D Dimer Preanalytical Analytical Postanalytical Variables and Clinical Applications

Critical Reviews in Clinical Laboratory Sciences
ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: https://www.tandfonline.com/loi/ilab20
D-dimer: Preanalytical, analytical, postanalytical

variables, and clinical applications
Julien Favresse, Giuseppe Lippi, Pierre-Marie Roy, Bernard Chatelain,

Hugues Jacqmin, Hugo ten Cate & François Mullier
To cite this article: Julien Favresse, Giuseppe Lippi, Pierre-Marie Roy, Bernard Chatelain, Hugues
Jacqmin, Hugo ten Cate & François Mullier (2018) D-dimer: Preanalytical, analytical, postanalytical
variables, and clinical applications, Critical Reviews in Clinical Laboratory Sciences, 55:8, 548-577,
DOI: 10.1080/10408363.2018.1529734
To link to this article: https://doi.org/10.1080/10408363.2018.1529734
© 2019 The Author(s). Published by Informa

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CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
2018, VOL. 55, NO. 8, 548–577
https://doi.org/10.1080/10408363.2018.1529734
REVIEW ARTICLE
D-dimer: Preanalytical, analytical, postanalytical variables, and clinical

applications
Julien Favressea, Giuseppe Lippib , Pierre-Marie Royc, Bernard Chatelaina, Hugues Jacqmina, Hugo ten
Cated and François Mulliera
a
CHU UCL Namur, Namur Thrombosis and Hemostasis Center, Hematology Laboratory, Universite Catholique de Louvain, Yvoir,
Belgium; bSection of Clinical Biochemistry, University Hospital of Verona, Verona, Italy; cDepartement de Medecine d’Urgence, CHU
d’Angers, Institut MITOVASC, Universite d’Angers, Angers, France; dDepartment of Internal Medicine, Cardiovascular Institute,
Maastricht University Medical Center, Maastricht, the Netherlands
ABSTRACT ARTICLE HISTORY

D-dimer is a soluble fibrin degradation product deriving from the plasmin-mediated degradation Received 23 June 2018
of cross-linked fibrin. D-dimer can hence be considered a biomarker of activation of coagulation Revised 31 August 2018
and fibrinolysis, and it is routinely used for ruling out venous thromboembolism (VTE). D-dimer Accepted 25 September 2018
is increasingly used to assess the risk of VTE recurrence and to help define the optimal duration Published online 10 January
2019
of anticoagulation treatment in patients with VTE, for diagnosing disseminated intravascular
coagulation, and for screening medical patients at increased risk of VTE. This review is aimed at KEYWORDS
(1) revising the definition of D-dimer; (2) discussing preanalytical variables affecting the measure- D-dimer; preanalytical;
ment of D-dimer; (3) reviewing and comparing assay performance and some postanalytical varia- analytical; postanalytical;
bles (e.g. different units and age-adjusted cutoffs); and (4) discussing the use of D-dimer venous thromboembolism
measurement across different clinical settings.
Abbreviations: AAD: acute aortic dissection; ACCF: American College of Cardiology Foundation;
AD: aortic dissection; AHA: American Heart Association; AMI: acute mesenteric ischemia; APTT:
activated partial thromboplastin time; ASA: American Stroke Association; BMI: body mass index;
CAP: College of American Pathologists; CDR: clinical decision rule; CLIA: chemiluminescent
enzyme immunometric assay; CLSI: Clinical Laboratory Standards Institute; CPR: clinical prediction
rules; CT: computed tomography; CTPA: CT pulmonary angiogram; CV: coefficient of variation;
CVT: cerebral venous thrombosis; DD/E: D-dimer/fragment E complex; DDU: D-dimer units; DIC:
disseminated intravascular coagulation; DiPEP: Diagnosis of PE in Pregnancy; DVT: deep venous
thrombosis; eGFR: estimated glomerular filtration rate; ELFA: enzyme-linked fluorescence
immunoassay; ELISA: enzyme-linked immunosorbent assays; ESC: European Society of Cardiology;
ESTES: European Society for trauma and Emergency Surgery; F/T: freezing/thawing; F1 þ 2: pro-
thrombin fragments 1 þ 2; FACT: fibrin assay comparison trial; FDA: Food and Drug
Administration; FDP: fibrin degradation products; FEU: fibrinogen equivalent units; FV: factor V;
FVIII: factor VIII; G: Gauge; GP: general practitioner; HMWF: high molecular weight fibrinogen; HR:
hazard ratio; HV: healthy volunteers; ISO: International Organization for Standardization; ISTH:
International Society on Thrombosis and Haemostasis; IV: intravenous; LMWF: low molecular
weight fibrinogen; NNT: number needed to test; NPV: negative predictive value; PE: pulmonary
embolism; PET: polyethylene terephthalate; POC: point of care; PPV: positive predictive value; PT:
prothrombin time; PTS: pneumatic tube system; RS: risk score; RT: room temperature; TAT: turn-
around time; t-PA: tissue plasminogen activator; TT: thrombin time; URL: upper reference limit;
VKA: vitamin K antagonist; VTE: venous thromboembolism; WHO: World Health Organization
Introduction: what is D-dimer? [1–4]. The first step of D-dimer formation involves the
D-Dimer is a generic term referring to multiple peptide thrombin-catalyzed conversion of fibrinogen into fibrin
fragments deriving from plasmin-mediated degradation monomers (Figure 1). Human fibrinogen is a soluble
of cross-linked fibrin. Their presence reflects concomi- plasma glycoprotein composed of three different pairs
tant activation of both coagulation and fibrinolysis of polypeptide chains (Aa-, Bb-, and c-) connecting
CONTACT François Mullier francois.mullier@uclouvain.be CHU UCL Namur, Hematology Laboratory, Universite Catholique de Louvain, Avenue G.
Therasse, 1, 5530 Yvoir, Belgium
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in
any way.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 549
Figure 1. Mechanism of D-dimer production [1,3,4,164].
two-outer D-domains to the central E-domain [5,6]. breakdown of both fibrinogen and fibrin by plasmin is
Thrombin enzymatically cleaves two cryptic polymeriza- counterbalanced by multiple enzymatic modulators
tion sites located on the E-domain, thus leading to the (thrombin-activatable fibrinolysis inhibitor, a-2-antiplas-
generation of both highly self-adhesive fibrin monomers min, and a-2-macroglobulin), whose functions are to
and fibrinopeptides A and B [1]. Fibrin monomers then limit fibrinolysis at the injury site [3].
bind to one another to form a soluble network [1]. The term “fragment D-dimer” was initially used to
Simultaneously, the complex between soluble fibrin pol- describe the final plasmin digestion products (resistant
ymers, thrombin, and plasma factor XIII promotes the to further plasmin breakdown) of the factor XIIIa–cross-
formation of factor XIIIa, which catalyzes covalent cross- linked fibrin clot, which is the fragment DD/E [1,10].
linking of fibrin polymer via intermolecular bonds However, the actual D-dimer antigen that can be
formed between lysine and glutamine residues, thus detected by current immunoassays is not necessarily the
enabling the generation of stable and insoluble clots DD/E complex [1]. In fact, the term D-dimer comprises a
[1,4]. Later, the fibrinolytic pathway leads to degradation broad mixture of degradation products of cross-linked
of stabilized clots through plasmin activation [4]. Plasmin fibrin with molecular weights likely to range from 190 to
is activated from plasminogen by tissue plasminogen over 10,000 kDa [9]. D-dimer fragments are mainly elimi-
activator (t-PA) at the fibrin surface and cleaves fibrin at nated by renal clearance and reticuloendothelial system
specific sites [7]. The products of this reaction are the catabolism. The plasma half-life of D-dimer is 8 h [11],
conventionally-defined fibrin degradation products which is much longer than that of the thrombin-antith-
(FDP), and display a vast array of molecular weights. In rombin complex (10–15 min) or prothrombin fragments
the first steps of fibrinolysis, FDP are large. Continual 1 þ 2 (F 1 þ 2; 90 min). In normal physiological condi-
breakdown generates the fragment D-dimer/fragment E tions, around 2–3% of fibrinogen is converted into fibrin,
complex (DD/E), which has two covalently-bound D- which instantly enters the fibrinolytic pathway. Thus, D-
domains [4]. Because of variable degrees of plasmin- dimer is measurable in small amounts in healthy subjects
mediated proteolysis, plasma samples then contain a and tends to increase with aging [4,12].
mixture of fibrin fragment complexes containing one or Fibrinogenolysis and fibrinolysis have to be consid-
several D-dimer motifs whose molecular weights range ered differently; fibrinogenolysis leads to generation of
from 228 kDa (DD/E) to several thousand kDa (X-oligom- fragments X, D, Y, and E (fibrinogen degradation prod-
ers) [8,9]. These FDPs can derive both from the insoluble ucts), whilst fibrinolysis leads to more complex frag-
fibrin clot and from soluble fibrin polymers [1]. The ments called X-oligomers that contain the D and E
550 J. FAVRESSE ET AL.
fragments in various sequences [1,4]. Only fibrin et al. during a 1-year period revealed that clotted sam-
polymers that have undergone factor XIII-mediated ples were the major source of preanalytical errors in
cross-linking will produce fragments containing covalent their hematology laboratory (43.2%), whilst samples
bonds between two adjacent D domains (i.e. D-dimer). with inappropriate volume were less frequent [23].
Therefore, D-dimer (DD/E fragment) should be consid- Recently, Dikmen et al. performed a similar study during
ered as a specific FDP that is recognized by most of the a 1-year follow-up and observed that clotted samples
reagent antibodies used in the laboratory assessment of (35%) and inadequate volume (13%) were the major
thrombosis [3]. Commercial D-dimer immunoassays are causes of sample rejection [24]. They found that the
currently predicted to detect an epitope on the degrad- specimen rejection rate for coagulation tests was higher
ation products of factor XIIIa–cross-linked fibrin. (13.3%) than all other tests (e.g. 3.2% for biochemistry
Monoclonal antibodies employed should not detect tests, 9.8% for blood gases and 9.8% for urinalysis) [24].
fibrinogen degradation products (e.g. X, Y, D, E) on non- The source of preanalytical variables impairing sam-
cross-linked FDP, but they predictably do detect only ple quality can be divided into three main categories: (i)
epitopes present in the factor XIIIa–cross-linked fragment sample collection (e.g. needle size, collection tubes), (ii)
D domain of fibrin [1,4]. Gaffney et al. found that anti- sample delivery to the laboratory (e.g. pneumatic tube
genic determinants of D-dimer assays are a part of the system (PTS), temperature), and (iii) sample processing
polypeptides in the D-domain, which are conformation- (e.g. centrifugation, hemolyzed samples). The storage
ally reactive only after factor XIIIa and plasmin have and stability of samples, as well as the freeze-thaw
modified the protein [10]. Notably, each monoclonal effect, are also part of the preanalytical process [25].
antibody has its own specificity toward FDP [13]. Importantly, each preanalytical step is vulnerable to
errors. Therefore, following preanalytical recommenda-
tions at each step of the process is essential for preserv-
Preanalytical variables ing sample integrity [25]. The impact of each of these
According to ISO 15189:2012 Medical laboratories– preanalytical variables on D-dimer measurement in cur-
Requirements for quality and competence (ISO, rent hemostasis laboratories will be discussed.
International Organization for Standardization), the ISO Through a review of the literature of the effects of pre-
analytical variables on D-dimer, it appeared that statistical
standard that is used for laboratory accreditation, the
analysis was frequently used to assess a significant pre-
preanalytical phase is defined as “processes that start, in
analytical change. The absence of significant bias is con-
chronological order, from the clinician’s request and
sidered a robust indicator that a pre-analytical variable has
include the examination request, preparation and identi-
not significantly impacted a particular analyte. However, a
fication of the patient, collection of the primary sam-
significant difference does not inherently correspond to
ple(s), and the transport to and within the laboratory,
clinical significance. Therefore, a clinical criterion is also
and end when the analytical examination begins” [14].
recommended to accurately assess the effect of preanalyt-
ical variables on a particular analyte. Regarding D-dimer, a
Preanalytical phase in hemostasis laboratories clinically acceptable cutoff of 10% has frequently been
used to assess a significant preanalytical change (espe-
It is now clearly established that most errors encountered
cially to assess the effect of interfering substances and for
in the hemostasis laboratory are related to preanalytical
performing stability studies). Unfortunately, this 10% cut-
activities [15–20]. Preanalytical errors have a frequency of
off has not been derived from biological variation data
60–70%, a frequency that is much higher than that occur-
(i.e. reference change values [26]) because such data are
ring in the analytical (e.g. 10–15%) and postanalytical
still lacking in the hemostasis field. Further validation of
phase (e.g. 15–20%) [20]. Preanalytical errors are related
this empirical 10% cutoff and establishment of biological
mainly to intensive manual activities [20,21].
variation studies are hence needed [27].
During a 2-year follow-up period, Salvagno et al.
studied all preanalytical errors detected in the local
coagulation laboratory, and found that the most fre- Preanalytical variables affecting the D-dimer
quent preanalytical problems were related to samples measurement
not received in the laboratory (49.3%), hemolysis Sample collection
(19.5%), clotting (14.2%), and inadequate sample vol-
Butterfly devices and needle bore size
ume (13.7%) [22]. They also showed that preanalytical
problems could be identified in up to 5.5% of all coagu- It is currently recommended to use straight needles
lation samples [22]. Another study performed by Grecu with a diameter ranging from 19 to 22 gauge (G) [21].
BD and VacuetteV, Greiner Bio-one (polypropylene)) and

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Blood samples should be obtained with relatively non-
traumatic venipuncture. Excess vein manipulation with glass citrated collection tubes (3.2%) [36]. More recently,
the needle should be avoided to limit the risk of devel- Yavas et al. compared three plastic citrate (3.2%) tubes
(VacutainerV, BD; VacutainerV Plus Plastic, BD; and
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oping clots [25], which have been found to potentially
VacuetteV, Greiner Bio-one) with a standard glass tube
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impact D-dimer concentration [28]. The use of butterfly
(VacutainerV, BD), and also failed to find significant differ-
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devices (small needle attached to flexible plastic wings
and connected with a flexing tube) is usually discour- ences [37]. Therefore, the quality of D-dimer measure-
aged because the passage of blood along the tubing ment seems to be not substantially dependent on the
might be a cause of enhanced shear stress, thus poten- type of collection tube.
tially triggering hemostasis activation, erythrocyte
injury (i.e. hemolysis); they are also more expen- Anticoagulant sample (or tube additive)
sive [21,29,30].
The use of collection tubes containing 3.2%
Interestingly, Lippi et al. found that the use of butter-
(105–109 mmol/L) buffered sodium citrate anticoagulant
fly devices (21 G, 300 mm-long tubing) did not introduce
is now recommended by the Clinical Laboratory
a significant bias in D-dimer values measured with the
Standards Institute (CLSI) and by the World Health
Mini VidasV Immunoanalyzer (bioMerieux, Marcy l’Etoile,
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Organization (WHO) for the vast majority of hemostasis

France) compared to conventional straight needles
tests [25,34]. A blood to anticoagulant ratio of 9/1 is rec-
(21 G) [31]. This observation implies that the potential
ommended because the sodium citrate anticoagulant
activation of the hemostatic system occurring within the
can be used only in a liquid form [25]. Failure to cor-
tubing of the butterfly device, regardless of its compos-
rectly fill the citrate anticoagulant tube will typically pro-
ition, may be negligible for the D-dimer measurement.
long clotting times (PT, APTT, and TT), and may also lead
Lippi et al. also found a clinically negligible bias
to underestimating D-dimer and fibrinogen [28].
using butterfly devices with different needle bore sizes
Therefore, the sample should be checked for quality (e.g.
(21, 23, or 25 G), when D-dimer was again measured
clotting, underfilling, or overfilling) [4]. Blood should also
with the Mini VidasV Immunoanalyzer [32]. Taken
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flow freely into the tube and be promptly mixed within

together, the use of butterfly devices, even with small
30 s after phlebotomy (i.e. from 3 to 6 complete inver-
size needles, seems to represent a viable option to
sions), thus ensuring the complete distribution of anti-
standard straight needles [31,32]. However, straight
coagulant activity [25]. Serum and heparinized/EDTA
needle venipunctures are preferable, especially when
plasma samples cannot be accepted because these addi-
other coagulation tests are requested [33]. The use of
tives dramatically interfere with clot-based assays (e.g.
butterfly devices may, therefore, be considered in par-
PT, APTT, FV, FVIII) [25,28].
ticular individuals (geriatrics, oncology, pediatrics, or in
However, several D-dimer assays (including POC tests)
emergency settings) [21,33]. When blood is collected
allow the use of citrated, heparinized or EDTA plasma
using a winged collection system (or IV catheters), a dis-
(PathfastV, Tina-quantV (Roche, Switzerland)), AQT-90V
R R R
card tube is mandatory for removing air contained

(Radiometer, Denmark), SimplifyV (Agen Inc, Australia)),
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within the tubing, which may be associated with the

whilst others recommend only citrate anticoagulated
collection of an inadequate volume of blood [34].
blood (e.g. VidasV, BCSV, STA-LiatestV (Diagnostica
R R R
Stago, Asnieres, France), ImmuliteV (Siemens, USA)) [38].

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Tube material
Schutgens et al. observed a higher mean D-dimer con-
centration (Tina-quantV D-dimer) in heparin collection
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Non-activating material (silicone-coated glass or poly-
propylene plastic) is preferred for hemostasis testing tubes (2510 mg/L), compared to citrate (2060 mg/L) albeit
because this will prevent initiation of clotting due to the difference did not achieve statistical significance
spurious hemostasis activation in the blood collection [39]. The higher D-dimer concentration in heparin
tube [25]. plasma is probably attributable to the introduction of a
Several studies were performed for comparing the use dilution factor in citrate-containing blood tubes.
of different collection tube materials. Leroy-Matheron Although Vukovich et al. reported that heparinized and
et al. found no significant differences in measured D- citrated samples yielded identical D-dimer values (D-
dimer values (AsserachromV ELISA assay) using glass or dimer GoldV, Agen Biochemical) samples, they recom-
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polyethylene terephthalate (PET) plastic collection tubes mended using a correction factor of 0.84 to take into
(VacutainerV, Becton Dickinson (BD)) [35]. Gosselin et al.
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account the dilution of the citrate anticoagulant, a pro-
also failed to find significant differences for D-dimer val- cess that may be confusing in routine practice [40].
ues (Advanced D-Dimer) when using plastic (VacutainerV,
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Local evaluation of other matrices should be validated
before they are used in clinical practice, especially when extent of in vitro microparticle generation [21,42].
the manufacturer recommends using only citrated sam- Although the presence of blood collection facilities
ples. Lippi et al. found only a modest bias of D-dimer close to the laboratory is the best option to limit pre-
values (ImmuliteV 2000 D-dimer) between lithium-
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analytical variability, many hospitals deliver blood sam-
heparin and sodium citrate blood tubes [41]. ples with a PTS [21]. PTS has the great advantage of
The main advantage of using other anticoagulants reducing the turnaround time (TAT), but these systems
(e.g. heparin) is the possibility to measure other analy- need to be validated before use, because excessive
tes (e.g. electrolytes, cardiac troponins). However, the acceleration/deceleration, radial gravity forces, vibra-
dilution factor may be a confounding factor and may tion, and changes in air pressure may trigger platelet
require setting specific algorithms for correcting data. activation and hemolysis [21,43,44].
Therefore, other anticoagulants should be locally vali- Schutgens et al. failed to find significant difference
between D-dimer values (Tina-quantV D-dimer) meas-
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dated or their use should be reserved for special cir-
cumstances; thus buffered sodium citrate (3.2%, or ured in samples delivered by PTS (100 m long) com-
105–109 mmol/L) remains the recommended sample pared to those carried by hand [39]. Wallin et al. also
matrix [33]. found no substantial difference in D-dimer values
(MediRoxV assay, Nyko €ping, Sweden) delivered to the
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Notably, D-dimer was first measured in serum sam-
ples with preliminary removal of fibrinogen to avoid laboratory by PTS (500 m long) compared to those dir-
cross-reactivity with polyclonal antibodies [4]. However, ectly collected in the laboratory [44]. Unlike these stud-
false-positive results were frequently encountered in ies, Le Quellec et al. observed significant difference of D-
dimer values (DDi-HS 500V, HemosILV) in samples con-
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patients under anticoagulant treatment, whilst false
negative values were also seen when FDPs were veyed by PTS (2000 m long) compared to those trans-
entrapped in the clot. The loss of fibrinopeptide A has ported with a motor vehicle (mean difference of 7.4%)
also been associated with degradation of non-cross- [43]. Nevertheless, a disagreement at the 500 mg/L D-
linked FDP [4]. dimer cutoff could be observed only in 1 out of the 39
samples (510 lg/L with PTS and 490 lg/L by motor
Tourniquet use vehicle) and the overall difference was still within the
intra-assay imprecision of the D-dimer assay. Although
Tourniquets are conventionally used to temporarily
the data in the literature seemingly is reassuring about
obstruct the vein flow and thereby assist the phlebot-
the low impact of PTS on D-dimer test results, it is advis-
omist to identify vein access. It is commonly recom-
able that each laboratory assesses its local PTS because
mended that the tourniquet should be removed as
systems are heterogeneous in terms of length, internal
soon as the needle is in the vein or when the first tube
diameter, maximal acceleration force, and speed [43].
starts to fill and that it should never remain in place for
more than 1–2 min [21,25]. The most important draw-
backs of prolonged tourniquet placement include Sampling processing
hemoconcentration and clot formation, which may
Prior to being analyzed, samples should be carefully
jeopardize the quality of coagulation testing [21,25,32].
checked for the presence of inappropriate additive,
More specifically, Lippi et al. observed that D-dimer val-
identification errors, insufficient volume (9/1 ratio not
ues (Mini VidasV Immunoanalyzer) were significantly
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respected), and even for the presence of clots.

increased, by 13.4%, when measured in samples col-
Unsuitable samples should then be rejected and test
lected after 3 min of venous stasis [32]. Lower differen-
results suppressed to preserve patient safety [21].
ces were also observed after a 1-min stasis (mean
increase of 7.9%) [32].
Centrifugation
Except for point of care (POC) whole blood D-dimer
Sample delivery to the laboratory
analyzers, samples are centrifuged at room temperature
Samples should be delivered to the laboratory at ambi- at 1500g for at least 15 min, so that plasma can be
ent temperature (15–22 C), in the shortest possible reliably separated from the cellular components [34].
time (usually <1 h) after collection [34]. The stability of According to Bernard et al., however, no difference
could be observed in D-dimer values (VidasV assay)
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D-dimer will be discussed below. Blood samples have
to be delivered in a vertical position and the cap should when samples were centrifuged at 4500g for 2 min
not be removed [21,25]. The transport of blood tubes in rather than at 1500g for 15–20 min [45]. The use of a
a vertical rather than a horizontal position limits the shorter centrifugation time will be indeed useful in
emergency settings to reduce the TAT. The samples can hemoglobin) was necessary to achieve the same level
be kept at room temperature for up to 8 h and at 4 C of D-dimer decrease (5%) with the AcuStar D-dimer
for up to 24 h (unspun citrated blood samples) before assay [49]. However, the bias never exceeded the clinic-
measurement of D-dimer [4,46,47]. Elf et al. centrifuged ally acceptable cutoff of 10%. D’Angelo et al. more
blood samples at 4 C and measured D-dimer with two recently reported a significant difference of D-dimer
quantitative immunoassays (InnovanceV and AxSYMV
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values with mild hemolysis (cell-free hemoglobin of
(Abbott, USA)) [48], although there was no evidence 0.5 g/L) obtained by either heat-shock at 80 C
supporting cold centrifugation for D-dimer testing. (þ6.2%) or by mechanical injury (rotating blade hom-
ogenizer for 30 s) (þ14.9%)) when measured on a
Interfering substances BCSxpV assay (Siemens Healthcare) [54].
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The comparison of studies assessing hemolysis inter-

The types of interferences occurring in the preanalytical
phase of coagulation testing are typically classified into ference is challenging. First, different D-dimer assays
paraproteinemia, icterus, lipemia, and, the most with different reagent formulations were used.
studied, hemolysis [20]. Secondly, different techniques to obtain hemolyzed
In vitro hemolysis still represents one of the most fre- specimens were used (freezing and thawing whole anti-
quent causes of preanalytical problems in clinical labo- coagulated blood, mechanical lysis of whole anticoagu-
ratories, with a prevalence ranging between 30–70% of lant blood by aspiration through a fine needle or by a
all unsuitable specimens [22,49–52]. Causes of hemo- rotating blade homogenizer). While spiking samples
lyzed samples could result from patient’s characteristics with hemolysate or pure hemoglobin solution is consid-
or diseases (e.g. hemolytic anemia, metabolic disorders, ered unsuitable (lysis of leukocytes and platelets is
infectious agents, hemoglobin-based blood substitute), taken into account), mechanical hemolysis obtained
phlebotomy (e.g. needle G, tourniquet time, traumatic with a syringe equipped with a fine needle is preferred
draw, no mixing or vigorous mixing of blood tubes), (i.e. it more closely reproduces the breakdown of cells
sample transport (e.g. PTS, time, emergency or inten- due to traumatic blood collection) to the freeze-thaw
sive care units), processing (e.g. delay before centrifuga- method, which also suffers from a lack of standardiza-
tion, specimen re-spun, time/temperature of transport), tion across studies (e.g. different temperatures (70 C
and storage (e.g. temperature and duration) [50,51]. [53] or 80 C [54]) and duration of freezing [51].
The interference will also depend on the analytical Thirdly, the use of a clinical cutoff derived from biologic
method used (photometric, clotting, immunoassay). variability studies should be preferred to the analytical
The CLSI guideline H21-A5, collection, transport, and bias assessed [51]. Fourth, the selection of blood sam-
processing of blood specimens for testing plasma- ples may produce different results and conclusions;
based coagulation assays and molecular hemostasis data generated in healthy volunteers may not necessar-
assays, advises against analyzing samples with visible ily be similar to that obtained in intensive care unit
hemolysis because of possible bias in coagulation tests patients, or in those undergoing multiple therapies
from release of pro-coagulant factors from injured (namely, antiplatelet or anticoagulant drugs) [51].
cells [34]. The rejection of all hemolyzed samples is recom-
Lippi et al. showed that a significant increase in D- mended by the CLSI. The majority of hemolyzed sam-
dimer level (VidasV assay) could be observed in samples ples (±95%) from clinical laboratories are only mildly
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containing a final whole blood lysate concentration of hemolyzed (cell-free hemoglobin 0.3–0.6 g/L) [49,50,55].
at least 2.7% obtained with a freeze-thaw cycle (70 C) Thus, when the concentration of cell-free hemoglobin
[53]. However, a clinically significant variation (10%) is below a non-interference limit (i.e. <3 g/L), D-dimer
was observed only above cell-free hemoglobin concen- values may still be reliable and may be safely reported
trations of 13.6 g/L (or final lysate concentration of to the clinicians [49]. Notably, avoiding rejection of all
6.4%) [51,53]. Another study by the same authors hemolyzed specimens will reduce additional blood
evaluated the impact of mechanical hemolysis (syringe sampling and may shorten the clinical decision-making
equipped with a fine needle (30 G, 0.3 8 mm)) on two process, thereby favorably impacting staff workload,
different D-dimer assays (HemosIL AcuStarV (Werfen,
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patient comfort, and costs [49,51].
USA) and HemosIL HSV (Werfen, USA)) [49]. The D-dimer
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The effect of lipemia, icterus, and paraproteinemia
concentration measured with the HemosIL HSV reached
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has been less widely discussed in the literature
a clinically significant decrease (5%) from a hemolysis [51,56,57]. In an early report, Pittet et al. failed to find
index between 5.5–7.0 g/L of cell-free hemoglobin. any impact of lipemia (triglycerides concentration of
Greater hemolysis (i.e. 11.5–15.0 g/L of cell-free 9.7 g/L) and icterus (bilirubin concentration of 485 mg/L,
829.5 mmol/L) on the Vidas D-dimer assay [58]. More D-dimer analysis to samples already delivered from the
recently, La’ulu et al. found similar findings on the emergency room.
AxSYM D-dimer assay, with no clinical significant differ- Detailed information on D-dimer stability is shown
ence (<10% of deviation from the baseline) following in Table 1. With the exception of the publication of
addition of bilirubin (final concentration of 292 mg/L, Schutgens et al., citrated specimens were used to
499.4 mmol/L) and triglycerides (final concentration of assess the stability of this analyte [39]. The D-dimer
41.6 g/L) [59]. stability was studied extensively in both plasma and
The Innovance D-dimer assay on the Sysmex CA- whole blood. In brief, D-dimer is stable for at least 24 h
7000 also appeared to be unaffected by bilirubin (both (in plasma/whole blood) at room temperature (RT) or
free and conjugated bilirubin up to 1000 mg/L, at 2–8 C with many different D-dimer immunoassays.
1710.4 mmol/L) [60]. However, spurious D-dimer results Only Bo €hm-Weigert et al. and Toulon et al. suggested,
were observed on the STA analyzer after adding high respectively a 6- and 8-h D-dimer plasma stability, but
concentrations of free bilirubin (from 800 mg/L, this was because they did not extend their analysis for
1368.3 mmol/L) [60]. According to Chen et al., D-dimer, longer periods [47,69]. Frozen samples may also be
as measured with the Sysmex CS-5100 analyzer, was used when longer storage periods (i.e. months or
also free from triglycerides and total bilirubin interfer- years) are needed, especially for research pur-
ences [61]. Moreover, the HIL check was able to detect poses [69–72].
lipemic samples (triglycerides concentrations >4 g/L) Several criteria are available to assess stability of ana-
on the CS-5100 D-dimer assay in three out of four sam- lytes [73]. As discussed above, statistical analysis is fre-
quently used, and the absence of significant bias is
ples [62]. The undetected sample had a very high con-
considered a robust indicator of stability. However, a
centration of triglycerides (9.43 g/L).
significant difference does not inherently correspond to
Regarding the paraproteinemia interference, Mugler
clinical significance [74]. Therefore, a clinical criterion is
et al. reported a case of a patient with Castleman dis-
also recommended to accurately assess stability of a
ease associated with an elevated D-dimer level [57].
particular analyte. It has also been demonstrated that
During investigation of this finding, no D-dimer band
the use of different stability criteria may strongly impact
could be noticed on Western blot compared to D-dimer
the result of stability studies [73]. Therefore, the use of
measured with the STAR coagulation instrument. The
multiple criteria is encouraged. All studies published so
patient’s Castleman disease-associated monoclonal
far used multiple stability criteria except that performed
gammopathy was identified to be the source of the
by Betsou et al. [70]. The review of D-dimer stability
false positive D-dimer result. Paraproteinemia interfer-
studies showed that a clinical cutoff of 10% was most
ence was also hypothesized in another report published
frequently used [69,71,72,75–78]. However, this 10%
by Roller et al. and it could not be excluded from the
cutoff has not been derived from biological variation
reports of Wu et al. and Huang et al. [63–65].
data. Further validation of this empirical 10% cutoff is
Spurious D-dimer concentrations due to heterophilic
needed, even though no discordance could be found in
antibodies have also been reported in the literature
the statistical approach available in the literature. The
[56,66,67]. Potential interferences in D-dimer testing analysis of discordance based on the cutoff used to rule
should always be suspected whenever clinical discrep- out venous thromboembolism (VTE), as evaluated by
ancies arise. Heterophilic blocking reagents could be Caliezi et al., Bo €hm-Weigert et al., and Toulon et al.
used to overcome the problem [56,57]. The comparison [47,69,79], is also a criterion that seems reasonable.
with another method to point out interferences repre- The studies of Schutgens, Zu €rcher, and Gosselin also
sents also a good choice to search for interfer- showed that the freeze-thaw procedure did not signifi-
ence [57,68]. cantly affect D-dimer concentration measured with
three different D-dimer assays [39,75,80]. Bo €hm-Weigert
et al. evaluated the impact of four freeze-thaw cycles
Stability, storage, and freeze-thaw effects
(60 C or less) and failed to find clinically significant
It is currently recommended that samples should be changes of plasma D-dimer values (mean deviation
maintained at room temperature (15–25 C) for no always <10%) [69].
more than 4 h before testing [21]. However, and as dis- In conclusion, while D-dimer is mainly used for emer-
cussed below, several studies have shown that D-dimer gency diagnoses and when rapid measurement is
may be stable for a longer period. This is of particular needed, stability information is required when samples
concern because clinicians frequently ask to add the are stored in central laboratories and when they are
Table 1. Stability of D-dimer.
Plasma/whole
Stability Conditions Anticoagulant blood D-dimer assay Subjects Stability criteria Reference
R
24 h RT Heparin Plasma Tina-quantV (Roche) 17 patients Student t-test and regression equation [39]
R
24 h RT Citrate Plasma Tina-quantV (Roche) 15 patients Student t-test and regression equation [39]
R
6h RT Citrate Plasma InnovanceV (Siemens) 40 patients 10% deviation from baseline, regression equation and [69]
discordance at the cutoff level of 0.5 mg/L FEU
R
24 h RT Citrate Plasma InnovanceV (Siemens)a 80 patients 10% deviation from baseline, analysis of variance, regres- [78]
sion equation and Pearson correlation coefficient
R
V
24 h RT Citrate Whole blood Vidas (bioMerieux) 117 patients Spearman correlation coefficient, regression equation [79]
and discordance at the cutoff level of 500 ng/mL FEU
R
24 h RT Citrate Whole blood InnovanceV (Siemens) 44 patients 10–20% deviation from baseline, Student t-test, and [76]
regression equation
R
V
24 h RT Citrate Whole blood ACL-TOP (Werfen) 26 patients Wilcoxon’s paired t-test, regression equation, and [46]
bias plot
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52 h RT Citrate Whole blood AsserachromV (Stago) 59 patients Analysis of variance, 10% deviation from baseline [75]
R
8h RT Citrate Whole blood ACL-TOPV (Werfen) 144 patients Analysis of variance, Student t-test or Wilcoxon signed [47]
rank test, Bland-Altman plot and discordance at the
cutoff level of 0.5 ng/mL FEU
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24 h 2–8 C Citrate Plasma InnovanceV (Siemens) 40 patients 10% deviation from baseline, regression equation, and [69]
R
V a
24 h 4 C Citrate Plasma Innovance (Siemens) 80 patients 10% deviation from baseline, analysis of variance, regres- [78]
sion equation and Pearson correlation coefficient
R
24 h 4 C Citrate Plasma VidasV (bioMerieux) 20 patients Wilcoxon’s paired t-test, 10% deviation from baseline [77]
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24 h 4 C Citrate Whole blood ACL-TOPV (Werfen) 26 patients Wilcoxon’s paired t-test, regression equation, and [46]
bias plot
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24 months 24 and 75 C Citrate Plasma STA-LiatestV (Stago) Plasma pool Statistical changeb, 5–10% deviation from baseline [72]
(6 patients)
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V

2 weeks 20 C Citrate Plasma STA-Liatest (Stago) 23 hV and 18 patients Paired t-test, 10% deviation from baseline [71]
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36 months 60 C (or less) Citrate Plasma InnovanceV (Siemens) 40 patients 10% deviation from baseline, regression equation, and [69]
R
9 years 80 C Citrate Plasma STA-LiatestV (Stago) 60 patients Wilcoxon’s paired t-test [70]
RT: room temperature; FEU: fibrinogen equivalent units; HV: healthy volunteers.
a R
using the SysmexV CA 7000 platform.
b
specific test not mentioned.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
555
Table 2. Specific pre-analytical data regarding D-dimer testing.

General recommendations in hemostasis
Pre-analytical variables laboratories Specific data regarding D-dimer
Sample collection
Needle bore size 19–22 G 23–25 G also tolerated
Butterfly devices Discouraged Tolerated
Tube material Non-activating material Glass or plastic
(silicone-coated glass or polypropylene plastic)
Anticoagulant sample Sodium citrate 3.2% (105–109 mmol/L) Sodium citrate or heparina
Tourniquet use Removed as soon as the needle is in the vein Longer tourniquet use (i.e. 3 min) not tolerated
(max 1–2 min)
Sample delivery to the laboratory At RT (15–22 C), in vertical position, usually <1 h PTS tolerated
Sample processing
Centrifugation At RT, 1500g for at least 15 min Faster protocol allowed (at RT, 4500g for 2 min)
Interfering substances Do not analyze samples with hemolysis Cell-free hemoglobin i.e. <3 g/L tolerated
Stability, storage and F/T effects At RT (15–22 C), no more than 4 h At least 24 h at RT or at 2–8 C or years at 60 to
80 C
No impact of F/T procedure
RT: gauge; RT: room temperature; PTS: pneumatic tube system; F/T: freezing/thawing.
a
correction factor needed (dilution).
used in a research environment [33]. Table 2 summarizes inspection of agglutination [1,3,4,82]. The development
specific pre-analytical data regarding D-dimer testing. of automated second-generation latex agglutination
immunoassays (or the latex-enhanced immunoturbidi-
metric assay) allowed quantitative measurement of D-
Analytical variables
dimer through recording aggregation rate in response
D-dimer assays to D-dimer [1,2]. Once D-dimer was added, latex micro-
particles agglutinated, thus preventing the light to pass
The first generation of D-dimer assays, which were
through the solution. The increase of light absorbance
developed in the 1970s, was capable to detect both
measured with a photometer was directly proportional
fibrinogen and FDP by means of polyclonal antibodies
to the D-dimer concentration [3]. Latex-enhanced
[2]. These immunoassays could be performed only in
immunoturbidimetric assays are rapid and achieve com-
serum to avoid the cross-reactivity of fibrinogen
parable sensitivity to conventional enzyme-linked
present in high concentrations in plasma. However,
immunosorbent assays (ELISA) [3].
false-positive results were encountered in patients
Microplate ELISA assays were employed before latex
taking anticoagulants, and false negative results were
agglutination assays, mainly for research purposes [1]. In
encountered in samples that developed clots and when
these assays, a capture antibody binds D-dimer antigen
degradation products absorbed into the clot. on a plate. After incubation, a labeled antibody is added
The loss of fibrinopeptide A during serum preparation to the well and binds the immobilized D-dimer antigen
impairs detection of non-cross-linked FDP [4]. The meas- (sandwich assay) to promote a colorimetric reaction
urement of FDP was initially performed using (among [1,2]. Although microplate ELISAs are very sensitive to D-
others) methods based on latex fixation and agglutin- dimer and have long been considered the reference
ation, hemagglutinin inhibition, staphylococcal clump- method [1–3], they are manual, require technical skills,
ing, immunoelectrophoretic, and immunodiffusion [3]. are time-consuming (i.e. around 2–4 h), and are plagued
A significant gain of specificity and sensitivity was by a high degree of analytical imprecision [2,83].
reached with the introduction of monoclonal antibodies In the mid-1990s, bioMerieux developed an ELISA-
targeting D-domains in the early 1980s [3]. These based assay with fluorescence end-point detection
immunoassays allowed the specific targeting of D- (enzyme-linked immunofluorescence assay (ELFA)) [3,58].
dimer epitopes, which were lacking in both FDP and This assay displayed sensitivity and specificity similar to
non-cross-linked fragments of fibrin [4,81]. The use of those of microplate ELISAs, with the great advantage of
plasma was, therefore, possible because of minimal being automated, thus generating more precise results
fibrinogen (or FDP) cross-reactivity. Since the first in a shorter time (30 min) [3,58]. The VidasV assay is still
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monoclonal antibody developed by Rylat et al. in 1983 considered the reference commercial quantitative
(3B6), more than 20 monoclonal antibodies have been immunoassay [84–86] and is the most clinically validated
produced and used in laboratory practice [9,81]. First D-dimer measurement technique [38,48,85,87–89].
generation methods were represented mainly by quali- More recently, chemiluminescent enzyme immuno-
tative latex agglutination immunoassays using anti- metric assays that display similar sensitivity to ELISAs
body-coated latex microparticles and needing visual and latex-enhanced immunoturbidimetric assays have
been developed [3,41,84]. Magnetic particles coated
Readily available, fast,
observer depend-
higher specificity,
SimplifyV (Agen)
SimpliRedV (Agen),
with monoclonal antibodies specific to D-dimer are
Sensitivity, not all

POC assay
whole blood
FDA cleared,
ent, manual
quantitative
used; incubation of anti-D-dimer antibody labeled with
Clearview
R
Qualitative/
R
isoluminol generates a chemiluminescent reaction that
2–20 min
is directly proportional to the D-dimer concentration [3].
Utilization of POC D-dimer assays is especially
attractive for general practitioners (GP) because quanti-
tative D-dimer assays are not always readily available in
immunoturbidimetric assay
HemosIL HSV (Werfen)

rural areas, during weekends, or at home visits [90–92].
STA-LiatestV (Stago),
Sensitivity, automated,
Latex-enhanced
Tina-quantV (Roche),
In such settings, the GP had to refer the patient to a
Moderate specificity
rapid, observer-
(Dade-Behring)
central laboratory facility [91]. The main interest in POC
R
independent
InnovanceV
R
Quantitative
D-dimer assays is the possibility to rapidly screen
ELISA: Enzyme-linked immunosorbent assay; ELFA: Enzyme-linked immunofluorescence assays; CLIA: Chemiluminescent enzyme immunometric assay; POC: Point of care.
patients for thromboembolic disease, thus leading to a
15 min
decrease in overcrowding in urgent care facilities
[3,90,93,94]. The majority of POC tests use whole blood,
are homogenous, use monoclonal antibodies and have
ImmuliteV (Siemens)
a short TAT [3,90]. In hemagglutination assays (e.g.
Sensitivity, rapid, auto-
Lack clinical validation,

moderate specificity
SimplyREDV), bivalent antibodies are specific to red
mated, observer-
R
AcuStarV (Werfen),
independent
CLIA
blood cells and D-dimer. In the presence of D-dimer in
R
Quantitative
the blood, red cell agglutination occurs and provides a
25–40 min
R
qualitative result [3,4]. Detection based on immuno-
chromatography (e.g. Clearview SimplifyV), fluorescence
R
(e.g. VidasV mini analyzer, TriageV, Stratus CSV), and

R R R
chemiluminescence (e.g. PathfastV) also exist. A few

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Moderate sensitivity, man-
DimertestV (Siemens)
semi-quantitative assays remain in use (e.g. Dade
agglutination assay
Unenhanced latex
(bioMerieux), Dade
Dimertest latexV (IL),
Dimertest, NyoCardV) [3,95]. The recent review of Riley
R Rapid, inexpensive
ver dependent
FibrinosticonV
Qualitative/semi-
R
R
et al. compiled the different POC D-dimer assays avail-
quantitative
ual, obser-
R
able on the market [3]. Table 3 summarizes the main
characteristics of D-dimer assays.
Rapid
Inter-laboratory variation
tivity, automation, wider linear
most validated method, sensi-
Considered as reference method,
Variability across assays

range, automated, observer-
Table 3. Characteristics of D-dimer assays [2,3,41,84,91,165,174].
The fibrin assay comparison trial (FACT) study, pub-

lished in 2001, evaluated 23 quantitative D-dimer assays
Moderate specificity
ELFA
VidasV (bioMerieux)
(15 latex-enhanced immunoassays, 6 ELISAs, and 2

independent
membrane-based immunoassays) [96]. The authors

Quantitative
35–40 min
found that the mean values obtained in 39 samples var-

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ied from 630 mg/L to 13,350 mg/L (21-fold), with two

assays displaying significant cross-reactivity toward
fibrinogen degradation products. It was also found that
not optimal linear range,
ELISAs and latex-enhanced immunoassays were more

skills, time-consuming,
observer-independent
Highly manual, technical
reactive to low-molecular-weight cross-linked fibrin and

AsserachromeV (Stago),
Considered as the gold
standard, sensitivity,
moderate specificity
high molecular weight fibrin, respectively [96].

(Dade Behring)
ELISA
Accordingly, the study of Meijer et al., based on 357

R
EnzygnostV
R
Quantitative
laboratories using the 7 frequently-used D-dimer immu-

noassays, found that some assays gave 20 times higher
2–4 h
D-dimer concentrations compared to others [97].

Another study from 423 laboratories comparing D-
dimer values also showed high variability in results
Examples
close to the cutoff used for VTE exclusion [98]. In 2014,

Cons
Type
Pros
TAT
in a survey involving 3800 laboratories, the Coagulation

Resource Committee of the College of American prepare pools were also measured to verify that this
Pathologists (CAP) found that the inter-method coeffi- model could be broadly implemented. The squared
cient of variation (CV) was as high as 42% [99]. Based regression coefficient values ranged from 0.7 to 0.92,
on four UKNEQAS reports (April 2017, July 2017, which led the authors to conclude that the use of a
September 2017, and January 2018), the inter-method conversion factor may be a feasible approach to
CVs (after the exclusion of outliers) were reported to be harmonized data generated with different assays [102].
33.4%, 38.2%, 41.8%, 30.9% and 18%, 19.3%, 17.7% and The second attempt was published by Dempfle et al.
19% for non-FEU and FEU units, respectively; these four in 2001. Overall, 39 individual samples with 23 D-dimer
surveys included between 736 and 752 laboratories. assays (including microlatex-enhanced, membrane-
based and ELISA assays) were tested [96]. For each
Calibrators assay, a conversion factor was calculated by using the
median value for each sample measured with all assays,
The controlled lysis of fibrin clots is used mainly to
obtain calibration materials [9]. Hence, manufacturers and for each assay, the median value obtained with all
have to ensure that lysis is reproducible in order to samples. The multiplication of individual sample assay
maintain the same variety of size degradation products, values by the corresponding conversion factor was
because the sensitivity of the assay may change accord- found to be effective to improve the correlations
ing to the relative amounts of high molecular weight among most assays.
fibrinogen (HMWF) or low molecular weight fibrinogen The third attempt was conducted by Meijer et al. in
(LMWF) [9,96]. D-dimer assays may be calibrated with 2006 [97]. A plasma pool of 50 patients diluted with nor-
purified fibrin fragment D-dimer equivalents or accord- mal plasma was used to prepare five different samples
ing to the fibrinogen amount used to prepare the cali- that were then distributed in an external quality control
brator [9]. D-dimer units thus may vary according to the survey to 502 participants using seven different D-dimer
type of calibrator used, i.e. D-dimer units (DDU) or assays. For each D-dimer assay, the mean results of each
fibrinogen equivalent units (FEU) [9,96,100]. sample were plotted against the amount of pool added
(assay-specific regression line). The median results of the
mean results obtained with each D-dimer assay were
Standardization versus harmonization also plotted against the amount of pool added (refer-
The heterogeneity of fragments derived from plasmin ence regression line). Harmonized results could, there-
digestion of cross-linked fibrin (from LMWF to HMWF) fore, be calculated from the measured result by using
[9], the use of monoclonal antibodies with different the intercept and the slope of both regression lines
specificities towards D-dimer epitopes [1,96,101,102], (assay-specific and reference). The authors showed that
the lack of international certified internal controls or a significant decrease in between-assays variation was
calibrators [103] and the use of different units and clin- observed by introducing this model.
ical cutoffs are the leading sources of the large inter- The fourth attempt was published by Jennings et al.
laboratory variability, thus making D-dimer assay stand- in 2007 [100]. Three calibrators and two test samples
ardization a challenging, if not impossible, target were delivered to more than 500 laboratories that used
[9,13,99,101,102,104–108]. Because the requirements 9 different D-dimer techniques and that participated in
for D-dimer assay standardization cannot be met, less the UK NEQAS external quality survey. Individual labora-
stringent harmonization procedures, based on the use tory results for calibrators were plotted against the
of mathematical models, have been proposed to make median results obtained with all the D-dimer immunoas-
results obtained with different assays more compar- says. The individual regression line was used to convert
able [96,97,100,102]. data generated on the two test samples into harmon-
The first attempt to achieve D-dimer assays harmonized results. This approach was effective in improving
ization was published in 1997 by Nieuwenhuizen et al. the between-center agreement after calibration, with
[102]. Different pools of patients with various diseases significant improvement in inter-laboratory variability for
were prepared and tested with 5 different D-dimer the two samples (from 25.9% to 11.6% and from 22.4%
assays (one microlatex and four microplate ELISA), and to 7.7% for results reported as FEU; from 55.3% to 21.6%
a “pool consensus value” (corresponding to the mean and from 40.8% to 11.6% for results reported as DDU).
D-dimer result of each assay) was assigned to each Harmonization of D-dimer assays may be particularly
pool. Results of each pool obtain from the individual useful for diagnosis and monitoring of disseminated
assays were then divided by the corresponding “pool intravascular coagulation (DIC), because the high inter-
consensus value” and averaged. Samples used to variability between D-dimer assays [96,98] may directly
STA-LiatestV). This may be a consideration when

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impact the DIC score and lead to misclassifications.
Harmonization of D-dimer results would provide better choosing a D-dimer assay [1,4,48,85,87,88,107,112].
consistency and improve the calculation of DIC scores [9]. Otherwise, comparison with validated assays is encour-
However, the inherent heterogeneity and the challenges aged. Manufacturers should stay abreast of the recent
of the harmonization efforts make the identification of a literature regarding the use of their immunoassays and
unique diagnostic cutoff even more difficult and clinically update their cutoffs when indicated [113].
questionable, because false-negative results have an A CAP survey showed that 488 laboratories out of
unfavorable impact on patient management [9]. 1506 in the USA were using cutoff values higher than
Nevertheless, the above-mentioned approaches indi- those recommended in the literature or by the manu-
cate that harmonization is feasible, and it should be facturer [99]. A European survey also highlighted that
considered on a larger scale [103]. Further validation of 24% and 55% of participants used lower or higher cut-
these models with more assay systems is needed to offs, respectively, than those recommended [9].
confirm whether such models may be generalized to all A recent Italian consensus document recommends
D-dimer immunoassays [9,97,100]. Recently, a call for the use of certified, quantitative immunoassays for test-
harmonization was raised by the Fibrinolysis and DIC ing the plasma of emergency department patients [33].
Standardization Subcommittees of the ISTH [103]. It was also recommended that D-dimer immunoassays
Utilization of a large number of samples from patients should have an imprecision <10% close to the diagnos-
with various clinical conditions may be useful to pro- tic cutoff and a measurement range and linearity of
duce a stable freeze-dried reference material containing 50–5000 mg/L FEU. Importantly, D-dimer assays should
high concentrations of D-dimer derived from both not react with fibrinogen or FDP, and preferably should
LMWF and HMWF [103]. A range of D-dimer values not react with fibrin and fibrinogen fragments released
could be generated from this reference material, to from proteolysis mediated by various enzymes [103].
obtain a reference regression line for D-dimer immuno- Clinicians need to be aware of the performance char-
assays. The use of monoclonal antibodies displaying acteristics of the D-dimer assay they are using because
high affinity to low and middle molecular weight FDP D-dimer comprises a mixture of degradation products
species combined with monoclonal antibodies target- of cross-linked fibrin with heterogeneous molecular
ing higher molecular weight forms may also represent weights [1,9,105]. Moreover, the laboratory should
a feasible alternative [101]. Continuing discussion always use clinically validated cutoff values, because
among manufacturers, scientists, and clinicians is essen- this diagnostic threshold plays a crucial role in clinical
tial to achieve better harmonization. decision making [1].
Recommendations on the performance of D- Postanalytical variables

dimer assays
According to the ISO 15189:2012 standard, the postana-
Because of the heterogeneity of the different D-dimer lytical phase is defined as “processes following the
immunoassays (i.e. format, antibodies) and the lack of examination including systematic review, formatting
standardization/harmonization, the analytical and clin- and interpretation, authorization for release, reporting
ical performances of the local method should always be and transmission of the results, and storage of samples
evaluated before implementation in management strat- of the examinations” [14].
egies for VTE [109,110]. Although it is now clearly established that most
The cutoff value used to exclude VTE needs to be problems in the hemostasis laboratory emerge from
confirmed locally, using, as suggested by the British the preanalytical phase (60–70%), [20] the postanalyti-
Committee for Standards in Haematology guidelines, a cal phase is still the cause of a large number of diagnos-
minimum of 200 subjects [1,111]. Several manufacturers tic errors (15–20%) that can jeopardize patient safety
also recommend local validation of cutoffs [1]. [105]. Here we discuss postanalytical issues that are
However, this approach may not be practical for all lab- specific to D-dimer measurement.
oratories; thus the cutoff proposed by the manufacturer
may be used [4], provided that reliable validation stud-
D-dimer units
ies have been conducted elsewhere and that no signifi-
cant batch-to-batch variability has been found [9]. Two units of D-dimer measurement are currently used
Cut-offs have been clinically validated in prospective in clinical laboratories: FEU and DDU. On the one hand,
studies for a number of assays (e.g. VidasV, AxSYMV,
R R
FEU compares the mass of D-dimer to that of
fibrinogen, and the calibrators are prepared from plas- (PPV) without significantly impairing the negative pre-
min degradation of purified fibrinogen clotted in the dictive value (NPV), and ultimately improve the clinical
presence of factor XIII. On the other hand, DDU deter- usefulness of D-dimer measurement in elderly patients
mine the mass of the estimated weight of D-dimer, and (i.e. aged 50 years or older) with low clinical probability
the calibrators are composed of purified D-dimer [4]. [12,33,118–120]. The test most validated for this pur-
pose is the VidasV assay (bioMerieux) [120]. However,
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FEU (340 kD) and DDU (195 kD) may be used inter-
changeably if multiplied by 2 as FEU is roughly two the age-adjusted strategy seems to be less useful with
the STA-LiatestV assay [121]. The analysis of different
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times the mass of one DDU [2–4,9,33]. Another difficulty
arises from using as many as 7 different measure units populations with different immunoassays may explain,
(i.e. ng/mL, mg/L, lg/L, lg/mL, g/L, g/mL, and mg/dL) at least in part, such discordances. This emphasizes that
for reporting D-dimer results [3]. Furthermore, the same the results obtained with specific D-dimer assays can-
D-dimer assay is currently reported in different units not be extrapolated from one test to another [121].
[2,114,115]. Therefore, at least 14 combinations for D- However, an international survey on the reporting of
dimer measurement coexist. D-dimer test results showed that published recommen-
As recently studied by Olson and Lippi, the majority dations on the use of age-adjusted cutoffs have not
of clinical laboratories (59% and 60%, respectively) used been broadly implemented (i.e. less than 10% of clinical
FEU for D-dimer measurement, whilst the leading meas- laboratories have implemented this approach) [105].
ure unit was mg/L, followed by ng/mL, in both surveys Along with the 14 combinations of D-dimer units,
[99,105]. Among the measure units that can be the use of age-adjusted cutoffs further complicates clin-
adopted, “mg/L” (or “ng/mL”) is probably the unit that ical decision making – there are nearly 30 different pos-
best approximates the Systeme Internationale (SI) and sibilities for reporting D-dimer test results [105].
is also recommended by the Italian Consensus docu- Worldwide efforts are needed to standardize D-dimer
ment [33,105]. Some laboratories (8% in the CAP sur- test result reporting, to pave the way for widespread
vey) did not even know the type of measurement unit implementation of age-adjusted cutoffs [106]. Specific
they were using [9,99,116]. cutoffs to be used for pregnant women and children
The use of different units (FEU or DDU, as well as the have also been proposed [106,122,123]. However, as D-
different measure units) is challenging for clinicians, dimer increases physiologically throughout pregnancy,
causes confusing and potentially leads to the misclassi- the identification of an accurate D-dimer diagnostic
fication or misdiagnosis of patients [3,4,105]. The fact threshold will be challenging [124]. The Diagnosis of PE
that 33% of 1500 US laboratories changed their D-dimer in Pregnancy (DiPEP) biomarker study recently showed
units from those recommended by the manufacturer that no biomarker has diagnostic efficiency for diagnos-
made the situation even more complicated [99]. These ing VTE in pregnancy or in the puerperium [125].
variations add additional confusion to diagnostic rea-
soning, especially when neighbor laboratories use other
Turnaround time
units [99]. Clinical laboratories should communicate any
change in D-dimer measurement units to their stake- The TAT is a crucial aspect in D-dimer reporting
holders in a timely manner. Even more importantly, because this test is mostly used in urgent clinical condi-
reaching a universal agreement on a uniform measure tions. The Italian consensus document has recently set
unit is a compelling need to achieve harmonization of an overall TAT <1 h, which seems suitable for managing
D-dimer immunoassays [99,103]. the vast majority of urgent test requests [33].
The use of D-dimer immunoassays characterized by
a wide linearity range (i.e. up to 5000 mg/L) without per-
Age-specific cutoffs
forming additional external dilutions, along with the
D-dimer values typically increase in parallel with aging, use of faster and validated centrifugation processes,
thus leading to a high proportion of elderly patients PTS, or reliable POC analyzers present valuable
with D-dimer levels higher than the conventional cutoff approaches to reduce the TAT [3,33,39,43–45,92,107].
set at 500 lg/L FEU [1,4,33,99,103,105,117,118]. The use Manual ELISAs do not meet the recommended TAT,
of age-adjusted cutoffs (i.e. [age-adjusted cutoff, lg/L whilst recent central laboratory D-dimer assays, with
FEU] ¼ [age in years]) 10) is now universally recom- analytical process times ranging between 15–40 min,
mended for reporting results of D-dimer testing (see are more likely to fulfill the 1-h criterion (Table 3).
below) [33,118–120]. Use of these cutoffs would enable Notably, owing to a half-life of 6–8 h for D-dimer,
a substantial increase in the positive predictive value repeating the test within this time interval has no clinical
basis [33]. In a European study, 81% of participants Future prospective studies are needed to confirm these
stated that they measure D-dimer 24 h per day [98]. findings. The recent meta-analysis performed by Alons
et al. (eight studies included; 636 patients) showed that
D-dimer has a high NPV in low-risk patients with isolated
Clinical applications
headache for excluding CVT [132]. Low-risk patients were
More than 30 years ago, D-dimer was found to be a reli- defined by normal neurological examination, normal
able biomarker of both coagulation activation and fibrin standard head CT (computed tomography), and absence
digestion [8]. Since then, D-dimer has been used in sev- of risk factors such as puerperium and pregnancy.
eral clinical conditions. Most widely, D-dimer is used for Sensitivity, specificity, PPV, and NPV for diagnosing CVT
ruling out VTE in patients with low-medium clinical prob- were 97.8%, 84.9%, 33.1%, and 99.8%, respectively [132].
ability, where D-dimer is now considered the biochem- Normal D-dimer levels may, therefore, reduce unneces-
ical gold standard [1,4,33,98,126]. D-dimer testing is also sary neuroimaging investigations.
useful for assessing the risk of recurrent thrombosis and
for guiding anticoagulant therapy, for diagnosing and Acute aortic dissection
monitoring DIC, for excluding acute aortic dissection
Symptoms of AAD typically include back and/or
(AAD), and for predicting and managing thrombotic
abdominal pain, acute onset of tearing chest, asymmet-
complications in patients with severe infections and sep-
sis [1,4,127]. Other applications have been studied (e.g. ric blood pressure and widened mediastinum on chest
prognostication of peripheral artery disease, identifica- x-ray [133]. However, in many patients, symptoms are
tion of vaso-occlusive crisis in sickle cell disease, screen- nonspecific and a missed diagnosis may be fatal [133].
ing of intracardiac thrombus, prediction of VTE in sleep The diagnosis of AAD currently relies on imaging tech-
apnea), but no clinical validation has so far been niques (i.e. magnetic resonance imaging, echocardiog-
achieved for these conditions [4,128,129]. raphy, contrast-enhanced CT) (Class I; Level of evidence
Below, we discuss the role of D-dimer for exclusion B) [133,134]. The potential usefulness of D-dimer lies in
of VTE, for prediction of VTE recurrence, for prediction the possibility to rule out AAD in patients with low clin-
of thrombosis in medically hospitalized patients, and ical probability because D-dimer has been reported to
for diagnosis and monitoring of DIC. be persistently elevated in AAD [4,133,135]. D-dimer
We briefly present here some indications discussed testing is also rapid, economical, and more accessible
in the literature, namely cerebral venous thrombosis than imaging investigations [136]. The meta-analysis of
(CVT), AAD, and acute mesenteric ischemia (AMI). Cui et al. (five studies included; 743 subjects) found a
sensitivity and specificity for the diagnostic of AAD of
Some indications for measuring D-dimer 94.5% and 69.1%, respectively [133]. Similar performan-
ces were obtained in the meta-analysis of Watanabe
Cerebral venous thrombosis et al. (12 studies included; 2827 subjects; sensitivity and
According to the statement of the American Heart specificity of 95.2% and 60.4%, respectively) [137]. D-
Association (AHA) and of the American Stroke dimer levels can, therefore, be used to rule out AAD in
Association (ASA) guidelines, a normal D-dimer level patients with low likelihood of the disease [133,137].
obtained with a sensitive assay may help to identify According to the American College of Cardiology
patients with a low probability of CVT. However, the class Foundation (ACCF) and to the AHA guideline, D-dimer
and level of evidence are low (Class IIb; Level of Evidence cannot be used to rule out the disease in high-risk
B) and the use of D-dimer is considered superfluous patients and they do not recommend D-dimer screen-
when the clinical suspicion of CVT is high [130]. Because ing for all patients being evaluated for aortic dissection
of the variability of presentation, the diagnosis of CVT is (AD) [134]. The sensitivity and the failure rate of D-
rarely confirmed by neuroimaging investigations, which dimer in patients with AD detection risk score (ADD-RS)
remains, however, the gold standard for the diagnosis of 0, 1, and 1 (high risk of AD) were 100% and 0%,
CVT [131]. Pretest clinical probability scores validated to 98.7% and 0.8%, and 97.5% and 4.2%, respectively
assist clinician in the diagnosis of CVT are also lacking [138]. The ADD-RS allows standardized assessment of
[131]. The meta-analysis of Dentali et al. (14 studies pretest probability for acute aortic syndromes [139].
included; 1134 patients) found a mean sensitivity and The combined ADD-RS (0 or 1) with negative D-dimer
specificity for D-dimer of 93.9% and 89.7%, respectively, was superior to D-dimer alone in AAD diagnosis
in patients with suspected CVT. The risk of false-negative [138,140,141]. However, the accuracy of D-dimer was
D-dimer results included longer duration of symptoms, found to be lower (4% failure rate) in patients at high
limited sinus involvement and isolated headache [131]. risk (ADD-RS of 1); therefore D-dimer was not
acceptable to rule out a diagnosis of AD in such set- fibrin production or breakdown may also generate
tings [141]. In conclusion, the absence of ADD risk increased D-dimer values [2]. These conditions, listed in
markers combined with a negative D-dimer result Table 4, include infections, cancer, chronic inflamma-
argues strongly against the diagnosis of AD [138,141]. tion, aging, pregnancy, recent surgery, and trauma. A
Integration of ADD-RS with D-dimer may be considered large retrospective study of 1647 patients showed that
to standardize the diagnosis of AD [141]. the most common causes of positive D-dimer results
were related to infections, followed by VTE, syncope,
Acute mesenteric ischemia heart failure, trauma, and cancer [126]. This is of par-
The interest in D-dimer in the diagnosis of AMI has also ticular concern for hospitalized patients, because D-
been acknowledged in the literature [142–145]. Four dimer may be increased for reasons other than VTE and
etiologies of AMI exist: arterial thrombosis (15–20%), DIC [1]. Brotman et al. found that only 22% of hospital-
venous thrombosis (5%), arterial embolism (50%), and ized patients had normal D-dimer levels [150].
non-occlusive mesenteric ischemia (20–30%) [146,147]. Therefore, D-dimer testing should not be considered
Failure to recognize AMI is responsible for a high mor- specific for VTE [1,2,4,33], and it should be used mainly
tality rate [147]. The sensitivity and specificity of spiral to exclude VTE because low D-dimer values reflect the
CT for the detection of AMI have been determined to lack of significant (ongoing) activation of blood coagu-
be 93.3% and 95.9%, respectively [148]. According to lation [1,151].
the guideline of the European Society for Trauma and
Emergency Surgery (ESTES), published in 2016, D-dimer Epidemiology of VTE
does not discriminate patients with AMI from non-AMI VTE, including both DVT and PE, is a life-threatening
patients [147]. Moreover, no correlation between serum condition, with an incidence ranging between 104 to
D-dimer levels and the severity of AMI was observed 183 per 100,000 person-years in Europe [152]. The inci-
[143]. However, the guideline cited only the paper of dence rate is generally higher in men after 45 years, as
Chiu et al. (prospective study on 67 patients), which
well as in women in the childbearing years [152]. The
was published in 2009 [143,147]. In 2017, a meta-ana-
prevalence of VTE has been estimated at 422 cases per
lysis performed by Sun et al. included more studies (12
100,000 individuals with isolated DVT (69.9%), PE
studies; 1300 patients), eight of which concerned AMI.
(23.7%) or both (6.4%) [117]. The frequency of VTE cases
The four other studies dealt with acute strangulated
appears to be slightly higher in women than in men in
intestinal obstruction, acute intestinal necrosis and
the United States [117]. The incidence of VTE is also
mixed types of acute intestinal ischemia, which could
known to increase sharply with age [153].
be gathered into the term “acute intestinal ischemia”;
Of note, during the last decades, the overall inci-
the calculated sensitivity and specificity were 94% and
dence of VTE decreased significantly [154]. The wider
50%, respectively [149]. D-dimer measurement could
introduction of thromboprophylaxis in hospitalized
hence be useful for identifying patients with AMI.
patients may, at least in part, explain this result.
Further validations of these results in large multi-center
Specifically, the incidence of isolated DVT also
clinical studies are needed.
decreased while the incidence of isolated PE increased
[154]. This discordance may be explained by the lack of
Exclusion of the diagnosis of venous major improvement in the accuracy of ultrasonographic
thromboembolism
diagnosis of DVT whereas improved resolution imaging
Although D-dimer levels are increased in almost all has been used for the diagnosis of PE (i.e. multi-
cases of acute VTE, any other condition that increases detector CT scan) [154].
Table 4. Causes of D-dimer elevation [1,126,127,164].

Acute respiratory distress syndrome Disseminated intravascular coagulation Pancreatitis
Advance age Heart failure Post transplantation complications
Alzheimer HELLP syndrome Pregnancy or puerperium
Aneurysm Hemolysis (falciform anemia) Recent surgery
Aortic dissection Hemorrhage Renal disease
Arthritis Hospitalization Severe chronic urticaria
Atrial fibrillation Inflammatory bowel disease Thrombolytic therapy
Burns Ischemic cardiopathy Trauma
Cancer Liver disease Venous or arterial thrombosis
Chronic inflammation Localized or systemic Infection
Disability Neonatal period
Several factors have been associated with an (median, 76%) [165]. These results were also confirmed
increased risk of VTE, including major surgery, hospital- in the meta-analysis of Stein et al., which included 78
ization for acute medical illness (e.g. heart failure, dia- studies [151]. Similarly, Roy et al. found that ELFA assays
betes or pneumonia), trauma/fracture, thrombophilia, exhibit the highest exclusion value (the lowest negative
obesity, active cancer, pregnancy or postpartum, likelihood ratio, see below) and whole blood agglutin-
extended periods of immobility, and oral contraceptives ation assays, the lowest [166]. The specificity of whole
[152,153,155]. Overall, the survival after PE is predict- blood POC D-dimer assays is overall higher than central
ably worse than after DVT alone. The survival rate for laboratory D-dimer assays (71% and 69% for DVT and
DVT at 3 months and 5 years is 91.9% and 72.7%, PE, respectively), meaning that the number of patients
respectively, whilst for PE, it is 62.8% and 47.5%, in whom VTE could be excluded increases [91].
respectively [152,156]. However, the sensitivity is lower (83% and 87% for DVT
VTE is a major healthcare issue worldwide, with an and PE, respectively) as compared to central laboratory
estimated incidence of 3.0–3.3 cases per 100 hospital- D-dimer assays [165]. More recent studies also found
izations per year and a rate of hospital readmission of optimal diagnostic performance for current laboratory
D-dimer immunoassays (e.g. STA-LiatestV, HemosIL HS
R
5–14% [152,157–159]. Compared to hospitalized con-
500V, HemosIL AcuStarV, InnovanceV (Siemens, USA),
R R R
trols, adjusted mean predicted costs were 1.5–2.5 times
Tina-QuantV) [38,48,84,112,114,167,168]. Not all POC D-
R
higher for patient with VTE. Cost differences were great-
est within the first 3 months [160,161]. The total annual dimer assays are appropriate to be used to exclude VTE
health care cost for VTE in 2007 ranged from $7594 to [107]. Some POC assays have been approved only as an
$16,644 per patient in the US [158]. The VTE-attribut- aid to diagnosis and still require additional tests to rule-
able cost has been estimated to be up to $10 billion out VTE reliably [107]. It is also important to know if the
[162]. It has also been estimated that the number of FDA has cleared the assay for exclusion of VTE [107]. Up
cases of VTE among adults will at least double by 2050 to now, few POC D-dimer assays meet the performance
(0.95 million in 2006 to 1.82 million predicted in characteristics recommended by CLSI [95,107].
2050) [117]. Quantitative POC assays have been found to be more
reliable than qualitative POC assays, especially in
D-dimer assay performance patients with low pretest probability of VTE
[91,107,169]. Taken together, GPs should weigh the
According to the US Food and Drug Administration
benefit of rapid POC D-dimer assay results over the
(FDA), the specifications for sensitivity and NPV of a D-
drawback of overall lower performance [91].
dimer assay applied to rule out VTE should be 95%
Notably, the NPV is directly influenced by the preva-
(with lower limit of CI 90%) and 97% (with lower
lence of a disease [38]. For example, the prevalence of
limit of CI 95%), respectively [163]. The CLSI provides
DVT ranges between 10–50% [38]. An accurate inter-
slightly more stringent recommendations for sensitivity
pretation of D-dimer immunoassay performance is
and NPV, entailing 97% (with lower limit of CI 90%)
hence needed. The negative likelihood ratio may be the
and 98% (with lower limit of CI 95%), respectively
most accurate criterion to assess the diagnostic per-
[163]. Based on these requirements, D-dimer assays can
formance of a D-dimer test [170,171]. The negative like-
be classified into two main categories: (1) those with
lihood ratio represents the extent of change in the
high sensitivity (>95%) and low specificity (<40%), and
odds or the probability of the disease after a test result
(2) those with moderate sensitivity (80–94%) and high
[172]. The number needed to test (NNT) index, or the
specificity (up to 70%) [164]. Quantitative laboratory
number of patients in whom D-dimer must be meas-
assays are more likely to have a higher NPV compared
ured to rule out one VTE, may also be used to compare
to semi-quantitative and qualitative assays [3]. The
the diagnostic yield of commercial D-dimer immunoas-
meta-analysis published by Di Nisio et al., which
says [168,173,174].
included 113 studies, showed that ELFAs (e.g. VidasV,
R
Stratus DSV (Siemens, USA), AxSYMV), microplate ELISAs

R R
Clinical prediction rules (CPR)

(e.g. AsserachromV, EnzygnostV), and latex quantitative
R R
immunoassays (second generation of latex-based assays It has been demonstrated extensively that assessment
[2]) (e.g. Tina-quantV, STA-LiatestV) had higher sensitiv-
R R
by means of a clinical score should be combined with
ity (median, 95%) but lower specificity (median, D-dimer testing to improve the diagnostic specificity of
±50%) [165]. They also found that whole blood agglu- the D-dimer result and enhance the diagnostic accuracy
tination, latex semi-quantitative or qualitative assays [2,4,33,120,153,175]. The aim of clinical probability
had low sensitivity (median, 84%) and high specificity assessment is to deal with the prevalence dependence
Table 5. Clinical prediction rules for PE (adapted from Righini et al. [174]).
Wells score Geneva score Revised Geneva score
Items Score Items Score Items Score
Previous PE or DVT 1.5 Previous PE or DVT 2 Age >65 1
Heart Rate >100 1.5 Heart Rate >100 1 Previous PE or DVT 3
Recent surgery or immobilization 1.5 Recent Surgery 3 Surgery or fracture within 1 month 2
Clinical signs of DVT 3 Age Active malignancy 2
Alternative diagnosis less likely than PE 3 60–79 1 Unilateral lower limb pain 3
Hemoptysis 1 80 2 Hemoptysis 2
Cancer 1 Arterial blood gases Heart rate
CO2 (kPa) 75–94 3
<4.8 2 95 5
4.8–5.19 1 Pain on lower limb deep vein 4
palpation and unilateral edema
O2 (kPa)
<6.5 4
6.5–7.99 3
8–9.49 2
9.5–10.99 1
Chest X-ray
Atelectasis 1
Elevated hemidiaphragm 1
Clinical probability Clinical probability Clinical probability
Low <2 Low 0–4 Low 0–3
Intermediate 2–6 Intermediate 5–8 Intermediate 4–10
High >6 High 9 High 11
Dichotomized
PE unlikely 4
PE likely >4
for accurate interpretation of diagnostic tests of D- inexperienced physicians) [1,155]. However, pretest prob-
dimer assays by identifying subgroups of patients ability calculation may still be subjective [181,182]. These
according to different levels of VTE prevalence: low algorithms are considered clinically validated when the
(<10%), moderate (around 30%), high (>50%), or number of thromboembolic event after 3 months is as
“unlikely” (±10%) and “likely” (±35%) [155]. There are high as 1–2% compared to a gold standard test (pulmon-
additional aspects that support the use of D-dimer in ary angiography for PE and venography for DVT) [183].
combination with CPR, such as the fact that some Different algorithms combining CPR and D-dimer
patients with thrombosis may present a false-negative measurement have been proposed and endorsed by
D-dimer concentration due to a hypofibrinolytic state, international guidelines [33,155,164,184]. A “low” or
small thrombi (i.e. distal DVT or isolated subsegmental “unlikely” probability is generally followed by D-dimer
PE), anticoagulant therapy or D-dimer testing per- testing. VTE can be ruled out when D-dimer values are
formed too early or too late after the throm- below the method-specific cutoff. In these conditions,
bosis [1,2,4,33,48]. the use of time-consuming, expensive and less safe (i.e.
CPR have been derived from clinical studies and due to radiation exposure and/or injection of contrast
include a number of signs, symptoms, and risk factors of products) tests would be unnecessary [2]. However,
VTE [2]. The use of two-level scores is typically preferred, when the clinical probability is “high or likely” or
because it is straightforward and because three-level “moderate/high”, D-dimer testing is not useful and
scores depend on the sensitivity of the D-dimer assay radiologic imaging should be used [2,185]. A positive
(which is rarely known by the clinicians) [153,175]. The D-dimer test result may also trigger imaging tests [2].
Well’s and Geneva scores (modified or not) are the two For DVT, this would entail ultrasonography [183] or
CPRs mainly used for PE, whilst the Well’s score is espe- Doppler flow studies [183]. For PE, the most used tech-
cially used for DVT [155,169,174,176]. These scores for PE niques include CT pulmonary angiogram (CTPA) [183]
are shown in Table 5. The diagnostic performances of or contrast-enhanced or unenhanced magnetic reson-
these CPR were found to be almost comparable ance imaging, especially when CTPA is unadvisable
[119,177–179]. Other CPR have also been validated and [183]. Some studies based on specific algorithms con-
may be used routinely (e.g. Empiric, Charlotte or cluded that a negative D-dimer result in patients with a
Simplified Geneva) [1,177,180]. A good CPR should not “moderate” score could also safely rule out a VTE epi-
contain subjective variables, and it should be accurate, sode, thus enhancing the usefulness of D-dimer testing
reproducible, easy to remember, and offer a standardized and decreasing the use of imaging testing [155,180].
approach compared to clinical assessment (especially for Several other studies, including a meta-analysis of 1660
patients, showed that a normal D-dimer concentration 80 years) is lower compared to younger patients
along with a low CPR score had a NPV of up to (49–67%, <50 years). Elderly patients are therefore less
95% [3,116,119,186]. likely to have negative D-dimer results when VTE is
However, in current practice, such clinical algorithms absent [12,118], and a high number of these patients
are not always followed [181,182]. In a recent survey with a low clinical score may undergo unnecessary imag-
that included 487 clinicians from six countries, only ing testing because of high D-dimer values [118]. This is
70.3% used pretest probability scores, and 10% because consolidated evidence shows that VTE preva-
excluded or confirmed DVT based only on D-dimer test lence increases with age [12,119,152,174].
results. Moreover, a significant number of clinicians still Age-adjusted cutoffs have subsequently been vali-
order D-dimer testing in patients with a high probabil- dated and recommended because their application
ity of VTE, whilst others order imaging testing in cases increases diagnostic specificity without significantly
of low pretest probability. This waste of resources may impairing the NPV [33,119,173,191–193]. The calculation
lead to a negative impact on health care resources of the age-adjusted cutoff is based on the following
[181,182]. Moreover, in a large study that included 117 equation: “age-adjusted cutoff, lg/L FEU ¼ age in years
centers and 1529 patients with suspected PE, the diag- 10 [119]”. Thus, the age-adjusted cutoff of a 70 year-old
nostic management was inappropriate in 662 patients patient is 700 lg/L FEU (rather than 500 lg/L FEU). The
(43%): in 36 of 429 (8%) patients with confirmed PE and multiplication factor of “10” has been slightly rounded to
in 626 of 110 (57%) patients in whom PE was ruled out facilitate practicability and clinical usefulness [119].
[187]. One of the independent risk factors for inappro- The ADJUST-PE study, which included over 3000
priate management was the lack of written algorithms patients, prospectively demonstrated that the combin-
that included a clinical probability score. Major efforts ation of age-adjusted cutoff and a clinical score (Wells or
are hence needed to harmonize the use of the different Revised Geneva score) was associated with a higher pro-
CPR algorithms. portion of patients in whom PE could be safely ruled out
A recent prospective study based on 808 patients without additional CTPA [120,194]. Many other studies
found that a low D-dimer level (<750 lg/L FEU) meas- have demonstrated that the number of patients in
ured with the MDA D-dimer assay (quantitative latex whom VTE could be ruled out increased remarkably after
agglutination assay, bioMerieux) could be used alone for applying age-adjusted cutoffs, and this approach has
ruling out PE, without the need of a CPR score [188]. The also been found to be cost-effective because a lower
theoretical advantage of limiting the use of CPR is a number of patients with low clinical score will need to
decrease in the cost attributable to imaging techniques undergo expensive imaging tests [120,168,174,183,194].
(e.g. CTPA and ventilation-perfusion lung scanning) and A recent meta-analysis evaluated the clinical
prevention of radiation exposure [188]. However, the effectiveness of age-adjusted cutoffs in 12497 older
MDA assay is no longer being marketed. Moreover, the patients and concluded that the sensitivity was >97% in
number of patients with a high pretest probability was all age categories [118]. The gain in specificity (up to
quite low in that study [188]. On the other hand, D-dimer 23% better specificity compared to a fixed cutoff) was
testing has limited clinical value in patients with a high especially favorable for patients aged 75 years or older
CPR score in whom the prevalence of VTE is high (i.e. low [118,120]. This strategy has been validated for both PE
NPV) [164]. Additional studies with other D-dimer assays and DVT with many commercial D-dimer immunoassays
are needed to confirm these findings. Nevertheless, the [38,119,120,168,191]. Specifically, Oude Elferink et al.
use of a D-dimer assay as a stand-alone test has not been found that the NPV of 9 D-dimer immunoassays was
widely endorsed by most guidelines and consensus always >97% [38]. Another study found a NPV >99% in
documents [1,2,4,33,38,127,164,175,183,189]. This is
using five different D-dimer immunoassays [168].
understandable because the high percentage of deaths More recently, Farm et al. reported that 4 D-dimer
in the untreated patients is the major risk of underdiag-
assays display sensitivity >95% and NPV >97%, whilst
nosing VTE [183,185].
only one method (MediRoxV, Sweden) displayed slightly
R
lower sensitivity (94%) [191]. Although age-adjusted

Age-adjusted cutoff
cutoffs seem straightforward in most cases, caution
As earlier discussed, D-dimer values tend to increase with should be used with non- or insufficiently validated D-
age, thus leading to a greater proportion of older patients dimer immunoassays [106]. More recently, the use of
(60%) with D-dimer values higher than the conventional age-adjusted cutoffs for ruling out PE has also been
cutoff of 500 lg/L FEU [1,4,33,105,117,118,190]. The speci- endorsed by the European Society of Cardiology
ficity of D-dimer for VTE in elderly patients (0–18%, (ESC) [184].
Interestingly, a recent study demonstrated that age- PE as the most likely diagnosis) and various D-dimer
adjusted cutoffs may also be useful in younger patients. cutoffs in 3465 patients with suspected PE. Among the
Prochaska et al. [192] performed a clinical study that 2946 (85%) patients in whom PE was ruled out (no
included 500 patients with suspected DVT, and YEARS CDR and D-dimer <1000 mg/L or at least one
reported that a D-dimer cutoff of 500 mg/L FEU had YEARS CDR and D-dimer <500 mg/L), the rate of VTE
lower sensitivity in patients <60 years (76.1%) com- during a 3-month follow-up period was only 0.61%
pared to older patients (92.9%) A lower sensitivity was (95% CI: 0.36%–0.96%) [180]. Furthermore, a 14%
found especially in women, and in patients with unpro- decrease in CTPA was observed [180]. Based on this lat-
voked DVT, with low thrombotic burden, and with dis- ter study, van der Pol et al. found that the age-adjusted
tal DVT [192]. However, the use of a lower cutoff in cutoff added no value to the YEARS algorithm [199].
younger patients (250 mg/L FEU) was effective in Takach Lapner et al. recently compared both the
increasing the sensitivity of this test remarkably, up to age-adjusted and the clinical probability-adjusted strat-
92.4%. Nevertheless, further studies will be needed to egies [197]. For that purpose, 1649 patients with low or
confirm the results of this preliminary observa- moderate clinical probability after assessment for a first
tional study. VTE event were studied retrospectively. The authors
found that the NPV was similar in both groups (99.7%
Clinical probability-adjusted cutoffs versus 99.6%), but also observed a significantly higher
rate of negative D-dimer test results (STA-LiatestV) in
R
The use of clinical probability-adjusted cutoffs has also

the clinical probability-adjusted strategy compared to
been proposed [2,195–197]. Linkins et al. used a low
the age-adjusted strategy (56.1% versus 50.9%) [197].
(200 mg/L FEU), an intermediate or “conventional”
According to these recent findings, it can be concluded
(500 mg/L FEU) and a high (2000 mg/L FEU) D-dimer cut-
that the clinical probability-adjusted cutoffs may be
off (MDA D-dimer assay) for high, moderate, and low
more efficient than the age-adjusted cutoffs for limiting
pretest probability patients, respectively [196]. Overall,
the number of imaging tests. However, this conclusion
571 patients were studied retrospectively. Although the
needs to be supported by further prospective studies
authors failed to find different sensitivities and NPVs
using different D-dimer immunoassays.
across the groups for diagnosing VTE, the use of clinical
probability-adjusted cutoffs increased the D-dimer test
VTE diagnosis in specific populations
specificity compared to the conventional approach (i.e.
44.7% versus 60.4%) [196]. Another study published by In patients with recent surgery, D-dimers are usually
Kabrhel et al. in 7940 patients with suspected PE and increased and the diagnosis of VTE is hence challeng-
based on six different D-dimer assays reported similar ing. No CPR is validated and only the global clinical
data [195]. The use of higher (conventional 2), con- judgment of the clinician can be used to assess CPR
ventional (identical), and lower (conventional/2) cutoffs [200]. However, clinical judgment is criticized for its lack
for low, intermediate, and high pretest probability of standardization and the difficulty in teaching.
patients displayed a higher specificity (75% versus 58%) Therefore, clinicians perform imaging whenever VTE is
and a similar NPV (99.1% versus 99.5%) [195]. Kline suspected. Nevertheless, Penaloza et al. developed a
et al. also suggested using a higher D-dimer cutoff score composed of 9 variables to assess the usefulness
(1000 mg/L instead of 500 mg/L) in younger patients (i.e. of D-dimer measurement, using data from 4537
aged <50 years) with a low clinical score for PE (clinical patients that had high sensitive D-dimer testing (VidasV
R
for 2079 patients, STA-LiatestV for 1783 patients, and

R
probability-adjusted strategy). The authors found a sig-
MDAV for 675 patients) [201]. These 9 variables were
R
nificant decrease in the need for performing unneces-
sary pulmonary angiograms but also observed a slight independently associated with a risk of false positive D-
decrease in diagnosis of isolated subsegmental PE dimer value (sex female: þ1, age 65–84 years: þ4, age
[198]. Likewise, Linkins et al. reported that modifying D- 85 years: þ8, heart rate 95/min: þ1, pulse oxygen
dimer cutoffs according to the clinical pretest probabil- saturation <95%: þ2, temperature 38.5 C: þ3, per-
ity of DVT (<500 mg/L and <1000 mg/L for moderate sonal history of VTE: þ1, surgery under general anes-
and low pretest probability, respectively) may be associ- thesia within 4 weeks: þ2, active malignancy: þ3,
ated with a reduced need to perform ultrasonography, pregnancy or postpartum within 4 weeks: þ4). In
without impairing the NPV of the test [185]. patients with non-high CPR and a relevance score 8,
Recently, van der Hulle et al. prospectively validated at least 10% had a quantitative D-dimer assay below
a diagnostic algorithm incorporating the YEARS clinical 500 mg/L, meaning that, in patients with recent surgery,
decision rule (CDR) (clinical signs of DVT, hemoptysis, D-dimer measurement may still be useful, at least if
they have no other risk factors for false positive results. decrease according to the renal failure status [212,213].
Indeed, a negative D-dimer result keeps its sensitivity The possibility of using renal function-adjusted cutoffs
and NPV [200]. should be verified in dedicated studies [212].
Similarly, D-dimer levels increased physiologically in
pregnancy and in the postpartum period. In a study
Prediction of recurrence of VTE
that included 1343 pregnant women with D-dimer
measurements using a turbidimetric method (STA- The risk of recurrence during a 1-year follow up after a
LiatestV), the rate of D-dimer test results below the
R
first VTE episode is higher in men (9.5%) than in women
usual cutoff (500 mg/L) in healthy pregnant women was (5.3%), and tends also to increase over time (9.1% and
85%, 29% and 4.1% during the first, the second and the 19.7%, for men and women after 3 years, respectively)
third trimesters, respectively [202]. Similar results were [214]. Interestingly, a high D-dimer value has been asso-
observed with other assays (MDAV and HemosIL HS
R
ciated with increased risk of recurrent VTE [127]. It has
assayV) [203,204]. In the post-partum period, D-dimer
R
hence been suggested that the duration of anticoagu-
returns to normal levels around the 6th week. In case of lant therapy in VTE patients may be combined with
suspicion of PE, because imaging tests may expose the results of D-dimer testing [1,215]. In the PROLONG
mother and the fetus to radiation, the ability to rule-out study, the D-dimer value was increased 1 month after
PE with non-radiologic tests is crucial [107]. To improve cessation of vitamin K antagonist (VKA) in 608 VTE anti-
the usefulness of D-dimer results, specific cutoffs have coagulated patients who were treated for at least
been proposed, but not yet validated, in a large pro- 3 months [215]. Patients with normal D-dimer values
measured with the Clearview SimplifyV D-dimer assay
R
spective study [124]. However, once again, a negative
D-dimer result keeps its sensitivity and NPV [107]. Two (whole blood qualitative assay) did not resume anti-
large studies are currently ongoing on this topic and coagulation, whilst those with abnormal D-dimer values
may give important information on the best algorithm were randomly assigned to receive or not receive a
to exclude VTE in pregnant women [205]. Currently and new anticoagulant therapy. An abnormal D-dimer con-
in order to avoid imaging tests, D-dimer measurement centration was observed in 37% of patients. Among
is still recommended in pregnant women with suspi- these, a new VTE episode occurred in 15% of patients
cion of VTE and a non-high CPR, at least in the suspi- who stopped VKA, whilst VTE events occurred only in
cion of PE [206]. 2.9% of those receiving VKA therapy (adjusted hazard
In patients known to have cancer, the relevance of ratio [HR] 4.26; 95% CI 1.23–14.6; p ¼ .02). Legnani et al.
D-dimer in the diagnosis of VTE is decreased [107]. The reanalyzed the data of the PROLONG study to assess D-
prevalence of VTE is increased (up to 20% of cancer dimer cutoff values for several quantitative assays
(VidasV, InnovanceV, HemosILV, and STA-LiatestV) [216].
R R R R
patients develop VTE) and the NPV is therefore reduced
[107,207]. A large meta-analysis of 10002 patients Based on this post-hoc analysis, they determine age-
showed that the prevalence of both a low Wells score specific cutoffs and identified a higher threshold in
and a negative D-dimer value among patients with can- older patients (i.e. aged 70 years or older) [216]. The use
cer was only 9% [208]. It has also been shown that of a strategy based on method-specific cutoff values,
88–94% of patients with malignancy require additional adjusted for age and gender, is therefore advisable for
tests to rule out VTE [209]. However, the D-dimer meas- more accurate prediction of VTE recurrence [217,218].
urement as included in a score may be useful to iden- The use of quantitative methods rather than qualitative
tify cancer patients at low or high risk of VTE [210]. tests results (i.e., positive or negative) may be more
The exclusion of VTE in patients with renal disease is suitable to achieve this goal [216–218]. The meta-ana-
also challenging, given that D-dimer fragments are lysis of Douketis et al., which included 1818 patients
eliminated mainly by renal clearance and reticuloendo- from seven prospective studies, found that the D-dimer
thelial system catabolism [11]. Accordingly, Robert- value was a significant predictor of VTE (HR 2.59; 95%
Ebadi et al. showed that D-dimer concentrations CI 1.90–3.52; p < .001) [214]. Overall, a D-dimer value
increased according to renal status, and the proportion higher than the diagnostic cutoff after 3-months of
of negative D-dimer results decreased from 46% to 11% anticoagulant therapy in patients with a first unpro-
with normal (90 mL/min) and moderate renal failure voked VTE event was associated with double the risk of
(30–59 mL/min), respectively [211]. In addition, Lindner recurrence compared to patients with lower D-dimer
et al. showed that 100% of patients with low eGFR values [2,219]. The risk of VTE recurrence was similar
(<30 mL/min; n ¼ 29) had a positive D-dimer value among young and elderly patients. More recently,
[212]. The specificity will therefore proportionally Nagler et al. showed that increased D-dimer values
(VidasV and InnovanceV) observed after discontinuing

R R
[229]. However, caution has to be used when translat-
anticoagulant therapy after 1 month (HR 3.3; 95% CI ing these findings to other D-dimer immunoassays.
1.8–6.1), male sex (HR 2.8; 95% CI 1.5–5.1), and use of Moreover, controversy remains about the most appro-
oral contraceptives (HR 0.1; 95% CI 0.0–0.9), were sig- priate timing of D-dimer monitoring during or after dis-
nificant predictors of recurrent DVT in 479 patients continuing anticoagulation. Finally, the accurate
diagnosed with proximal DVT [220]. These authors also definition of “positive” or “negative” D-dimer (i.e. pre-
found that high factor VIII values (HR 2.2; 95% CI cise cutoff) is still an unmet requirement [2,214].
1.2–4.0) could be predictors of recurrent DVT [220]. The
distinction between provoked and unprovoked VTE is
Prediction of risk of VTE in hospitalized patients
also important. Patients with provoked VTE (after sur-
gery, lower limb trauma, and orthopedic immobilization Recently, a baseline D-dimer level measured in 7441
or hospitalization for an acute medical illness) have a hospitalized patients was found to be independently
very low risk of recurrence as compared to patients associated with symptomatic VTE during a period of
with unprovoked VTE who have a higher risk of recur- 3 months (adjusted HR 2.22; 95% CI 1.38–1.58;
rence at two years (±20%) [221]. p < .001) [230,231]. Patients hospitalized for acute ill-
Several models have been developed to predict the ness (ischemic stroke, heart failure, respiratory failure,
risk of recurrence of unprovoked VTE. In the real-life rheumatic disorders, infection) have an increased risk of
study of Tosetto et al., performed with different D- developing VTE [231–234]. A risk assessment for VTE is
dimer assays in nine centers, an increased D-dimer hence needed before initiating thromboprophylaxis
value after stopping anticoagulation (cutoff 500 mg/L [231]. The International Medical Prevention Registry on
with a quantitative assay or “positive” with a qualitative Venous Thromboembolism (IMPROVE) assessment tool
assay), a younger age (<50 years), male sex, and VTE has been developed with the aim of risk stratification in
not associated with hormonal therapy in women were hospitalized, medically ill patients. The score encom-
identified as the best predictors of VTE recurrence (area passes many clinical variables such as previous VTE,
under the curve, 0.71) [222]. The diagnostic perform- known thrombophilia, current lower-limb paralysis, cur-
ance of a model including all these variables had a rent cancer, immobilization 7 days, intensive care or
higher diagnostic efficiency compared to D-dimer test- coronary care units, and age >60 years [231]. Because
ing alone (area under the curve 0.61; p < .001). This D-dimer is an independent predictor of symptomatic
score may hence be useful to assess whether the anti- VTE, Gibson et al. combined D-dimer testing with the
coagulant treatment should be discontinued or IMPROVE assessment tool [231]. A D-dimer level 2
resumed after the usual 3-month period. The DASH the upper reference limit (URL) measured with STA-
score has been retrospectively validated in a recent Liatest platform was assigned two more points in the
independent cohort study, but the score appeared to new scoring system (IMPROVEDD). The population of
perform better in younger subjects (<65 years old) the APEX study was used to validate this approach. The
[223]. To our knowledge, the DASH score has not been combination of clinical variables already included in the
validated in an interventional prospective study. IMPROVE score with D-dimer test results was effective
Additional algorithms, such as the Vienna and in substantially improving risk discrimination and
HERDOO2 prediction models, have also been devel- patients risk reclassification at both 42 and 77 days.
oped [224]. The Vienna prediction model included D-
dimer (Asserachrom assayV), sex, and site of index event
R
Diagnosis and monitoring of disseminated

[224,225]. It has been validated in external populations
intravascular coagulation
but, again, not in an interventional study [226].
Conversely, the HERDOO2 model has been recently vali- DIC is a life-threatening condition characterized by per-
dated in an interventional study that included 3155 sistent activation of the hemostatic system with intra-
patients from several countries [227]. This model vascular thrombin generation, fibrin formation, and
included D-dimer (measured with VidasV assay; cutoff,
R
increased fibrinolysis [3]. Patients with DIC may present
250 mg/L), age, body mass index (BMI), and post-throm- with bleeding, thrombosis, or both [164]. Early recogni-
botic signs [224,228]. In line with these findings, the tion of DIC is paramount to initiate the appropriate
Scientific and Standardization Committee of the treatment, which generally entails eliminating the
International Society on Thrombosis and Hemostasis underlying condition (e.g. sepsis, malignancy, trauma or
recommends that D-dimer testing should be performed burns, obstetrical diseases, toxins, drugs, immunological
in all patients with clinical suspicion of recurrent VTE disorders, and other inflammatory diseases) [1]. The
International Society of Thrombosis and Haemostasis readily available. However, not all POC D-dimer assays
(ISTH) [235] and the Japanese Ministry of Health and are appropriate to be used to exclude VTE, and only
Welfare [236] have proposed two different scoring sys- POC assays that have been validated in clinical trials
tem for diagnosis and management of DIC. Comparison and cleared by the FDA should be used.
of these scoring systems yielded similar diagnostic D-dimer measurement is also useful to predict the
effectiveness for DIC [1,237]. The laboratory parameters risk of VTE recurrence and the risk of VTE in hospitalized
included in these scoring systems are platelet count, patients and to diagnose and monitor DIC. Other
fibrinogen, prothrombin time, and FDP [235,236,238]. emerging indications (i.e. prognostication of peripheral
Although D-dimer is the most common FDP measured artery disease, screening of intracardiac thrombus)
in clinical laboratories, some authors suggested that need further investigations.
soluble fibrin may be more specific to detect intravas- Pre-analytical requirements are paramount in the
cular clot generation. D-dimer is considered positive hemostasis laboratory. Nevertheless, the literature
when its value is 2 times the URL [1,4]. As for VTE, appears to be quite reassuring regarding several prea-
there is now consolidated evidence that a “normal” D- nalytical variables that affect D-dimer results (needle
dimer value excludes a diagnosis of DIC [127]. However, bore size, butterfly devices, use of PTS, in vitro hemoly-
a “positive” D-dimer value cannot be considered spe- sis, stability). In some specific settings (i.e. emergency,
cific for DIC, because D-dimer may be increased in a older patients, delay before centrifugation), D-dimer
vast array of clinical conditions (Table 4). Repeated D- results may be released to the clinician without impair-
dimer analysis (i.e. 3 times per day) is also important to ing patient management.
monitor DIC [236,238,239]. The combination of D-dimer A major drawback of D-dimer assays is the high vari-
and fibrin monomer allowed the identification of ability observed between immunoassays. This variability
patients with septic shock with a worse survival [240]. is explained by the fact that D-dimers comprise a broad
mixture of degradation products of cross-linked fibrin,
by the utilization of different monoclonal antibodies, by
Conclusions
the lack of international certified internal controls or
Today, D-dimer has become one of the most commonly calibrators, and by the use of different units and clinical
requested coagulation tests. D-dimer, considered a bio- cutoffs. Further studies are needed to achieve harmon-
marker of activation of coagulation and fibrinolysis, is ization of D-dimer measurements. Ongoing discussion
mainly employed for the exclusion of VTE. A negative among manufacturers, scientists and clinicians is essen-
D-dimer value along with a low clinical probability can tial for achieving this goal.
safety exclude VTE in suspected individuals. Currently, it
is recommended to employ D-dimer assays with a very
Acknowledgments
high sensitivity and NPV to safety exclude VTE (95%
and 97%, respectively). However, a positive D-dimer The authors would like to thank Mr. Nicolas Bailly for the
value should always trigger imaging tests to confirm editing of the figures and tables.
VTE, given that a wide array of diseases and conditions
are characterized by an increase in D-dimer (i.e. aging, Disclosure statement
inflammation, cancer, renal failure). More recently, the
No potential conflict of interest was reported by the authors.
use of age-adjusted cutoffs to safely rule out VTE has
been proposed to substantially increase the PPV with-
out significantly impairing the NPV, so ultimately ORCID
improving the clinical usefulness of D-dimer measure- Giuseppe Lippi http://orcid.org/0000-0001-9523-9054
ment in elderly patients with low clinical probability. François Mullier http://orcid.org/0000-0001-6947-6099
Even though these adjusted cutoffs have been
endorsed in several guidelines, many laboratories are
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D Dimer Preanalytical Analytical Postanalytical Variables and Clinical Applications

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Critical Reviews in Clinical Laboratory Sciences

ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: https://www.tandfonline.com/loi/ilab20

D-dimer: Preanalytical, analytical, postanalytical

Julien Favresse, Giuseppe Lippi, Pierre-Marie Roy, Bernard Chatelain,

To link to this article: https://doi.org/10.1080/10408363.2018.1529734

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Published online: 10 Jan 2019.

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D-dimer: Preanalytical, analytical, postanalytical variables, and clinical

ABSTRACT ARTICLE HISTORY

Figure 1. Mechanism of D-dimer production [1,3,4,164].

BD and VacuetteV, Greiner Bio-one (polypropylene)) and

Organization (WHO) for the vast majority of hemostasis

flow freely into the tube and be promptly mixed within

card tube is mandatory for removing air contained

within the tubing, which may be associated with the

Stago, Asnieres, France), ImmuliteV (Siemens, USA)) [38].

respected), and even for the presence of clots.

The comparison of studies assessing hemolysis inter-

Table 2. Specific pre-analytical data regarding D-dimer testing.

been developed [3,41,84]. Magnetic particles coated

Readily available, fast,

Sensitivity, not all

HemosIL HSV (Werfen)

Sensitivity, rapid, auto-

Lack clinical validation,

(e.g. VidasV mini analyzer, TriageV, Stratus CSV), and

chemiluminescence (e.g. PathfastV) also exist. A few

Moderate sensitivity, man-

Variability across assays

The fibrin assay comparison trial (FACT) study, pub-

(15 latex-enhanced immunoassays, 6 ELISAs, and 2

membrane-based immunoassays) [96]. The authors

found that the mean values obtained in 39 samples var-

ied from 630 mg/L to 13,350 mg/L (21-fold), with two

ELISAs and latex-enhanced immunoassays were more

Highly manual, technical

reactive to low-molecular-weight cross-linked fibrin and

high molecular weight fibrin, respectively [96].

Accordingly, the study of Meijer et al., based on 357

laboratories using the 7 frequently-used D-dimer immu-

D-dimer concentrations compared to others [97].

close to the cutoff used for VTE exclusion [98]. In 2014,

in a survey involving 3800 laboratories, the Coagulation

STA-LiatestV). This may be a consideration when

Recommendations on the performance of D- Postanalytical variables

Table 4. Causes of D-dimer elevation [1,126,127,164].

Stratus DSV (Siemens, USA), AxSYMV), microplate ELISAs

Clinical prediction rules (CPR)

lower sensitivity (94%) [191]. Although age-adjusted

The use of clinical probability-adjusted cutoffs has also

for 2079 patients, STA-LiatestV for 1783 patients, and

(VidasV and InnovanceV) observed after discontinuing

Diagnosis and monitoring of disseminated

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