COVID‐19‐associated pseudothrombocytopenia

Coronavirus disease 19 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be associatedwith changes in platelet count [1,2]. Thrombocytopenia hasbeen reported in up to 40% of COVID-19 infections [3–5] and is an important marker for morbidity and mortality [1,2,5]. Hence, monitoring of platelet counts is important in diagnosis and treatment of COVID-19 patients. Thrombocytopenia can be a result of the COVID-19 infection itself (septicaemia), diffuse intravascular coagulation (DIC),medication or a COVID-19-associated immune thrombocytopenic purpura (ITP) [6]. A rare and oftenmissed alternative explanation of thrombocytopenia is pseudothrombocytopenia [7]. Pseudothrombocytopenia or spurious thrombocytopenia is an in vitro phenomenon of platelet agglutination caused by an anticoagulant, usually ethylenediaminetetraacetic acid (EDTA), resulting in a falsely lowered automated platelet count [8]. The mechanism of pseudothrombocytopenia is not clearly defined, but it is suggested to be an immunologically mediated phenomenon of platelet clumping due to the formation of immune complexes between naturally occurring autoantibodies and cryptic epitopes of the glycoprotein IIb/IIIa complex on the platelet membrane that are exposed by the EDTA anticoagulant used for routine blood sample collections [9]. This phenomenon has been previously reported to be associated with autoimmune diseases and infections [10], such as hepatitis A [11], mononucleosis [12] and Plasmodium falciparummalaria [13]. It has a reported incidence between 0.03% and 0.27% among the general population [14]. Here, we report the first patient with pseudothrombocytopenia related to COVID-19 infection and its natural course. Our patient is a 54-year-old woman with a history of sarcoidosis diagnosed in October 2019, for which she was still being treated with daily prednisolone 7.5 mg (December 2019) and weekly methotrexate 12.5 mg (January 2020). She previously had a stable normocytic anaemiaandnormal platelet counts.Approximately10daysafteronset

Coronavirus disease 19 , caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be associated with changes in platelet count [1,2]. Thrombocytopenia has been reported in up to 40% of COVID-19 infections [3][4][5] and is an important marker for morbidity and mortality [1,2,5]. Hence, monitoring of platelet counts is important in diagnosis and treatment of COVID-19 patients. Thrombocytopenia can be a result of the COVID-19 infection itself (septicaemia), diffuse intravascular coagulation (DIC), medication or a COVID-19-associated immune thrombocytopenic purpura (ITP) [6]. A rare and often missed alternative explanation of thrombocytopenia is pseudothrombocytopenia [7]. Pseudothrombocytopenia or spurious thrombocytopenia is an in vitro phenomenon of platelet agglutination caused by an anticoagulant, usually ethylenediaminetetraacetic acid (EDTA), resulting in a falsely lowered automated platelet count [8]. The mechanism of pseudothrombocytopenia is not clearly defined, but it is suggested to be an immunologically mediated phenomenon of platelet clumping due to the formation of immune complexes between naturally occurring autoantibodies and cryptic epitopes of the glycoprotein IIb/IIIa complex on the platelet membrane that are exposed by the EDTA anticoagulant used for routine blood sample collections [9]. This phenomenon has been previously reported to be associated with autoimmune diseases and infections [10], such as hepatitis A [11], mononucleosis [12] and Plasmodium falciparum malaria [13]. It has a reported incidence between 0.03% and 0.27% among the general population [14]. Here, we report the first patient with pseudothrombocytopenia related to COVID-19 infection and its natural course.
Our patient is a 54-year-old woman with a history of sarcoidosis HIV-serology test was negative. There were no clinical signs of bleeding or thrombosis. On day 10 after presentation, platelet count in a citrate blood sample was 129 × 10 9 /L. Analysis of a peripheral blood film at the same moment showed platelet agglutination in EDTA as well as in citrate, although much less evident ( Figure 2). Hence, the diagnosis of pseudothrombocytopenia was confirmed, which also marked the misdiagnosis of a true thrombocytopenia at first in this patient.  Figure 1): EDTA 28 × 10 9 /L and citrate 217 × 10 9 /L in week 6 after nadir, EDTA 99 × 10 9 /L and citrate 249 × 10 9 /L in week 8 after nadir.
The phenomenon seems transient, as it was reported to be in the only other publication describing a similar case, although platelet transfusion was given in this case [7]. SARS-CoV-2 IgM and total antibodies were first measured, using the Wantai ELISA-test (WS-1196 and

WS-1096), in week 7 after diagnosis of COVID-19 infection, already
showing sufficient SARS-CoV-2 seroconversion. In COVID-19-related pseudothrombocytopenia, we suggest a possible link with SARS-CoV-2 IgM antibodies and hypothesize an EDTA-dependent immune-complex formation with cryptic platelet membrane epitopes. To test this, we incubated patient serum with EDTA-blood of a universal donor, but this did not induce platelet agglutination. Taken together, this may suggest that generation of cryptic epitopes is patient specific.
In conclusion, we illustrate the importance of considering pseudothrombocytopenia in COVID-19-associated thrombocytopenia.
This is the first case of COVID-19-associated pseudothrombocytopenia in which we also describe the transience of this diagnosis. It is essential to recognize this in vitro phenomenon, as this falsely lowered automated platelet count is not associated with a clinical bleeding tendency, does not have any therapeutic consequences (platelet transfusion nor discontinuation of essential medication) and is self-limiting, as shown in our patient. In the differential diagnosis of COVID-19associated thrombocytopenia, exclusion of pseudothrombocytopenia is therefore critical.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.