Ethylene Diamine Tetra Acetate-Induced Pseudo Thrombocytopenia (EDTA-PTCP) in an Adolescent: A Case Report

Ethylene diamine tetra acetate-induced pseudo thrombocytopenia (EDTA-PTCP) is a fictitious laboratory condition that is associated with platelet clumping, leading to falsely low platelet counts. This fictitious occurrence can lead to expensive, time-consuming, and invasive diagnostic procedures. It may also result in the application of unnecessary therapies, although it is not linked to any hemorrhagic symptoms or platelet malfunction. This emphasizes the necessity of verifying laboratory results from automated analyzers in every patient with thrombocytopenia with a peripheral smear, particularly when they are out of proportion when compared to the clinical features. When using hematology analyzers, EDTA-induced pseudo thrombocytopenia can be missed. In cases of isolated thrombocytopenia, this can be easily avoided by performing a simple visual peripheral blood smear check, hence making it an important differential for thrombocytopenia on an automatic analyzer report, which has to be ruled out. Here, we present the case of an adolescent who presented to us with low platelet counts and was diagnosed with EDTA-PTCP after proper evaluation.


Introduction
Ethylene diamine tetra acetate-induced pseudo thrombocytopenia (EDTA-PTCP) is a spurious laboratory phenomenon that is present in 0.1%-0.2% of the general population [1][2][3]. This in vitro phenomenon is characterized by platelet aggregates in EDTA-anticoagulated blood due to antiplatelet autoantibodies [4]. It may result in the application of unnecessary therapies and diagnostic procedures, such as platelet transfusions [5], bone marrow aspiration and biopsy [6], and occasionally long-term steroid therapy or even splenectomy [7][8][9], although it is not linked to any hemorrhagic symptoms or platelet malfunction. Several case studies where emergency treatments were withheld have been described [10]. Additionally, it could trigger an unwelcome alarm in the patient's mind, which might prompt precautionary and restrictive behaviors to stop bleeding manifestations [5]. Hence, it is essential for healthcare professionals to be aware of this phenomenon, especially when managing patients with low platelet counts.

Case Presentation
A 17-year-old girl from a small-town hospital presented with a low platelet count and a recent episode of fever. The patient was healthy and had no complaints of weakness, rashes, or bleeding. Her blood counts were tested as a part of routine investigations, which revealed thrombocytopenia but no other symptoms such as bleeding or easy bruising. She was subjected to repeated laboratory testing of platelet counts in different labs to rule out possible disparities between the methods used. Figure 1 shows a summary of her platelet counts. Tests for scrub typhus serology, rapid testing for the malarial parasite antigen, rapid testing for the nonstructural protein 1 antigen (NS-1), and immunoglobulin M (IgM) and IgG antibodies for dengue were all negative. To rule out the possibility of marrow dysfunction, bone marrow aspiration and biopsy were performed, revealing a hypercellular, normoblastic bone marrow with adequate iron reserves and megakaryocytic hyperplasia. A summary of her laboratory test results is presented in Table 1.  Her immature platelet fraction was 39.2. The platelet count as determined using an automated analyzer and the EDTA anti-coagulated sample was 8000/mm3, and the platelet histogram exhibited a typical "sawtooth" pattern rather than a smooth curve, indicating the necessity of a peripheral smear ( Figure 2). When EDTA was used as the anticoagulant, a visual examination of the peripheral smear revealed normocytic and normochromic RBCs, while the platelets were sufficient in number and were primarily organized in clumps (Figures 3, 4).

FIGURE 4: Direct peripheral smear from the sample without using any anticoagulant
Consequently, the analysis was repeated with sodium citrate as the anticoagulant, which revealed a normal platelet distribution and no platelet clumping ( Figure 5).

FIGURE 5: A peripheral smear performed using sodium citrate as the anticoagulant showed no platelet clumping and a normal platelet count
The platelet count was 202,000/mm3 when it was determined using sodium citrate as the anticoagulant on an automated analyzer, with a platelet histogram showing a normal smooth curve ( Figure 6). Considering the absence of any clinical symptoms in the patient, the presence of platelet clumps coupled with low platelet counts when EDTA was used as the anticoagulant, and the normalization of platelet counts when citrate was used as the anticoagulant, a diagnosis of EDTA-induced pseudo thrombocytopenia was made. The patient was made aware of her diagnosis and was also informed to let any further investigating physician know that she has EDTA-PTCP, which can decrease unwanted excessive evaluation.

Discussion
EDTA-induced pseudo thrombocytopenia (EDTA-PTCP) is a laboratory phenomenon that is characterized by platelet clumping, which can be seen on a peripheral smear, and an abnormal platelet histogram with a typical sawtooth pattern. The incidence is about 0.1-0.2% in the general population [1][2][3], which makes it an important differential for thrombocytopenia without any pathological causes. It is more commonly seen in women under the age of 50, and there is a slight male predominance in age groups over 50 [5]. Studies have shown that this phenomenon is not necessarily associated with any disease, and the patients do not show any clinical features of thrombocytopenia, but it can rarely be a serendipitous finding in association with a disease or syndrome [5]. It is important for physicians to have a high degree of suspicion about this phenomenon, especially when dealing with asymptomatic patients showing thrombocytopenia on repeated tests.
A frequently asked question is the possibility of transmission of PTCP to the recipient after blood transfusion, plateletpheresis, or peripheral blood stem cell transplant. As per the available data and case reports, it has been repeatedly concluded that PTCP should not be a reason to withhold any of the aforementioned procedures and is usually not transferred to the recipient [11][12][13][14]. However, theoretically, there is a chance of developing PTCP in peripheral blood stem cell transplantation, especially after the development of the B-cell repertoire, which might take a significant amount of time and was observed in a case reported by Di Francesco et al. [15]. Hence, it is also important to examine the patient's peripheral smear after a certain period following the stem cell transplant, which will allow mature B-cells to repopulate the recipient's bloodstream. Neonates can also display pseudo thrombocytopenia due to the transplacental transmission of maternal EDTA-dependent antibodies [16,17].
Scrub typhus was found to be associated with PTCP, which was transient for a few weeks after treatment [18,19]. This is an important association because scrub typhus can also be associated with true thrombocytopenia, which might require platelet transfusions. Hence, one has to be vigilant in such a pathological setting. Abciximab, an anticoagulant that acts by preventing fibrinogen and von Willebrand factor from binding to the glycoprotein (GP) IIb/IIIa receptor on platelets, has also been associated with PTCP [20]. There was a significant association noted between PTCP and viral infections, especially hepatitis A, followed by cytomegalovirus and H1N1 influenza (swine flu) [21]. Apart from the abovementioned scenarios, PTCP is also observed in patients with bladder cancer [22], autoimmune thrombocytopenia [23], SARS CoV2 infection, post-Oxford-AstraZeneca COVID-19 vaccine [24,25], and Graves' disease [26].
EDTA-PTCP is primarily precipitated by the presence of EDTA-dependent anti-platelet antibodies (27). The following flowchart (Figure 7) summarizes the pathophysiology of this phenomenon (27,28). IgG and IgM-class antibodies are most frequently produced in response to the abovementioned phenomenon, although IgA antibodies can also be found and can lead to cold agglutination [5]. Among the IgG subtypes, IgG1 is seen most frequently [4]. It is to be noted that there is a progressive thrombocyte count decline as the duration between sample collection and testing increases due to platelet clumping, and the thrombocyte count is more stable at 4 o C compared with other temperatures. This shows that the period between sample collection and testing is an important parameter to consider when trying to alleviate PTCP. Hence, it is important to measure platelet counts as early as possible after sample collection [29].
Peripheral smear examination is the gold standard to identify platelet clumping and PTCP, which was evident in our case report [1]. Another typical finding of the automatic analyzer report is a sawtooth pattern of platelet histogram with an abnormal height, which was also observed on the histogram presented previously in our case report [1]. This is an easier way to identify this spurious laboratory phenomenon, as it can become difficult to perform peripheral smears in every case. Proper awareness about platelet clumping, the use of smear examination, and a high degree of suspicion can guide physicians in preventing unnecessary treatment in patients with PTCP. It should be noted that PTCP is not limited to EDTA. It can also be seen with heparin, oxalate, hirudin [30,31], and, in some cases, citrate [5]. The following step-by-step evaluation of blood samples (as described by Waseem et al.) can help in identifying the anticoagulants causing PTCP [32]: Step 1: A blood sample is collected in EDTA. If clumping persists, continue to step 2.
Step 2: The blood sample is collected in sodium citrate. If step 2 is not possible, proceed to step 3.
Step 3: Obtain a sample in lithium heparin. If clumping is still present, then perform step 4.
Step 4: Obtain a sample in sodium fluoride. If clumping is still present, then perform step 5.
Step 5: Obtain a sample in ammonium oxalate and count platelets using a Neubauer chamber, if available.
Steps 3, 4, and 5 are usually needed in rare circumstances where steps 1 and 2 do not solve the issue of platelet clumping.
Vortex-mixing of samples with platelet clumps has also shown a reduction in platelet clumping and platelet satellitism without any changes in RBC, WBC, or schistocyte counts [33].
Magnesium sulfate has been known to be a decent replacement for EDTA as an anticoagulant in cases of EDTA PTCP [27,28]. Magnesium sulfate does not cause GP IIb/IIIa dissociation by preventing the exposure of the hidden GP IIb component and maintaining the integrity of the complex. This prevents the binding of EDTA-dependent antiplatelet antibodies to the gp IIb subunit, resulting in a reduction in platelet clump formation [34]. It has also been noted that prewarming blood samples to 37 o C and adding kanamycin to the sample reduces platelet clumping and PTCP [35,36]. Other aminoglycosides might also have similar effects on PTCP [5,36]. A novel method of platelet clump dissociation, which is as efficacious as kanamycin, has been devised using a combination of 9 mmol/L CaCl2 and 0.1 unit/L sodium heparin [37]. Apart from these, a mixture of citrate, pyridoxal-5-phosphate, and Tris (CPT) can also be used to prevent PTCP [5].

Conclusions
It is crucial to double-check lab results obtained with automated analyzers in each patient with thrombocytopenia (especially when it is unrelated to any clinical features) by examining the peripheral smear in order to find any platelet aggregates that can help confirm EDTA-PTCP and avoid unnecessary investigatory procedures.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.