Br J Haematol - 2020 - Baker - Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.pdf

Guidelines on the laboratory aspects of assays used in
haemostasis and thrombosis
Peter Baker,1
Sean Platton,2
Claire Gibson,3
Elaine Gray,4
Ian Jennings,5
Paul Murphy,6
Mike Laffan7
and On behalf
of British Society for Haematology, Haemostasis and Thrombosis Task Force
1
Oxford Haemophilia and Thrombosis Centre, Oxford University Hospitals NHS Foundation Trust, Oxford, 2
Haemophilia Centre,
Barts Health NHS Trust, London, 3
Specialist Haemostasis, Cambridge University Hospitals NHS Foundation Trust, Cambridge,
4
Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Controls, Hertfordshire, 5
UK NEQAS
for Blood Coagulation, Sheffield, 6
Department of Haematology, the Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle
upon Tyne, and 7
Centre for Haematology, Imperial College and Hammersmith Hospital, London, UK
Summary
This guideline amalgamates and updates two previous guide-
lines1,2
published on behalf of the British Society of Haema-
tology (BSH).
Methodology
The guideline was compiled according to the BSH process at
https://b-s-h.org.uk/guidelines. The writing group produced
the first draft of the manuscript which was revised by wider
members of the Haemostasis and Thrombosis Task Force
before being reviewed by an extended group of UK haema-
tology medical and scientific members of the BSH; amend-
ments were made accordingly. The ‘GRADE’ system (http://
www.gradeworkinggroup.org) does not apply (due the lack
of clinical trials to support the best practice recommenda-
tions) and was therefore not used. Appendix 1 details the lit-
erature search process undertaken.
General introduction
This guideline is intended to help clinical laboratories per-
form high quality valid assays for the basic procoagulants
and anticoagulants as part of a routine diagnostic service.
Areas that overlap with or have been included in other BSH
(https://b-s-h.org.uk/guidelines/) or United Kingdom Hae-
mophilia Centre Doctors Organisation (UKHCDO) (http://
www.ukhcdo.org/guidelines/) guidelines have been omitted,
including guidance on: heparin-induced thrombocytopaenia
(HIT); lupus anticoagulant (LA) testing; D-dimer assays; pla-
telet function testing; diagnosis of von Willebrand disease
(VWD); measurement of factor replacement in haemophilia
A and B; monitoring of anticoagulants [vitamin K antago-
nists (VKA) and direct oral anticoagulants (DOAC)]; and
global assays of haemostasis (e.g. TEG, ROTEM, thrombin
generation).
Preanalytical variables
Preanalytical errors account for the majority of errors in the
haemostasis laboratory and it is essential that they are well
understood and minimised.3
To produce accurate and mean-
ingful results and increase quality and standardisation within
haemostasis laboratories, correct procedures must be fol-
lowed (Table I). Phlebotomy staff must be well trained and
laboratory staff must understand the tests used and potential
sources of error. Result interpretation requires understanding
of the potential effect of patient factors on the assays.
The correct filling of tubes (especially in patients with a high
haematocrit), effects of haemolysis, and presence of anticoagu-
lant drugs all need to be considered. Before processing, sam-
ples should be inspected visually to ensure they are labelled
correctly, correctly filled (using manufacturers guides) and not
clotted or haemolysed. Samples should be rejected if <90%
filled, unless shorter collection volumes have been validated
for a particular test.4
Gross icterus and lipaemia may affect
results by interfering with optical absorbance or impeding light
transmittance, but analyses using mechanical end points are
not affected. The use of analysers with automated spectropho-
tometric detection of haemolysis, icterus and lipaemia (HIL)
and with the ability to check fill volume may improve stan-
dardisation and quality control as well as prevent over or
under-rejection of haemolysed samples.5–7
Guidelines from the Clinical & Laboratory Standards Insti-
tute (CLSI) suggest samples with gross haemolysis should
not be used because of possible clotting, factor activation
and interference with end-point measurement.8
They recom-
mend rejecting all haemolysed samples but the degree to
which the haemolysis has a significant effect on results varies
according to the assay being performed.9–12
Modern
Correspondence: BSH Guidelines Administrator, British Society for
Haematology, 100 White Lion Street, London, N1 9PF, UK.
E-mail: bshguidelines@b-s-h.org.uk
guideline
ª 2020 The Authors. British Journal of Haematology published by British Society for Haematology
and John Wiley & Sons Ltd. British Journal of Haematology, 2020, 191, 347–362
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
First published online 14 June 2020
doi: 10.1111/bjh.16776
13652141,
2020,
3,
Downloaded
from
https://onlinelibrary.wiley.com/doi/10.1111/bjh.16776
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
(https://onlinelibrary.wiley.com/terms-and-conditions)
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

coagulometers now provide optical quantitation of free hae-
moglobin which removes any subjectivity in sample assess-
ment and rejection. Therefore it may be necessary to locally
validate the impact of haemolysis and define rejection cut-
offs.13
Causes of in vivo haemolysis still need to be excluded
in patients when repeated flagging occurs.
We suggest that a coagulation screen, prothrombin time
(PT) and activated partial thromboplastin time (APTT) be
performed on all samples referred for haemostasis assays.
This serves as confirmation of the quality of the sample and
may also detect the presence of anticoagulants if this is not
known from the clinical details provided. However the sensi-
tivity of PT and APTT reagents to anticoagulants varies and
these tests may not detect concentrations that can interfere
with other assays; so if there is doubt about their presence
an assay using specific calibrators (if known) should be per-
formed.14
If indicated, the presence of a normal thrombin
time (TT) effectively excludes the presence of a thrombin
inhibitor or significant contamination with unfractionated
(but not low molecular weight) heparin. Commercial prod-
ucts such as DOAC-Stop (Haematex, Hornsby, NSW, Aus-
tralia),15–17
DOAC-Remove (5-Diagnostics, Basel,
Switzerland),18
20 mg/ml of activated charcoal19
or Adex-
anet alfa20
have been shown to remove the effect of DOAC.
However, if used, results should be interpreted with caution
as there may be differing limits to the concentration of the
DOAC they can remove.15,17
Most routine tests should be performed within 4 h of col-
lection with the exception of APTT for monitoring unfrac-
tionated heparin (UFH) which preferably should be tested
within 1 h and the PT which has a stability of 24 h.9
There
is now more evidence that the 4-h window can be extended
for many tests; however, if local processes make this neces-
sary validation is required (Table II).3,21–23
Other patient factors have been shown to interact with
haemostasis testing. The list is too extensive to report here but
examples include the presence of rheumatoid factor or parapro-
teins that may interfere with clot-based assays (i.e. routine PT/
APTT) and acquired anti-mouse antibodies that have been
reported to cross react with immunoassays (for example D-
dimer) with the potential to produce erroneous results.6,24–26
Recommendations
Laboratories should minimise the effects of preanalyti-
cal variables and be aware that inappropriate sample
handling and testing can lead to incorrect results
being generated.
Coagulation screens should be performed prior to spe-
cialist haemostasis assays for investigation of haemo-
static disorders. Screening tests including the use of a TT
may detect some anticoagulants but specific assays may
also be required to definitively exclude contamination.
Table I. Summary of recommendations for blood collection, han-
dling and storage.
Blood collection
Perform clean venepuncture with minimal stasis.
Use a 21-gauge needle (19 gauge may be used in adults with
good veins, 23 gauge may be required for infants). If a butterfly
is used and the coagulation tube is the first tube drawn then a
discard tube should be used.
Do not use heparin-contaminated venous lines. Where this is
unavoidable because of poor venous access, flush the line with
saline and use a labelled discard tube.
Use 0105–0109 mol/l (32%) tri-sodium citrate (9 volumes
blood to 1 volume anticoagulant).
Adjust volume of citrate if haematocrit is over 055 using pub-
lished algorithm.155
Use plastic or siliconised glass tubes.
Ensure correct order of drawing (generally the coagulation tube
should be the first drawn).
Ensure the correct filling of citrate tubes as per manufacturer’s
recommendation (for example against minimum fill line) and
mix immediately by gentle inversion 3–6 times. Never transfer
blood from one tube to another.156
Ensure samples are labelled correctly.
Sample handling
Whole blood samples should be transported at room temperature
to the laboratory as soon as possible, within 1 h if possible.
Prior to centrifugation samples should be examined for correct
fill and clots.
Centrifugation and analysis should be undertaken as soon as pos-
sible. Optimally centrifuge at 18–25°C for 10 min at 1500–2000 g
in a centrifuge that has a rotor with swing out buckets. Centrifu-
gation for 5–10 min (2000 g) is acceptable if only routine assays
(for example PT/INR/APTT) are to be performed. Either method
should be locally validated to generate platelet poor plasma
(10 9 109
/l).3,9
After centrifugation samples should be examined for haemolysis,
icterus and lipaemia.
Storage and preparation
Most tests should be performed within 4 h of sample collection.9
For plasma that is to be frozen the sample should be stored in a
screw cap polypropylene tube with an ‘O’-ring. Samples should
be stored below 70°C, although storage below 24°C can be
used for short periods (up to 3 months).157
Storage below
70°C is essential for periods longer than 3 months. Freezers
with auto-defrost cycles must not be used for storing haemostasis
samples. Frozen plasma shipped to another laboratory should be
sent on dry ice to ensure that it remains frozen during transport.
Prior to analysis, thaw in confirmed 37°C water bath for 5 min
or until completely thawed and mix gently and thoroughly by
inversion before testing. Samples should be monitored to avoid
inadequate or excessive incubation at 37°C as either may lead to
loss of clotting factor activity either due to formation of cryopre-
cipitate or heat inactivation.Once thawed, do not refreeze plasma
unless data are available to demonstrate that results are unaf-
fected.157
BAKER et al.
348 ª 2020 The Authors. British Journal of Haematology published by British Society for Haematology
and John Wiley Sons Ltd. British Journal of Haematology, 2020, 191, 347–362
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Techniques
End-point clot detection
Haemostasis assays using a clotting end-point can be per-
formed by photo-optical and electro-mechanical methodolo-
gies, forming the basis of the majority of commercial
analysers. Parameters generated from clot waveform analysis
in photo-optical systems may give additional information
over electro-mechanical systems [examples include its ability
to detect changes in patients with underlying disseminated
intravascular coagulation (DIC) or monitor factor (F)VIII
replacement therapy] but optical systems are also prone to
interfering substances (i.e. lipaemia) so in practice, as these
parameters have not been universally adopted for clinical
use, both systems are acceptable.27,28
The procurement and
validation of commercial coagulation analysers or reagents is
outside the scope of this guideline.
Recommendation
Either electro-mechanical or photo-optical end-point
detection is acceptable in routine automated clot-based
coagulation analysers.
Calibration and control
Regardless of method and target analyte, quantitative assays
rely on a general principle of establishing a calibration curve
using a sample with known concentration, to provide a clini-
cally useful dose–response curve. For many assays, a parallel-
line assay using multiple test dilutions should be employed.
The accuracy and precision of the assay should be deter-
mined for each assay, and these should inform the assay
design for clinical samples (for example whether duplicate
testing is required).29,30
Calibrators and quality control samples
Commercial plasma calibrators, lyophilised or frozen, trace-
able to the relevant World Health Organization (WHO)
International Standards (IS) when available, should be used
as reference standards for quantitative haemostasis assays.
Where IS are available, activities or antigenic values of coag-
ulation factors and inhibitors should be expressed in Interna-
tional Units (iu), except fibrinogen which is routinely
reported in g/l. Normal and pathological quality control
(QC) material obtained from commercial sources or pre-
pared in-house is essential to evidence the quality of perfor-
mance of the assay and should be included in each assay run
or sample batch to ensure adequate assay precision and safe-
guard against reporting invalid results. Commercial QC
material is available for some tests with stated values towards
critical cut-off points (i.e. D-dimers) when not covered by
normal and pathological preparations. Clinical laboratories
should participate in accredited external quality assurance
(EQA) schemes for each analyte to monitor performance and
assess comparability with peer group laboratories.
Calibration/reference curve
When establishing an assay, laboratories should determine
the linear portion of the dose–response curve, which will
require multiple dilutions (at least three dilutions are
required to establish linearity) e.g. serial doubling dilutions.
A wide-ranging dilution curve is desirable as this maximises
the quantitation capability of the assay. However, for some
assays, linearity of responses can only be achieved over a lim-
ited range (Fig 1). In these cases, separate reference curves
should be established for high and low concentrations of the
analyte. Re-dilution of test samples may also be useful. Ide-
ally, a fresh calibration curve should be carried out for each
batch of assays, but a stored calibration curve is acceptable
with prior validation and proof of stability [defined by
acceptable internal quality control (IQC) and EQA perfor-
mance]. Calibration curves should be renewed on a regular
basis, the frequency determined by the local laboratory work-
load, but always when changing reagent lots. Multiple dilu-
tions of test samples should be assayed where one-stage
factor assays are used, to demonstrate parallelism. The cut-
off may be predefined locally by the instrument/reagent com-
bination; however agreement of 20% deviation from each
other is often considered linear or parallel.31
As this value is
sometimes perceived as too high as a single parameter, other
values such as calibrator and test r values, and calibrator to
slope test ratios may be of use to identify sample activation
or the presence of inhibitors; this is not required for chro-
mogenic assays insensitive to this type of interference, or for
fibrinogen activity assays.
Table II. Summary of recommendations for maximum analyte sta-
bility in citrated whole blood at 18–25°C.
Assay Analyte stability
APTT or anti-Xa for monitoring
UFH
1 h (4 h if centrifuged
within 1 h)
APTT 4 h
Factor VIII
Antithrombin
Protein C
Anti-Xa for monitoring LMWH 4–6 h
Factor V 8 h
Protein S activity
Anti-Xa for monitoring of DOAC
APC resistance
Prothrombin time/INR 24 h
D-Dimer
Factor II, VII, IX, X, XI
VWF antigen and activity
Lupus anticoagulant
Fibrinogen 7 days
Protein S antigen
Guideline
349
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Assay detection limits
The limits of detection must be established for each assay. In
clotting assays, a lower indication is obtained from the buffer
blank time. However, to establish an accurate quantitation
limit in clotting, chromogenic and immunoassays, the lower
limit of quantification (LLOQ) i.e. the minimum dilution of
the analyte that reliably gives a value statistically significant
from zero should be determined.30,32
When clinical samples
are tested, results below the quantitation limit should be
reported as being less than this value.
Reference ranges and cut-off values
Normal reference ranges should be established locally with
an appropriate number of healthy normal subjects; appar-
ently normal patients should not be used. A minimum num-
ber of 120 subjects is recommended for establishing reference
ranges.33
Transference of the reference range may be accepted
from ranges quoted by manufacturers or other sources
(where resources are limited) using 20–40 normal subjects,
depending on the required accuracy by defining acceptable
concordance in advance.29,30,33
Reference ranges may vary for
different reagent and analyser combinations. For some assays
age, sex and/or blood group-related ranges may be employed,
and should be considered in the interpretation of results;
examples including D-dimer, free protein S antigen and von
Willebrand factor (VWF) respectively. Pregnancy is also
known to affect levels, particularly of free protein S antigen,
FVIII and VWF. Specific and separate ranges are required for
paediatric populations as differing factors reach adult levels
at differing times.34–36 Generating local reference ranges for
each assay for each age range is likely to be unfeasible for
most laboratories. Published ranges should be considered if
based upon similar analyser/reagent combinations, otherwise
referral to a specialist centre should be considered.37
For data fitting a normal or Gaussian distribution,
mean 196 standard deviation (SD) is a usual definition
employed for a reference range and commonly rounded to
2 SD. If data are known to be skewed then normalisation, for
example by log transformation or the use of non-parametric
evaluation, may be used to calculate the interval.33
Clinical sig-
nificance of results is an important part of data interpretation,
and clinical definitions (e.g. for haemophilia and VWD) do
not always match the statistically calculated reference range.38
(A)
(B)
Fig 1. (A) Schematic representation of a coag-
ulation assay dose–response curve. (B) Graphi-
cal solution of a coagulation assay. Coagulation
times of a reference preparation (diamonds);
coagulation times of a test sample (squares).
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

For some analytes, cut-off values derived from normal and
patient populations are more clinically useful. Receiver oper-
ating characteristic (ROC) analysis is usually required, for
example when studying D-dimer for deep vein thrombosis
(DVT) exclusion.39
If it cannot be generated locally due to
the numbers or resource then the manufacturer’s recom-
mended cut-off should be locally validated on a smaller sam-
ple (20 is often suggested).
Laboratories should report reference ranges with every
assay result.
Recommendations
Reference ranges or clinical cut-off values should be locally
verified for the reagent/analyser combination in use.
Stored calibration curves generated with a different
reagent batch should not be used.
At least three patient sample dilutions should be used
to determine linearity and parallelism in one-stage
factor assays.
Sample and calibrant dilutions should be adjusted so
that the test sample clotting times/absorbance changes
fall within the range of the standard curve.
Standards, calibrators and controls with potencies
traceable to an IS should be used (for example iu/ml
or iu/dl for factors) when available.
At least two levels of internal quality control samples
(normal and pathological) should be used: these can be
from the instrument/reagent suppliers or a third party.
Laboratories must participate in an accredited EQA
scheme, if available, for each analyte and assay type
that they routinely use.
For analytes without an available accredited EQA scheme,
laboratories must consider alternative measures to ensure
accuracy and precision of the assay (e.g. regular local sam-
ple exchange of normal and abnormal samples and/or
participation in shared data interpretation schemes).
Investigation of a bleeding tendency
One-stage clotting assays
Clinical laboratories predominantly use one-stage clotting
assays (OSCA) to measure factor levels based on the ability of
test samples to correct the clotting time of factor-deficient
plasma. PT-based OSCA are predominantly used for FII, FV,
FVII and FX, and APTT-based OSCA for FVIII, FIX, FXI, FXII,
high molecular weight kininogen (HMWK) and prekallikrein
(PRK). An APTT-based OSCA can also be used to measure FII,
FV40
and FX,41
and snake venom-based OSCA assays can be
used to measure FII42
and FX.41
Some rare coagulation factor
variants may only be detected by these alternative assays.40
Prothrombin time reagents can contain heparin neutralisers
and vary in the source of tissue factor (TF) and phospholipids
used. The source and composition of TF can influence
measured levels in some cases43,44
and whether the reagent is
sensitive to direct factor Xa inhibitors (DFXaI)45
or LA.46
Reagents for the APTT vary in the type of contact activator
(e.g. ellagic acid, silica, kaolin), which can influence the sensi-
tivity of the reagent to contact factors (including FXI and
FXII,47
HMWK and PRK48,49
). Modifying the incubation time
of the APTT has been reported as increasing the sensitivity to
contact factor deficiencies and may be of use as a screen in cen-
tres where specific deficient plasmas are not available (subject
to local validation).50
The source and concentration of phos-
pholipid can also influence how sensitive the reagent is to
LA.51
Reagents for APTT do not contain heparin neutralisers
but vary in sensitivity to heparin and DFXaI.45
Commercially available factor-deficient plasmas can be from
inhibitor-free congenitally deficient patients, or immunode-
pleted or immuno-absorbed normal plasmas, and can be lyo-
philised or frozen. Although it may be seen as a cost saving in
underresourced circumstances it is not recommended to use
in-house patient sourced plasma. The deficient plasma should
have 1 iu/dl of the deficient factor (locally confirmed for each
batch), and normal levels (above 50 iu/dl) of the non-deficient
factors. Factor VIII-deficient plasmas used for the Nijmegen
modification of the Bethesda assay for FVIII inhibitors should
also have normal levels (above 50 iu/dl) of VWF, as anoma-
lous results have been reported without it.52
Chromogenic (amidolytic) assays
Chromogenic assays (CA) are available for various clotting fac-
tors53
but are predominantly used for FVIII and FIX assays.
These are two-stage assays where test plasma is added to a mix-
ture containing the appropriate co-factors, FX, thrombin or
prothrombin, calcium ions and phospholipid. After incubation
the amount of activated FX (FXa) generated is proportional to
the amount of functional FVIII or FIX in the sample.54
The
FXa is then assayed in the second step using a chromogenic
substrate. The results obtained by the OSCA and CA are con-
cordant in most instances, but in certain situations can differ
significantly.55–58
Up to 10% of non-severe haemophilia A
cases have a normal APTT and normal FVIII by OSCA, but a
reduced FVIII by CA.59
The opposite discrepancy (FVIII
reduced in OSCA but normal in CA) has also been
described.60,61
As CA rely on the formation of FXa, DFXaI may
interfere with the assay and cause falsely reduced results.45
These assays tend to be LA-insensitive.62
Monitoring of factor VIII or factor IX replacement therapy
Laboratory monitoring of FVIII or FIX replacement therapy
for treatment of haemophilia A or B is performed to ensure
optimal therapy, and OSCA or CA of FVIII and FIX are used
for this purpose. It is beyond the scope of this guideline to dis-
cuss the new modified molecules that are available as treatment
options but is worth noting that in many cases these assays are
not suitable in their unmodified form for measuring them
Guideline
351
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

accurately.63,64
Factor VIII can be measured in the presence of
emicizumab (Hemlibra
) using a CA but only if the compo-
nents are bovine in origin. A modified form of FVIII OSCA
assay can be used to estimate emicizumab concentration in
combination with a product-specific calibrator.65
Screening for inhibitors against procoagulant coagulation
factors
Plasma samples found to have an abnormal screening PT or
APTT may be further investigated to define the abnormality
by performing mixing tests: abnormal screening tests are
repeated on equal volume mixtures (50:50) of normal and
test plasma. Correction of the prolonged result into the refer-
ence range suggests the absence of an immediate-acting inhi-
bitor, but it is important to note that some FVIII inhibitors
are slow-acting (and therefore mixing may correct if the
APTT is performed immediately after mixing) and that the
dilution of the antibody in a 50:50 mixing study may also
normalise the result into the reference range. A lack of cor-
rection in mixing studies suggests the presence of an
immediate-acting inhibitor, the most common of which is
LA. It should also be noted that FIX inhibitors are typically
fast-acting,66
as are some FVIII inhibitors in acquired hae-
mophilia A. The results of immediate or incubated mixing
studies are often not clear-cut and formulae such as those of
Rosner or Chang may be helpful. However, no approach is
both 100% sensitive and specific.67,68
If the result of more than one OSCA is reduced, this may
indicate the presence of a non-specific inhibitor (LA) or a high-
titre specific inhibitor. The OSCAs should be examined for
non-parallelism and repeated at higher plasma dilutions if nec-
essary to dilute the inhibitor and confirm its specificity (Fig 2).
Factor VIII or FIX inhibitors should be quantified with a
Bethesda assay,69
with Nijmegen modification for FVIII.70
Incubation at 37°C for 2 h is required. This should be repeated
with a porcine substrate replacing the human normal plasma
to quantify cross-reactive inhibitors to porcine FVIII.71,72
Commercial enzyme-linked immunosorbent assays (ELISA)
are available and may be useful to detect anti-factor FVIII anti-
bodies if a LA is present or inhibitors that increase clearance
rather than inhibiting activity.73
(A)
(B)
Fig 2. Detection of activated samples and sam-
ples containing inhibitory activity in a parallel-
line bioassay system. (A) Activated test samples
tend to show an apparent decrease in relative
potency as the sample is diluted. (B) Test sam-
ple containing inhibitory activity show an
apparent increase in relative potency as the
sample is diluted. Coagulation times of a refer-
ence preparation (diamonds); coagulation
times of test sample (squares).
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Acquired deficiencies of FII,74
FV,75
FVII,76,77
FX76,78
and
FXI66
have all been described and when due to neutralising
antibodies can be distinguished from congenital deficiencies
by PT and/or APTT mixing studies. For slow-acting inhibi-
tors such as those associated with FVIII inhibition, incuba-
tion at 37°C may be required before testing. Acquired FII
deficiency should be recognised as often linked with the LA
hypoprothrombinaemia syndrome.74
Details of laboratory investigation for inhibitors in congenital
and acquired bleeding disorders can be found elsewhere.79,80
Recommendations
Laboratories should perform assays on factor-deficient
plasmas to ensure that each new batch has 1 iu/dl of
the deficient factor and normal levels of VWF in
FVIII-deficient plasmas being used for Nijmegen-mod-
ified Bethesda inhibitor assays.
In-house patient plasma should not be used as a
source of reagent material.
Laboratories should locally determine the effect of
lupus anticoagulant on the PT and APTT reagents
used in OSCA. Results should be scrutinised for paral-
lelism which may also be lost in patients with other
immediate-acting inhibitors.
If non-severe haemophilia A is a possibility a two-
stage chromogenic assay (CA) for FVIII should be per-
formed in addition to an OSCA, to ensure detection
of all cases and to correctly assess the severity.
Factor assays (OSCA or CA) should be avoided in
patients taking direct FXa or direct thrombin inhibi-
tors. Charcoal-based reversal agents may be of use.
Haemophilia centres should ensure that appropriate
laboratory assays are available for FVIII and FIX
products in local clinical use.
Fibrinogen
Plasma fibrinogen is a hexameric glycoprotein, which behaves
differently in different assays.81
As an acute phase protein, fib-
rinogen can initially be raised in sepsis and underlying malig-
nancy before being consumed. Routine PT- and APTT-based
assays are not sensitive enough for the estimation of fibrinogen
concentration. Afibrinogenaemia, hypofibrinogenaemia and
dysfibrinogenaemia are all associated with a bleeding tendency;
in addition, some dysfibrinogenaemias may lead to a pro-
thrombotic tendency or have no clinical phenotype.
Clauss fibrinogen activity assay
A high concentration of thrombin (typically ~100 U/ml) is
added to buffer-diluted test plasma and the clotting time is mea-
sured.82
The test result is compared with a calibration curve pre-
pared by clotting a series of dilutions (at least three, ideally five)
of a reference sample of known fibrinogen concentration. Sam-
ples with clotting times falling outside the linear part of the stan-
dard curve should be re-diluted and retested. Fibrinogen
sialylation may be increased in neonates and in liver disease
which may prolong the TT but does not alter the Clauss assay.
High levels of fibrin degradation products (FDPs) or direct
thrombin inhibitors (DTI) may inhibit some Clauss assays
depending on the thrombin concentration in the reagent, result-
ing in variable underestimation of fibrinogen concentra-
tion.69,83,84
The Clauss assay is not usually affected by
therapeutic levels of heparin although reports of variability in
fibrinogen estimation have been reported in cardiac patients on
bypass.85
Fibrinogen antigen assay
Fibrinogen concentration in plasma can be measured using an
immunoassay, with similar results to a Clauss assay.86
These
assays are essential in differentiating between dysfibrino-
genaemia (with bleeding or thrombotic tendencies), where
antigen levels could be approximately twice that of the Clauss
activity assay, and hypofibrinogenaemia, where both antigen
and activity are concordantly reduced. The assays must be per-
formed on aliquots from the same sample to avoid discrepan-
cies due to fibrinogen being an acute phase protein.
Derived fibrinogen
If the PT is measured using a photo-optical system then a
‘derived fibrinogen concentration’ can be derived from a cali-
bration curve. The results vary according to the PT method. In
one EQA exercise, PT-based estimations had 117% higher fib-
rinogen values than Clauss assays, and five reagent–instrument
combinations had ≥20% bias,87
a finding exaggerated in war-
farinised and critically ill patients and those with dysfibrino-
genaemia in whom the derived fibrinogen is closer to the
antigenic value and five times higher than the Clauss assay.88,89
The derived fibrinogen is not recommended for clinical use.90
TEG/ROTEM
Thromboestography (TEG) and thromboelastometry
(ROTEM) can be used to monitor fibrinogen levels during
the management of major bleeding. Their use is reviewed in
the recent BSH guideline.91
Recommendations
Fibrinogen activity should be measured using the
Clauss assay.
Fibrinogen antigen assays should be used to distin-
guish between hypofibrinogenaemia and dysfibrino-
genaemia.
PT-derived fibrinogen is not recommended for clinical
use.
Guideline
353
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Factor XIII
Historically the clot solubility assay for factor XIII has had
the widest uptake being relatively cheap and simple to per-
form. It involves the exposure of plasma clotted with throm-
bin and calcium ions to either 5 M urea, 2% acetic or 1%
monochloroacetic acid. This semi-quantitative assay has been
shown to be poorly standardised and subject to inter-labora-
tory variation.92,93
Abnormal results may only be seen in sev-
ere cases (5 iu/dl) and International Society on Thrombosis
and Haemostasis Scientific and Standardization Committee
(ISTH SSC) guidance is that it is not used.94
Quantitative assays are based upon the catalytic activity of
FXIIIa in the transglutaminase reaction stabilising fibrin clot
formation. Three assays based on this have been described
including amine incorporation, isopeptidase activity and
ammonia release.95
Amine incorporation and isopeptidase
activity assays are time-consuming and difficult to standard-
ise and may be susceptible to falsely raised results in the
presence of the most common FXIII-A Val34-Leu polymor-
phism.94
CA based upon ammonia release have been success-
fully adapted for use on routine coagulation analysers and
are reported to be sensitive to approximately 5–10 iu/dl.
However FXIII-independent background ammonia release is
thought to contribute to overestimation of values at levels
20 iu/dl,96
so laboratories should confirm a blanking step
(usually based on 1 mmol/l iodoacetamide) is included in
the protocol.
FXIII antigen levels can be assayed using immunoassay to
classify subtypes of FXIII-A, FXIII-B and the FXIII–A2B2-
complex deficiency and distinguish between them if necessary
but should not be used as a screen on its own.
Molecular analysis of the FXIII-A (F13A1) and FXIII-B
(F13B) genes has revealed that over 95% of reported severe
FXIII deficiencies result from F13A1 variants, and targeted
genotyping may be warranted in areas of high consanguinity
where the specific mutation/s is known.97
Recommendations
Clot solubility tests should not be used for the diagno-
sis of factor XIII deficiency.
Automated ammonia release assays are recommended
for measuring factor XIII activity, taking into account
the need for background blanking at values of 20 iu/dl.
Immunoassays should be considered to further cate-
gorise the deficiency.
Inhibitors of fibrinolysis
Fibrinolytic parameters are subject to acute phase as well as
diurnal and seasonal fluctuation resulting from changes in
tissue plasminogen activator (tPA) and plasminogen activator
inhibitor-1 (PAI-1), and so collection and handling of sam-
ples must be preplanned.98,99
Alpha-2-plasmin inhibitor and PAI-1. Alpha-2-plasmin and
PAI-1 deficiencies are rare but potentially significant bleeding
disorders. Commercial CAs and ELISA are available for mea-
surement of activity and antigenic levels respectively, which
should be considered in the investigation of a patient with a
bleeding diathesis if other tests appear normal.100
In the case
of plasminogen activator inhibitor however, the lower end of
the normal range is often near zero which makes it difficult
to distinguish possible cases of deficiency from the normal
population. Deficiency requires genetic confirmation.101,102
Recommendations
Chromogenic and ELISA should be used for the detec-
tion of alpha-2-plasmin inhibitor and plasminogen
activator inhibitor.
Investigation of a thrombotic tendency
The choice of tests for inherited thrombophilia is described
in a previous BSH guideline and may include assays of pro-
tein C, protein S and antithrombin, plus tests for the F2
G20210A (c.*97GA, prothrombin) and F5 G1691A
[c.1601GA, factor V Leiden (FVL)] variants.103
As when
performing investigations into a bleeding tendency, a routine
clotting screen should be performed to exclude the effects of
underlying conditions or anticoagulation. This may include a
Clauss fibrinogen and the addition of a TT to exclude the
presence of UFH or DTI. Specific sequencing of antithrom-
bin (SERPINC1), protein C (PROC) and protein S (PROS1)
genes may add value. Screening for LA if often included as
part of a thrombophilia screen programme. Guidance on
performing LA testing is given elsewhere.104–107
Recommendations
The significance of the results should be interpreted
by an experienced clinician/scientist who is aware of
all relevant variables that may influence individual test
results in each case.
Preanalytical variation due to anticoagulation with
heparin, VKA and DOAC must be excluded prior to
testing.
Phenotypic thrombophilia testing should not be
undertaken close to an acute thrombotic event due to
the possibility of consumption-reducing values, and
testing during pregnancy avoided if possible.
A Clauss fibrinogen activity assay should be consid-
ered to screen for dysfibrinogenaemia. A low result
should be followed up with an antigenic assay and
genetic analysis.
Testing for Protein C (PC) and protein S (PS) in not
recommended in patients taking VKA. PT and APTT
should be measured prior to testing to exclude VKA
therapy. PC or PS deficiency should not be diagnosed
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

or excluded on the basis of low results performed
when the patient is taking VKA.
A reduction in antithrombin, PC or PS should be con-
firmed on two or more separate samples. Deficiency
should not be diagnosed on a single abnormal result.
Specific gene sequencing of antithrombin, PC and PS
may add value when low results are identified.
Antithrombin
Antithrombin activity assays. Chromogenic antithrombin
(AT) heparin cofactor assays are simple and precise, are
easily automated and are recommended.103
Thrombin (FIIa)
or FXa is incubated with diluted plasma and heparin, and
residual enzyme activity is measured using a chromogenic
substrate.
Heparin cofactor II influences human FIIa-based assays
and therefore these are not recommended.108,109
Bovine FIIa
and human or bovine FXa-based assays should be used.
Antithrombin activity may be reduced due to defects in
the heparin-binding site (HBS), reactive site (RS) or muta-
tions with pleiotropic effects (PE).103,110
In type II deficiency,
AT activity assays may be discrepant with levels measured by
bovine thrombin-based assays both being reported as lower
or higher than those measured with FXa-based assays.111,112
Shortening the incubation time of the sample dilution with
the enzyme may increase the sensitivity of assays to type II
AT defects; however no single assay can be guaranteed to
detect all type II AT defects.113–115
Antithrombin may be overestimated in FIIa-based assays
in the presence of DTI, and by FXa-based assays in the pres-
ence of DFXaI.116
AT antigen assays. Antithrombin antigen may be measured
by immunoassay, with ELISA and immunoturbidimetric
assays having the greatest precision.117
The assay is only
needed to identify type II AT deficiency, which has clinical
relevance, particularly for HBS defects which have a low
thrombotic risk in heterozygotes.110,118
An alternative is
molecular analysis of the AT gene (SERPINC1).
Recommendations
A chromogenic antithrombin heparin cofactor activity
assay using FXa or bovine FIIa should be performed to
screen for antithrombin deficiency. A confirmed low
result should be followed by an antigenic assay, and
discrepancies followed up with genetic investigation.
Laboratories must be aware of the target enzyme used
in the local AT activity assay (Xa or IIa) and if
patients are on DOACs before testing. If necessary
under these circumstances an alternative assay with a
different substrate may add value or pretreatment of
the sample with a removal agent should be consid-
ered.
Protein C
Protein C activity assays. To measure activity, PC is con-
verted to activated PC (APC) by the enzyme Protac (Pen-
tapharm, Basle, Switzerland) from Southern Copperhead
snake venom.119
The APC generated can be measured using
a CA or clotting assay.
Protein C chromogenic assays. Chromogenic assays are recom-
mended for functional PC assays because they are more specific
than clotting assays. However, the APC substrates have relatively
poor specificity so the assays include a number of inhibitors but
in some circumstances the substrate can be cleaved by various
other serine proteases (e.g. factor XIa) leading to overestimation
of PC.103
In kinetic assays, this may be difficult to detect, but
may be identified by poor linearity of the reaction rate. Activa-
tor blanks for patient samples, calibrators and a control should
be considered where increased effects of non-specific proteases
are anticipated, for example with paediatric samples, and in
patients with DIC or pathological fibrinolysis.
Protein C may be overestimated by CA in patients receiving
VKA, because of the presence of acarboxy-PC, which has similar
reaction kinetics to fully carboxylated PC cleaving the sub-
strate.120
Protein C coagulation assays. Although PC CAs are more
specific than clotting assays, type IIb PC deficiency can only
be detected in the clotting assay. The consequences of failing
to detect this rare functional defect by CA must be balanced
against the poorer specificity and generally poorer precision
of clotting assays.121,122
In the clotting assay APC prolongs the clotting time of PC-
depleted plasma through destruction of FVa or FVa and FVIIIa
(depending on assay type): if the circulating PC levels are
reduced the clotting time will be shorter. The phospholipid
composition of the reagent in APTT-based assays is important,
as it influences sensitivity to APC, FV and FVIII. There is also
variability in the PS dependence of the assay.123,124
Underesti-
mation of PC may occur in samples with high FVIII or in the
presence of FVL.125,126
Predilution of patient plasma in PC-de-
pleted plasma may reduce or remove the influence of FVL.126
Clotting assays can also be activated by Russell’s viper venom
(RVV) which has the benefit of not being influenced by high
levels of FVIII although still maybe be impacted by FVL and
LA.127
As for all clotting assays, ideally at least three dilutions of
patient plasma should be tested so that parallelism can be
assessed. Assays may be affected by LA depending on the reagent
phospholipid concentration and composition, DTI and DFXaI
can interfere with results. Results from activated samples due to
poor venepuncture may cause overestimation of PC and should
be repeated if activation is suspected.122
Patient age is important
to confirm as PC is reduced in neonates and children.35,128
PC antigen assays. There is no accepted difference in throm-
botic risk between type I and II deficiencies129
and therefore
the antigen assay is not recommended for routine use.
Guideline
355
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Recommendations
Pre-activated samples may lead to overestimation of
PC and need to be excluded prior to testing.
A chromogenic PC activity assay should be performed
to screen for PC deficiency. A confirmed low result
does not require follow-up by an antigenic assay.
Clotting PC activity assays are not recommended for
routine use but may add value for identifying familial
thrombophilia due to type IIb PC deficiency.
Protein S
Plasma PS exists in two forms: bound to C4b-binding pro-
tein (C4BP); and free unbound (FPS). Around 40% of PS is
in FPS form and functions as a cofactor to APC130
and tissue
factor pathway inhibitor (TFPI).131
The proportion of bound
and unbound forms is regulated by the availability of C4BP.
The diagnosis of genetically confirmed PS deficiency is com-
plex when based upon plasma phenotype alone with many
interacting factors.132
Protein S antigen assays
Free protein S antigen. Under normal circumstances free PS
antigen concentration usually reflects functional PS activity
in plasma and can be measured by immunoassays. Latex-
based immunoturbidimetric assays use either: two mono-
clonal antibodies specific for FPS; or latex particles coated
with C4bBP to capture FPS, then monoclonal antibody-
coated particles, which agglutinate in the presence of cap-
tured FPS.
Delay in addition of sample to the microplate or pro-
longed incubation can result in overestimation of FPS by
ELISA, so dilutions should be tested immediately and incu-
bation times and temperatures controlled appropriately.133
Total protein S antigen. Bound and free (Total) PS antigen
(TPS) can be measured by immunoassay. In some TPS
ELISA assays, C4bBP must be dissociated from PS by high
plasma dilution as well as prolonged incubation (18 h) with
capture antibody,134
but in others the use of monoclonal
antibodies negates these requirements.133
Due to its addition
cost and limited contribution to the diagnosis of PS defi-
ciency it is often not routinely included in a diagnostic algo-
rithm.135
Protein S activity assays. These assays measure the ability of
PS to inactivate FVIIIa and/or FVa in the presence of APC,
detected using PT, APTT or RVV-based coagulation times.132
Hereditary or acquired APC resistance and LA can result in
under- or overestimation of functional PS.136
As for all clot-
ting assays, ideally at least three dilutions of patient plasma
should be tested to confirm low results so that parallelism
can be assessed. Marked differences have been seen in PS
activity results between different kits complicating the inter-
pretation of quality assurance assessment for these assays.137
Some DOAC have been reported to impact on the results of
these assays138
but not others.139
Coagulation-based PS activity assays are not recom-
mended for routine use, although type II PS deficiency can
only be detected in the clotting assay. Recently however, the
type II variant PS Tokushima has been reported as not
detected using a clot-based activity assay so further limiting
its use.140
Consequences of failing to detect rare functional
defects must be balanced against the difficulties of perform-
ing the assay, the poor specificity and generally poorer preci-
sion.103
Recommendations
An immunoassay for free PS antigen should be per-
formed to screen for PS deficiency. A confirmed low
result can be followed by an immunoassay for total PS
antigen. Genetic analysis should be done in cases of
suspected heritable deficiency.
Clot-based PS activity assays are not recommended for
routine use but may add value for subtyping or unex-
plained familial thrombophilia due to type II defi-
ciency.
Protein S activity testing on DOAC and VKA therapy
should be avoided.
Activated PC resistance (APCr) assays
Resistance to APC may be hereditary or acquired. The most
common hereditary defect is due to FVL, and samples may
be screened for FVL using a clot-based assay, with abnormal
results being verified by polymerase chain reaction (PCR)
assay. A number of rare low incidence variants of the FV
gene demonstrate increased thrombotic risk, and a mismatch
between clotting and PCR assays may identify this: additional
genotyping may be required.
Predilution of samples in FV-deficient plasma improves
specificity of the assay for FV variants, after which two APTT
assays are performed: one in the presence of APC and one
without APC. The result is expressed as a ratio of the two
tests, with a significant distinction between wild type,
heterozygous and homozygous factor V subtypes. Alternative
assays have been described using snake venoms to initiate
clotting via FXa and FVa, as well as the use of a chromogenic
Xa assay.141–143
DOAC and DTI interfere with the baseline
results and need to be excluded prior to testing.142
Factor V and factor II mutation analysis
The FVL variant causes activated factor V to be resistant to
inactivation by APC, and the prothrombin variant results in
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

hyperprothrombinaemia: both are independently associated
with thrombosis.144
PCR-based testing for both mutations
alongside suitable control material [WHO international
genomic DNA (gDNA) available] should be part of a routine
screen for heritable thrombophilia.103
An APC resistance
assay is unnecessary if a direct genetic test for F5G1691A
(c.1601GA) is used initially.103
There are several other low
incidence FV mutations known to generate mild to moderate
APC resistance that have not been demonstrated to exhibit a
thrombotic tendency in isolation but could cause a mismatch
between APC testing and FVL genotyping.145,146
Recommendations
If an APC resistance assay is used to detect F5G1691A
(c.1601GA) then predilution of the test sample in
factor V-deficient plasma should be used.
The presence of anticoagulants that may impact on
interpretation needs to be excluded prior to testing.
An APC resistance assay is unnecessary if a direct
genetic test for F5G1691A (c.1601GA) is used ini-
tially.
Thrombotic thrombocytopenic purpura
The acute presentation of thrombotic thrombocytopenic pur-
pura (TTP) is a clinical emergency and the initiation of
treatment in suspected cases is currently made without wait-
ing for an ADAMTS13 assay.
VWF protease assays
Measurement of the VWF protease (ADAMTS13) activity
and associated auto-antibodies forms the basis of screening
for inherited or acquired TTP. Most commonly used are
commercial chromogenic or fluorogenic end-point ELISA
assays assessing the ability of ADAMTS13 to cleave the syn-
thetic molecule GST-VWF73.147,148
Auto-antibodies can be
inhibitory or non-inhibitory and therefore the use of an
ELISA-based assay is recommended for their detection.149–151
These assays are relatively time-consuming and labour inten-
sive although recent transition of this assay onto an auto-
mated platform has been described152
; however, single
patient testing in an emergency may be prohibitively expen-
sive locally. A rapid semi-quantitative screen is available153
but further studies are required to confirm its suitability as
an alternative or adjunct to quantitative assays.
Recommendations
Validated commercial or in-house methods should be
employed for quantitation of ADAMTS-13 activity and
auto-antibodies for the diagnosis of TTP using calibra-
tors traceable to the WHO International Standard.
Molecular testing
The International Society on Thrombosis and Haemostasis
maintains a list of all genes known to cause haemostatic dis-
ease (https://www.isth.org/page/GinTh_GeneLists). Rapid
high throughput sequencing of these genes is now available
and can provide additional information about the clinical
phenotype. Currently genetic testing should be used along-
side, rather than in place of, phenotypic assays. In cases
where laboratory assays show no abnormality, genetic testing
is unlikely to uncover any potentially pathogenic variants but
could still be considered a broader approach in research
studies. It can also be of value for prenatal diagnosis and
identification of affected relatives.154
Acknowledgements
The authors wish to thank Gareth Hardy from Niche Science
and Technology (www.niche.org.uk) for help in undertaking
the initial literature review.
The BSH Haemostasis and Thrombosis Task Force mem-
bers that reviewed this guideline were Dr Raza Alikhan, Dr
Julia Anderson, Dr Deepa Jayakody Arachchillage, Dr Tina
Biss, Dr Keith Gomez, and Dr Will Lester. The authors
would like to thank them, the BSH sounding board, and the
BSH guidelines committee for their support in preparing this
guideline.
Conflicts of interest
The BSH paid the expenses incurred during the writing of
this guidance. All authors have made a declaration of inter-
ests to the BSH and Task Force Chairs which may be viewed
on request, and have no conflicts of interest to declare.
Review process
Members of the writing group will inform the writing group
Chair if any new evidence becomes available that would alter
the strength of the recommendations made in this document
or render it obsolete. The document will be reviewed regu-
larly by the relevant Task Force and the literature search will
be re-run every three years to search systematically for any
new evidence that may have been missed. The document will
be archived and removed from the BSH current guidelines
website if it becomes obsolete. If new recommendations are
made an addendum will be published on the BSH guidelines
website https://b-s-h.org.uk/
Disclaimer
While the advice and information in this guidance is believed
to be true and accurate at the time of going to press, neither
the authors, the BSH, or the publishers accept any legal
responsibility for the content of this guidance.
Guideline
357
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

References
1. Mackie IJ, Kitchen S, Machin SJ, Lowe GD; Haemostasis and Thrombosis
Task Force of the British Committee for Standards in Haematology.
Guidelines on fibrinogen assays. Br J Haematol. 2003;121:396–404.
2. Mackie I, Cooper P, Lawrie A, Kitchen S, Gray E, Laffan M, et al. Guide-
lines on the laboratory aspects of assays used in haemostasis and throm-
bosis. Int J Lab Hematol. 2013;35:1–13.
3. Adcock Funk DM, Lippi G, Favaloro EJ. Quality standards for sample
processing, transportation, and storage in hemostasis testing. Semin
Thromb Hemost. 2012;38:576–85.
4. Lippi G, Salvagno GL, Montagnana M, Lima-Oliveira G, Guidi GC,
Favaloro EJ. Quality standards for sample collection in coagulation test-
ing. Semin Thromb Hemost. 2012;38:565–75.
5. Arora S, Kolte S, Dhupia J. Hemolyzed samples should be processed for
coagulation studies: the study of hemolysis effects on coagulation param-
eters. Ann Med Health Sci Res. 2014;4:233–7.
6. Lippi G, Ippolito L, Tondelli MT, Favaloro EJ. Interference from hetero-
philic antibodies in D-dimer assessment. A case report. Blood Coagul Fib-
rinolysis. 2014;25:277–9.
7. Nagant C, Rozen L, Demulder A. HIL interferences on three hemostasis
analyzers and contribution of a preanalytical module for routine coagula-
tion assays. Clin Lab. 2016;62:1979–87.
8. CLSI. Procedures for the collection of diagnostic blood specimens by
venepuncture; Approved guideline. CLSI. 4th ed. Wayne, PA: Clinical
and Laboratory Standards Institute; 1998.
9. CLSI. Collection, transport and processing of blood specimens for testing
plasma-based coagulation assays and molecular haemostasis assays;
Approved guideline. CLSI. 5th ed. Wayne, PA: Clinical and Laboratory
Standards Institute; 2008.
10. D’Angelo G, Villa C, Tamborini A, Villa S. Evaluation of the main coag-
ulation tests in the presence of hemolysis in healthy subjects and patients
on oral anticoagulant therapy. Int J Lab Hematol. 2015;37:819–33.
11. Hernaningsih Y, Akualing JS. The effects of hemolysis on plasma pro-
thrombin time and activated partial thromboplastin time tests using
photo-optical method. Medicine. 2017;96:e7976.
12. Woolley A, Golmard JL, Kitchen S. Effects of haemolysis, icterus and
lipaemia on coagulation tests as performed on Stago STA-Compact-Max
analyser. Int J Lab Hematol. 2016;38:375–88.
13. Novelli C, Vidali M, Brando B, Morelli B, Andreani G, Arini M, et al. A
collaborative study by the Working Group on Hemostasis and Thrombo-
sis of the Italian Society of Clinical Biochemistry and Clinical Molecular
Biology (SIBioC) on the interference of haemolysis on five routine blood
coagulation tests by evaluation of 269 paired haemolysed/non-haemol-
ysed samples. Biochem Med. 2018;28:030711.
14. Douxfils J, Ageno W, Samama CM, Lessire S, ten Cate H, Verhamme P,
et al. Laboratory testing in patients treated with direct oral anticoagu-
lants: a practical guide for clinicians. J Thromb Haemost. 2018;16:209–19.
15. Exner T, Michalopoulos N, Pearce J, Xavier R, Ahuja M. Simple method
for removing DOACs from plasma samples. Thromb Res. 2018;163:117–
22.
16. Favresse J, Lardinois B, Sabor L, Devalet B, Vandepapeliere J, Braibant
M, et al. Evaluation of the DOAC-stop(R) procedure to overcome the
effect of DOACs on several thrombophilia screening tests. TH Open.
2018;2:e202–9.
17. Platton S, Hunt C. Influence of DOAC Stop on coagulation assays in
samples from patients on rivaroxaban or apixaban. Int J Lab Hematol.
2019;41:227–33.
18. Cox-Morton S, Macdonald S, Thomas W. A diagnostic solution for
haemostasis laboratories for patients taking direct oral anticoagulants
using DOAC-Remove. Br J Haematol. 2019;187:377–85.
19. Frans G, Meeus P, Bailleul E. Resolving DOAC interference on aPTT, PT
and lupus anticoagulant testing by the use of activated carbon. J Thromb
Haemost. 2019;17:1354–1362.
20. Favaloro EJ, Gilmore G, Bonar R, Dean E, Arunachalam S, Mohammed
S, et al. Reducing the effect of DOAC interference in laboratory testing
for factor VIII and factor IX: A comparative study using DOAC Stop
and andexanet alfa to neutralize rivaroxaban effects. Haemophilia.
2020;26:354–62.
21. Kitchen S, Olson JD, Preston FE. Quality in laboratory hemostasis and
thrombosis. Oxford: Blackwell publishing Ltd; 2013.
22. Magnette A, Chatelain M, Chatelain B, ten Cate H, Mullier F. Pre-analyt-
ical issues in the haemostasis laboratory: guidance for the clinical labora-
tories. Thromb J. 2016;14:49.
23. Toulon P, Metge S, Hangard M, Zwahlen S, Piaulenne S, Besson V.
Impact of different storage times at room temperature of unspun citrated
blood samples on routine coagulation tests results. Results of a bicenter
study and review of the literature. Int J Lab Hematol. 2017;39:458–68.
24. Gardiner C, Pennaneac’H C, Mackie IJ, Sheldrake A, Harrison J, Machin
SJ. Falsely elevated D-dimer results in a healthy patient on account of
heterophiletul antibodies. Br J Haematol. 2003;122:871–3.
25. Kricka LJ. Human anti-animal antibody interferences in immunological
assays. Clin Chem. 1999;45:942–56.
26. Marks V. False-positive immunoassay results: a multicenter survey of
erroneous immunoassay results from assays of 74 analytes in 10 donors
from 66 laboratories in seven countries. Clin Chem. 2002;48:2008–16.
27. Sevenet PO, Depasse F. Clot waveform analysis: where do we stand in
2017? Int J Lab Hematol. 2017;39:561–8.
28. Tekkesin N, Kilinc C. Optical and mechanical clot detection methodolo-
gies: a comparison study for routine coagulation testing. J Clin Lab Anal.
2012;26:125–9.
29. Kitchen S, McGraw A, Enchenagucia M. World Federation of Hae-
mophilia. Diagnosis of haemophilia and other bleeding disorders: a Labo-
ratory manual. WFH; 2010.
30. Westgard JO. Basic method validation. Madison, WI: Westgard Quality
Corporation; 2008.
31. CAP. Hematology and coagulation checklist. Northfield, IL: College of
American Pathologists; 2017.
32. CLSI. Protocols for determination of limits of detection and limits of
quantitiation; Approved guideline. EP17 ed. Wayne, PA: Clinical and
Laboratory Standards Institute; 2004.
33. CLSI. Defining, establishing, and verifying reference intervals in the clini-
cal laboratory; Approved Guideline. CLSI. 3rd ed. Wayne, PA: Clinical
and Laboratory Standards Institute; 2008.
34. Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM,
et al. Development of the human coagulation system in the full-term
infant. Blood. 1987;70:165–72.
35. Andrew M, Vegh P, Johnston M, Bowker J, Ofosu F, Mitchell L. Matura-
tion of the hemostatic system during childhood. Blood. 1992;80:1998–
2005.
36. Lippi G, Franchini M, Montagnana M, Guidi GC. Coagulation testing in
pediatric patients: the young are not just miniature adults. Semin Thromb
Hemost. 2007;33:816–20.
37. Ignjatovic V, Kenet G, Monagle P; Perinatal and Paediatric Haemostasis
Subcommittee of the Scientific and Standardization Committee of the
International Society on Thrombosis and Haemostasis. Developmental
hemostasis: recommendations for laboratories reporting pediatric sam-
ples. J Thromb Haemost. 2012;10:298–300.
38. Vanoverschelde L, Kelchtermans H, Musial J, de Laat B, Devreese KMJ.
Influence of anticardiolipin and anti-beta2 glycoprotein I antibody cutoff
values on antiphospholipid syndrome classification. Res Pract Thromb
Haemost. 2019;3:515–27.
39. Keeling DM, Mackie IJ, Moody A, Watson HG; Haemostasis and Throm-
bosis Task Force of the British Committee for Standards in Haematology.
The diagnosis of deep vein thrombosis in symptomatic outpatients and
the potential for clinical assessment and D-dimer assays to reduce the
need for diagnostic imaging. Br J Haematol. 2004;124:15–25.
40. Mumford AD, Ackroyd S, Alikhan R, Bowles L, Chowdary P, Grainger J,
et al. Guideline for the diagnosis and management of the rare coagula-
tion disorders: a United Kingdom Haemophilia Centre Doctors’ Organi-
zation guideline on behalf of the British Committee for Standards in
Haematology. Br J Haematol. 2014;167:304–26.
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

41. Girolami A, Vettore S, Scarparo P, Lombardi AM. Persistent validity of a
classification of congenital factor X defects based on clotting, chro-
mogenic and immunological assays even in the molecular biology era.
Haemophilia. 2011;17:17–20.
42. Dumont MD, Tapon-Bretaudiere J, Fischer AM, Bros A, Chassevent J,
Aufeuvre JP. Prothrombin Poissy: a new variant of human prothrombin.
Br J Haematol. 1987;66:239–43.
43. Girolami A, Scarparo P, Bonamigo E, Treleani M, Lombardi AM.
Homozygous FVII deficiencies with different reactivity towards tissue
thromboplastins of different origin. Hematology. 2012;17:350–4.
44. Smith SA, Comp PC, Morrissey JH. Phospholipid composition controls
thromboplastin sensitivity to individual clotting factors. J Thromb Hae-
most. 2006;4:820–7.
45. Gosselin RC, Adcock DM, Bates SM, Douxfils J, Favaloro EJ, Gouin-Thi-
bault I, et al. International Council for Standardization in Haematology
(ICSH) recommendations for laboratory measurement of direct oral anti-
coagulants. Thromb Haemost. 2018;118:437–50.
46. Tripodi A, Chantarangkul V, Clerici M, Negri B, Galli M, Mannucci PM.
Laboratory control of oral anticoagulant treatment by the INR system in
patients with the antiphospholipid syndrome and lupus anticoagulant.
Results of a collaborative study involving nine commercial thromboplas-
tins. Br J Haematol. 2001;115:672–8.
47. Toulon P, Eloit Y, Smahi M, Sigaud C, Jambou D, Fischer F, et al. In
vitro sensitivity of different activated partial thromboplastin time
reagents to mild clotting factor deficiencies. Int J Lab Hematol.
2016;38:389–96.
48. Hathaway WE, Assmus SL, Montgomery RR, Dubansky AS. Activated
partial thromboplastin time and minor coagulopathies. Am J Clin Pathol.
1979;71:22–5.
49. Turi DC, Peerschke EI. Sensitivity of three activated partial thromboplas-
tin time reagents to coagulation factor deficiencies. Am J Clin Pathol.
1986;85:43–9.
50. Asmis LM, Sulzer I, Furlan M, Lammle B. Prekallikrein deficiency: the
characteristic normalization of the severely prolonged aPTT following
increased preincubation time is due to autoactivation of factor XII.
Thromb Res. 2002;105:463–70.
51. Kumano O, Ieko M, Naito S, Yoshida M, Takahashi N. APTT reagent
with ellagic acid as activator shows adequate lupus anticoagulant sensitiv-
ity in comparison to silica-based reagent. J Thromb Haemost.
2012;10:2338–43.
52. Verbruggen B, Giles A, Samis J, Verbeek K, Mensink E, Novakova I. The
type of factor VIII deficient plasma used influences the performance of
the Nijmegen modification of the Bethesda assay for factor VIII inhibi-
tors. Thromb Haemost. 2001;86:1435–9.
53. Gallimore MJ, Friberger P. Chromogenic peptide substrate assays and
their clinical applications. Blood Rev. 1991;5:117–27.
54. Adcock DM, Strandberg K, Shima M, Marlar RA. Advantages, disadvan-
tages and optimization of one-stage and chromogenic factor activity
assays in haemophilia A and B. Int J Lab Hematol. 2018;40:621–9.
55. Bowyer AE, van Veen JJ, Goodeve AC, Kitchen S, Makris M. Specific and
global coagulation assays in the diagnosis of discrepant mild hemophilia
A. Haematologica. 2013;98:1980–7.
56. Duncan EM, Duncan BM, Tunbridge LJ, Lloyd JV. Familial discrepancy
between the one-stage and two-stage factor VIII methods in a subgroup
of patients with haemophilia A. Br J Haematol. 1994;87:846–8.
57. Kihlberg K, Strandberg K, Rosen S, Ljung R, Astermark J. Discrepancies
between the one-stage clotting assay and the chromogenic assay in hae-
mophilia B. Haemophilia. 2017;23:620–7.
58. Pouplard C, Trossaert M, Le querrec A, Delahousse B, Giraudeau B,
Gruel Y. Influence of source of phospholipids for APTT-based factor IX
assays and potential consequences for the diagnosis of mild haemophilia
B. Haemophilia. 2009;15:365–8.
59. Rodgers SE, Duncan EM, Sobieraj-Teague M, Lloyd JV. Evaluation of
three automated chromogenic FVIII kits for the diagnosis of mild dis-
crepant haemophilia A. Int J Lab Hematol. 2009;31:180–8.
60. Lyall H, Hill M, Westby J, Grimley C, Dolan G. Tyr346–Cys mutation
results in factor VIII: C assay discrepancy and a normal bleeding pheno-
type – is this mild haemophilia A? Haemophilia. 2008;14:78–80.
61. Mumford AD, Laffan M, O’Donnell J, McVey JH, Johnson DJ, Manning
RA, et al. A Tyr346–Cys substitution in the interdomain acidic region
a1 of factor VIII in an individual with factor VIII: C assay discrepancy.
Br J Haematol. 2002;118:589–94.
62. Moser KA. Adcock Funk DM. Chromogenic factor VIII activity assay.
Am J Hematol. 2014;89:781–4.
63. Gray E, Kitchen S, Jenkins PV, Bowyer A, Riddell A, Platton S, et al. Lab-
oratory measurement of factor replacement therapies in the treatment of
congenital haemophilia. A United Kingdom Haemophilia Centres Doc-
tors’ Organisation guideline. Haemophilia. 2020;26:6–16.
64. Young GA, Perry DJ; International Prophylaxis Study Group (IPSG).
Laboratory assay measurement of modified clotting factor concentrates: a
review of the literature and recommendations for practice. J Thromb
Haemost. 2019;17:567–73.
65. Jenkins PV, Bowyer A, Burgess E, Gray E, Kitchen S, Murphy P, et al.
Laboratory coagulation tests and emicizumab treatment A United King-
dom Haemophilia Centre Doctors’ Organisation guideline. Haemophilia.
2020;26:151–155.
66. Collins PW, Chalmers E, Hart DP, Liesner R, Rangarajan S, Talks K,
et al. Diagnosis and treatment of factor VIII and IX inhibitors in congen-
ital haemophilia: UK Haemophilia Centre Doctors Organization. Br J
Haematol. 2013;160:153–70.
67. Chang SH, Tillema V, Scherr D. A percent correction formula for eval-
uation of mixing studies. Am J Clin Pathol. 2002;117:62–73.
68. Rosner E, Pauzner R, Lusky A, Modan M, Many A. Detection and quan-
titative evaluation of lupus circulating anticoagulant activity. Thromb
Haemost. 1987;57:144–7.
69. Adam S, Karger R, Kretschmer V. Influence of different hydroxyethyl
starch (HES) formulations on fibrinogen measurement in HES-diluted
plasma. Clin Appl Thromb Hemost. 2010;16:454–60.
70. Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M,
Mauser-Bunschoten E. The Nijmegen modification of the Bethesda assay
for factor VIII: C inhibitors: improved specificity and reliability. Thromb
Haemost. 1995;73:247–51.
71. Barrow RT, Lollar P. Neutralization of antifactor VIII inhibitors by
recombinant porcine factor VIII. J Thromb Haemost. 2006;4:2223–9.
72. Robinson MM, Tiefenbacher S, Adcock DM, Schlager M, Gritsch H. Val-
idation of a FVIII Nijmegen Bethesda Assay for the Detection of Inhibi-
tors to Porcine FVIII. ISTH2017. Berlin.
73. Sahud MA, Pratt KP, Zhukov O, Qu K, Thompson AR. ELISA system
for detection of immune responses to FVIII: a study of 246 samples and
correlation with the Bethesda assay. Haemophilia. 2007;13:317–22.
74. Mazodier K, Arnaud L, Mathian A, Costedoat-Chalumeau N, Haroche
J, Frances C, et al. Lupus anticoagulant-hypoprothrombinemia syn-
drome: report of 8 cases and review of the literature. Medicine.
2012;91:251–60.
75. Ortel TL. Clinical and laboratory manifestations of anti-factor V antibod-
ies. J Lab Clin Med. 1999;133:326–34.
76. Granger J, Gidvani VK. Acquired factor VII deficiency associated with
Wilms tumor. Pediatr Blood Cancer. 2009;52:394–5.
77. Mullighan CG, Rischbieth A, Duncan EM, Lloyd JV. Acquired isolated
factor VII deficiency associated with severe bleeding and successful treat-
ment with recombinant FVIIa (NovoSeven). Blood Coagul Fibrinolysis.
2004;15:347–51.
78. Uprichard J, Perry DJ. Factor X deficiency. Blood Rev. 2002;16:97–110.
79. Franchini M, Castaman G, Coppola A, Santoro C, Zanon E, di Minno G,
et al.; on behalf of the AICE Working Group. Acquired inhibitors of clot-
ting factors: AICE recommendations for diagnosis and management.
Blood Transfus. 2015;13:498–513.
80. Strandberg K, Astermark J. The role of the laboratory in diagnosis and
management of inhibitory antibodies in haemophilia. Haemophilia.
2018;24(Suppl 6):9–14.
Guideline
359
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

81. Henschen-Edman AH. On the identification of beneficial and detrimental
molecular forms of fibrinogen. Haemostasis. 1999;29:179–86.
82. Clauss A. Rapid physiological coagulation method in determination of
fibrinogen. Acta Haematol. 1957;17:237–46.
83. Adam S, Karger R, Kretschmer V. Photo-optical methods can lead to
clinically relevant overestimation of fibrinogen concentration in plasma
diluted with hydroxyethyl starch. Clin Appl Thromb Hemost.
2010;16:461–71.
84. Zhang L, Yang J, Zheng X, Fan Q, Zhang Z. Influences of argatroban on
five fibrinogen assays. Int J Lab Hematol. 2017;39:641–4.
85. Solomon C, Baryshnikova E, Tripodi A, Schlimp CJ, Schochl H, Cada-
muro J, et al. Fibrinogen measurement in cardiac surgery with cardiopul-
monary bypass: analysis of repeatability and agreement of Clauss method
within and between six different laboratories. Thromb Haemost.
2014;112:109–17.
86. Peters SA, Woodward M, Rumley A, Koenig W, Tunstall-Pedoe H, Lowe
GD. Direct comparisons of three alternative plasma fibrinogen assays
with the von Clauss assay in prediction of cardiovascular disease and all-
causes mortality: the Scottish Heart Health Extended Cohort. Br J Hae-
matol. 2013;162:392–9.
87. Cunningham MT, Olson JD, Chandler WL, van Cott EM, Eby CS, Ter-
uya J, et al. External quality assurance of fibrinogen assays using normal
plasma: results of the 2008 College of American Pathologists proficiency
testing program in coagulation. Arch Pathol Lab Med. 2012;136:789–95.
88. Lawrie AS, McDonald SJ, Purdy G, Mackie IJ, Machin SJ. Prothrombin
time derived fibrinogen determination on Sysmex CA-6000. J Clin Pathol.
1998;51:462–6.
89. Miesbach W, Schenk J, Alesci S, Lindhoff-Last E. Comparison of the fib-
rinogen Clauss assay and the fibrinogen PT derived method in patients
with dysfibrinogenemia. Thromb Res. 2010;126:e428–33.
90. Mackie J, Lawrie AS, Kitchen S, Gaffney PJ, Howarth D, Lowe GD, et al.
A performance evaluation of commercial fibrinogen reference prepara-
tions and assays for Clauss and PT-derived fibrinogen. Thromb Haemost.
2002;87:997–1005.
91. Curry NS, Davenport R, Pavord S, Mallett SV, Kitchen D, Klein AA,
et al. The use of viscoelastic haemostatic assays in the management of
major bleeding: a British Society for Haematology Guideline. Br J Hae-
matol. 2018;182:789–806.
92. Hsu P, Zantek ND, Meijer P, Hayward CP, Brody J, Zhang X, et al. Fac-
tor XIII Assays and associated problems for laboratory diagnosis of factor
XIII deficiency: an analysis of International Proficiency testing results.
Semin Thromb Hemost. 2014;40:232–8.
93. Jennings I, Kitchen S, Woods TA, Preston FE, Uk N. Problems relating
to the laboratory diagnosis of factor XIII deficiency: a UK NEQAS study.
J Thromb Haemost. 2003;1:2603–8.
94. Kohler HP, Ichinose A, Seitz R, Ariens RA, Muszbek L, Factor X, et al.
Diagnosis and classification of factor XIII deficiencies. J Thromb Haemost.
2011;9:1404–6.
95. Durda MA, Wolberg AS, Kerlin BA. State of the art in factor XIII labora-
tory assessment. Transfus Apher Sci. 2018;57:700–4.
96. Ajzner E, Muszbek L. Kinetic spectrophotometric factor XIII activity
assays: the subtraction of plasma blank is not omissible [corrected]. J
Thromb Haemost. 2004;2:2075–7.
97. Dorgalaleh A, Farshi Y, Alizadeh SH, Naderi M, Tabibian SH, Kazemi A,
et al. Challenges in implementation of ISTH diagnostic algorithm for
diagnosis and classification of factor XIII deficiency in Iran. J Thromb
Haemost. 2015;13:1735–6.
98. Longstaff C. Measuring fibrinolysis: from research to routine diagnostic
assays. J Thromb Haemost. 2018;16:652–62.
99. Macy EM, Meilahn EN, Declerck PJ, Tracy RP. Sample preparation for
plasma measurement of plasminogen activator inhibitor-1 antigen in
large population studies. Arch Pathol Lab Med. 1993;117:67–70.
100. Pozzi L, Della Valle P, D’angelo A. May restricted specificity of commer-
cially available plasmin inhibitor activity assays affect correct diagnosis of
antiplasmin deficiency? Blood Coagul Fibrinolysis. 2019;30:52–3.
101. Agren A, Wiman B, Stiller V, Lindmarker P, Sten-Linder M, Carlsson A,
et al. Evaluation of low PAI-1 activity as a risk factor for hemorrhagic
diathesis. J Thromb Haemost. 2006;4:201–8.
102. Kluft C, Verheijen JH. Leiden fibrinolysis working party: blood collection
and handling procedures for assessment of tissue-type plasminogen acti-
vator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Fibrinolysis.
1990;4:155–61.
103. Baglin T, Gray E, Greaves M, Hunt BJ, Keeling D, Machin S, et al. Clini-
cal guidelines for testing for heritable thrombophilia. Br J Haematol.
2010;149:209–20.
104. CLSI. Laboratory testing for the lupus anticoagulant; approved guideline.
Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
105. Cohen H, Mackie IJ, Devreese KMJ; International Society for Thrombosis
and Haemostasis Scientific and Standardization Committee for Lupus
Anticoagulant/Antiphospholipid Antibodies. Clinical and laboratory prac-
tice for lupus anticoagulant testing: an International Society of Thrombo-
sis and Haemostasis Scientific and Standardization Committee survey. J
106. Keeling D, Mackie I, Moore GW, Greer IA, Greaves M; British Commit-
tee for Standards in Haematology. Guidelines on the investigation and
management of antiphospholipid syndrome. Br J Haematol. 2012;157:47–
58.
107. Pengo V. ISTH guidelines on lupus anticoagulant testing. Thromb Res.
2012;130(Suppl 1):S76–7.
108. Demers C, Henderson P, Blajchman MA, Wells MJ, Mitchell L, Johnston
M, et al. An antithrombin III assay based on factor Xa inhibition pro-
vides a more reliable test to identify congenital antithrombin III defi-
ciency than an assay based on thrombin inhibition. Thromb Haemost.
1993;69:231–5.
109. Tran TH, Duckert F. Influence of heparin cofactor II (HCII) on the
determination of antithrombin III (AT). Thromb Res. 1985;40:
571–6.
110. Kottke-Marchant K, Duncan A. Antithrombin deficiency: issues in labo-
ratory diagnosis. Arch Pathol Lab Med. 2002;126:1326–36.
111. Kovacs B, Bereczky Z, Olah Z, Gindele R, Kerenyi A, Selmeczi A, et al.
The superiority of anti-FXa assay over anti-FIIa assay in detecting hep-
arin-binding site antithrombin deficiency. Am J Clin Pathol.
2013;140:675–9.
112. Ungerstedt JS, Schulman S, Egberg N, Antovic J, Blomback N. Discrep-
ancy between antithrombin activity methods revealed in Antithrombin
Stockholm: do factor Xa-based methods overestimate antithrombin activ-
ity in some patients? Blood. 2002;99:2271–2.
113. Corral J, Hernandez-Espinosa D, Soria JM, Gonzalez-Conejero R, Ordo-
nez A, Gonzalez-Porras JR, et al. Antithrombin Cambridge II (A384S): an
underestimated genetic risk factor for venous thrombosis. Blood.
2007;109:4258–63.
114. Kristensen SR, Rasmussen B, Pedersen S, Bathum L. Detecting
antithrombin deficiency may be a difficult task–more than one test is
necessary. J Thromb Haemost. 2007;5:617–8.
115. van Cott EM, Orlando C, Moore GW, Cooper PC, Meijer P, Marlar R,
et al. Recommendations for clinical laboratory testing for antithrombin
deficiency; Communication from the SSC of the ISTH. J Thromb Hae-
most. 2020;18:17–22.
116. Ruhl H, Reda S, Muller J, Oldenburg J, Potzsch B. Activated factor X-
based versus thrombin-based antithrombin testing in thrombophilia
workup in the DOAC Era. Thromb Haemost. 2018;118:381–7.
117. CAP. CG2-A survey (comprehensive coagulation). Northfield, IL: College
of American Pathologists; 2000.
118. Rossi E, Chiusolo P, Za T, Marietti S, Ciminello A, Leone G, et al.
Report of a novel kindred with antithrombin heparin-binding site variant
(47 Arg to His): demand for an automated progressive antithrombin
assay to detect molecular variants with low thrombotic risk. Thromb Hae-
most. 2007;98:695–7.
119. Stocker K, Fischer H, Meier J, Brogli M, Svendsen L. Protein C activators
in snake venoms. Behring Inst Mitt. 1986;37–47.
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

120. Guglielmone HA, Vides MA. A novel functional assay of protein C in
human plasma and its comparison with amidolytic and anticoagulant
assays. Thromb Haemost. 1992;67:46–9.
121. Chalmers E, Cooper P, Forman K, Grimley C, Khair K, Minford A, et al.
Purpura fulminans: recognition, diagnosis and management. Arch Dis
Child. 2011;96:1066–71.
122. Cooper PC, Pavlova A, Moore GW, Hickey KP, Marlar RA. Recommen-
dations for clinical laboratory testing for protein C deficiency, for the
subcommittee on plasma coagulation inhibitors of the ISTH. J Thromb
Haemost. 2020;18:271–7.
123. Cooper PC, Siddiq S, Morse C, Nightingale J, Mumford AD. Marked dis-
crepancy between coagulometric protein C activity assays with the pro-
thrombotic protein C Asn2Ile substitution. Int J Lab Hematol.
2011;33:451–6.
124. D’Angelo SV, Gilardoni F, D’Angelo A. Evaluation of coagulometric
assays in the assessment of protein C anticoagulant activity; variable sen-
sitivity of commercial APTT reagents to the cofactor effect of protein S.
125. de Moerloose P, Reber G, Bouvier CA. Spuriously low levels of protein C
with a Protac activation clotting assay. Thromb Haemost. 1988;59:543.
126. Jennings I, Kitchen S, Cooper PC, Rimmer JE, Woods TA, Preston FE.
Further evidence that activated protein C resistance affects protein C
coagulant activity assays. Thromb Haemost. 2000;83:171–2.
127. Cooper PC, Cooper SM, Goodfellow KJ, Hickey KP, Kitchen S, Makris
M. Evaluation of a new venom-based clotting assay of protein C. Int J
Lab Hematol. 2008;30:437–43.
128. Toulon P, Berruyer M, Brionne-Francois M, Grand F, Lasne D, Telion C,
et al. Age dependency for coagulation parameters in paediatric popula-
tions. Results of a multicentre study aimed at defining the age-specific
reference ranges. Thromb Haemost. 2016;116:9–16.
129. Haemostasis and Thrombosis Task Force, British Committee for Stan-
dards in Haematology. Investigation and management of heritable
thrombophilia. Br J Haematol. 2001;114:512–28.
130. Said JM, Ignjatovic V, Monagle PT, Walker SP, Higgins JR, Brennecke
SP. Altered reference ranges for protein C and protein S during early
pregnancy: implications for the diagnosis of protein C and protein S defi-
ciency during pregnancy. Thromb Haemost. 2010;103:984–8.
131. Hackeng TM, Sere KM, Tans G, Rosing J. Protein S stimulates inhibition
of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl
Acad Sci USA. 2006;103:3106–11.
132. Marlar RA, Gausman JN. Protein S abnormalities: a diagnostic night-
mare. Am J Hematol. 2011;86:418–21.
133. Persson KE, Hillarp A, Dahlback B. Analytical considerations for free
protein S assays in protein S deficiency. Thromb Haemost. 2001;86:1144–7.
134. Bertina RM. Protein S antigen. In: Jesperson J, Bertina RM, Haeverkate
F, editors. Laboratory techniques in thrombosis – a manual; 2nd revised
edition of the ECTA assay procedures. Dordrecht: Kluwer Academic Pub-
lishers; 1999.
135. Marlar RA, Gausman JN. Laboratory testing issues for protein C, protein
S, and antithrombin. Int J Lab Hematol. 2014;36:289–95.
136. Faioni EM, Franchi F, Asti D, Sacchi E, Bernardi F, Mannucci PM. Resis-
tance to activated protein C in nine thrombophilic families: interference
in a protein S functional assay. Thromb Haemost. 1993;70:1067–71.
137. Jennings I, Kitchen S, Woods TA, Preston FE. Multilaboratory testing in
thrombophilia through the United Kingdom National External Quality
Assessment Scheme (Blood Coagulation) Quality Assurance Program.
Semin Thromb Hemost. 2005;31:66–72.
138. Maryamchik E, Rosenbaum MW, van Cott EM. Rivaroxaban causes
missed diagnosis of protein S deficiency but not of activated protein C
resistance (factor V Leiden). Arch Pathol Lab Med. 2018;142:70–4.
139. Maryamchik E, van Cott EM. Apixaban does not interfere with protein S
or activated protein C resistance (factor V Leiden) testing using aPTT-
based methods. Arch Pathol Lab Med. 2020.
140. Noguchi K, Nakazono E, Tsuda T, Jin X, Sata S, Miya M, et al. Plasma
phenotypes of protein S Lys196Glu and protein C Lys193del variants
prevalent among young Japanese women. Blood Coagul Fibrinolysis.
2019;30:393–400.
141. Herskovits AZ, Morgan EA, Lemire SJ, Lindeman NI, Dorfman DM. An
improved algorithm for activated protein C resistance and factor V Lei-
den screening. Am J Clin Pathol. 2013;140:379–86.
142. Moore GW, van Cott EM, Cutler JA, Mitchell MJ, Adcock DM; Subcom-
mittee on Plasma Coagulation Inhibitors. Recommendations for clinical
laboratory testing of activated protein C resistance; communication from
the SSC of the ISTH. J Thromb Haemost. 2019;17:1555–61.
143. Quehenberger P, Handler S, Mannhalter C, Kyrle PA, Speiser W. The
factor V (Leiden) test: evaluation of an assay based on dilute Russell
Viper Venom Time for the detection of the Factor V Leiden mutation.
Thromb Res. 1999;96:125–33.
144. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic
variation in the 3’-untranslated region of the prothrombin gene is associ-
ated with elevated plasma prothrombin levels and an increase in venous
thrombosis. Blood. 1996;88:3698–703.
145. Norstrom E, Thorelli E, Dahlback B. Functional characterization of
recombinant FV Hong Kong and FV Cambridge. Blood. 2002;100:524–30.
146. van Cott EM, Khor B, Zehnder JL. Factor V Leiden. Am J Hematol.
2016;91:46–9.
147. Kokame K, Matsumoto M, Fujimura Y, Miyata T. VWF73, a region from
D1596 to R1668 of von Willebrand factor, provides a minimal substrate
for ADAMTS-13. Blood. 2004;103:607–12.
148. Kokame K, Nobe Y, Kokubo Y, Okayama A, Miyata T. FRETS-VWF73, a
first fluorogenic substrate for ADAMTS13 assay. Br J Haematol.
2005;129:93–100.
149. Chiasakul T, Cuker A. Clinical and laboratory diagnosis of TTP: an inte-
grated approach. Hematology Am Soc Hematol Educ Program.
2018;2018:530–8.
150. Rieger M, Mannucci PM, Kremer Hovinga, J.A., Herzog, A., Gersten-
bauer, G., Konetschny, C., et al. ADAMTS13 autoantibodies in patients
with thrombotic microangiopathies and other immunomediated diseases.
Blood. 2005;106:1262–7.
151. Thomas MR, de Groot R, Scully MA, Crawley JT. Pathogenicity of anti-
ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic
purpura. EBioMedicine. 2015;2:942–52.
152. Favresse J, Lardinois B, Chatelain B, Jacqmin H, Mullier F. Evaluation of
the fully automated HemosIL acustar ADAMTS13 activity assay. Thromb
Haemost. 2018;118:942–4.
153. Griffiths M, Vetr H, Binder NB. Performance evaluation of a new screen-
ing assay for ADAMTS-13 Activity. GTH, Poster presentation, P06-7.
154. Lillicrap D. Molecular testing for disorders of hemostasis. Int J Lab
Hematol. 2013;35:290–6.
155. Marlar RA, Potts RM, Marlar AA. Effect on routine and special coagula-
tion testing values of citrate anticoagulant adjustment in patients with
high hematocrit values. Am J Clin Pathol. 2006;126:400–5.
156. Lippi G, von Meyer A, Cadamuro J, Simundic AM. Blood sample quality.
Diagnosis. 2019;6:25–31.
157. Woodhams B, Girardot O, Blanco MJ, Colesse G, Gourmelin Y. Stability
of coagulation proteins in frozen plasma. Blood Coagul Fibrinolysis.
2001;12:229–36.
Appendix 1
Search strategy
The strategy was developed to identify articles suitable for
updating the BSH guidelines. Searches were performed using
the online search engine Medline (PubMed). Search terms
were: [coagulation assay AND immunoassay]; [coagulation
assay AND immunoturbidimetric]; [amidolytic assay]; [chro-
mogenic assay AND coagulation]; [chromogenic assay AND
haemostasis]; [haemostasis assay AND technique]; [haemo-
philia assay AND technique]; [thrombophilia assay AND
Guideline
361
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

technique]; [coagulation assay AND technique]; [fibrinogen
assay AND technique]. Filters were applied to include only
publications written in English, studies carried out in
humans, clinical trials or clinical studies, comparative studies,
evaluation studies, guidelines, meta-analyses, multicentre
studies, observational studies, practice guidelines, reviews,
systematic reviews, validation studies, and published between
1 January 2011 and 21 April 2020, inclusive for all but the
final search term which included publications dated between
1 January 2002 and 21 April 2020 inclusive. Additional rele-
vant articles were identified by screening reference lists and
by publications known to the writing group.
Searches of individual journals were not implemented
because it was felt that publications not captured during the
database search process would have had limited availability
and would have had little impact on the scientific community.
BAKER et al.
13652141,
2020,
3,
Downloaded
from
by
Cochrane
Saudi
Arabia,
Wiley
Online
Library
on
[15/03/2023].
See
the
Terms
and
Conditions
on
Wiley
Online
Library
for
rules
of
use;
OA
articles
are
governed
by
the
applicable
Creative
Commons
License

Br J Haematol - 2020 - Baker - Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.pdf

Recommended

Recommended

More Related Content

Similar to Br J Haematol - 2020 - Baker - Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.pdf

Similar to Br J Haematol - 2020 - Baker - Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.pdf (20)

Recently uploaded

Recently uploaded (20)

Br J Haematol - 2020 - Baker - Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.pdf