Coagulation Case Study
John T. Brandt, MD
Table 1 | Figure 1
| Figure 2
This is the sixth in a periodic series of articles written
by members of the CAP Coagulation Resource Committee and focusing
on laboratory evaluation of coagulation disorders.
Case history. A 67-year-old woman with a 20-year history
of type 2 diabetes mellitus was admitted for therapy of acute exacerbation
of her peripheral vascular disease. Several years ago she underwent
a revascularization procedure of the right leg that failed in the
immediate postoperative period, necessitating a below-the-knee amputation.
The amputation was subsequently revised to an above-the-knee amputation
due to tissue necrosis. Two months prior to the current admission
the patient developed nonhealing ulcers of the left lower extremity.
An arteriogram at the time of admission revealed diffuse arterial
disease. A left femoral-anterior tibial artery graft was placed.
On the first postoperative day she became cyanotic and short of
breath, with a pO2 of 40 mm Hg and an arterial pH of
7.17. She was resuscitated and did well until the 10th postoperative
day, when progressive cyanosis of both hands was noted. The lab-oratory
was requested to eval-uate the patient for acute disseminated intra-vas-cular
coagulation (DIC). Laboratory studies at that time showed the values
listed in Table
Consider the following questions:
Case summary. Although the platelet count is decreased and
the D-dimer concentration is elevated, the remainder of the coagulation
studies do not support a diagnosis of acute DIC. The patient does
have a history consistent with a pulmonary embolism in association
with heparinization during vascular surgery. Review of the chart indicated
the patient continued to receive subcutaneous heparin through-out
her hospital stay. The onset of cyanosis of the hands could be an
indicator of vascular compromise/thrombosis of the upper extremities
related to heparin-induced thrombocytopenia (HIT).
- Do the results of the laboratory studies indicate a diagnosis
of acute DIC?
- What other diagnoses should be considered?
- What additional testing would you recommend to establish your
A platelet aggregation assay for the diagnosis of HIT was performed
and was strongly positive. Intermittent subcutaneous heparin was
discontinued without improvement of the platelet count over two
days. A heparin-coated catheter was then removed and the platelet
count increased over the next two days. Unfortunately, the patient
developed multiple organ failure and died on the 15th postoperative
Overview of heparin-induced thrombocytopenia. Heparin-induced
thrombocytopenia is a serious clinicopathologic syndrome characterized
by immune-mediated activation and clearance of platelets. Immune-mediated
platelet activation may lead to significant arterial and/or venous
thromboembolism while enhanced platelet clearance may contribute
to moderate to severe thrombocytopenia.1 Typically the
question of whether a patient has HIT arises when either a decrease
in the platelet count or onset of thrombosis is noted in the setting
of heparin therapy.
The basic pathogenesis of HIT is now reasonably well characterized.2-4
Upon exposure to exogenous heparin, multimolecular complexes composed of platelet
factor 4 (PF4) and heparin form (Figure 1). The binding
of PF4 to heparin in these complexes is associated with a conformational change
in PF4, exposing a neoepitope. This heparin-dependent neoepitope elicits an
immune response in some patients; the antibody response is usually an IgG type
of antibody. This IgG binds to the PF4/heparin complex, creating an immune complex
that binds to the receptor for IgG on the platelet surface (FcyRIIa). Aggregation
of platelet FcyRIIa receptors by the immune complex triggers platelet activation
and aggregation through transmembrane signaling. In addition, during the process
of platelet activation, platelets release phospholipid microparticles derived
from the cell membrane.5 These microparticles support the enzymatic
reactions of the coagulation cascade, leading to thrombin formation; consequently,
the release of these microparticles into the circulation is thrombogenic. In
addition, the PF4/heparin immune complexes can bind to the endothelium, leading
to endothelial injury.6 The endothelial damage may also participate
in the “thrombotic storm” that can be associated with this syndrome.
As the PF4/heparin complex is necessary for the pathogenesis of
this disorder, cessation of all sources of exogenous heparin is
an essential component of the therapeutic approach to these patients.
However, it should be remembered that increased thrombin generation
is a component of this syndrome and simple cessation of heparin
may not immediately reverse this process. Thus, it may also be critical
to introduce an alternate immediate--acting anticoagulant, such
as lepirudin or danaparoid, to control the risk or propagation of
thrombosis.1,7,8 Recent clinical data demonstrating a
high rate of venous thromboembolic disease in the period following
cessation of heparin without immediate alternate anticoagulation
support this hypothesis.9 Warfarin should be avoided
in patients with newly diagnosed HIT as it may be associated with
venous limb gangrene.10 The pathogenesis of venous limb
gangrene appears to be related to the combination of a rapid decrease
in protein C due to warfarin and the continued generation of thrombin
related to HIT.
Laboratory methods for diagnosing HIT. The laboratory is
often called on to confirm the diagnosis of HIT to justify the continued
use of alternate anticoagulants and guide future exposure to heparin.
Two basic types of assays are used for diagnosing HIT. One group
of assays, the functional assays, depends on detecting platelet
activation following exposure of normal plate-lets to patient serum
or plasma in the presence of heparin. The second group of assays,
the antigenic assays, depends on detection of antibody binding to
PF4/heparin (or heparin substitute). Recent clinical experience
suggests these two different assay approaches are complementary,
as neither alone is 100 percent sensitive.
The functional assays are based on the ability of patient immunoglobulin
to activate normal platelets in the presence of heparin. The source
of the patient immunoglobulin may be either serum or plasma. Detection
of platelet activation may be based on platelet aggregation, release
of granular contents (for example, serotonin), or change in membrane
properties (flow cytometry). There are advantages and disadvantages
to each of the major functional methods described.
The platelet aggregation method is the most widely used functional
assay.11 It is based on the observation that adding patient
platelet-poor plasma (as a source of immunoglobulin) to normal platelet-rich
plasma in the presence of heparin can induce platelet aggregation.
Attention to technical details is important for the assay to be
used successfully.12 Donor platelets vary in their response
to HIT-positive plasmas. Therefore, it is important to use platelets
that are known to respond to HIT plasma and to use platelets from
more than one donor. The final concentration of heparin in the assay
is also important. If a single concentration of heparin is used,
it should be about 0.4-0.5 U/mL. A common weakness of studies comparing
the platelet aggregation assay to other functional assays is that
insufficient heparin (for example, 0.1 U/mL) is used in the aggregation
assay. We have found it useful to use two concentrations (0.2 and
0.5 U/mL) to increase the assay sensitivity. The platelet aggregation
assay should be allowed to run for a sufficient perioda minimum
of 10 to 15 minutesbefore it is called negative. An aggregation
response >20 percent greater than a buffer control (no
heparin) is usually regarded as a positive result. A positive result
is specific for HIT, but a negative result does not exclude HIT.
The serotonin release assay is a well-characterized assay that
is technically demanding for the routine hospital laboratory. Donor
platelets must be isolated from platelet-rich plasma by differential
centrifugation and incubated with radiolabeled serotonin. The platelets
must then be washed to remove free serotonin. The labeled platelets
are then incubated at room temperature with patient serum (heat-treated)
in the presence of low (0.1 U/mL) and high (100 U/mL) concentrations
of heparin. A positive assay is defined as greater than 20 percent
release in the presence of the low concentration of heparin and
less than 20 percent release in the presence of the high concentration
As with the platelet aggregation assay, selection of donor platelets
is critical for success with this assay. It may also be important
to run controls (positive HIT samples) to ensure the platelets are
still functional after the isolation and serotonin labeling procedure.
The serotonin release assay is thought to be more sensitive than
the platelet aggregation assay.14 Again, a positive result
is specific for HIT, but a negative result does not exclude the
The heparin-induced platelet agglutination (HIPA) assay is similar
to the serotonin release assay in that it uses washed donor platelets
and heat-treated patient serum.15,16 In contrast to the
serotonin release assay, the endpoint for the HIPA assay is macroscopic
agglutination of platelets in a microtiter well. The results of
the HIPA assay appear to compare well with functional and antigenic
assays for HIT.16 The sensitivity and specificity of
this assay appear to be similar to those of the serotonin release
Flow cytometric assays for the diagnosis of HIT were developed recently.17-20
The assays are based on incubating patient plasma with normal donor
platelets in the presence or absence of heparin and then measuring
a change in the platelet surface, such as binding of annexin to negatively
charged phospholipids or expression of an activation marker such as
P-selectin (CD62P). These assays appear to have a similar sensitivity
and specificity for the diagnosis of HIT as does the serotonin release
assay. Of particular interest, the assay described by Jy et al suggests
it may be possible to distinguish patients at high risk for thrombosis
from other patients with HIT.18 These assays do not require
use of radioactive material and can be performed fairly rapidly in
an efficient mode.19 This approach, however, requires a
skilled flow cytometry laboratory.
The second group of assays
measures the presence of antibodies capable of binding to PF4 bound
to heparin or a heparin-like molecule.21-23 The detection
system can be modified to detect IgG, IgM, and/or IgA antibodies.24
These assays are usually performed in an enzyme-linked immunosorbent
assay (ELISA) format and thus are less amenable to single patient,
rapid turnaround testing.
The ELISA assays appear to be more sensitive for the detection
of PF4/heparin antibodies than the functional assays. However, it
is now apparent that not all patients with antibodies detectable
by the ELISA method have clinically evident HIT. For example, 40
to 60 percent of patients undergoing cardiopulmonary bypass procedures
develop antibodies to PF4/heparin detectable by the ELISA method,
yet few of these patients develop HIT.25-27 In addition,
it is apparent that other proteins can substitute for PF4 in the
immunogenic complex.28 These antibodies are not detected
by the PF4/heparin ELISA method but may cause clinically significant
HIT. Thus the ELISA method may be negative in some patients with
HIT or give false positive results in patients without HIT.
Strategy for diagnosing HIT. The laboratory needs to provide access
to appropriate testing to establish the diagnosis of HIT. There are two components
to this issue: identifying the appropriate patients to evaluate and selecting
the appropriate assays to perform. Laboratory testing should be performed whenever
there is clinical evidence of HIT. This may include any of the following (Figure
There is generally no reason to screen patients for HIT prior to their
receiving heparin. If a patient has a past history of HIT, it is preferable
to use an alternate anticoagulant rather than re-expose the patient
to heparin even if the antibody is no longer detectable. This is because
the antibody can recur rapidly following re-exposure to heparin. Likewise,
a negative laboratory evaluation prior to heparin exposure does not
rule out previous heparin exposure with antibody formation and the
potential for an anamnestic response. As noted earlier, a positive
ELISA assay for antibodies to PF4/heparin in the absence of clinical
manifestations of HIT does not mean the patient will develop clinically
evident HIT upon exposure to heparin. For these reasons, testing for
HIT should be limited to patients who are suspected of having the
disease based on clinical and laboratory manifestations.
- A greater than 50 percent decrease in the platelet count from
baseline (pre-heparin) in association with exposure to heparin.
The platelet count should be monitored at least every other day
(q. 48 hours) in patients receiving heparin.
- A decrease in the platelet count to <100,000/µl associated with
exposure to heparin.
- Evidence of a new throm-bo-embolic event (venous or arterial)
while the patient is exposed to heparin.
the possibility of HIT is suspected on clinical grounds, the most
important next step is to discontinue heparin, including removing
heparin-coated catheters. The diagnosis of HIT should be confirmed
using appropriate laboratory studies. As no method is 100 percent
sensitive for the diagnosis of HIT, it is now recommended that a
functional assay and an ELISA-type of method be performed before
excluding the diagnosis.2 A positive result with a functional
assay is specific for the diagnosis of HIT, so it makes sense to
perform the functional assay first. If a positive result is obtained,
then the diagnosis is established and no further testing is required.
If the functional assay is negative, then an ELISA may provide useful
information. If both tests are negative, then HIT is unlikely. If
the ELISA is positive, the patient should be treated as positive
Occasionally patients with HIT may be negative using both functional
and antigenic assays. These patients may develop recurrent thrombocytopenia
or thromboembolism when re-exposed to heparin. The recurrence of
either of these manifestations of HIT upon re-exposure to heparin
should be taken as positive evidence for HIT, and the patient should
be treated accordingly. Among patients with HIT, the platelet count
usually starts to rise toward normal within a couple of days of
stopping the heparin. Failure of the platelet count to rise following
cessation of heparin should prompt a search for alternate sources
of heparin, including heparin-coated catheters. If present, such
catheters should be exchanged for nonheparin-coated catheters. Occasionally,
the return of the platelet count may be delayed for weeks, even
in the absence of heparin exposure.
Patients identified as having HIT should be educated about their
condition, and the condition should be documented in their medical
records. Patients with HIT should also consider carrying some indicator
of their heparin “allergy” for use in emergency situations.
Finally, patients should be strongly counseled against future exposure
Dr. Brandt is senior clinical research pathologist, Lilly Research
Laboratories, Indianapolis, and a member of the CAP Coagulation Resource
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- Warkentin TE, Hayward CP, Boshkov LK, et al. Sera from patients
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- Jy W, Mao WW, Horstman LL, Valant PA, et al. A flow cytometric
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