Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies

Research output: Contribution to journalArticle


Monoclonal antibodies ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) which are approved for usage in patients with chronic lymphocytic leukemia (CLL), efficiently activate the classical complement pathway. However complement is an exhaustible component and high doses of its activators may deplete complement-dependent cytotoxicity (CDC) potential, thus reducing the effect of repeated mAb dosing. Widely used method to measure CDC activity of patients' serum is hemolytic assay (CH50) on sheep erythrocytes. Despite its simplicity, such CH50 assay may not reflect pivotal interactions between patient serum and human complement inhibitors on the surface of target cells. We propose calcein release assay performed on tumor cells similar to those targeted by therapeutic antibodies as an alternative method. We analyzed serum samples collected from 12 patients participating in the clinical study, receiving s.c. 30 mg alemtuzumab three times per week combined with i.v. ofatumumab at an initial dose of 300 mg in week 3 further escalated to 2000 mg every other week. All serum samples were measured by hemolytic assay on sheep erythrocytes as well as using calcein release assay on CD20-positive Raji cells. Our data show that results obtained from both assays are related to each other at the level of the whole group (n = 96 samples, Spearman r = 0.504, p < .001) but may substantially differ when analyzing individual patients. Furthermore, by using CDC assay on Raji cells, we found that in the presented clinical study CDC serum potential was not significantly affected when measured before consecutive administrations in most of the patients.


  • Grzegorz Stasiłojć
  • Anna Felberg
  • Aleksandra Urban
  • Daria Kowalska
  • Shuo Ma
  • Anna M. Blom
  • Jeanette Lundin
  • Anders Österborg
  • Marcin Okrój
External organisations
  • Medical University of Gdansk
  • Northwestern University
  • Karolinska University Hospital
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area


  • Alemtuzumab, CLL, Complement system, Ofatumumab
Original languageEnglish
Article number112675
Pages (from-to)1-5
JournalJournal of Immunological Methods
Early online date2019
Publication statusPublished - 2020
Publication categoryResearch