Ethylenediaminetetraacetic Acid-induced Pseudothrombocytopenia in Association with Graves' Hyperthyroidism

By Jing Jing, Jingwen Li, Minmin Yu

Affiliations

1. Department of Endocrinology, Qingdao Municipal Hospital, Qingdao, Shandong, China

doi: 10.29271/jcpsp.2022.Supp.S146

ABSTRACT
Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is an in vitro phenomenon of EDTA-induced platelet aggregation. A number of mechanisms have been proposed to account for this phenomenon. EDTA-PTCP has been found in healthy subjects and patients with a variety of conditions, including viral infections, cardiovascular disease, liver disease, neoplastic diseases and autoimmune diseases. In the present case, a 66-year male had a 5-month history of palpitations. The patient presented with Graves' hyperthyroidism and was diagnosed with transient EDTA-PTCP. With the improvement of hyperthyroidism, the phenomenon of EDTA-PTCP disappeared. So far, EDTA-PTCP related to Graves' hyperthyroidism has not been reported in the literature.

Key Words: Hyperthyroidism, Thrombocytopenia, Autoimmune diseases, Platelet aggregation, Edetic acid.

INTRODUCTION

Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is an abnormal low platelet count on an automatic analyzer. Since the platelets are large in this condition, the automated platelet window counts them as white blood cells, thereby resulting in a high white blood cell count and a low platelet count. A configurational change in glycoprotein (GP) IIb/IIIa by EDTA and the presence of certain specific autoantibodies lead to EDTA-PTCP.¹ A transient EDTA-PTCP case appearing in a patient with Graves' hyperthyroidism is presented here. EDTA-PTCP related to Graves' hyperthyroidism has not been reported thus far.

CASE REPORT

A 66-year male presented with a 5-month history of palpitations. Despite a good appetite, he has lost weight for eight months and experienced quick bowel movements. In the four months leading up to his admission, he developed a taste for cold weather and began to overly sweat.

Vital signs included: oral temperature of 36.8°C, respiratory rate, 16 breaths per minute, heart rate, 95 beats per minute, blood pressure, 110/70 mmHg, height, 170 cm, body mass index (BMI), 19.3 kg/m², and weight, 56 kg. The baseline weight was 61 kg with a BMI of 21.1 kg/m². The skin was warm and diaphoretic. Eye examination was normal. Neck examination revealed a diffusely enlarged non-tender goitre without masses or nodules. No lymphadenopathy was detected. There was no thyroid bruit. The laboratory results including liver function tests and serum chemistry were normal. The test of thyroid function revealed thyrotoxicosis: thyrotropin, <0.01 mIU/mL (Normal: 0.49–4.91 mIU/mL); free T4, 40.5 pmol/L (Normal: 7.64–16.3 pmol/L); and free T3, 15.42 pmol/L (Normal: 3.28–6.47 pmol/L). Thyrotropin receptor antibody was elevated at 26 IU/L (normal, <1.75 IU/L). Thyroperoxidase antibody titer was 109 IU/mL (normal, <9 IU/mL). Human immunodeficiency virus, HbsAg, and hepatitis C virus were negative. Antinuclear antibody profile was negative ruling out autoimmune causes. Bone marrow examination revealed normal functioning marrow. The levels of Vitamin B12, folate and iron were normal. The haemoglobin was 12 g/dL, the red blood cell index and the white blood cell count were normal. Platelet counts varied from 40 to 90´ 10⁹/L. The patient had thrombocytopenia but there were no findings suggesting bleeding diathesis, e.g., petechiae, purpura and ecchymosis in the physical examination. The patient had no family history of hemorrhagic disease and no history of bleeding. Peripheral blood smear was performed using EDTA and citrate blood samples.

Table I: Trend of thyroid function tests and platelets after starting antithyroid therapy.

Date	Serum TSH (normal range: 0.49-4.91 mIU/mL)	Free T4 (normal range: 7.64-16.3 pmol/L)	Free T3 (normal range: 3.28-6.47 pmol/L)	Platelet counts [normal range: (125-350)´ 10^9/mL]		Treatment received
				EDTA	Citrate
August 02	0.01	40.5	15.42	40	144	Methimazole 30 mg/day
September 15	0.01	30.53	10.42	70	174	Methimazole 20 mg/day
November 02	1.99	11.39	5.7	212	202	Methimazole 10 mg/day
TSH: Thyroid-stimulating hormone; T3: Triiodothyronine; T4: Thyroxine.

Figure 1: Representative images of platelet aggregation in blood smear of an EDTA-anticoagulated sample (A) and no platelet aggregation in blood smear of non-anticoagulated (B) and citrate-anticoagulated (C) samples.

Platelets were counted at ambient temperature. Citrate sample yielded a normal platelet count while EDTA sample yielded a platelet count of 40´ 10⁹/L. Platelet clumping was observed in blood smears made from EDTA sample (Figure 1), it was absent in samples using citrate. Based on these findings, the diagnosis of EDTA-PTCP and Graves' hyperthyroidism was made. After we started methimazole, 10 mg, three times a day, his platelet counts gradually improved. During subsequent follow-up, her thyroid function tests and platelet counts became normal. Trends in the platelet count and thyroid function tests are shown in Table I.

DISCUSSION

EDTA-PTCP is an abnormal low platelet count on an automatic analyzer, which results from an in vitro phenomenon that involves aggregation of large number of platelets in a blood sample. Since platelets are large, the automated platelet window does not count them as platelets, but counts as white blood cells, thereby resulting in a high white blood cell count and a low platelet count.² EDTA-PTCP is detected in 0.1% of patients referring to hospitals that serve routine healthcare, while the rate increases to 15% in facilities evaluating thrombocytopenia aetiology. EDTA-PTCP has been found in healthy subjects and patients with a variety of conditions, including viral infections, cardiovascular disease, liver disease, gastrectomy, autoimmune diseases and neoplastic diseases.^3-6 The mortality of EDTA-PTCP patients was significantly higher than that of non-EDTA-PTCP patients matched by age and gender.⁷

The mechanism by which EDTA causes thrombocytopenia is unclear. A hypothesis suggests that antibodies activated in the presence of EDTA may cause in vitro platelet aggregation by binding to Gp IIb/IIIa receptors on the platelet surface.⁸ Calcium is required to maintain the heterodimeric structure in Gp IIb/IIIa. It is believed that activation of EDTA-related calcium cascade is responsible for interaction between calcium and platelet antibodies by removing calcium from Gp IIb/ IIIa binding sites.³ Meanwhile, these antiplatelet autoantibodies, mainly in the form of immunoglobulins IgM, IgG, or IgA subsets, recognise the exposure of the epitopes on GP IIb/IIIa leading to platelet clumps.⁹ When clinical symptoms of severe thrombocytopenia are absent, EDTA-PTCP should be considered in the differential diagnosis, which can be verified by peripheral blood smears that demonstrate platelet aggregation. Citrate is used as an anticoagulant to confirm EDTA-PTCP.

EDTA-PTCP is a result of the presence of certain specific autoantibodies. Hyperthyroidism, as an autoimmune disease, is also prone to concur with other autoimmune diseases such as immune thrombocytopenic purpura (ITP). In one longitudinal study, hyperthyroidism occurred in 8-14% of ITP patients. Platelet-associated IgG and specific platelet antibodies were detected in 83% and 86% of patients having ITP with and without autoimmune thyroid disease, respectively, while anti-thyroid antibodies were found in 89% of patients with ITP. Many case reports indicated that patients with ITP and hyperthyroidism responded to thyroid disease control rather than standard ITP treatment.¹⁰ With regards to this case, the appearance of EDTA-PTCP can be attributed to the damage of platelets by autoimmune factors, resulting from the exposure of cryptic antigens. Our case was similar to hyperthyroidism combined with ITP, in that, with the improvement of hyperthyroidism, EDTA-PTCP was also alleviated.

ETHICAL APPROVAL:
This work was carried out in accordance with the Declaration of Helsinki and was approved by the institutional review board of Qingdao Municipal Hospital.

PATIENT^’S CONSENT:
The patient gave his informed consent for publication of this report.

COMPETING INTEREST:
The authors declared no competing interest.

AUTHORS' CONTRIBUTION:
JJ: Collect the data and write the manuscript.
JL: Record the data.
MY: Review and edit the manuscript.
All the authors have approved the final version of the manuscript to be published.

REFERENCES

1. Mohan B, Sheeja S, Prabhalekshmy KK. EDTA dependent pseudothrombocytopenia in a patient with scrub typhus - A case report. BMH Med J 2016; 3(1):15-8.
2. Rajajee S, Subbiah E, Krishnamurthy N, Paranjothi S, Lohiya N. Pseudothrombocytopenia and usefulness of platelet aggregates in peripheral smear in the diagnosis of scrub typhus. Indian J Pediatr 2019; 86(1):93-4. doi: 10.1007/ s12098-018-2712-2. 
3. Shi X, Lin Z, He L, Li W, Mo L, Li Y, et al. Transient appearance of EDTA-dependent pseudothrombocytopenia in A postoperative patient with sepsis: A case report. Medicine (Baltimore) 2017; 96(11):e6330. doi: 10.1097/ MD.00000000 00006330.
4. Sahin C, Kirli I, Sozen H, Canbek TD. EDTA-induced pseudothrombocytopenia in association with bladder cancer. BMJ Case Rep 2014; 2014:bcr2014205130. doi: 10.1136/bcr- 2014-205130.
5. Lippi G, Plebani M. EDTA-dependent pseudothrombo-cytopenia: Further insights and recommendations for prevention of a clinically threatening artifact. Clin Chem Lab Med 2012; 50(8):1281-5. doi: 10.1515/cclm-2012- 0081.
6. Schuff-Werner P, Steiner M, Fenger S, Gross HJ, Bierlich A, Dreissiger K, et al. Effective estimation of correct platelet counts in pseudothrombocytopenia using an alternative anticoagulant based on magnesium salt. Br J Haematol 2013; 162(5):684-92. doi: 10.1111/bjh.12443.
7. Ohashi-Fukuda N, Inokuchi R, Sato H, Nakamura K, Iwagami M, Wada T, et al. Poorer prognosis with ethylenediamine-tetraacetic acid-dependent pseudothrombocytopenia: A single-centRE case-control study. Medicine (Baltimore) 2015; 94(15):e674. doi: 10.1097/MD.0000000000000674.
8. Sousa SM, Sousa TM, Silva CF, Mendes CC. Pseudo-thrombocytopenia: A case of platelet satellitism and phagocytosis by neutrophils. Platelets 2020; 31(4):541-3. doi: 10.1080/0953 7104.2019.1664733.
9. Bai M, Feng J, Liang G. Transient EDTA-dependent pseudothrombocytopenia phenomenon in a patient with antiphospholipid syndrome. Clin Lab 2018; 64(9):1581-3. doi: 10.7754/Clin.Lab.2018.180337.
10. Shekhda KM. The association of hyperthyroidism and immune thrombocytopenia: Are we still missing something? Ci Ji Yi Xue Za Zhi 2018; 30(3):188-90. doi: 10.4103/tcmj. tcmj_139_17.