboratories that process large volumes of speci-
tion of PPP can generally be achieved by centrifuging speci- mens. Nonrefrigerated centrifuges are adequate providing
mens at 1500 gravity (g) forces for no less than 15 minutes.10 they do not overheat. Alternatively, refrigerated centrifuges
To prevent the remixing of plasma and reintroduction of the may be used but should be set to maintain ambient temper-
cellular component, a swing-out bucket (angle) rotor should atures, rather than low temperatures, which can lead to
be used and the brake should not be applied at the end of platelet activation and activation of select clotting factors.53
centrifugation. It has been documented, however, that rou- Nevertheless, refrigerated centrifugation does not appear to
tine coagulation assays such as APTT, PT/INR, and thrombin affect routine coagulation tests when testing is performed
time, are not affected by platelet counts up to 200  109 soon after centrifugation.
(200,000/µL) when testing is performed on fresh Some samples, such as those for LA testing or UFH moni-
samples.52,53 toring, should be double centrifuged (“double spun”), to

Figure 2 Fibrin clots. (A) Visual inspection of a coagulation sample shows a fibrin clot in the tube (arrow). (B) This clot (arrow) can be fished out of
the tube using a wooden stick. However, this is still a cause for sample rejection; as such a clot indicates (partial) clotting has already occurred in
this sample, thereby compromising performance of any subsequent clot-based assay. Subsequent nonclot–based assays may also be affected,
depending on the test being performed.

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al. 583

ensure platelet-depleted preparations.58 A single freeze– FV and FVIII as well as PS activity, lose activity following
thaw cycle will result in lysis of any platelets present in the multiple freeze–thaw cycles.62 In an unpublished study by
postcentrifuged specimen. This may alter results of certain Gosselin, it was demonstrated that most other coagulation
assays, especially those where significant intraplatelet stores factors, specifically FII, FVII, FX, FIX, FXI activities, and anti-
of the analyte exist, such as plasminogen activator inhibitor-1 thrombin, protein C (PC), VWF, and plasminogen activities are
antigen. In the case of LA, the freeze-thawed platelets release stable through multiple freeze–thaw cycles.
phospholipids which may mask any LA present in samples
and lead to false-negative test results. The process of double
Considerations When Using Laboratory
centrifugation is performed as follows: the original specimen
Automation Modules or Systems
tube is centrifuged using standard techniques, using forces
around 1500 to 2000 g.10,58 Plasma is carefully removed using Several clinical pathology laboratories have implemented
a plastic disposable pipette making certain not to disturb the different elements or levels of laboratory automation in an
buffy coat layer and transferred into a nonactivating aliquot effort to improve quality, reduce turnaround times, save
tube. This second tube is capped and centrifuged again using money, and enhance staff productivity. To this end, several
standard techniques, and again using similar gravity forces. in vitro diagnostics manufacturers are developing or have
Plasma from this second tube is removed using care to not introduced laboratory automation solutions and instrument
disturb any pellet of cellular material that may be present in features, with a variety of different capacities.63 A fully
the bottom of the tube and transferred to a nonactivating automated system is one that can perform most laboratory
aliquot tube.6 As all plasma-based hemostasis tests can safely tasks without human interaction, while a modular approach
be performed on “double-spun” material, it is prudent to automates only targeted functions. Modular devices are
institute this process as a general laboratory policy for any especially well suited for automating front-end processes
plasma that will be frozen before testing. Use of micropore such as labeling, centrifugation, sorting, and aliquoting speci-
filters, such as 0.2-µm filters, to achieve PPP is not recom- mens. Due to the many preanalytical variables associated
mended. The passage of plasma through a micropore filter with sample collection, processing, and transportation, spe-
may result in the selective removal of several factors, includ- cial care must be taken when adapting samples for hemosta-
ing VWF, FVIII, FIX, and FV.59 Lastly, some tests require sis assays to automated lines.9 Some of the following potential
additional special differential processing (e.g., platelet func- or realized capabilities of automated systems and instrumen-
tion testing, as previously noted). tation should be considered. Certain types of hemostasis
Following centrifugation, samples should be inspected for samples, however, such as those for platelet function analysis,
the presence of potentially interfering substances, such as are not appropriate for automated modules or systems.
hemolysis, icterus, or lipemia as well as the presence of fibrin Centrifugation may be a component of some automated
clots. lines, which may allow centrifugation of 100 samples at a
time. Issues surrounding on-line sample centrifugation
would be the same as those discussed above, specifically
Controlled Thawing of Previously Frozen
using speeds to achieve PPP but prevent platelet activation
Plasma Samples
and cell lysis. An additional issue to be considered in the
Previously frozen samples should be rapidly thawed in a 37°C automatic processing of primary blood tubes is the potential
water bath for 5 to 10 minutes or until completely thawed.10 dishomogeneous separation of citrated plasma. Lippi et al
Close monitoring during this time is necessary to avoid showed that PT is significantly shortened and fibrinogen
inadequate or excessive incubation at 37°C. Sample integrity values significantly higher in the lower than in the upper
may be compromised if samples are either not completely part of the tube, whereas results of APTT are mostly un-
thawed or if maintained too long at 37°C. Furthermore, water changed throughout the tube.64 As such, it seems reasonable
baths must be properly maintained to make certain they are to suggest that citrate plasma should be removed from the
not inadvertently upheld at a higher temperature because collection tube and appropriately mixed before analysis or
this may lead to deterioration of coagulation factor activities aliquoting.
and spurious coagulation test results. Once samples are Some systems include a “point-in-space” arm that samples
thawed, it is imperative that they are thoroughly and ade- directly from the automation line rather than requiring that
quately mixed before testing (e.g., by means of a vortex, use of the sample be transported to the instrument. Point-in-space
a rocker, or adequate end-over-end conversions). sampling is more time-efficient and can therefore hasten
the time between sample processing and analysis, which may
Sample Stability of Previously Frozen Samples be better suited to samples with limited stability, such as
It is generally accepted that samples for coagulation testing samples for hemostasis testing. Cameras may be used on
can safely undergo one freeze–thaw cycle, but multiple some lines to determine fill volume and also to detect optical
freeze–thaw cycles may affect the functional portion of interferences such as hemolysis, icterus, and lipemia. Some
hemostasis proteins causing variations in activity results. systems provide a liquid level sense to confirm fill volume
Exceptions to this rule may be FVIII, which has been reported when a primary tube is sampled. Sysmex® (Siemens Health-
to lose activity after one such cycle and FXI which may care Diagnostics Inc., Marburg, Germany) has introduced a
increase activity.60,61 It has been demonstrated that both feature on their new coagulometer that allows detection of

Seminars in Thrombosis & Hemostasis Vol. 38 No. 6/2012

584 Sample Processing, Transportation, and Storage in Hemostasis Testing Funk et al.

hemolysis, icterus, and lipemia, by measuring the sample 11 van Geest-Daalderop JH, Mulder AB, Boonman-de Winter LJ,
simultaneously at multiple wavelengths; 340, 405, 575, and Hoekstra MM, van den Besselaar AM. Preanalytical variables and
660 nm.65,66 Based on the specific interference detected, off-site blood collection: influences on the results of the pro-
thrombin time/international normalized ratio test and implica-
analysis is performed using an appropriate wavelength to
tions for monitoring of oral anticoagulant therapy. Clin Chem
avoid the discerned interference. Instrumentation Laborato- 2005;51(3):561–568
ries ACL TOP® coagulation analyzer overcomes the optical 12 Clinical and Laboratory Standards Institute. Tubes and Additives
interference of hemolysis, icterus, and lipemia by measuring for Venous Blood Specimen Collection; Approved Standard. 5th ed.
clot detection at a wavelength above 600 nm. Sample analysis CLSI Document H1–A5. Wayne, PA: Clinical Laboratory Standards
Institute; 2003
using wavelengths that do not interfere with hemoglobin,
13 Clinical and Laboratory Standards Institute. Platelet Function
however, do not account for the shortening in clotting times
Testing by Aggregometry; Approved Guideline. CLSI Document
that may occur in hemolyzed samples due to release of H58-A. Wayne, PA: Clinical Laboratory Standards Institute; 2008
intracellular and thromboplastic substances that occur with 14 Cattaneo M. Light transmission aggregometry and ATP release for
lysis.67 the diagnostic assessment of platelet function. Semin Thromb
Hemost 2009;35(2):158–167
15 Harms CS. Coagulation pretesting variables and quality control. In:
Conclusion Triplett DA, ed. Laboratory Evaluation of Coagulation. Chicago, IL:
ASCP Press; 1982:350–366
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