IgG Platelet Antibodies in EDTA-Dependent Pseudothrombocytopenia Bind to Platelet Membrane Glycoprotein IIb

Abstract

A b s t r a c t EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serumIn EDTA-dependent pseudothrombocytopenia (PTCP), autoantibodies present in serum samples react with platelets in blood anticoagulated with EDTA causing platelet agglutination and an incorrect low platelet count. The minimal EDTA concentration (0.3-mmol/L of Na,-EDTA) required to produce platelet agglutination is much lower than that needed to prevent clotting and is independent from the cationic composition of the EDTA solution used as anticoagulant. Platelet agglutination also has been reported to occur in blood anticoagulated with citrate or heparin and is time-and temperature-dependent. 1 The majority of antibodies isolated in EDTA-dependent PTCP act as cold agglutinins with a heat range from 4°C to 20°C and are of the IgG or IgM class. Time course studies showed that platelet agglutination in EDTA anticoagulated blood generally occurs within a few minutes and becomes more conspicuous when blood samples are kept at room temperature; at 37°C, agglutination is abolished or, in a minority of cases, less evident. In patients with PTCP with EDTA and citrate-dependent platelet clumping, agglutinins are reactive at room temperature and at 37°C and belong mainly to the IgM class. Although some indirect observations suggest that these antibodies might recognize the platelet membrane glycoprotein (GP) complex Ilb/IIIa,'^ only a few reports directly demonstrate the antigenic determinant recognized by these antibodies: by means of crossed Immunoelectrophoresis, van Vliet et al 5 observed an IgM antibody directed against the membrane GP lib, and De Caterina et al 6 found an antibody directed against a protein with a 78-kd molecular weight.To assess any potential role of PTCP-associated IgG antibodies, we assayed plasma levels of glycocalicin (GC), which is the external carbohydrate-rich hydrophilic fragment of the cc-subunit of GP lb 7 ; it is readily cleaved from GP lib by 1 7 8

Co-existent Platelet Phagocytosis, Satellitism and Clumping Causing Spurious Thrombocytopenia

“…Platelet clumping in blood samples collected in EDTA occurs due to the chelating effect of EDTA, which alters the GPIIb-IIIa molecule on the platelet surface, thereby leading to binding of a naturally occurring IgG antibody to GPIIb [12]. Other anticoagulants, such as sodium citrate or heparin, do not affect GPIIb-IIIa, and therefore do not lead to platelet agglutination in these cases [13].…”

EDTA-dependent pseudothrombocytopenia in a patient with the prediagnosis of idiopathic thrombocytopenic purpura

“…Psödotrombositopeniyi saptamak için farklı yöntem-ler bulunmuştur. EDTA yerine diğer antikoagülanların (sodyum sitrat, oksalat ve heparin) kullanılması [12,13], EDTA ile antikoagüle edilmiş kanın 37 0 C'ye getirilerek incelenmesi [1,4], mikroskop altında trombosit kü-meleşmesinin saptanması [5], mekanizması henüz bilinmemesine rağmen kanamisin eklenmiş kan örnekleri-nin kullanılması bu yöntemlerdendir [8]. Çalışmamızda psödotrombositopeniyi saptamak amacıyla EDTA yerine antikoagülan olarak ilk aşamada sitratı seçtik.…”

“…Eğer sitratlı örnekte gerçek trombosit sayısına ulaşılamazsa heparin veya diğer antikoagülanlar düşünülebilir. Kan örneğinin 37 0 C'ye getirilerek incelenmesinde temel mekanizma, glikoprotein IIb/IIIa kompleksinin bu sıcaklıkta ayrışmasıyla trombositlerin kümeleşememe-sine bağlanmıştır [4]. Ancak psödotrombositopeniye sahip bazı vakalarda bilinmeyen bir mekanizma nedeniyle kümeleşmenin 37 °C'de de devam ettiği bildirilmiştir [1].…”

“…Düşük sıcaklık ve EDTA'nın kalsiyum iyonlarına bağlanırken oluşturduğu kombine etki, normalde saklı olan ve trombosit yüzeyinde bulunan glikoprotein IIb/IIIa molekülünü etkileyerek glikoprotein IIb epitopunu açığa çıkarır. Eğer kişide bu epitopa karşı otoantikor mevcutsa, antikor trombositlere bağlanarak trombositlerin kümeleşmesine neden olur [3][4][5]. Bu tedavi gerektirmeyen EDTA'ya bağlı psödotrombositopeni (EDTA-PTP) fenomeni, çoğu zaman gerçek trombositopenilerle karıştırılır.…”

The platelet count in EDTA‐anticoagulated blood from patients with thrombocytopenia may be underestimated when measured in routine laboratories

“…Variations in PC appear to be only partly due to the intrinsic variability of the test, because, at t 90 , (i) the percentage of patients exhibiting a decrease in PC was significantly higher than that of patients exhibiting an increase of PC, both in EDTA-and CPT-samples; (ii) the prevalence of platelet agglutinates was highest in blood smears of patients exhibiting a decrease in PC; (iii) the percentage of patients shifting from a lower to a higher bleeding risk category was higher than that of patients shifting from a higher to a lower risk category. The mechanisms responsible for the observed artifact are likely the same as those responsible for pseudothrombocytopenia [18], because the use of CPT instead of EDTA attenuated the severity of this artifact, albeit it did not completely prevent it [19].…”