Analysis of Procoagulants Phospholipids in Plasma of Paroxysmal Nocturnal Hemoglobinuria Patients,Processed in Differents Preanalytic Conditions

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an infrequent hematopoietic stem cell disorder characterized by a higher risk of Tromboembolic disease. This complication is associated with hemolysis, Fibrinolysis and platelet activation. The increase in plasma of procoagulants microparticles from platelets has been incriminated in the pathophysiology of this complication. Up to now the analysis of microparticles has been made by Cytometry, ELISA, Electronic Microscope and thrombin generation, all of them very time-consuming and expensive techniques. Recently, an automatic quantification of microparticles has been introduced for procoagulant phospholipids time coagulation measurement. The objective of this study is to compare the procoagulant phospholipids levels in PNH and Aplasia/PNH overlap disease patients compared with twenty healthy subjects, in samples processed by different preanalytic conditions.

After an informed consent, twenty healthy subjects (blood donors) matched for age and sex were selected as controls. Eighteen patients (15 PNH and 3 AA /PNH) followed in our Reference Unit. From these, 15 were men and 3 women. Median age of 45,5 yrs. (16–68), 12 were treated with different types of treatments (five of them with Eculizumab) and 6 were not treated. To measure microparticles, after double centrifugation 2500 g × 15 min, and separation in three aliquots, were stored at −80°C and −40°C, and a third sample was processed in fresh. A FXa based coagulative technique was used.

The media in controls was 86,3±11,0 seg in fresh, 78,3±13,5 seg at −40°C and 79,3±12,3 seg at −80°C. These differences were significant between the fresh samples at −40°C (p<0,01) and −80°C (p<0,01). In not treated PNH patients the media was 78,91 ±10,9 seg in fresh, 67,4±13,6 at −40°C, and 69,1±19,2 at −80°C. Treated patiens shaw microparticles values of 88,1±12 in fresh, 80,7±19 at-40, and 78,6±15 at −80°C.

Through this automated coagulative technique based of FXa, a significant increased of microparticles has been observed in controls, at – 40°C and in less significance at −80°C respect fresh samples. In not treatment PNH patients a no significant microparticles levels increased has been measured (because of the reduced sample size) about controls. These differences disappear in treatment PNH patients. There is an increased in microparticles levels in PNH patients before treatment, that becomes normal in treatment patients. Moreover the freezing show an increasing of the plasma procoagulant activity higher at −40°C about −80°C.

No relevant conflicts of interest to declare.