Collection, transport, and processing of blood specimens for testing plasma based coagulation assays

Collection, Transport, and Processing of Blood Specimens
for Testing Plasma-Based Coagulation Assays
Dr. Tamer Soliman
CLSI H21-A5

Why it is important?
A procedural guideline for the collection, transport, and processing of specimens for
plasma-based coagulation is necessary, as many preanalytical variables may affect
test results (e.g. concentration and volume of anticoagulant or additive; specimen and
sample storage time and temperature).
Because important diagnostic and therapeutic decisions are based on the results of
hemostasis assays, a procedural guideline for the collection, transport, and processing
of specimens for the general performance of plasma-based coagulation is warranted.
Collection, Transport, and Processing of Blood Specimens
for Testing Plasma-Based Coagulation Assays
CLSI H21-A5

Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays
PROCESSINGCOLLECTION TRANSPORT STORAGE
2 3 41

Specimens should be collected, labeled, handled, and stored
in a manner that respects patient privacy
with respect to their medical records and medical data.
Specimen Collection

1. Specimen Labeling
Specimen label
should include at least the following:
̶ Patient’s full name
̶ A unique identification number
̶ The date and time of collection
̶ Name of the person collecting the specimen
̶ Specimen type if a secondary or aliquot tube is used
(i.e. anticoagulant type vs serum)
̶ Assay(s) performed (optional).
̶ If a bar-coded label is used, attach the label
according to established institutional policy.
Specimen Collection

1. Specimen LabelingSpecimen Collection
Request Form
The following information should be included:
̶ Patient’s full name
̶ A unique identification number
̶ Patient’s Sex
̶ An accessioning number (if appropriate to operations at the facility)
̶ Date of birth
̶ Date and time of collection
̶ Specimen type (whole blood, plasma, etc.)
̶ Assay(s) performed
̶ Relevant clinical and laboratory information
̶ The health care provider’s name
̶ The location (department) where the specimen was collected;
̶ Billing information (if applicable)

Examples of Bad Labeling

Specimen Collection
2. Methods of Obtaining Specimen
Gloves should be worn when handling human tissue or body fluids
Gloves can prevent transmission of bloodborne pathogen infection from a specimen

Specimen Collection
Devices
2. Syringe
3. Vascular Access Device (VAD) (e.g. Vascular catheters)
1. Blood Collection System (Vacutainer System)

Specimen Collection
Devices 1. Blood Collection System (Vacutainer System)

Specimen Collection
• Recommended
• Collects the specimen directly into a glass or plastic
evacuated tube of non-activating containing the
appropriate additive.
• The blood collection system typically includes a
straight needle attached to an adapter used to hold
and pierce the collection container(s).
Straight
needle
Adapter
Holder

Specimen Collection
• A winged device can also be used and may be preferable
in babies, children, patients with small veins, or those
requiring frequent venipuncture.
• A winged set includes a small needle with pliable plastic
phalanges attached to the needle for easy insertion into
the vein. The needle is connected to flexible tubing of
variable length, which can connect to a collection
container.

Specimen Collection
• Syringes could be used for collection, rather than an vacutainer system.
• The syringe must have a non-activating surface (e.g. polypropylene).
• The blood is added to the appropriate volume of anticoagulant within 1
minute of completion of draw
• The specimen immediately and properly mixed
• Hypodermic needle/syringe may increase the risk of hemolysis.
• Larger syringes, may  chance that clotting may occur during the collection.
Devices 2. Syringe
‫اﻷﻃﻔﺎ‬ ‫ﺳﻦ‬ ‫ﺳﺮﳒﺎت‬‫ل‬

Specimen Collection
Devices 2. Syringe

Specimen Collection
• If a syringe is used, the blood can be transferred to an evacuated tube by
removing and discarding the needle and safety device.
• Apply a safety transfer device to the syringe, and puncture the
diaphragm of the rubber stopper. Allow the correct amount of blood to
flow slowly into the tube. Rapid filling may induce hemolysis, potentially
impairing the integrity of the results. The tube should be placed in a
fixture and should not be hand-held when puncturing the top.
• Blood should never be forced into an evacuated tube by exerting
pressure on the syringe plunger. This may cause the tube stopper to pop
off, spraying blood.
Devices 2. Syringe

• Ensure No air leaks
(may cause hemolysis and incorrect draw volumes)
• Avoid blood collection from lines previously flushed with heparin.
• If the blood must be drawn through a VAD, possible heparin
contamination and specimen dilution should be considered.
• In this case, the line should be flushed with 5 mL saline and the first
5 mL blood discarded.
Specimen Collection 2. Methods of Obtaining Specimen
Devices 3. Vascular Access Device (VAD)

• If heparin contamination is suspected, when the specimen
is obtained from a VAD, the option of heparin neutralization
(e.g. heparinase) of the sample should be considered.
• The laboratory should have a policy in place to notify the
client, or address using an algorithm for specimens suspected
of heparin contamination.
• The plasma-based hemostasis test may be repeated after the
specimen is treated with a method that removes or neutralizes
heparin.
Devices 3. Vascular Access Device (VAD)

All anticoagulant-containing collection containers should immediately
be gently mixed by 3 to 6 complete end-over-end inversions to assure
thorough mixing of the specimen with anticoagulant
Mixing
• DO NOT shake or mix vigorously.
• Excessive mixing can cause hemolysis and/or platelet clumping
and activation, leading to erroneous results.
= 1 complete end-over-end inversions

3. Collection Containers and Additives
Composition of Containers (Tubes and Syringes)
Specimen Collection and storage in containers that have non-activating surfaces
Glass
Manufactured from high-quality glass
must be siliconized to render their
surface “non-activating.”
Plastic
Composed of a non-activating
material such as polypropylene
Specimen Collection
Vs

Composition of Containers (Tubes and Syringes)
• When switching between glass or plastic tubes or between tube manufacturers,
careful parallel study should be conducted to assess the effect of the different tube
material or manufacturer on the plasma-based coagulation test result.
• A difference or variability attributable to the different tube container or manufacturer
may not be apparent for specimens with values within the reference range and may
only become apparent when specimens with prolonged values are tested.
• Secondary or Aliquot Tubes should be also composed of a non-activating material
Specimen Collection
Specimen Collection and storage in containers that have non-activating surfaces

Na3C6H5O7 • 2H2O
Anticoagulant
Specimen Collection
Tri sodium citrate
Dihydrate form | buffered or non-buffered
3.2%
109 mmol/L
Other anticoagulants (e.g. oxalate, heparin, or EDTA) are unacceptable
2 available concentrations either 3.8%
129 mmol/L

Laboratories should standardize to one concentration of Na citrate,
as variation of normal ranges may occur between these 2 concentrations (3.2% vs 3.8%).
APTT and PT results tend to vary between citrate concentrations
(especially when results fall outside the reference interval)
As 3.8% Na citrate concentration binds more assay-added Ca2+ than 3.2%,
clotting times tend to be longer in 3.8% vs 3.2% sodium citrate.
CTAD: Use of 109 mmol/L buffered Citrate containing Theophylline, Adenosine,
and Dipyridimole, which may minimize platelet activation, is also acceptable.
Anticoagulant
Specimen Collection

Examples
• 0.45 mol/L Na citrate buffer, pH 4.3, and a strongly acidic citrate additive, which were
developed to stabilize plasma for the measurement of fibrinolytic factors as well other
markers in the clotting system, including soluble thrombomodulin and vWF.
• Na citrate in combination with protease inhibitor(s), such as PPACK (D-phenylalanine-
proline-arginine-chloromethyl ketone), were formulated to protect the integrity of the
plasma sample from protease activity before performing non-routine coagulation assays.
If these special anticoagulants are used for routine assays,
a normal range specific for that tube matrix should be determined.
Anticoagulant
Specimen Collection
More recently, new anticoagulants have been developed that are
also acceptable for the collection of plasma for specific hemostasis assays.

For plasma-based coagulation testing,
the proportion of blood to the liquid Na citrate volume
is
Blood : Anticoagulant Ratio
Specimen Collection
9:1
liquid Na citrate
Blood
Minimum
Maximum
Pediatric (e.g. 2 mL volume) evacuated collection tubes
may be more sensitive to variances in fill volume than
the standard 5 mL tubes such that clinically significant,
yet spurious, INR prolongation was reported with < 90%
fill volume.

For plasma-based coagulation testing,
the proportion of blood to the liquid Na citrate volume
is
Specimen Collection
9:1 Same manufacturer
Same concentration (3.2%)
Different Volumes
4.5 ml : 0.5 ml 1.8 ml : 0.2 ml

Inadequate filling of the collection device will decrease this ratio (< 9:1),
may  clotting times giving inaccurate results.
This effect is greater when samples are drawn into 3.8% citrate as compared to 3.2%
The effect of fill volume on clotting time results may also be dependent on:
• The size of collection tube
• The particular APTT and/or PT reagent
Unless the package insert indicates otherwise or in-house studies were performed,
90% fill volume is recommended.
Specimen Collection

Citrate Concentration Adjustments
In samples with  HCT, the blood-to-citrate ratio drops below 9:1, this means
Excess citrate for the volume of plasma present in the tube.
 This leads to excess binding of the Ca2+ added to the clotting test reaction
Possible dilutional effect due to the volume of liquid anticoagulant present,
 tending to increase the clotting time.
Specimen Collection
In patients with HCT values > 55%,
the final citrate concentration in the blood should be adjusted

The citrate conc. must be adjusted by removing a portion of the volume of the citrate solution.
C = (1.85 x 10-3) (100 – HCT) (VBlood)
C The volume of citrate remaining in the tube
HCT Patient’s hematocrit
V The volume of blood added (if a 5 mL tube is used, then the volume is 4.5 mL)
1.85 x 10-3 Constant (taking into account the citrate and blood volumes and citrate conc.)
where
Specimen Collection
To calculate the amount of citrate required in the collection tube or syringe, use the following formula:

In common practice, 0.1 mL citrate can be removed for the majority of samples, since the
majority of high HCT values are between 55% and 65%.
This will allow for the laboratory to prepare a number of adjusted blood collection tubes in advance
of the physician order.
• Sample is mixed and processed in the same fashion as the other coagulation samples.
• A NOTE should be added to the laboratory record and patient record stating that the HCT was
elevated and the citrate concentration was adjusted.
Specimen Collection

For hematocrits < 20%
There are No current data available
to support a recommendation for adjusting the citrate concentration
Specimen Collection

4. Needle Gauge
Manufacturers of needles and blood collection sets designed
to eliminate inner surface roughness, which may
contribute to possible hemolysis and may activate clotting in
vitro.
Before obtaining the specimen,
the phlebotomist must determine the appropriate needle
gauge for use based on
• The amount of blood drawn
• Subject age
• Size of the subject’s veins
Specimen Collection

4. Needle Gauge
To obtain blood from an antecubital vein,
18- to 21-gauge needles are ideal,
although smaller needles may be necessary in newborns
and children or in the adult with compromised veins.
18
20
21
25
16
Specimen Collection
Large-gauge needles (16-gauge or larger),
May cause turbulence-induced hemolysis
Small-gauge needles (25 or smaller),
May induce hemolysis or activate platelets, leading to
erroneous results in plasma-based coagulation assay

4. Needle Gauge
Winged blood collection sets,
because of longer path length between vein and anticoagulant, when used in
combination with smaller-gauge needles (25-gauge or smaller),
should be used with caution to avoid platelet and coagulation activation.
Specimen Collection

Specimen Transport
• Transportation of whole blood specimens on ice is not recommended because of
possible cold activation of FVII, loss of vWF, and platelet disruption.
• Extremes of temperature (high and low) during transportation must be avoided
Following phlebotomy, samples should be transported at room temperature
Temperature

Specimen Transport
The acceptable time delay before processing depends on the coagulation test performed.
Ideally, transport to the processing site should occur within 1 hour of collection
PT assay
PTT assay when monitoring UFH therapy
up to 24 hours
(potential for heparin neutralization by platelet factor 4)
< 1 hour if collected on Na citrate
< 4 hour if collected on CTAD
Acceptable Delay (at Room Temp.)Coagulation test
Acceptable
Delay

Specimen Transport
When transporting specimens by pneumatic
tube, specimen tubes should be protected
from vibrations and shock to avoid protein
denaturation and platelet activation through
foaming of the specimen.
Agitation

Specimen Transport
It is important to NOTE the following for every specimen:
• Date shipped
• Date received by the laboratory
• Approximate temperature of the specimen when received

Specimen Transport
In addition,
laboratories may rely upon their own experiences
regarding optimum specimen handling
when dealing with the different types of specimens.

Specimen Processing
Inspection of
Blood clot Centrifugation
Inspection of
Interference

Specimen Processing
Inspection of
Inspection of
Interference
Before centrifugation,
check the whole blood specimen for blood clots
• Gross clot formation by either
• Gentle inversion and observation, or
• Removing the cap and inserting and then removing 2 wooden
sticks to detect the presence of a clot.
• Micro clots, may not be detected by these methods; if present, they
may alter results.

Specimen Processing
 Capped specimen tube
 A swing-out bucket rotor to minimize contamination of the plasma
with platelets and other blood cells
Centrifugation
Inspection of
Inspection of
Interference
Required to produce platelet-poor plasma (platelet count < 10 x 103/μL)
The centrifugal speed and duration must be established by the laboratory,
but the most common conditions are Speed 1500 g
Duration for no less than 15 minutes
Temperature at room temperature
Higher speed and shorter duration centrifuges, commonly known as “Statfuge” may be used.

Specimen Processing
Centrifugation
Inspection of
Inspection of
Interference
Again, it is imperative that the speed and duration are tested to determine
the optimum conditions for producing PPP.
The reliability of the centrifugation procedure, to ensure plasma platelet
counts are within acceptable limits, should be validated every 6 months or
after modification of the centrifuge.

Specimen Processing
Centrifugation
Inspection of
Inspection of
Interference
• Double centrifugation of the sample may be performed to ensure that
the sample is PPP.
• Transferring the plasma following the initial centrifugation process to a
plastic centrifuge tube using a plastic pipette, then re-centrifuging the
sample for an additional 10 minutes.
• When aliquoting to a secondary tube, take care to not include the
residual platelets that may have collected at the bottom of the
centrifuge tube.

Specimen Processing
Visible hemolysis, as evidenced by a pink to red-tinge to the plasma, should be noted.
Lysis of RBCs and resultant release of intracellular or membrane components may
cause clotting factor activation. This activation of coagulation factors may impact clotting
time results, whether using an optical or mechanical end-point detection system.
+ Interference with light transmittance
Plasma may be tinged when there is RBCs lysis or when the patient has been administered
a hemoglobin substitute. Samples that appear hemolyzed due to the presence of
hemoglobin substitutes are not a cause of specimen rejection, and these samples should
ideally be tested with an instrument that uses mechanical end-point detection.
Hemoglobin substitute
Inspection of
Inspection of
Interference

Specimen Processing
• Some current instruments using an optical end-point determination may have
problems with samples that are icteric, lipemic, or contain substances that
interfere with light transmission.
• Ultracentrifugation has been suggested in some circumstances; however, there
are no published studies. In a small, un-published study of lipemic samples,
 PT and APTT assays
 Fibrinogen levels by ~ 10 – 15%.
• Until published studies are performed or the laboratory has validated the
procedure, mechanical and/or electromechanical methods for clot detection
should be utilized when possible for these samples that are icteric, lipemic, or
contain substances that interfere with light transmission.icteric lipemic
Inspection of
Inspection of
Interference

Short-Term Storage of Plasma for Coagulation Testing
Specimen Storage

Specimens for PT assays
For whole blood samples avoid mechanical agitation during storage
that may cause spurious elevation of the PT/INR through an unknown mechanism.
Specimen Storage
Centrifugation

In all instances,
Specimens should be stored capped unless sitting for short periods (< 30 minutes)
Specimen Storage
Capping

Room FreezeRefrigerate
Storage at refrigerated temperatures or placing on ice is not recommended,
 May cause cold activation of Factor VII
 alter PT results.
Specimen Storage
Storage Temperature

24Hours
Storage is acceptable for up to 24 hours
from time of specimen collection
Specimen Storage
Storage Duration

24Hours
Sample Integrity during short-term storage:
o May be dependent on the system (thromboplastin reagent, instrument, collection container,
combination) used; therefore, each laboratory should confirm stability with its system.
o Enhanced sample integrity if samples are centrifuged immediately after blood collection.
Specimen Storage

24Hours
If the patient is on both heparin and a coumarin-based oral anticoagulant therapy,
the PT may vary with time of storage of the specimen, unless the PT reagent used
for specimen testing contains a heparin neutralizer.
Specimen Storage

Specimens for aPTT assays
Specimens can be maintained un-centrifuged or centrifuged
from non-heparinized patients
Specimen Storage
(Preferable)

Specimens for aPTT assays from non-heparinized patients
un-opened tube
Specimen Storage

kept at room temperature
Placing whole blood specimens directly on ice or in an ice water bath
simulates refrigeration and is also not recommended.
Specimen Storage

(better stored at room temperature)
Samples that will be subjected to testing of APTT — (especially includes analysis of F VIII
or vWF activity, or for evaluation of vWF multimers) — Should NOT be stored refrigerated
Specimen Storage

COLD temperatures may lead to a gradual loss of vWF and factor VIII activity,
potentially leading to a spurious diagnosis of vWD, vWD misclassification,
or spurious diagnosis of factor VIII deficiency.
Specimen Storage

4Hours
Storage for up to 4 hours
from time of specimen collection
Specimen Storage

Specimens for aPTT assays
If laboratories choose to maintain APTT samples for > 4 hrs
before testing, in-house studies should be performed.
These studies should evaluate normal and abnormal APTT
samples, and the study should include factor activity (V and
VIII) measurements.
from non-heparinized patients
>4
Hours
Specimen Storage

Specimens for aPTT assays from heparinized patients (UFH)
Specimen Storage
• Should be kept at room temperature
• Should be centrifuged and tested within 1 hours from time of specimen collection.
• The plasma tested within 4 hours from time of specimen collection.
If agitation of the specimen is likely after centrifugation, such as transportation to a
remote testing site, the plasma should be removed within 1 hour of collection and
tested within 4 hours from the time of specimen collection.
If samples from patients on unfractionated heparin cannot be centrifuged within 1 hour
of collection, the use of CTAD tubes may enhance stability. However, therapeutic
APTT heparin ranges must be determined for that matrix.

Specimens for most other assays TT, Protein C, Protein S and anti-F Xa
Specimen Storage
• Should be kept at room temperature,
• Should be centrifuged and tested within 4 hours from time of specimen collection.
• Although there are limited data to support removal of the plasma from the cellular
component, this is preferred.
for monitoring anticoagulant therapy other
than unfractionated heparin
If laboratories choose to accept specimens maintained for > 4 hrs at room temp. for
these special hemostasis assays, in-house validation studies should be performed.
Specific data regarding stabilities with time at room temp. for many other clot-based,
chromogenic, and immunologic assays are limited (with the exception of FVIII & vWF).

Specimen Storage
Platelet-poor plasma should be removed without disturbing the sedimented cells
(buffy coat) and frozen at – 20 °C or below for short-term storage (up to two weeks)
or – 70 °C or below for long-term storage.
If the testing is NOT completed within 24 hours for PT specimens
and 4 hours for APTT and other assay(s),
• Freezers that require manual defrosting should be used.
• A freezer that undergoes automatic freeze/thaw cycles (frost-free)
is not acceptable, as it may result in cold activation of F VII.

Specimen Storage
Platelet-poor plasma should be removed without disturbing the sedimented cells
(buffy coat) and frozen at – 20 °C or below for short-term storage (up to two weeks)
or – 70 °C or below for long-term storage.
If the testing is NOT completed within 24 hours for PT specimens
and 4 hours for APTT and other assay(s),
For testing, Frozen plasma specimens should be
• Rapidly thawed at 37°C, gently mixed and tested immediately.
• Resuspend precipitations by thorough mixing immediately after thawing & before testing.
• Mixing is critical before testing, as precipitation of certain proteins may occur with freezing.

Whole Blood Processed Plasma Aliquoted
Room Refrigerated Frozen Room
aPTT for UFH
aPTT
aPTT for vWF & FVIII
PT
Refrigerated -20°C -70°C
Others
Up to 24 hrs
4 hrs
1 hr
4 hrs
4 hrs
Up to 24 hrs
4 hrs
4 hrs
4 hrs
4 hrs
2 weeks
4 hrs
4 hrs
4 hrs
4 hrs
12 month
2 weeks 12 month
2 weeks 6 month
2 weeks
PPP PPP
= Unacceptable
= Unknown
PPP = Platelet-Poor Plasma

In contrast to the abundant clinical data available in support of the short-term
storage of plasma for coagulation testing, data demonstrating long-term storage
of plasma are not supported by well-documented scientific studies.
Specimen Storage
Long-Term Storage of Plasma for Coagulation Testing
Published literature indicates that the majority of clinical data available was
collected in blood banks and transfusion centers, where stability testing of a
limited number of coagulation assays (FV, FVIII, FXI, and antithrombin activity)
was performed on thawed plasma previously collected in CPDA and CPD
anticoagulant rather than in Na citrate.

From Woodhams B, Girardot O, Blanco MJ, Colesse G, Gourmelin Y. Stability of coagulation proteins in frozen plasma. Blood Coag
Fibrinol. 2001;12(4):229-236. Reprinted with permission from Lippincott Williams & Wilkins.)
Summary of Data Showing
the Variation of Results
From the Baseline Value
With Time of All Analytes
Measured

• Clotted
• Collected in the wrong anticoagulant
• Wrong blood/anticoagulant ratio (under- or overfilled)
• Un-labelled or Mis-labelled
• Collected or stored in containers with other than a non-activating surface
• Severely hemolyzed
• Whole blood samples that were frozen before processing
• Whole blood or plasma samples for PT testing that were refrigerated before testing
• Whole blood samples for vWF or FVIII analysis that were refrigerated before processing.
Causes for Specimen Rejection

Collection, transport, and processing of blood specimens for testing plasma based coagulation assays

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