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