Sta immunodef 8

Peripheral blood samples drawn at baseline and after 12 h are stained within 1 h of collection for activation markers relevant to sickle vaso-occlusion^{12 (link),25 (link)–28 (link)} and assessed by flow cytometry (BD FACSCanto™). For CD16b⁺ (1D3, Beckman Coulter) neutrophils: activated β2 integrin (clone 24, abcam), activated Mac-1 (CBRM1/5, eBioscience), E-selectin-Fc chimera (724-ES, R&D Systems), L-selectin (DREG-56, eBioscience), Mac-1/CD11b (ICRF44, BD Pharmingen), and LFA-1/CD11a (HI111, BD Pharmingen). For CD14⁺ (M5E2, BD Pharmingen) monocytes: tissue factor (HTF-1, BD Pharmingen). For CD41⁺ (HIP2, BD Pharmingen) platelets: CD16b (1D3, Beckman Coulter) and CD14 (M5E2, BD Pharmingen). The percentages of positive cells and median fluorescent intensity (MFI) are assessed for each patient, with calculation of the absolute number of positive cells by multiplying the percentage of positive cells by the relevant number of cells obtained from a concurrent complete blood count. For plasma studies, whole blood is centrifuged at 2500 rpm for 15 min at 4°C and plasma frozen at ≤ −80°C until batch testing. The following coagulation system activation markers relevant to sickle vaso-occlusion are tested:^{26 (link),29 (link)} prothrombin fragment 1.2 (Enzygnost F1⁺2, Dade-Behring) and factor VIII (STA^®−ImmunoDef VIII, Stago).

Plasma samples from Leiden were measured for VWF:Ag and VWF and FVIII activity by the clinical chemistry laboratory at the LUMC, Leiden. VWF:Ag in plasma was determined using the STA LIA VWF:Ag test (Stago) and was analyzed on the Sta‐R Max analyser (Stago) with a commercial STA VWF:Ag calibrator (STA Unicalibrator, Stago) as reference. VWF activity was determined with the VWF ristocetin‐triggered GPIb binding assay (VWF:GPIbR) with HemosIL AcuStar VWF:RCo reagent (Werfen IL). Samples were analyzed on the BIO‐FLASH (Werfen) and a commercial calibrator (supplied with the HemosIL AcuStar VWF:RCo) was used as reference. FVIII activity was determined using an automated one‐stage clotting assay on the STA‐R MAX analyzer (Stago) with Sta‐immunodef VIII (Stago) and STA‐CK Prest 5 (APTT; Stago) reagents. Commercial normal pool plasma (STA Unicalibrator, Stago) was used as reference. At Kingston, only VWF:Ag was measured in the plasma samples, and this was done by ELISA as previously described.²²