Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab.

Objectives: A matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time. Methods: Serum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 µl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results. Results: The automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT). Conclusions: Mass-Fix is ready for implementation in a high-throughput clinical laboratory.

The impact of eculizumab on routine complement assays.

BACKGROUND: Eculizumab (ECU) blocks complement C5 cleavage, preventing the formation of C5a and the cytolytic effects of the membrane attack complex. The presence of ECU in blood impacts routine complement tests used to monitor treatment. METHODS: Residual serum samples with normal total complement (CH50) and residual citrate plasma with normal PT/APTT were spiked with ECU at varied concentrations ranging from 25 to 600 µg/mL. In addition, seventy-one samples from patients on ECU were obtained. Artificial and patient samples were analyzed for CH50 and C5 function (Wako Diagnostics), C5 concentration (Quidel), AH50 (Wieslab ELISA) and sMAC (Quidel). ECU concentration was measured by mass spectrometry for all patients. RESULTS: Complement blockage by ECU was evident in spiked artificial samples. At 25 µg/mL ECU, partial complement blockage was observed in CH50, AH50 and C5 function in serum. Complete blockage defined by undetectable AH50 (<10%) occurred at 100 µg/mL ECU. C5 concentrations remained the same regardless of ECU. sMAC results stayed around 81% of baseline in serum and 47% in citrate plasma with 50µg/mL ECU. Patient samples had ECU ranging from <5 to 1220 µg/mL. In all patients with ECU >100 µg/mL, C5 function was <29 U/mL. CONCLUSIONS: The spiked sera and patient samples showed complement blockage with CH50, AH50 and C5 function assays when ECU >100 µg/mL. CH50, AH50 or C5 function assays can serve as indicators for the pharmacodynamic effects of eculizumab. Allied to ECU concentration, laboratory studies may be helpful to tailor therapy.