Anda di halaman 1dari 37

The Importance of Pre-Analytical

in Hemostasis Testing
Dr. Yetti Hernaningsih, dr., Sp.PK(K)
Clinical Pathology Department, Faculty of
Medicine, Universitas Airlangga

17th ASEAN Congress of Clinical Laboratory Medicine


April 25th, 2018

1
Types and potential outcomes of laboratory
errors

2 2
Lippi G & Favaloro EJ. Quality in laboratory hemostasis and thrombosis. 2 ed., 2013
nd
Types and rates of error in the three stages of the laboratory
testing process

3 3
Plebani M. Clin Chem Lab Med 2006;44(6):750-759
The laboratory cycle

4
Olson JD. Quality in laboratory hemostasis and thrombosis. 2nd ed., 2013
Salvagno GL, Lippi G, Bassi A, Poli G, Guidi GC.
(J Eval Clin Pract 2008;14:351–353)

Recent data have determined that preanalytical


problems can be identified in up to 5.5% of
coagulation specimens,

the more frequent being samples not received in the


laboratory after physician’s order (49%), hemolysis
(20%), sample clotting (14%), and inappropriate
blood-to-anticoagulant ratio (14%)

5 5
Leading causes of preanalytical errors

6
Lippi G & Favaloro EJ. Quality in laboratory hemostasis and thrombosis. 2nd ed., 2013
Pre-Analytical Factors

7
Sample Collection
• Tubes should be made of non-activating surfaces
(e.g. siliconized glass or polypropylene)
• A winged-device, syringe, and vacutainer system
can be used to draw blood samples for coagulation
testing
• Syringe  small volume (<20 ml) is recommended
and blood should be added immediately into the
citrated tube and mix well
• Winged needle non-activating and non-additive
discard tube is needed to fill the dead space
8
Sample Collection
•Clean venipuncture with minimal stasis.
Do not prolonged tourniquet application. The
tourniquet may be applied for up to 1 minute.
Tourniquet causes venous stasis, which falsely
elevates the concentration of large molecules
(vWF/FVIII)

•Prolonged use of a tourniquet or considerable


manipulation of the vein by needle, may develop a
clot in vitro, which the blood is slow to fill the
collection container

9
About citrate?
•Most samples referred for coagulation testing must
be drawn into citrate-based anticoagulant tubes
(generally 105-109 mM or 129 mM sodium citrate,
also referred to as 3.2% or 3.8%), respectively

•CLSI guidelines favor the use of the lower citrate


concentration, except for specific applications.

10
About citrate?
• Specimens collected in 129 mM (3.8%) buffered sodium
citrate may overestimate the PT and APTT and
underestimate fibrinogen if the normal range is based on
3.2% citrated samples

• Conversely, samples collected into 129 mM (3.8%) citrate


may provide a more stable sample for assessing antiplatelet
(eg, aspirin) therapy response using the PFA-100

11
About citrate?
• Ensure that citrate tubes are completely filled and
comply with a 9:1 blood to anticoagulant ratio. A
minimum 90% is recommended unless otherwise
indicated by manufacturer or internal validation

• The APTT test is the most sensitive to variations in


the final citrate concentration
Variables Lengthens (%) Underfilling (%)
APTT 3 10
10 20
PPT 5 20
15 30 12
Anticoagulant/Blood Ratio
•The presence of significant anemia has not been
shown to influence test result
•Adjust the volume of the citrate anticoagulant
when collecting samples from patients with
hematocrit of >0.55 L/L (55%)
•As recommended by the NCCLS,

Anticoagulant volume(ml) =
blood volume (ml) x [100 – hematocrit(%)]
[595 – hematocrit (%)]

13
Needle gauge
•Use 19 – 21 gauge needle. Avoid smaller (> 25
gauge) or bigger needles (< 16 gauge)

•>25 gauge:
As the slower rate of blood flow through the small
bore may induce clotting or activation of the
sample

•Very large bore needle (<16 gauge):


May induce hemolysis of the sample due to
turbulence of flow through the needle

14
The way to mix
• Samples should be mixed thoroughly (but gently) by 3 to 6
end-over-end tube inversions to ensure adequate mixing of
test sample with anticoagulant and to prevent sample
clotting.

• Conversely, too vigorous mixing (eg, by shaking of tubes)


might lead to in vitro hemolysis or spurious factor activation
resulting in false shortening of test clotting times and even
possible false elevation of clotting factor activity (eg, FVII).

15
How the sample ordering?
• If multiple evacuated tubes will be collected, the
coagulation tube should be the first tube drawn
• If only one evacuated tube will be collected, the
blue stopper tube can be the only tube drawn and
a discard tube is not necessary
• If using an evacuated tube collection system, the
tube for hemostasis testing should not be collected
following collection of an additive tube
16
About drawing sample?
• The old dogma that the first collection tube should be
discarded may not generally be required, as evidence for
differential effects on coagulation assays are lacking

• Nevertheless, a discard tube is needed if the sample is


drawn using a winged collection with variable tubing
length so air in the tubing is not introduced into blood
collection tubes leading to under-filling

17
About drawing sample?

• Tubes should be adequately filled (to the mark noted on the


tube if provided) or to no less than 90% of this total volume.
Under-filling may cause significant sample dilution and may
also provide falsely prolonged clotting times due to the excess
calcium-binding citrate present
• This effect depends on the citrate concentration, the tube
size, and the test performed being more pronounced with
3.8% citrate tubes and small volume (pediatric) collection
tubes
18
The effect of tourniquet
• Application of a tourniquet for 3 minutes vs 1
minute
Value (%) Effect Variables

3.1 shortening PPT

10.1 increase fibrinogen

13.4 increase D-dimer

10.6 increase FVII activity

10.2 increase FVIII activity

19
Influence of stress
• In situation of stress (a child is very tearful):
vWF
Factor VIII
Fibrinogen
may increase, as these are acute phase reactan
proteins

•This may cause spurious shortening of the APTT or


bring low levels of these factors into the normal
range resulting in a missed diagnosis, for example
of hemophilia or von Willebrand disease

20
Pre-Analytical Factors

21
Transportation
• Sample should be preferably delivered non
refrigerated, at room temperature (18 – 25oC) as
short a time as possible

• Whenever delay in transport is expected , local


centrifugation and separation of plasma followed by
freezing and frozen transport of the plasma should
be considered

• The effect of freez—thawing of plasma might


determine the loss of some labile factor (FV, FVIII)
22
Transportation
• Transportation of whole blood sample in ice should
NOT be done due to possible cold activation of
platelet and FVII, loss of FVIII and vWF

• When transporting via a pneumatic system, protect


tubes from excessive agitation or vibration

23
Transportation
• Ideally, testing for routine coagulation tests like the
PT and the APTT should be accomplished within 4
hours of collection, although allowable tolerances
may be greater than this

• However, APTT testing for unfractionated heparin


monitoring should preferably be processed within 1
hour due to the potential for heparin neutralization
by platelet releasates.
24
Transportation

• Extremes of temperature (ie, both refrigerated or


high) should be avoided

• Delays in transport may affect in particular the


labile factors (FV, FVIII), leading to prolonged
clotting times and in vitro loss of factor activity

25
Pre-Analytical Factors

26
Processing
• To maintain the highest level of specimen integrity, citrated
blood samples should ideally be processed within the first
hour after collection

• Check for the presence of clot either by gentle inversion or by


using wooden sticks

• Centrifuge the citrated whole blood samples to produce a


platelet poor plasma with platelet count of <10,000/ul (<10 x
109/L). Residual platelet count can be verified using an
automated hematology cell counter

• The use of filters to obtain platelet poor plasma is NOT


recommended

27
Processing
• Coagulation tests when testing is performed soon
after centrifugation. Centrifugation should ideally be
at 1500 g for a minimum of 10-15 minutes, with
rotor swing out bucket

• Double centrifugation can be performed to ensure


the plasma is platelet-poor

• Using centrifugal forces greater than 1500 g are not


recommended as this may induce platelet activation
and lysis of RBCs

28
Processing
• The use of centrifuge breaks should also be
avoided to avoid remixing of test samples, since
there is a potential for hemolysis and platelet
contamination, which may subsequently affect
most hemostasis assays

• Samples that cannot be tested within 4 hours


should be centrifuged and the plasma aliquot
frozen

29
Pre-Analytical Factors

30
Variables Stability (hours) Temperature (0C) Sample can be Additional
stored information
PT/INR 24 room WB or in capped tube
centrifuged with and either
plasma on top of uncentrifuged or
the cellular centrifuged
elements
APTT do not 4 room
contain UFH
Sample 1 or Release PF4 from
containing UFH 4 (after platelet in vitro
centrifuge within will neutralize of
1 hour) heparin
Vit K dependent 24 room
factor

Protein S activity 8

Fibrinogen, 7 days
protein C,
antithrombin
activity 31
Storage
• If samples for coagulation testing are stored frozen,
they should not be maintained in freezer that has
automatic defrost cycles. The use of frost-free
freezers for patient samples is acceptable

• Storage as whole blood at cold temperatures (2-8o C)


is NOT recommended. Refrigeration of
separated/aliquoted plasma for up to 4 hours is
acceptable for APTT and other coagulation assays but
NOT for PT assay

• If testing cannot be completed as described above,


platelet-poor plasma (platelet-free plasma for LA
testing) should be removed and frozen at ≤ -20o C for
two weeks or at ≤ -70o C for longer storage
32
Storage
• Prior to testing, previously frozen plasma samples
should be thawed in 370C water bath for
approximately 5 minutes or until completely
thawed
• The vWF activity assay tends to decrease to a
greater extent than the antigen assay in response
to cold whole blood storage

33
Causes for Sample Rejection

1. Clotted Samples
2. Samples in wrong anticoagulant
3. Inappropriate blood fill volume
4. Mislabeled or unlabeled samples

34
Take home messages
• Pre-analytical issues in hemostasis testing are an important cause
of diagnostic error and can lead to significant adverse clinical
events

• Sample collection must be proper in vein puncture, needle bore,


blood: anticoagulant ratio, tourniquet application

• Transportation should be in room temperature, as short time as


possible

• Samples were processed for centrifugation in 1500 g not less than


15 minutes, at room temperature

• Storage stability depends on variables and temperature. Plasma


frozen at ≤ -20o C for two weeks or at ≤ -70o C for longer storage
35
References
• Olson JD. General quality planning in the hemostasis laboratory. In: Kitchen S, Olson JD,
Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed. Wiley-
Blackwell. New Delhi, India, 2013
• John D. Olson1,2Lippi G & Favaloro EJ. Causes of errors in medical laboratories. In: Kitchen
S, Olson JD, Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed.
Wiley-Blackwell. New Delhi, India, 2013
• Funk D (Adcock). Sample integrity and preanalytical variables. In: Kitchen S, Olson JD,
Preston FE (eds.). Quality in laboratory hemostasis and thrombosis. 2nd ed. Wiley-
Blackwell. New Delhi, India, 2013
• Favaloro EJ, Funk DM (Adcock), Lippi G. Pre-analytical variables in coagulation testing
associated with diagnostic errors in hemostasis. Labmedicine. Februaru 2012;43(2)
• Plebani M. Errors in clinical laboratories or errors in laboratory medicine? Clin Chem Lab
Med 2006;44(6):750-759
• Salvagno GL, Lippi G, Bassi A, Poli G, Guidi GC. Prevalence and type of pre-analytical
problems for inpatients samples in coagulation laboratory. J Eval Clin Pract
2008;14:351–353.

36
THANK YOU FOR YOUR
ATTENTION

37