292 Abstracts / Molecular Immunology 56 (2013) 240–316

eculizumab administration vs. “watch and wait” strategy) result-ing in all four patients achieving remission. Prompt availability ofthese diagnostic tests seems essential for therapy decision in theera of complement blocking treatment.

The study was supported by grant MZ NT1145 and by theproject (Ministry of Health, Czech Republic) for conceptual devel-opment of research organization 00064203 (University HospitalMotol, Prague, Czech Republic).

http://dx.doi.org/10.1016/j.molimm.2013.05.147

P CDT 13

Resampling model of the complement component functionalassay: Are we measuring what we think we are measuring?

I. Pavlov ∗, J. Delgado

ARUP Laboratories, R&D, Salt Lake City, United States

Introduction: Functional assays of individual complementcomponents are based on corresponding total complement activ-ity assays. Component functional assay employs patient serum andserum depleted by the component of interest. Test result is deter-mined by complex multi-component mix.

Objectives: To evaluate under which conditions such assay doesactually measure functional activity of the component of interest,authors introduced simple resampling model.

Methods: Following assumptions were accepted:

• Complement system contains 11 markers. Complement compo-nents build up a chain resulting in assay signal generation.

• Functional assay employs mix of the patient serum and serumdepleted by the component of interest at some ratio.

• Value of the test signal is determined by the component withminimal functional concentration.

• Components’ functional concentrations in patients’ populationhave Gaussian distributions.

• For each of the components, the mean value of the Gaussian dis-tribution is the concentration saturating depleted serum.

Virtual experiment of the functional test model consists of ran-dom sampling of components concentrations for depleted andpatient sera. For each resampling, lowest component concentrationwas compared with chosen concentration of the target component.

10,000 virtual assay runs were performed for the followingratios: 1:1, 2:1, 4:1 with standard deviations of complementcomponents: 10, 20, 30, 40 or 50. Real-life distributions of thecomponents were also evaluated.

Results: Percentage of random samples for which test resultsdiffer from the concentration of the target component for 5% ormore (called “wrong results”) is presented in Table:

devP = 20 devP = 30 devP = 40 devP = 50 devP = 50

Ratio 1:1 1:1 1:1 1:1 2:1devD = 10 0.04 2.9 11.9 23.5 0.3devD = 20 0.03 2.1 9.5 20.6 0.2devD = 30 0.01 2.1 8.9 19.2 0.2

Conclusions:

• We could only underestimate functional activity of the comple-ment component of interest.

• Underestimation could happen only when functional activity ofthe component of interest is above the population average mul-tiplied by the ratio of depleted to patient serum volumes.

• The chance to underestimate functional activity of the compo-nent is low for the real-life component distribution for the assayvolume ratio 2:1, and practically absent for higher assay ratios.

http://dx.doi.org/10.1016/j.molimm.2013.05.148

P CDT 14

Complement-dependent cytotoxicity exerted by two therapeu-tic anti-CD20 mAbs: Rituximab and ofatumumab in humanB-cell malignant cell lines and primary cultures

M. Okroj 1,∗, I. Eriksson 2, A. Österborg 2, A. Blom 1

1 Lund University, Malmö, Sweden2 Karolinska Institut, Department of Hematology, Stockholm, Sweden

Introduction: Complement system is one of the effector mech-anisms, which can be employed by immune system to combatB cell malignancies. Rituximab and ofatumumab are therapeuticantibodies routinely used in non-Hodkins lymphoma and chroniclymphocytic leukemia (CLL). They are both capable to activate com-plement and recognize CD20 molecule on the surface of B cells withofatumumab targeting the epitope located more proximally to thecell membrane.

Objectives: We analyzed complement-dependent cytotoxicity(CDC) of rituximab and ofatumumab as a function of serum concen-tration in the range from 5 to 50%. Unlike other studies comapringthe effective dose of these antibodies, our experiments were car-ried out at saturating concentrations of mAbs to model the limitedavailability of complement as a critical parameter.

Materials and methods: CDC was assessed by chromiumrelease assay. In parallel the level of CD20 expression as well asmembrane-bound complement inhibitors was assessed by flowcytometry.

Results: Efficacy of ofatumumab and rituximab was comparablein cell lines expressing substantial levels of CD20 and low levels ofCD59. However, ofatumumab used complement more efficientlythen rituximab when applied for cells expressing high levels ofCD59 and/or moderate levels of CD20.

Conclusions: Expression of CD20 and CD59 are two most impor-tant factors influencing CDC in B cell malignant cells treated withtherapeutic mAbs. Assessment of these two factors may be impor-tant for choosing a proper therapy, which relies on the complementsystem.

http://dx.doi.org/10.1016/j.molimm.2013.05.149