Two Independant Assays Demonstrate High Phospholipid Procoagulant Activity In Poor Platelet Plasma of Sickle Cell Disease Children.

A group of 30 SCD children aged between 2 – 20 years (median=9, 16 males, 14 females) were studied: 12/30 didn't receive any specific treatment, 6/30 were chronically transfused (TRX) and 12/30 were treated by hydroxyurea (HU). 11/30 were previously admitted because of a vaso-occlusive crisis (VOC). 24 SCD patients were HbSS, 4 HbSC, and 2 HbSβ.

18 remaining samples from children aged between 3 – 20 years (median=9, 11 males, 7 females) undergoing minor elective surgery, with no history of coagulation disorder, normal routine coagulation tests, normal CRP, liver and kidney functions tests. The controls were not matched for the age neither sex.

Venous blood was collected into 0,109M citrated tubes using a 23G butterfly needle. PPP was obtained after 2 steps of centrifugation (3200g for 15 min and 16000g for 2 min) within 2 hours following venipuncture, and stored at -80°C. Procoagulant activity of ePL was assessed using two independent assays: a commercial fully automated factor Xa-based clotting time assay (XACT: sec) as described by Exner et al (ProcoagPPL^® Stago, Asnières sur Seine, France), and a thrombin generation assay (TGA) using calibrated automated thrombinography (CAT) as described by Hemker et al (Thrombinoscope^®, Maastricht, The Netherland). TGA was triggered with 10pM tissue factor (TF) alone and with a combination of 10pM TF and 4μM synthetic PL (positive control). Four TGA parameters were analysed: lag time (LT), Peak, Velocity Index (VI) and Endogenous Thrombin Potential (ETP). For each parameter, the result was also expressed as a % of the positive control, to minimize the influence of age. Correlations were assessed between XACT and TGA parameters using the Spearman's coefficient. Group comparison was assessed using non parametric tests.

Significant correlations were found between XACT and Peak (r= -0,76, p<0,0001), VI (r= -0,73, p<0,0001), ETP (r= -0,47, p<0,0001) measured without addition of PL, and also between XACT and % Peak (r= -0,73, p<0,0001), %VI (r= -0,63, p<0,0001) and % ETP (r= -0,60, p<0,0001). No correlation was found between XACT and LT (r= 0,20, p = 0,096).

The table shows median and range values obtained from SCD patients in steady state as compared to those of normal controls, using both methods. The TGA values are those obtained with 10pM alone, and are also expressed as a percentage of positive control.

Concerning the clinical state and treatment, XACT and all CAT parameters failed to show significant differences between subgroups of SCD (steady vs. CVO, n=11), and their treatment (TRX vs. HU vs. No specific treatment). For the patients undergoing chronic exchange transfusion, no significant difference was observed between the samples collected before and few hours after transfusion exchange.

In SCD children in steady state, XACT is significantly shortened compared to normal controls. Velocity index and Peak thrombin are significantly increased when using a high TF concentration alone as trigger. Each test showed similar performance to detect a higher procoagulant activity of ePL in SCD patients, which could contribute to the hypercoagulable state observed in those patients. In our study, ePL procoagulant activity was not influenced by the clinical state, neither the type of treatment, but this could be due to the relatively small number of patients included till now. We are currently studying more patients to confirm our results in a larger group and in particular to analyze the effects of treatment.

Noubouossie:Belgian Kid's Fund: Grant's recipient of the Belgian Kid's Funds.