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Received: 18 December 2018    Revised: 25 January 2019    Accepted: 1 February 2019

DOI: 10.1111/ijlh.12993

REVIEW

Laboratory diagnosis of heparin‐induced thrombocytopenia

Theodore E. Warkentin1,2,3,4

1
Department of Pathology and Molecular
Medicine, Michael G. DeGroote School of
Medicine, McMaster University, Hamilton,
Ontario, Canada
2 laboratory testing for the pathogenic platelet‐activating antiplatelet factor 4 (PF4)/
Department of Medicine, Michael G.
DeGroote School of Medicine, McMaster
University, Hamilton, Ontario, Canada
3
Hamilton Regional Laboratory
Medicine Program, Hamilton Health
Sciences, Hamilton General Hospital,
Hamilton, Ontario, Canada
4
McMaster Centre for Transfusion Research,
Hamilton, Ontario, Canada
Correspondence
Theodore E. Warkentin, Hamilton Regional
Laboratory Medicine Program, Room
1-270B, Hamilton General Hospital, 237
Barton St. East, Hamilton, Ontario L8L 2X2
Canada
Email: twarken@mcmaster.ca
Abstract
Heparin‐induced thrombocytopenia (HIT) is a clinical‐pathological disorder; thus,
heparin antibodies is central for diagnosis. The “iceberg” model summarizes the inter‐
relationship between platelet activation assays and PF4‐dependent immunoassays,
with platelet‐activating antibodies comprising a subset of anti‐PF4/heparin antibod‐
ies. The platelet serotonin‐release assay (SRA), performed by reference laboratories,
has high sensitivity and specificity for HIT (~95% each), and is especially suited for
detecting highly pathogenic HIT sera containing both heparin‐dependent and hepa‐
rin‐independent platelet‐activating antibodies; this latter subgroup of antibodies ex‐
plains “autoimmune HIT” disorders (delayed‐onset, persisting, spontaneous, heparin
“flush,” fondaparinux‐associated). Recently, SRA‐negative HIT has become recog‐
nized, in which serum from some HIT patients contains subthreshold levels of plate‐
let‐activating antibodies (by SRA) that become detectable using a PF4‐enhanced
platelet activation assay. Unusual immunologic features of HIT include early antibody
detectability (at onset of platelet count fall) and antibody transience (seroreversion).
Widely available PF4‐dependent enzyme immunoassays (EIAs) have high sensitivity
but poor specificity for HIT, although specificity is enhanced with IgG‐specific EIAs
and strong positive results; unfortunately, EIA results are usually not available in real
time. Automated rapid immunoassays, such as the chemiluminescence immunoassay
(CLIA) and latex immunoturbidimetric assay (LIA), facilitate real‐time laboratory diag‐
nosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR−)
test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian
analysis, into real‐time posttest probabilities that are dramatically increased (test
positive) or decreased (test negative). Moreover, (semi‐)quantitative CLIA‐ and LIA‐
positive results (weak, moderate, strong positive) can further refine the posttest
probability of HIT.

KEYWORDS
(autoimmune) heparin‐induced thrombocytopenia, Bayesian analysis, chemiluminescence
immunoassay, latex immunoturbidimetric assay, rapid immunoassays, serotonin‐release assay

1 |  I NTRO D U C TI O N IgG class that recognize multimolecular complexes consisting of a

(positively charged) chemokine, platelet factor 4 (PF4), bound to
Heparin‐induced thrombocytopenia (HIT) is an immune‐mediated, (negatively charged) heparin or certain other platelet‐associated
prothrombotic disorder caused by platelet‐activating antibodies of polyanions.1 HIT is a clinical‐pathological syndrome: This means that

Int J Lab Hematol. 2019;41(Suppl. 1):15–25. © 2019 John Wiley & Sons Ltd |  15
wileyonlinelibrary.com/journal/ijlh  
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16       WARKENTIN

a diagnosis requires both a compatible clinical picture and laboratory

evidence of the pathogenic “HIT antibodies.”1 This concept under‐
scores the laboratory's key role in HIT diagnosis.
HIT is a distinct clinical syndrome. 2 To illustrate, contrast HIT
with classic drug‐induced immune thrombocytopenia (D‐ITP),
caused by drugs such as quinine and vancomycin. Whereas D‐ITP
features severe thrombocytopenia (<20 × 109/L) and bleeding, HIT
typically causes a moderate thrombocytopenia (median platelet
count nadir, ~60 × 109/L). Further, most patients develop thrombo‐
sis, reflecting HIT‐induced hypercoagulability (platelet and coagula‐
tion system activation).
Important clinical decisions are needed when HIT is suspected.
Should heparin be stopped? Should an alternative nonheparin an‐
ticoagulant be given? The importance of these decisions has led to
recent development of rapid assays for HIT, with potential for real‐
time incorporation of laboratory test results during the initial diag‐
nostic evaluation of HIT.
2 |  I C E B E RG M O D E L
The iceberg model views HIT as being caused by a subset of anti‐
PF4/heparin antibodies that cause platelet activation (Figure 1). 3
This model illustrates that platelet activation assays, such as the
serotonin‐release assay (SRA) and the heparin‐induced platelet
activation (HIPA) assay, have similar high sensitivity as PF4‐de‐
pendent immunoassays, such as enzyme immunoassays (EIAs), for
diagnosis of HIT, but that platelet activation assays have greater
specificity, as they do not detect nonpathogenic, nonactivating
antibodies.
2.1 | SRA versus EIA
Evidence for the superior biological relevance of the SRA compared
with EIAs for HIT diagnosis was found in a clinical trial of heparin
versus fondaparinux for treating venous thromboembolism. This
study4 found that all 4 patients who were SRA‐positive/EIA‐positive
developed rapid‐onset HIT when given therapeutic‐dose heparin, ei‐
ther unfractionated heparin (UFH) or low‐molecular‐weight heparin
(LMWH), whereas none of 15 SRA‐negative (but EIA‐positive) pa‐
tients who received heparin developed thrombocytopenia (4/4 vs
0/15; P < 0.001).
Other data supporting superiority of the SRA versus EIAs in‐
clude studies of serial blood samples in HIT patients who undergo
therapeutic plasma exchange5 or administration of high‐dose im‐
mune globulin6 for treating HIT: In both situations, the SRA becomes
negative first with the EIA showing persisting positivity, with such
patients able to receive heparin safely.
2.2 | Definition of thrombocytopenia
Assay sensitivity/specificity also reflect differences in HIT defini‐
tion. For example, HIT defined as a platelet count fall to less than
F I G U R E 1   “Iceberg model” of Heparin‐induced
thrombocytopenia (HIT). Clinical HIT, comprising HIT with (HIT‐T)
or without thrombosis, is represented by the portion of the iceberg
above the waterline; the portion below the waterline represents
subclinical anti‐PF4/heparin seroconversion. Three types of assays
are highly sensitive for the diagnosis of HIT: the washed platelet
activation assays (serotonin‐release assay [SRA] and heparin‐
induced platelet activation [HIPA]), the IgG‐specific PF4‐dependent
EIAs (EIA‐IgG), and the polyspecific EIAs that detect anti‐PF4/
heparin antibodies of the three major immunoglobulin classes (EIA‐
IgG/A/M). In contrast, diagnostic specificity varies greatly among
these assays, being the highest for the platelet activation assays
(SRA and HIPA) and lowest for the EIA‐IgG/A/M. This is because
the EIA‐IgG/A/M is most likely to detect clinically irrelevant, non–
platelet‐activating anti‐PF4/heparin antibodies. The approximate
probability of SRA‐positive status in relation to a given EIA result,
expressed in optical density (OD) units, is also indicated. HIPA+,
heparin‐induced platelet activation assay positive; SRA+, serotonin‐
release assay positive. Reprinted, with permission3
150 × 109/L occurred in only about one‐third of SRA‐positive pa‐
tients in a clinical trial of heparin thromboprophylaxis.7 However,
approximately twice as many patients were identified in the same
trial as having HIT when a more sensitive definition—50% or greater
platelet count fall from the postoperative peak platelet count—was
applied.8
2.3 | IgG antibody class
Only antibodies of IgG class are capable of activating platelets
(through platelet FcγIIa receptors), yet heparin‐exposed patients
often make anti‐PF4/heparin antibodies of IgA and IgM classes.9
Detecting nonpathogenic IgA and IgM class antibodies explains
lower diagnostic specificity of so‐called “polyspecific” EIAs that de‐
tect antibodies of any of the three major immunoglobulin classes,
IgG, IgA, and IgM.10
2.4 | Frequency of HIT: seroconversion,
breakthrough, and clinical factors
Heparin causes HIT through two distinct but interrelated mecha‐
nisms. First, heparin exposure triggers anti‐PF4/heparin antibody
formation (“seroconversion”). Second, among antibody‐forming pa‐
tients, heparin is usually needed for heparin‐dependent antibodies
WARKENTIN
to cause platelet activation and resulting thrombocytopenia (“break‐
through”). (However, I discuss unusual HIT antibodies that do not
require heparin for pathogenicity later in this review—see sec‐
tion 5.1) UFH causes HIT with about 10‐fold greater frequency
versus LMWH, through approximately threefold higher frequencies
for each of these two distinct phenomena, immunization and break‐
through.11 Other factors that influence HIT frequency include type
of patient population (orthopedic surgery/trauma > cardiac surgery
> medical) and patient sex (female > male).12
3 | TE M P O R A L FE AT U R E S O F H IT
HIT has highly characteristic profiles of antibody seroconversion and
seroreversion.
3.1 | Timing of onset of HIT
Patients immunized by intra‐/postoperative heparin usually develop
their platelet count fall during a narrow time period between days 5
and 10 (first day of heparin = day 0);13 this timing of “typical‐onset
HIT” is not influenced by past heparin exposure. In contrast, when
heparin administration causes an abrupt platelet count fall (“rapid‐
onset HIT”), recent heparin exposure is usually identified.13
3.2 | HIT antibody transience
HIT antibodies are remarkably transient: The median time to loss of
antibody detectability following an episode of HIT ranges from 40
to 80 days, depending on the test performed (SRA vs. EIA, respec‐
tively).13 HIT antibody transience explains why risk of rapid‐onset
HIT is generally limited to patients who have received heparin in the
recent past.13 This phenomenon also explains why it is important
to test patients for HIT using blood samples obtained either during
acute thrombocytopenia or soon after platelet count recovery. We
have observed some HIT patients in whom the platelet count recov‐
ers despite continued heparin exposure;11,14 in such cases, waning of
antibody reactivity (“seroreversion”) can be shown.14
3.3 | Antibody detectability at onset of HIT
HIT antibodies are readily detectable in patient serum/plasma—
either by EIA or SRA—at the onset of the HIT‐related platelet
count decline (Figure 2), even at such an early time point when
a diagnosis of HIT would not even be considered.15,16 I believe
this phenomenon is key to understanding the pathophysiology of
HIT antibody‐induced platelet activation. Newman and Chong17
showed that HIT antibody‐induced platelet activation is a dynamic
process, beginning with binding of antibodies via their Fab re‐
gions to platelet‐bound PF4/heparin, with progressive assembly
of in situ PF4‐heparin‐IgG immune complexes that over time en‐
gage more platelet Fc receptors, ultimately leading to Fc receptor
complexing and platelet activation. I believe it is a requirement for
|
      17
F I G U R E 2   Anti‐PF4/heparin antibodies by EIA‐IgG in the
evolution of HIT: comparison of 12 patients with HIT and 36
seropositive non‐HIT controls. Individual results of anti‐PF4/
heparin antibodies detected using an IgG‐specific anti‐PF4/heparin
EIA at baseline and 3 sequential time points for 12 patients with
HIT (solid circles) and 36 seropositive non‐HIT controls (open
circles). The 3 time points are as follows: (1) early postoperative
platelet count nadir (i.e, preceding the platelet count fall indicating
HIT), (2) earliest platelet count decrease associated with HIT,
and (3) platelet count fall of 50% or greater; in addition, (4) the
maximum OD value for each patient is also shown. The maximum
OD values were significantly greater for the 12 HIT patients
versus the 36 non‐HIT controls (median, 1.63 vs. 0.94; P < 0.001).
EIA‐IgG, IgG‐specific enzyme immunoassay; HIT, heparin‐
induced thrombocytopenia; OD, optical density. Reprinted, with
permission15
HIT antibodies to be “free” in plasma in order to permit gradually
progressive assembly of these platelet‐activating immune com‐
plexes. This mechanism could explain why early detection of anti‐
bodies in patient serum/plasma is a consistent finding when serial
blood samples obtained during HIT‐associated seroconversion are
studied.
4 | PL ATE LE T AC TI VATI O N A S SAYS
Several different platelet activation assays are available for HIT
diagnosis. They are all indirect assays; that is, they test patient
serum/plasma against normal donor platelets; in contrast, direct
assays that utilize patient platelets are not used to diagnose HIT.
Additional information is available in a comprehensive review of
laboratory testing for HIT.18 An advantage of platelet activation
assays is their unique role for diagnosis of “autoimmune HIT”
(aHIT) (see section 5.1).
4.1 | Serotonin‐release assay (SRA)
The SRA has traditionally been viewed as the gold standard test for
HIT, based on its high sensitivity (~95%) and specificity (~95%).3,18,19 A
positive test is indicated by serum‐induced platelet serotonin release
 |
18       WARKENTIN
at pharmacologic heparin concentrations (0.1‐0.3 U/mL UFH) that is
19,20
inhibited at high heparin (100 U/mL). However, the assay is tech‐
nically demanding (platelet washing) and requires selected (pedigree)
platelet donors whose platelets react well to HIT antibodies, 21 and so
laboratories that offer the SRA may not necessarily have good per‐
formance. Moreover, the usual platelet activation end‐point—release
from platelet dense granules of radioactive serotonin—limits testing
to laboratories permitted to use radiolabels. Recent controversies re‐
garding SRA sensitivity are discussed in Section 5.5.
4.2 | Heparin‐induced platelet activation assay
(HIPA)
The HIPA is another washed platelet activation assay (primar‐
ily used in Europe) that uses platelet aggregation, judged visually,
as the platelet activation end‐point.18 Whereas the SRA is usually
performed testing platelets in a shaker (reacted for 60 minutes), the
HIPA is performed using stirred platelets, and read visually in 5‐min‐
ute intervals (up to 45 minutes). Like the SRA, the HIPA can be used
to recognize aHIT. Recently, it has been observed that the HIPA is
more sensitive for detecting platelet‐activating antibodies than the
22
SRA; however, it is unclear whether this improves sensitivity for
true HIT or rather results in detection of nonpathogenic antibodies
(decreased diagnostic specificity).
4.3 | Platelet aggregometry (platelet‐rich plasma
[PRP])
Standard platelet aggregometry using PRP was the first platelet
18
activation test developed for HIT. Unfortunately, even when op‐
timized through donor selection, assay sensitivity is only ~80%. 23
Moreover, plasma from critically ill patients can cause false‐posi‐
tive test results.18 This assay is infrequently used nowadays for
HIT diagnosis.
4.4 | Heparin‐induced multiple electrode
aggregometry (HIMEA)
A technique known as heparin‐induced multiple electrode ag‐
gregometry (HIMEA) assesses platelet aggregation using whole
blood from a normal donor, in the presence of patient serum (or
citrated plasma), and heparin at two concentrations, low (0.25‐1.0
U/mL) and high (50‐100 U/mL). It is unclear how HIMEA operat‐
ing characteristics compare with washed platelet activation assays.
Galea and colleagues24 found the HIMEA to be superior to standard
platelet aggregometry. Favaloro and co‐workers25 found the assay
to have sensitivity intermediate between that of standard platelet
aggregometry and the SRA.
4.5 | PF4‐enhanced platelet activation assays
Traditionally, platelet activation assays have been performed test‐
ing patient serum (or plasma) with normal donor platelets in the
presence of various concentrations of heparin. Recently, it has been
shown that addition of increasing concentrations of PF4—rather
than heparin—can be used to perform the SRA. 26 Moreover, this
maneuver appears to increase sensitivity for detection of heparin‐
dependent platelet‐activating antibodies (although impact on test
specificity vis‐à‐vis the SRA remains unclear). Our laboratory has
found that up to one‐third of sera that test SRA‐negative/EIA‐posi‐
tive will yield a positive result in a PF4‐enhanced SRA, which we call
the “PF4‐SRA”; however, case review suggested the patients were
unlikely to have had HIT. 26
Padmanabhan and colleagues have also developed a PF4‐en‐
hanced platelet activation assay, named the PF4‐dependent P‐se‐
lectin expression assay (PEA). 27 In this assay, 62.5 μg/mL PF4 is
added to isolated donor platelets prior to addition of patient serum,
measuring platelet activation through flow cytometric detection of
P‐selectin expression. This assay reportedly detects HIT antibodies
1 to 2 days prior to the SRA. 28 Whereas the PF4‐SRA and the PEA
perform the assays at one or more concentrations of PF4 (without
use of heparin), Vayne and colleagues29 have recently developed a
hybrid assay in which addition of PF4 is used to enhance reactivity
in the SRA (which is otherwise performed using heparin as per the
usual fashion). To date, the utility of these various PF4‐enhanced
assays in improving the sensitivity‐specificity profiles of platelet ac‐
tivation assays for HIT remains uncertain.
4.6 | SRA‐negative HIT
The availability of PF4‐enhanced assays to detect subthreshold levels
of platelet‐activating antibodies not readily detectable in the (clas‐
sic) SRA implies existence of “SRA‐negative HIT.” Two groups have
described this phenomenon. Vayne et al29 reported an SRA‐nega‐
tive patient strongly suspected to have HIT; by adding PF4 (10 μg/
mL) to their conventional SRA (performed at 0, 0.1, 0.5, and 10 U/
mL heparin), these authors obtained a positive test result. Pandya
and colleagues30 reported 2 patients with clinical courses consistent
with HIT, and who tested EIA‐IgG positive but SRA negative; both
patients tested positive in the PEA. More work is needed to deter‐
mine how often SRA‐negative HIT occurs and to what extent this
phenomenon reflects differences in SRA performance among labo‐
ratories or rather reflect fundamental nuances in HIT pathogenesis.
The McMaster Platelet Immunology Laboratory quotes a ~95% SRA
test sensitivity,3 which implies that ~5% of patients with true HIT
have SRA‐negative HIT.
5 | AU TO I M M U N E H IT (a H IT )
aHIT refers to patients with atypical clinical features explainable by
unusual HIT antibodies that can activate platelets strongly even in
the absence of heparin (heparin‐independent platelet activation). 31
In general, when serum/plasma from such patients is diluted, hep‐
arin‐dependent platelet activation can also be demonstrated. These
highly pathogenic antibodies are able to bind and cross‐link PF4
WARKENTIN |
      19

TA B L E 1   Autoimmune HIT (aHIT)

Disorder Definition
disorders
Delayed‐onset HIT HIT that begins or worsens after stopping HIT
Persisting HIT HIT where thrombocytopenia persists despite
stopping heparin
Spontaneous HIT syndrome Disorder clinically and serologically identical to HIT
but without proximate heparin exposure to explain
the presence of HIT antibodies
Heparin “flush” HIT HIT triggered by exposure only to heparin flushes
Fondaparinux‐associated HIT HIT caused by exposure to fondaparinux

These five autoimmune HIT (aHIT) disorders feature HIT antibodies that activate platelets in the
absence of heparin both in vitro and in vivo. Thus, strong platelet activation is seen in platelet activa‐
tion assays (e.g, SRA, HIPA) even at 0 U/mL heparin, with greater platelet activation, using either
neat or diluted patient serum/plasma, in the presence of pharmacologic heparin concentrations
(0.1‐0.3 U/mL UFH). As with conventional HIT sera, platelet activation is inhibited in the presence of
high heparin concentrations (100 U/mL).

molecules with (nonheparin) platelet‐associated polyanions (chon‐
droitin sulfate, polyphosphates) or even in the absence of polyanion
altogether. Table 1 lists various presentations of HIT in which most
32-38
(or all) patients have aHIT antibodies.
Platelet activation assays performed both in the presence and
absence of heparin are important in supporting a diagnosis of aHIT:
Strong platelet activation at 0 U/mL heparin, which is enhanced in
the presence of pharmacological concentrations of heparin (0.1‐0.5
U/mL UFH)—either using neat or diluted serum/plasma—with in‐
31,38,39
hibition at 100 U/mL heparin, is the serological picture.
major U.S. reference laboratories that perform the SRA do not
perform the test in the absence of heparin,36 contributing to aHIT
underrecognition.
High‐dose intravenous gamma globulin (IVIG) interrupts HIT anti‐
body‐induced platelet activation, resulting in rapid platelet count
recovery.31,40
5.3 | Spontaneous HIT syndrome
This increasingly recognized aHIT disorder is clinically and serologi‐
cally indistinguishable from HIT, but occurs in the absence of a prox‐
imate immunizing heparin exposure.34-36 Precipitating events have
The included bacterial infection and knee replacement surgery (the latter
despite postoperative nonheparin thromboprophylaxis). A large pro‐
portion (~70%) of patients with post‐knee replacement spontaneous
HIT syndrome evince adrenal hemorrhagic necrosis.36

5.1 | Delayed‐onset HIT 5.4 | Heparin “flush”‐induced HIT

Delayed‐onset HIT—the first aHIT disorder recognized—was initially
defined as HIT with a platelet count fall that begins several days
after stopping heparin.32 More recently, the definition has been ex‐
panded to include patients whose platelet count continues to fall
after heparin discontinuation, 33,39 directly reflecting the heparin‐
independent nature of the platelet‐activating antibodies. The moni‐
ker “delayed‐onset” is a misnomer, as the platelet count fall usually
begins in the usual time frame of HIT, that is, 5 to 10 days after
the start of the heparin exposure that triggers HIT. Delayed‐onset
HIT has an especially high frequency of life‐ and limb‐threatening
thrombosis, including overt disseminated intravascular coagulation
(DIC).
Rarely, HIT can occur in a patient whose only exposure to hepa‐
rin is low doses (“flushes”) typically used to maintain intravascular
catheters. During the investigation of four cases of HIT that com‐
plicated stem cell harvesting for multiple myeloma (with heparin
exposure only through apheresis catheter management), we found
that the frequency of aHIT antibodies (with strong heparin‐inde‐
pendent platelet‐activating properties) was significantly higher than
in patients with HIT identified in the same hospital (4/4 vs. 34/100;
P = 0.0161).37 Moreover, there was an inverse relationship between
platelet counts and the magnitude of serum‐induced serotonin
release.

5.2 | Persisting HIT
Persisting HIT refers to patients with prolonged time to plate‐
let count recovery. For example, well‐documented cases of HIT
have been reported where the platelet count takes more than one
month to recover.32,33,39 Interestingly, there is an inverse relation‐
ship between the severity of thrombocytopenia and the degree of
heparin‐independent platelet activation induced by patient serum.39
5.5 | Fondaparinux‐associated HIT
An advantage of fondaparinux—a sulfated pentasaccharide anti‐
coagulant modeled after the antithrombin‐binding region of hepa‐
rin—is its low frequency (versus UFH and LMWH) of promoting
platelet activation in the presence of HIT antibodies.4 However,
HIT has been described in patients who have only received post‐
operative fondaparinux thromboprophylaxis. 38 In these cases,
aHIT antibodies have been observed where platelet activation
 |
20       WARKENTIN
was further enhanced in vitro in the presence of fondaparinux.
Rarely, persisting thrombocytopenia and laboratory features of
overt DIC have occurred in aHIT patients treated with fonda‐
parinux, in whom in vitro “cross‐reactivity” with fondaparinux was
demonstrated. 33,36
6 |  PF4 ‐ D E PE N D E NT I M M U N OA S S AYS
Various immunoassays have been developed based on detecting
binding of HIT antibodies to PF4/heparin (or PF4/polyanion).18 HIT
antibodies are stable in serum/plasma frozen at −70°C, and thus, fro‐
zen, well‐characterized sera/plasmas can be used to evaluate quickly
41
a new assay's operating characteristics. I will discuss immunoas‐
says currently available in the United States
The dominant PF4‐dependent immunoassay is the EIA, also
called enzyme‐linked immunosorbent assay (ELISA). The EIA is
usually performed in batches, in some clinical settings as often as
once‐daily. Although EIAs remain the most widely performed type
of immunoassay for diagnosis of HIT, there is growing use of rapid
immunoassays, allowing for “on‐demand” testing that can provide
42
results within an hour of blood collection.
6.1 | Enzyme immunoassays (EIAs)
The major advantage of PF4‐dependent EIAs is their high sen‐
sitivity: At least 97% of patients with HIT will test positive in
a solid‐phase EIA.10 Moreover, these assays are commercially
available and can be performed by hospitals that employ EIA
technology, allowing results to be obtained within a few hours
(however, testing is often batched, so test results can be de‐
layed). A major disadvantage is low diagnostic specificity, espe‐
cially when only a weak positive result is obtained. Thus, it is
recommended to consider strength of reactivity when interpret‐
ing an EIA result. For example, the frequency of a positive SRA is
less than 5% if the EIA is only weakly positive (0.40 to 0.99 opti‐
cal density [OD] units), whereas the probability of a positive SRA
43
exceeds 90% if the OD is > 2.00 units. Diagnostic specificity is
enhanced if an IgG‐specific EIA is performed (although a lower
cutoff of 0.40 units is suggested when interpreting IgG‐specific
EIAs).10
6.2 | Particle ImmunoFiltration Assay (PIFA)
The PIFA adds patient serum (fresh, not frozen/thawed) to a reaction
well containing dyed particles coated with PF4 (not PF4/heparin).
Subsequently, nonagglutinated—but not agglutinated particles—will
migrate through the membrane filter, so a negative test is shown by
a blue color in the result well, and no color indicates a positive test.
Despite the PIFA being approved in the United States, I am unaware
of any peer‐reviewed data supporting its utility for HIT diagnosis;
rather, available reports indicate lack of diagnostic value of this
assay.44,45
6.3 | Chemiluminescence immunoassay (CLIA)
This rapid, automated chemiluminescence assay (Instrumentation
Laboratory, Bedford, MA, US) detects binding of anti‐PF4/heparin
antibodies to magnetic particles coated with PF4/polyvinyl sulfonate
(PVS).18 After incubation, magnetic separation, and a wash step, a
tracer consisting of an isoluminol‐labeled antihuman IgG antibody
is added, which binds to the captured anti‐PF4/heparin antibodies
on the particles. After a second incubation, magnetic separation,
and washing, reagents that cause chemiluminescence are added,
with the emitted light (directly proportional to the concentration of
anti‐PF4/heparin antibodies) measured by the instrument. Testing is
performed “on‐demand” with results available in ~30 minutes after
preparation of serum (or citrated plasma).
In a study of 509 patient sera (n = 33 SRA‐positive) obtained
from a prospective study46 of the 4Ts scoring system,47 we found
the (IgG‐specific) CLIA to have remarkable agreement (~98%) with
the SRA.48 The CLIA's sensitivity was 97.0% (32/33), a value that
remained similarly high (166/168 = 98.8% [95% CI 95.7%‐99.8%])
when we also included sera from 135 consecutive SRA‐positive pa‐
tients diagnosed with HIT at our medical center. The CLIA's specific‐
ity was 98.5% (469/476). We also determined the likelihood ratios
(LRs) for a positive (LR+) and negative (LR−) SRA test result, which
at the manufacturer's recommended cutoff (positive test, ≥1.00 U/
mL) yielded LR+ and LR− values of 65.9 and 0.031, respectively. The
utility of LRs for real‐time diagnostic evaluation of HIT—including
for graded LR+ values that depend on the degree of test positivity
(weak, moderate, strong)—is discussed subsequently (see section 8).
6.4 | Latex immunoturbidimetric assay (LIA)
(functionalized immunoassay)
The latex immunoturbidimetric assay (LIA) is an automated assay
(Instrumentation Laboratory) classified as a “functionalized immu‐
noassay” as it detects HIT antibodies based upon their ability to in‐
hibit the aggregation of latex nanoparticles coated with a HIT-like
monoclonal antibody (KKO) following addition of PF4/PVS com‐
plexes (Figure 3). In theory, this assay will detect anti‐PF4/heparin
antibodies of IgG, IgA, and/or IgM classes (as any of these could in‐
hibit nanoparticle aggregation), yet the diagnostic specificity of the
LIA exceeds that of IgG‐specific EIAs.49 As with the CLIA, testing
is usually performed “on‐demand” with results available less than
20 minutes after preparation of patient plasma (serum is not used
for the LIA). Use of standardized equipment and monoclonal anti‐
body calibrator suggests that comparable results should be obtained
among different laboratories.
In a study of 429 patient plasma samples obtained from the 4Ts
scoring system evaluation,46 we found the LIA to have higher diag‐
nostic specificity (94%) than the IgG‐specific EIA (85.2%; P < 0.0001
vs. the LIA).49 Our estimate of LIA sensitivity, based on 31 SRA‐pos‐
itive patients identified in the 4Ts study and another 125 consec‐
utive patients with SRA‐positive HIT recognized at our institution,
was 97.4% (152/156; 95% CI, 93.6%‐99.3%). As discussed in the
WARKENTIN |
      21

F I G U R E 3   Schematic representation of the latex immunoturbidimetric assay (LIA) for detection of HIT antibodies. Latex nanoparticles
coated with a HIT‐like monoclonal antibody (KKO), upon addition of PF4/PVS complexes, will become maximally agglutinated if tested
with a blood sample that contains no PF4/heparin‐reactive antibodies (upper‐third of figure). In contrast, inhibition of particle agglutination
occurs if free KKO (positive calibrator [used to define the calibration curve, expressed in arbitrary U/mL] or positive control) is added
before PF4/PVS (middle‐third). HIT antibodies within patient plasma will also inhibit (to varying degrees) particle agglutination triggered by
PF4/PVS (lower‐third). The instrument automatically interpolates delta (Δ) absorbance with the calibration curve where the concentration of
antibodies in the sample is inversely proportional to the degree of agglutination.
*Positive control can be KKO or another HIT‐like monoclonal antibody. Δ, delta (change); HIT, heparin‐induced thrombocytopenia; HIT Abs,
HIT antibodies; IgGAM, antibodies of any of the immunoglobulin classes, IgG, IgA, and IgM; KKO, name of HIT‐like monoclonal antibody
used in the LIA; MAb, monoclonal antibody; PF4/PVS, platelet factor 4/polyvinyl sulfonate. Reprinted, with permission49

next section, the semi‐quantitative reporting of the LIA, permitting
graded classification of positive results (weak, moderate, strong), al‐
lows for nuanced, real‐time diagnostic evaluation of HIT.
7 | BAY E S I A N A N A LYS I S FO R H IT
D I AG N OS I S
Clinical tools such as the 4Ts scoring system47 are helpful in esti‐
mating the pretest probability of HIT. The 4Ts evaluates parameters
typically sought during clinical evaluation, for example, the timing of
onset and magnitude of the platelet count fall (in relation to hepa‐
rin exposure), thrombosis occurrence, and other clinical features
arguing for or against HIT. Traditionally, the clinician would make
treatment decisions based on either the 4Ts score or an empiric esti‐
mation of HIT likelihood, with subsequent treatment changes based
upon subsequent laboratory results (EIAs, platelet activation tests),
as they become available (perhaps several days later). However,
rapid, on‐demand immunoassays allow for real‐time incorporation
of HIT laboratory test results into the initial diagnostic evaluation.
Moreover, as some assays (eg, CLIA, LIA) provide (semi‐)quantita‐
tive data, fine‐tuning of clinical probability estimates is achievable.
Potentially, if the posttest probability for HIT (per combination of
clinical probability and results of a validated rapid assay) is very low
or very high, further diagnostic testing with traditional HIT tests
(EIA, SRA) might not necessarily be required.
In Bayesian analysis, pretest probability is modified by the test
result(s) to yield a revised posttest probability.46,48,50 The test result
 |
22      

TA B L E 2   Bayesian analysis: posttest probabilities for HIT per CLIA or LIA

(A) CLIA

Posttest probability of HIT (based on 4Ts pretest probability and CLIA test result)

Pretest probability CLIA‐neg (<1.0 U/ CLIA‐positive (any pos result, CLIA‐positive (weak) (1.0‐4.99 CLIA‐positive (moderate) CLIA‐positive (strong)
4Ts score of HITa mL) LR− = 0.031 i.e, ≥1.0 U/mL) LR+ = 65.9 U/mL) SSLR+ = 12.0 (5.0‐19.99 U/mL) SSLR+ = 144 (≥20.0 U/mL) SSLR+ = ∞

Low 1.9% <0.1% 56.1% 18.9% 73.6% 100%

Intermediate 6.7% 0.2% 82.6% 46.3% 91.1% 100%
High 36.6% 1.8% 98.4% 87.4% 98.3% 100%

(B) LIA

Posttest probability of HIT (based on 4Ts pretest probability and LIA test result)

LIA‐positive (strong)
Pretest probability LIA‐negative (<1.0 LIA‐positive (any pos result, i.e, LIA‐positive (weak) (1.0‐4.9 LIA‐positive (moderate) (≥16.0 U/mL)
4Ts score of HITa U/mL) LR− = 0.034 ≥1.0 U/mL) LR+ = 16.0 U/mL) SSLR+ = 5.7 (5.0‐15.9 U/mL) SSLR+ = 31 SSLR+ = 128

Low 1.9% <0.1% 24.2% 10.0% 37.5% 71.3%

Intermediate 6.7% 0.2% 53.5% 29.0% 69.0% 90.2%
High 36.6% 1.9% 90.2% 76.7% 94.7% 98.7%

4Ts, Four Ts scoring system; CLIA, chemiluminescence‐based immunoassay (IgG‐specific); HIT, heparin‐induced thrombocytopenia; LIA, latex immunoturbidimetric assay; LR−, negative likelihood ratio; LR+,
positive likelihood ratio; SSLR+, stratum‐specific likelihood ratio (for the positive result range shown); U, units.
Data shown are obtained from published sources.48,49 CLIA testing was performed in most cases using sera, whereas LIA testing was performed using citrated plasma. Data for the CLIA and LIA are based
on 509 and 429 subjects, respectively; the discrepancy relates to fewer patients having plasma samples available for testing in the LIA.
a
The pretest probabilities shown are based upon the frequency of SRA‐positive status for the three 4Ts scoring groups (low, intermediate, and high), per the clinician who performed the score at time of
enrollment into the 4Ts clinical trial. 4Ts score for pretest probability of HIT is based on the following 3 risk categories: 0 to 3 points, low probability; 4‐5 points, intermediate probability; and 6‐8 points,
high probability.
WARKENTIN
WARKENTIN
can be considered either as dichotomous (positive or negative), with
corresponding positive or negative likelihood ratios (LR+ or LR−, re‐
spectively), or the result can be considered in the context of a par‐
ticular “stratum,” for example, weak, moderate, or strong positive
results, with various associated stratum‐specific likelihood ratios
(SSLRs) that progressively increase to reflect the greater probabil‐
ity of HIT associated with a stronger test result. Table 2 summarizes
data from our institution that illustrates the power of Bayesian anal‐
ysis when using the CLIA (Table 2A) or LIA (Table 2B), based upon the
approximate pretest probabilities of HIT found in our 4Ts study.46
To illustrate, consider a patient judged to have a 30% proba‐
bility of HIT on clinical grounds; the corresponding “odds” of this
patient having HIT is 3 to 7 (ie, 3:7). Now, further consider that
the actual test result obtained was positive and of a magnitude
that corresponded to an SSLR+ of 31. (Per Table 2B, a moderate
positive LIA yields an SSLR+ of 31.) In this scenario, the posttest
odds can be easily calculated as (3 × 31):7, which is 93:7, that is,
a 93% probability for HIT. In contrast, if the test result was nega‐
tive, which corresponds to a low LR− value of 0.034 (ie, 1/0.034
or a 29‐fold decrease in odds of having HIT), that negative test
result would reduce the posttest probability of HIT as follows:
3:(7 × 29), or 3:203, which corresponds to a small possibility of
HIT of only 3/(203 + 3), or ~1.5%. This is a striking difference
in posttest probability (93% vs. 1.5%) and is based upon real‐
world data. 49 In other words, in the example shown, the differ‐
ence between a moderate positive result obtained with the LIA,
versus a negative result, corresponds to a ~900‐fold (!) change
in diagnostic probability (from 31‐fold higher to 29‐fold lower),
which in the context of a pretest probability for HIT of ~30%
corresponds to a major shift in posttest probability of HIT, from
93% vs. 1.5%.
With such a rapid test result (assuming local availability of the
CLIA or LIA), the physician can order the blood test while performing
the usual diagnostic workup for HIT (review heparin exposures, as‐
sess timing of platelet count changes, examine the blood film, order
duplex ultrasound for deep‐vein thrombosis, etc.), and have the HIT
test result back in time to impact diagnostic reasoning. Thus, cru‐
cial therapeutic decisions, such as whether to stop heparin and/or
commence alternative anticoagulation in prophylactic or therapeu‐
tic dosing, can be made—in most situations—in the context of much
greater clinician confidence as to whether HIT is likely to be present
or not.
8 | CO N C LU S I O N
The fundamental paradigm of HIT as an antibody‐mediated plate‐
let activation disorder accounts for the importance of the SRA and
other platelet activation assays in diagnosing HIT with high confi‐
dence. Moreover, platelet activation assays uniquely are able to
identify the heparin‐independent antibodies responsible for aHIT.
There are growing interest and availability of rapid immunoassays
that allow for real‐time diagnosis of HIT. Bayesian analysis of (semi‐)
|
      23
quantitative instrument‐based immunoassays such as the CLIA and
LIA is ideally suited for real‐time diagnostic evaluation of HIT.
AC K N OW L E D G E M E N T S
I thank Jo‐Ann I. Sheppard and other members of the McMaster
Platelet Immunology Laboratory for performing much of the work
described in this review.
C O N FL I C T O F I N T E R E S T
Dr. Warkentin reports having received consulting fees from Aspen
Global and Octapharma; research support and consulting fees from
W.L. Gore and Instrumentation Laboratory; royalties from Informa
(Taylor & Francis); and consulting fees related to medical‐legal
testimony.
ORCID
Theodore E. Warkentin  https://orcid.
org/0000-0002-8046-7588
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How to cite this article: Warkentin TE. Laboratory diagnosis
50. Raschke RA, Curry SC, Warkentin TE, Gerkin RD. Improved clinical
of heparin‐induced thrombocytopenia. Int J Lab Hematol.
interpretation of the anti‐platelet factor 4/heparin enzyme‐linked
immunosorbent assay for the diagnosis of heparin‐induced throm‐ 2019;41(Suppl. 1):15‐25. https://doi.org/10.1111/ijlh.12993
bocytopenia through the use of receiver operating characteristic