![Figure 1. Blocking fH-mediated cell surface protection of human red blood cells that have normal or decreased CD55 and CD59 function increases their susceptibility to autologous complement-mediated lysis. (A) EHs (5 × 106) in GVB were preincubated with a neutralizing anti-CD59 monoclonal antibody (0-7.5 μg/mL) for 20 minutes at 4°C. NHS (40% final) in the presence or absence of 14 μM rH19-20 (as an inhibitor of fH cell-surface protection) was added and the mix (24 μL total containing 5 mM MgEGTA) was incubated for 20 minutes at 37°C. Cold GVBE (200 μL) was then added to stop the reaction, and lysis was subsequently measured by hemoglobin release (A414) after centrifugation to remove unlysed cells. (B) Same as panel A, but the EHs were preincubated with anti-CD55 monoclonal antibody. Averages and standard deviations of 3 separate experiments are graphed in panels A and B. (C) CD59 profile analysis of PNH and normal erythrocytes after in vitro exposure to NHS + MgEGTA in the presence or absence of rH19-20. The PNH erythrocytes (patients 1-4) and normal EHs (one representative sample shown) were treated with 40% NHS (first column titled “Cells After NHS Treatment”) or with NHS in the presence of 17 μM rH19-20 (second column titled “Cells After NHS + rH19-20 Treatment”), for 20 minutes at 37°C. The CD59 profile of the remaining unlysed cells was determined by incubation with anti-CD59 monoclonal antibody (5 μg/mL) for 45 minutes at 0°C, followed by incubation with fluorescein isothiocyanate–conjugated rabbit anti–mouse IgG antibodies for 45 minutes at 0°C, and analyzed by fluorescence-activated cell sorting (FACS). The markers I, II, and III indicate the populations of CD59 normal, CD59 partially positive, and CD59-negative cells, respectively. In the histograms titled “Cells After NHS + rH19-20 Treatment,” the percent lysis of PNH II + III cells is indicated and was calculated as described17: 100 − ([(% PNH II + III Cells After NHS + rH19-20/% PNH I Cells After NHS + rH19-20)/(% PNH II + III Cells After NHS/% PNH I Cells After NHS)] × 100). At least 10 000 events were acquired per sample. The results shown are representative of 3 separate experiments. The samples that were treated with NHS (panels A-C) contained 5 mM MgEGTA to prevent classical pathway activation.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/6/10.1182_blood-2007-04-083170/7/zh80180707370001.jpeg?Expires=1724941587&Signature=gZlarni6npFy-wTws2PsPHxH0Xbl-maXdIAUmX0B9N8dCt2jCCXkz0hXjGxnBgeUS~46CKfPvuFUgNsrsPQqwQPDRRnNGX~ujHBa89WAkHPJgOM6ONrNKn~aYMnSu3zVHM~zywZniGgYoxhPSlJIuK4w7hh4IVSQVbMEmmjimhnKDP2fZh9VXmeze4T77SCLQNGydQKfzIpHDGa77lD1KrdPfV0TmrybFETvFCkZZCi0quxV1oMWWs0zjmtLPlTplrS4T9NBvIWQCW3KdQw55fJZZFpspq1olm9imkGbR3Yzv8HP2XS6aM7k2U~3a6fk5N8devojbK-2o-H0gVK3vg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Blocking fH-mediated cell surface protection of human red blood cells that have normal or decreased CD55 and CD59 function increases their susceptibility to autologous complement-mediated lysis. (A) EHs (5 × 106) in GVB were preincubated with a neutralizing anti-CD59 monoclonal antibody (0-7.5 μg/mL) for 20 minutes at 4°C. NHS (40% final) in the presence or absence of 14 μM rH19-20 (as an inhibitor of fH cell-surface protection) was added and the mix (24 μL total containing 5 mM MgEGTA) was incubated for 20 minutes at 37°C. Cold GVBE (200 μL) was then added to stop the reaction, and lysis was subsequently measured by hemoglobin release (A414) after centrifugation to remove unlysed cells. (B) Same as panel A, but the EHs were preincubated with anti-CD55 monoclonal antibody. Averages and standard deviations of 3 separate experiments are graphed in panels A and B. (C) CD59 profile analysis of PNH and normal erythrocytes after in vitro exposure to NHS + MgEGTA in the presence or absence of rH19-20. The PNH erythrocytes (patients 1-4) and normal EHs (one representative sample shown) were treated with 40% NHS (first column titled “Cells After NHS Treatment”) or with NHS in the presence of 17 μM rH19-20 (second column titled “Cells After NHS + rH19-20 Treatment”), for 20 minutes at 37°C. The CD59 profile of the remaining unlysed cells was determined by incubation with anti-CD59 monoclonal antibody (5 μg/mL) for 45 minutes at 0°C, followed by incubation with fluorescein isothiocyanate–conjugated rabbit anti–mouse IgG antibodies for 45 minutes at 0°C, and analyzed by fluorescence-activated cell sorting (FACS). The markers I, II, and III indicate the populations of CD59 normal, CD59 partially positive, and CD59-negative cells, respectively. In the histograms titled “Cells After NHS + rH19-20 Treatment,” the percent lysis of PNH II + III cells is indicated and was calculated as described17 : 100 − ([(% PNH II + III Cells After NHS + rH19-20/% PNH I Cells After NHS + rH19-20)/(% PNH II + III Cells After NHS/% PNH I Cells After NHS)] × 100). At least 10 000 events were acquired per sample. The results shown are representative of 3 separate experiments. The samples that were treated with NHS (panels A-C) contained 5 mM MgEGTA to prevent classical pathway activation.
