ESPN 53rd Annual Meeting

ESPN 2021


 
ECULIZUMAB THERAPY MONITORING: RESIDUAL HEMOLYSIS IN ALTERNATIVE PATHWAY TEST IS NOT CAUSED BY C5 ACTIVITY
BERT VAN DEN HEUVEL 1 NICOLE VAN DE KAR 1 ANDREI SARLEA 1 SANNE VAN KRAAIJ 1 THEA VAN DER VELDEN 1 WILHELMUS LIEBRAND 1 CAROLINE DUINEVELD 1 ROMY BOUWMEESTER 1 JACK WETZELS 1 TOM EIRIK MOLLNES 2 ELENA VOLOKHINA 1

1- Radboud university medical center, Nijmegen, The Netherlands
2- Oslo University Hospital, Oslo, Norway
 
Introduction:

Eculizumab is a C5-blocker used in the treatment of atypical hemolytic uremic syndrome (aHUS). Hemolytic assays are often used to monitor classical (CP) and alternative pathway (AP) blockade in these patients. The AP assay is known to show hemolysis in patient samples that attain target drug concentrations > 100 µg/mL. This suggests incomplete complement blockade and may lead to a change of therapy (higher dose or alternative drug). Therefore, we investigated whether the residual hemolysis detected by the AP assay in the presence of eculizumab is caused by incomplete C5 blockade by the drug.

Material and methods:

Normal human serum (NHS) spiked with eculizumab (100, 200 and 500 µg/mL), five aHUS samples containing 256-371 µg/mL of the drug and serum of C5 deficient donor were tested in CP and AP hemolytic assays and ELISA-based Wieslab® tests for complement activity. Eculizumab, C5 and complement activation markers were assessed by ELISAs. Mechanical fragility was assessed by exposing erythrocytes to low NaCl concentrations.

Results:

All eculizumab samples and C5 deficient serum had comparable high (up to 81%) hemolysis in AP test, but no measurable complement activity in CP hemolytic assay and ELISA-based AP and CP tests. Furthermore, NHS spiked with 100 µg/mL of eculizumab and stimulated by 100 µg/mL of zymosan A produced time-dependent increase of C3 activation markers C3bBbP, C3bc and C3a but not of the C5 activation marker sC5b-9. Erythrocytes remaining after AP assay of eculizumab samples were fragile in osmotic test. This fragility was abolished by adding C3-inhibitor compstatin.

Conclusions:

Hemolysis in AP hemolytic assay under eculizumab treatment is not caused by the residual C5 activity. Likely, it is caused by deposition of C3 activation products, which weaken the erythrocytes and make them more prone to hemolysis. ELISA-based methods are the better option to monitor C5 blockade during eculizumab treatment.