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EDTA-Dependent Pseudothrombocytopenia in a Child

Sinan Akbayram, Murat Dogan, Cihangir Akgun, Hseyin Caksen and Ahmet Faik Oner CLIN APPL THROMB HEMOST 2011 17: 494 originally published online 7 June 2010 DOI: 10.1177/1076029610373367 The online version of this article can be found at: http://cat.sagepub.com/content/17/5/494

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EDTA-Dependent Pseudothrombocytopenia in a Child

Sinan Akbayram, MD1, Murat Dogan, MD2, Cihangir Akgun, MD2, seyin Caksen, MD2, and Ahmet Faik Oner, MD1 Hu

Clinical and Applied Thrombosis/Hemostasis 17(5) 494-496 The Author(s) 2011 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1076029610373367 http://cath.sagepub.com

Abstract Ethylenediaminetetraacetic acid (EDTA)-dependent pseudothrombocytopenia (PTCP) is the phenomenon of a spurious low platelet count due to antiplatelet antibodies that cause platelet clumping in blood anticoagulated with EDTA. The aggregation of platelets in EDTA-dependent PTCP is usually prevented by other anticoagulants, such as sodium citrate or heparin. EDTAdependent PTCP has never been associated with hemorrhagic diathesis or platelet dysfunction. In this article, a 10-year-old boy with EDTA- and heparin-dependent PTCP is presented because of rare presentation. We report that EDTA and heparin can induce platelet clumping, and thus spuriously low platelet counts. However, aggregation of platelets was not detected in blood samples with sodium citrate, and platelet count was normal. Keywords ethylenediaminetetraacetic acid, heparin, citrate, pseudothrombocytopenia

Introduction
Although ethylenediaminetetraacetic acid (EDTA) is commonly used as an anticoagulant for complete blood cell counts, it may agglutinate the platelets in some patients.1 This EDTA-induced aggregation of platelets leads to pseudothrombocytopenia (PTCP).2 EDTA-dependent PTCP can be recognized by the presence of platelet clumps in the peripheral smear of blood anticoagulated with EDTA. The aggregation of platelets in EDTA-dependent PTCP is usually prevented by other anticoagulants, such as sodium citrate or heparin.3 Of these, EDTA is particularly useful for hematological tests because of its stability in blood cell counting, although platelet clumping sometimes occurs in EDTA solution.4 Platelets exhibit almost no clumping in solutions with sodium citrate, although this agent is also a calcium-ion chelator like EDTA. Heparin is known to induce platelet aggregation, but it does not chelate calcium ions.5 The mechanism of platelet activation in heparinized blood is different from that occurring in the absence of calcium ions, that is, in the presence of EDTA.5 The prevalence rate of EDTA-dependent PTCP was reported as 0.07% to 0.20% in the literature.2,6-9 EDTA-dependent PTCP has never been associated with hemorrhagic diathesis or platelet dysfunction.1 In this case report, we presented a child with EDTA-dependent PTCP to emphasize evaluating a patient with medical and family history and examination of the blood smear is very crucial and must be the first step.

no history of drug using, or no infection history recently. There was no history of ecchymosed, recent melena or weight loss. In his laboratory findings, white blood cell count was 10 000 /mm3, platelet count was 9.000 /mm3, and hemoglobin level was 14 g/dL. Family history for hemorrhagic disorders was unremarkable. On physical examination, neither signs of hemorrhagic diathesis, such as bruises, nor lymphadenopathy were detected. The patients platelet count in our laboratory was 6000/mm3, but the aggregation of platelets was noted on peripheral blood smear (Figure 1). We considered that this low platelet count was due to EDTA-induced aggregation of the platelets. Therefore, we measured platelet count of his blood samples obtained by venepuncture in different test tubes, each containing sodium citrate or standard heparin. Platelet counts of blood samples in test tubes containing sodium citrate or heparin were found to be 315 000/mm3 and 11 000/mm3, respectively. On the examination of blood smears, although aggregation of the platelets was detected in blood samples with heparin, it was not observed in blood samples with sodium citrate. We observed that heparin and EDTA failed in preventing the aggregation of platelets in this case.

1 2

Department of Pediatric Hematology, Yuzuncu Yil University, Van, Turkey Department of Pediatrics, Yuzuncu Yil University, Van, Turkey

Case Report
A-10-years-old boy was referred to our hospital because of thrombocytopenia detected incidentally. He had no complaints,
494

Corresponding Author: Sinan Akbayram, Department of Pediatrics, Faculty of Medicine, Yuzuncu Yl University, 65200, Van, Turkey Email: drsinanakbayram@gmail.com

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Akbayram et al

495 citrate. But we did not evaluate the aggregation of platelets by using low-molecular-weight heparin. However, careful sampling and processing in the collection of specimens are so important to obtain accurate platelet count in healthy and EDTA-dependent PTCP participants. In a study that was performed by Nishioka et al,5 the possibility of platelet activation in anticoagulated solutions was examined. In that study, whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. On flow cytometric examination of whole blood, platelets were identified using 2 different monoclonal antibodies, CD41/FITC and CD42b/FITC, and activated platelets were identified with CD62p/PE. They observed that blood treated with EDTA exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, with this report, we suggested that PTCP must be thought in cases with low platelet counts, who had no history or physical examination findings consistent with thrombocytopenia. Additionally, we think that expensive and sophisticated technics, which are helpful for diagnosing PTCP, are not available in all clinics, especially in developing countries, and these methods are not superior to examination of peripheral blood smear. So, we emphasized that evaluating a patient with medical and family history and examination of the blood smear is very crucial and must be the first step. Declaration of Conflicting Interests
The author(s) declared no conflicts of interest with respect to the authorship and/or publication of this article.

Figure 1. Increased clumping.

Discussion
EDTA is particularly useful for hematology tests because of its stability in blood cell counting and sizing, although platelet clumping sometimes occurs in EDTA solution.5 This situation was named as EDTA-dependent PTCP. EDTA-dependent PTCP is the phenomenon of a spurious low platelet count autoantibodies that cause platelet clumping in blood anticoagulated with EDTA.10 Commonly, this clumping is caused by an alteration of the platelet surface glycoproteins when they are incubated with a calcium chelator such as EDTA.11 These modified platelet antigens then react to antiplatelet autoantibodies, usually immunoglobulin G (IgG) or immunoglobulin M (IgM) and rarely immunoglobulin A (IgA) types recognize platelet antigens on the platelet membrane modified by EDTA.1,2,12 The platelet membrane glycoprotein complex IIb/IIIb might be involved in EDTA-dependent antibody reaction.1,13 Kunicki et al14 showed a prototype of autoantibodies associated with EDTA-PTCP. They cloned a human monoclonal IgM autoantibody, 2E7, which binds to the octapeptide on the heavy chain of GP IIb and demonstrated that the binding of 2E7 to normal platelets is facilitated by the presence of EDTA. The collection and examination of blood at 37 C and supplementation of aminoglycosides to anticoagulant can prevent the aggregation of platelets.2,13 Additionally, the aggregation of platelets in patients with EDTA-dependent PTCP can also be prevented by the use of other anticoagulants such as sodium citrate or heparin.1 Although sodium citrate is also a calcium ion chelator like EDTA, platelets exhibit almost no clumping in solutions with sodium citrate. Heparin could also cause platelet aggregation, but it does not chelate calcium ions.5 The mechanism of platelet activation in heparin blood is different from that occurring in the absence of calcium ions, that is, in the presence of EDTA.5 In the present report, appropriately to the literature, on examination of blood smears, aggregation of platelets was detected in blood samples with standard heparin, however, aggregation of platelets was not noted in blood samples with sodium

Funding
The author(s) received no financial support for the research and/or authorship of this article.

References
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