MgSO 4 anticoagulant prevents pseudothrombocytopenia by preserving the integrity of the platelet GPIIb‐IIIa complex

Ethylenediaminetetra-acetic acid (EDTA) is the in vitro anticoagulant commonly used to collect blood samples for cell counts.1 One drawback development of pseudothrombocytopenia (PTP), a spuriously low platelet count caused by clumping 0·07–0·27% EDTA during storage at room temperature.2 PTP binding (auto)antibodies cryptic epitope(s) glycoprotein (GP)IIb/IIIa complex that become(s) accessible presence EDTA.3, 4 Although an artefact without clinical relevance, failure diagnose it may trigger unnecessary and expensive diagnostic investigations potentially dangerous treatments.2 It has been proposed substitute with magnesium sulfate (MgSO4) which prevents PTP.5-7 Excess Mg2+ inhibits aggregation,8, 9 likely interfering essential role Ca2+,10, 11 this property advocated as mechanism PTP.5, 12 However, suggestion appears incongruous, because very potent inhibitor aggregation, strong Ca2+ chelator:11 if prevented inhibitors then would prevent, rather than cause it. We compared MgSO4 under different experimental conditions, characterize better prevention MgSO4. A detailed description methods published online supplement. First all, we confirmed MgSO4, when instead EDTA, effectively PTP:5, The counts three patients decreased time kept temperature, while they remained stable (Fig 1A). results additional experiments were not compatible effect mediated inhibition aggregation.5, Indeed, showed observed citrate platelet-rich plasma (PRP), was abolished EDTA-PRP any tested agonist [adenosine diphosphate (ADP), collagen arachidonic acid], ADP only partially inhibited MgSO4-PRP 1B). Were be more effective preventing Failure inhibit aggregation induced completely could attributed fact that, variance ADP, induces causing secretion directly,13 induce directly, thus providing from dense granules membrane level sufficiently high concentrations overcome inhibitory Mg2+. In experiments, serum subject PTP, but healthy subject, increase light transmission through EDTA-PRP, 1 µmol/l prostaglandin-E1 (not shown), indicating agglutination contrast, or did appreciable Therefore, platelets exposed antibody opposite picture agonists evident: allowed occur, addition before challenge sera slightly increased extent although difference effects statistically significant added restore result being unable chelate samples, due competing Mg2+, also chelated EDTA. Finally, monoclonal (MoAb) anti-CD41a (clone HIP8) 1B) anti-CD41 P2) bind GPIIb coupled GPIIIa form GPIIb/IIIa complex, significantly lower citrate, + anti-CD61 VI-PL2) 1B), binds independently integrity higher EDTA-anticoagulated other samples. These data are uncoupling chelates holds two glycoproteins disrupting it.14 This lead unmasking epitopes recognized antibodies,4 allowing easier accessibility MoAb against GPIIIa. contrast affect indicates does uncouple antibody. finding contributes accounting lesser on depends complex. Figure 2 summarizes anticoagulants divalent cations aim illustrating consequent antibodies. evidence prevent should continue using switch counts, suggested?5, wonder whether routine use imply relevant organizational consequences. addition, must emphasized stimulated released synthesized cumbersome procedures sampling. less respect, weaker believe most convenient way misdiagnosis well aware possibility encountering indicative PTP. suspected cases suggest repeated MgSO4-anticoagulated alternative do occur all cases,2, 15 again immediately after sampling, considering develops time-dependently.15 MS supported Fondazione Umberto Veronesi “Post-Doctoral Fellowship 2020”. authors like thank Giuseppina Vismara Elisabetta Sinigaglia their help collecting data. contributed design study, performed laboratory analyses, analysed data, writing manuscript critically reviewed it; EB study analyses; GMP enrolled subjects, manuscript; MC designed coordinated group, analysis interpretation wrote manuscript. All read approved final report no conflicts interest. Please note: publisher responsible content functionality supporting information supplied authors. Any queries (other missing content) directed corresponding author article.