European Journal of Cardiovascular Medicine

Platelets are quintessential for primary hemostasis. Platelet counts are measured by automatic cell counters which work on the basis of electrical impedance, optical and fluorescent methods using the size, refractive index and nucleic acid stain. Ethylene diamine tetra acetic acid (EDTA) is commonest used anticoagulant for haematological investigations. This can very rarely lead to a spuriously low platelet count called EDTA induced pseudo thrombocytopenia (PTCP). PTCP is an in-vitro problem and low platelet count can be alarming to the patient and clinician. It may lead to the use of unnecessary treatments and diagnostic procedures, such as platelet transfusions, bone marrow aspiration and biopsy, and sometimes long-term steroid therapy or even splenectomy. However, it is not associated with bleeding symptoms or platelet dysfunction. 1-3

Cross checking platelet distribution in a peripheral smear helps in identifying aggregated platelets and retest platelet count by a suitable method to confirm the diagnosis.  

Various methods have been employed to accurately determine the platelet count in individuals with EDTA-dependent pseudo thrombocytopenia (EDTA-PTCP) in routine clinical practice. These include the use of alternative anticoagulants (such as citrate, heparin, or magnesium sulfate), the addition of aminoglycoside antibiotics (like kanamycin), or analyzing EDTA-anticoagulated blood samples at 37 °C. 4  

This is a cross-sectional prospective study of 5600 cases related to platelet count in 3 years from 2021-2024 in a private diagnostic centre in Mandya, Karnataka. Among these, 12 cases of EDTA induced thrombocytopenia were observed. The female to male ratio was 3:1. The median age was 37 ± 14.08 years. All cases showed thrombocytopenia on cell -counters. There was no clinical suspicion of thrombocytopenia in these patients. PTCP leads to more pronounced thrombocytopenia even in mild cases of thrombocytopenia.
History and clinical examination of the patients revealed no underlying clinical condition like common viral infections, steroid or chemotherapy that would have caused thrombocytopenia.

The very first drawn EDTA samples were flagged for platelet aggregates in the automated analyser at 37°C. (Figure 1) The peripheral blood smears showed numerous aggregates of platelets. We suspected errors in mixing of blood with anticoagulant immediately after drawing if the universal order of blood collection was not followed, as few of the samples were received from outside in vacutainers.  The possibility of a pre-analytical error were excluded as universal precautions and order of blood collection are followed unerringly during the pre-analytical phase in the current private laboratory. All samples are routinely placed on a blood mixer machine immediately after collection till they’re analysed in the cell counter to prevent clumping of platelets. This method is employed to minimize such errors which can be drastic for patient management. Thus, a possibility of PTCP was considered. The blood was then collected in Heparin/Citrated vacutainer following which the platelet count was normal on cell counters and the platelet aggregates were not found in the peripheral smears.

Figure 1:

Figure 2: Comparison of Platelet counts in EDTA versus Sodium citrate

The cell counters showed abnormal platelet histograms in 5 cases (41.6%). On retesting with citrate, 2 cases showed moderate thrombocytopenia and 4 cases showed mild thrombocytopenia.

PTCP leads to more pronounced thrombocytopenia even in mild cases.

PTCP is an in vitro condition of aggregation of platelets due to mechanisms not clearly described, few of which are temperature changes, autoantibodies to platelet membrane glycoproteins, viral infections like COVID 19, Hepatitis B or Swine flu or autoimmune inflammatory conditions and stem cell transplantation. 4,5,6 It can be seen as an abnormal platelet histogram with a typical sawtooth pattern in few cases.7 The incidence is about 0.1-0.2% in the general population. 8

It is an important differential for thrombocytopenia without any pathological causes.

Other potential pre-analytical factors to consider when examining cases of platelet clumps in peripheral blood smears include the collection method, whether capillary, venous, or intravenous line draws. Capillary collections, in particular, are more susceptible to clotting and the formation of platelet clumps.9 This can be prevented by keeping the blood sample in a blood mixer immediately after transferring blood to anticoagulated vacutainer.

In this study, citrate-anticoagulated samples had higher platelet counts than those with heparin. Wu Wei et al. also identified citrate as a more effective anticoagulant in managing PTCP. Similarly, Werner et al. concluded that citrate is superior to EDTA in reducing PTCP. 10, 11
Only 1 case (8.3%) showed persistent clumping even in citrate sample. Bizzaro N found 20% of their samples showed such clumping in citrated sample. 1

In cases of thrombocytopenia, particularly when no clinical symptoms are present, it's important to confirm results from automated analyzers by examining a peripheral blood smear. This helps identify platelet aggregates, which can indicate EDTA-dependent pseudothrombocytopenia (EDTA-PTCP). In such cases, retesting with Citrate/heparin anticoagulated blood rules out PTCP and helps in avoiding unnecessary diagnostic tests. In the ongoing era of automation and upcoming era of artificial intelligence, it is necessary to not lose sight of our roots, the peripheral smear examination.

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