Baquar H.

Qureshi, MD

CLINICAL

Case Presentation
D
II
EDTA-Dependent Pseudothrombocytopenia
A 30-year-old man came to the hospital with
vomiting and diarrhea. Present and past history
• Artifacts of blood collection. Inadequate mix-
ing of blood with an anticoagulant leads to
microdot formation and a low platelet count.
c
were unremarkable. Clinical examination Overfilling of the vacuum tube can also cause 0
revealed no abnormality except moderate dehy- spurious thrombocytopenia.1 Thus, the sample
dration. Except for the platelet count (70 X should be properly collected and immediately Dr Qureshi is a
i
S
109/L), the routine chemistry and CBC were nor- mixed well. consulting w
immunologist with
mal. After the patient received standard therapy • Platelet satellitism, a rare cause of pseudo- the Ministry of
for gastroenteritis, the platelet count was mea- thrombocytopenia characterized by EDTA- Health and is
sured again, with proper collection of sample in dependent rosetting of platelets around director of
potassium-EDTA. The repeated platelet count neutrophils or monocytes.2'3 This in vitro phe- Laboratories and
was 60 X 109/L. Platelet clumping was noted on nomenon is probably caused by antibodies in
Blood Bank,
Al-Qassim region,
examination of a peripheral smear, and most cases, but EDTA-dependent interactions of Kingdom of Saudi
pseudothrombocytopenia was suspected (Fig 1). cryofibrinogen with platelets and the leukocyte Arabia.
A fresh sample was collected in citrate at room surface have been implicated.4
temperature and in EDTA at 37°C. The platelet • Platelet-reactive cold agglutination, an infre-
count was normal in both samples (235 X 109/L quent cause of pseudothrombocytopenia in which Fig 1. Platelet
and 270 X 109/L, respectively) (Fig 2). A periph- platelets clump in all types of anticoagulants.5,6 clumps in EDTA-
eral smear obtained from the capillary blood • Monoclonal platelet agglutinin. Anticoagulant- anticoagulated
showed no clumping. The patient was negative for specimen
and temperature-independent pseudothrombo-
antiphospholipid antibody. Based on this data, it maintained at room
cytopenia caused by a monoclonal M paraprotein temperature.
was concluded that the patient had pseudothrom-
bocytopenia. The patient and physician were
assured of the benign nature of this condition; no
further investigations took place.

Clinical Background
In a thrombocytopenic patient, the clinician must
determine the cause of thrombocytopenia and
assess the risk for bleeding. Many thrombocy-
topenic patients do not bleed. After assessment of
the bleeding risk, the clinician should start appro-
priate therapy or avoid inappropriate interven-
tion for asymptomatic thrombocytopenic
patients and prevent treatment-related morbid-
ity. Clinicians also should consider pseudothrom-
bocytopenia when managing a case of
thrombocytopenia. The following conditions
may be responsible for an erroneously low
platelet count.

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 Pseudothrombocytopenia occurs in up to 0.1% of
all CBCs and is usually caused by EDTA-depen-
dent platelet agglutinating antibody.9'10 These
ot antibodies behave like cold agglutinins and are
usually reactive only at room temperature, not at
37°C.U In vivo platelet counts are normal. These
antibodies are not specific for any of the known
platelet-specific antigens (eg, human platelet
antigen-1, -2, -3) associated with alloimmuniza-
tion.12 These agglutinins (usually IgM but some-
times IgG and IgA) have no pathogenic
significance, and in vivo platelet counts are nor-
mal. They appear to be autoantibodies directed
6: against cryptic epitopes (antigenic determinants)
on the platelet membrane that are unmasked at
low temperature in the presence of EDTA. EDTA
*'-*;Pi presumably causes a conformational change
because of its ability to chelate calcium. The cryp-

sCP* tic epitope is sometimes located on the GPII/IIIb

Fig 2. A, Blood has been described in one patient. In that case, an
drawn with sodium accurate platelet count could not be obtained.7
citrate at room
temperature; B,
• EDTA-dependent pseudothrombocytopenia.
EDTA-anticoagulated Gowland and colleagues first described two cases
blood at 37"C (no of EDTA-dependent pseudothrombocytopenia in
clumping observed). 1969. They determined that identical EDTA-
dependent platelet clumping was the cause.8 This
immunologically mediated phenomenon was
caused by the presence of EDTA-dependent cold
antiplatelet autoantibodies.
EDTA is the anticoagulant most commonly
used in specimens for determining the CBC.
receptor complex.13'14
These agglutinins can prevent accurate deter-
mination of platelet count. Pseudothrombocy-
topenia can also complicate accurate
determination of platelet count in a patient with
an underlying thrombocytopenic disorder.15
No clinical evidence suggests that
pseudothrombocytopenia is associated with spe-
cific diseases, problems of possible sensitization
(such as pregnancy or transfusion), or the use of
a specific drug. In addition, the condition has not
been associated with hemorrhagic diathesis or
platelet dysfunction. The practical importance of
pseudothrombocytopenia is that if it is not recog-
nized, it may cause unnecessary investigation or
treatment of thrombocytopenia. Patients with
pseudothrombocytopenia have been hospitalized
and subjected to unnecessary platelet transfu-
sions;10'16 others have undergone bone marrow
aspiration and biopsy.17 EDTA-dependent
pseudothrombocytopenia can persist for years
without clinical manifestations.
The exact nature and origin of these autoanti-
bodies are not yet clear. EDTA-dependent platelet
antibodies have been associated with antiphos-
pholipid antibodies. In a recent study, 56 patients
with EDTA-dependent platelet antibodies also
had anticardiolipin antibodies.18 Absorption
studies with platelets and cardiolipin suggest that
most EDTA-dependent platelet antibodies have
antiphospholipid activity but that not all
antiphospholipid antibodies can induce EDTA-
dependent platelet agglutination.18 Antiphospho-
lipid antibodies are commonly seen in patients

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 with systemic lupus erythematosus and are
believed to be responsible for thrombocytopenia.
Thus, it is important to exclude pseudothrombo-
cytopenia in patients with lupus who present
with thrombocytopenia.
It is not known why normal patients have this
autoantibody in their blood. The antiplatelet
antibody responsible for platelet clumping might
be a natural antibody, with a physiologic role in
the removal of circulating platelets. EDTA-depen-
dent antiplatelet autoantibodies may also be pre-
sent in the blood of many healthy patients
without causing pseudothrombocytopenia.11
The Role of the Laboratory
Pseudothrombocytopenia caused by platelet
clumping is an in vitro phenomenon that occurs
in EDTA-anticoagulated blood at room tempera-
ture. In these cases, automatic analyzers yield
falsely low counts because of the formation of
agglomerates. Examination of peripheral smear
reveals platelet agglutination and a normal
platelet count (Fig 1). EDTA-dependent platelet
agglutination may also occur in the presence of
citrate at room temperature or in the presence of
EDTA at 37°C.
Several methods can be used to accurately
measure the platelet count:
• Collect the blood sample into different antico-
agulants, such as sodium citrate or heparin.
• Collect the blood sample into a prewarmed
tube that is maintained at 37°C until testing.
• Determine the platelet count by phase-
contrast microscopy using a sample obtained by
finger prick.
• Determine the platelet count within 15 min-
utes of collection if the sample is obtained in
EDTA at room temperature.
In this patient, partial clumping was seen in
sodium citrate (Fig 2A), and platelet clumping
was eliminated in EDTA-anticoagulated blood at
37°C (Fig 2B).
Conclusion
Although platelet counts measured by electronic
particle counters are more accurate and less
expensive than counts obtained manually, unrec-
ognized spurious thrombocytopenia can lead to
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unnecessary tests or inappropriate therapy.
Pseudothrombocytopenia results from antibody-
mediated platelet clumping in the presence of
EDTA. The blood film must be examined in every
patient who has purported thrombocytopenia.®
Acknowledgments
The author thanks Hasan Iddin of the Training and Education
Department of King Saud Hospital for typing the manuscript. V
0
£
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