Complement factor serum and plasma levels measured by a MILLIPLEX^® Magnetic Bead Panel: A reply

Abstract

BACKGROUNDWe would like to thank you for the opportunity to respond to the discrepancies raised in Dr Sandholm's letter and to present our recent findings. Sandholm and colleagues previously described the rationale for testing patient samples for complement component levels for the study and treatment of numerous pathological conditions, as well as the advantages of multiplex assays over single enzyme immunoassays. 1 There is an expectation that commercially available immunoassay kits, including the research use only MILLIPLEX ® Human Complement Magnetic Bead Panel 2 (HCMP2MAG-19K), will perform accurately and consistently. These studies were initiated in response to the published results of Sandholm et al 1 to investigate the cause of aberrant sample values, particularly for C3. | MATERIALS AND METHODSWe tested the commercially available MILLIPLEX ® Magnetic Bead Panel, HCMP2MAG-19K (Merck, Darmstadt, Germany), which consists of a multiplexed immunoassay for complement factors C1q, C3, C3b/iC3b, C4, Factor B, H, and Properdin. This panel is for research use only, not for use in diagnostic procedures. Serum and plasma samples were tested at a 40 000-fold dilution. Samples were also tested with the MicroVue ™ Complement Factor H EIA (Quidel, San Diego, CA, USA).Blood samples included serum (n = 8) and K2EDTAplasma (n = 10) for which C3 values had been clinically determined using nephelometry (Discovery Life Sciences, Los Osos, CA, USA) and matched donor samples (n = 4) of serum, K2EDTA-plasma and lepirudin plasma (BioIVT, Westbury, NY, USA). Blood samples were stored at −80°C pending analysis, except during the heat-stress study described below. All assays were run according to the manufacturer's instructions.For the heat-stress study, four aliquots were taken from each sample type (serum, K2EDTA-plasma, lepirudin plasma) from each donor and incubated at 4°C for 24 hours, room temperature for 24 hours, or 37°C for 2 hours pending analysis. One aliquot was kept at −80°C as a control. While four donors are a small subset, the three sample types and four temperature conditions produced 96 data points with samples assayed in duplicate and were considered sufficient for the observation of any large deviation of sample values from acceptable C3 reference ranges as indicated by Sandholm et al.