Journal: Transfusion

Article Title: Neutrophil‐reactive autoantibodies in blood donors: Can we expect TRALI ?

doi: 10.1111/trf.70125

Figure Lengend Snippet: Binding of neutrophils autoantibodies to target antigens expressed on neutrophil fails to fix complement. Complement fixing capacities of human IgG from transfusion‐related acute lung injury (TRALI) sera with neutrophil autoantibodies were evaluated in flow cytometry. Isolated neutrophils (2 × 10 5 ) were incubated with human sera in presence of human C1q protein for 30 min. After washing, bound C1q protein was detected by fluorescein isothiocyanate (FITC)‐labeled anti‐human C1q antibody in flowcytometry. Control serum and serum containing complement fixing alloantibodies served as negative and positive controls, respectively Data are shown as means ± SEM for four separate donors. ** p < 0.01.

Article Snippet: Anti‐CD177 (clone MEM‐166, Serotec, Duesseldorf, Germany), anti‐CD11a (clone B‐B15, Medix Biochemica, Konstanz, Germany), anti‐CD11b (clone Mac‐1, Beckman Coulter, Krefeld, Germany), anti‐CD18 (clone ITGB2, Biozol Diagnostica, Hamburg, Germany), anti‐CD16b (clone LNK16, Santa Cruz Biotechnology, Heidelberg, Germany; clone 3G8, Beckman Coulter, Krefeld, Germany) and anti‐HLA class I (clone W6/32, Biolegend, Koblenz, Germany); ortho ‐pheneylendiamine (Dako, Hamburg, Germany); lymphocyte separation media (Anprotec, Bruckberg, Germany); CM‐H 2 DCFDA (Thermofisher, Darmstadt, Germany); human C1q complement protein (Merck, Darmstadt, Germany); polyclonal sheep anti‐human C1q FITC mAb (Bio‐Rad, Dreieich, Germany); fluorescein‐isothiocyanate‐labeled bovine serum albumin (FITC‐BSA, Taufkirchen, Germany); fibronectin (Merck, Darmstadt, Germany); FITC‐labeled rabbit anti‐human IgG antibody (Jackson ImmunoResources, Brussels, Belgium); Phorbol 12‐myristate 13‐acetate (Merck, Darmstadt, Germany); paraformaldehyde (PFA) (Carl Roth, Karlsruhe Germany); Dulbecco's Modified Eagle Medium (DMEM) (Anprotec, Bruckberg, Germany); and EBM‐2 bullet kit (Lonza, Cologne, Germany).

Techniques: Binding Assay, Flow Cytometry, Isolation, Incubation, Labeling, Control