Validation of automated fluorescent-based technology for measuring total nucleated cell viability of hematopoietic progenitor cell products.

BACKGROUND: A reliable rapid method for measuring total nucleated cell (TNC) viability is essential for cell-based products manufacturing. The trypan blue (TB) exclusion method, commonly used to measure TNC viability of hematopoietic progenitor cell (HPC) products, is a subjective assay, typically uses a microscope, and includes a limited number of cells. The NucleoCounter NC-200 is an automated fluorescent-based cell counter that uses pre-calibrated cartridges with acridine orange and DAPI dyes to measure cell count and viability. This study describes the validation of the NC-200 for testing HPC's viability. METHODS: Samples from 189 fresh and 60 cryopreserved HPC products were included. Fresh products were tested for viability after collection by both TB and NC-200. 7-aminoactinomycin D (7AAD) CD45+ cell viability results were obtained from a flow cytometry test. Cryopreserved products thawed specimens were tested for viability by both TB and NC-200. The NC-200 viability results were compared with the other methods. Acceptability criteria were defined as ≤10% difference between the NC-200 method and the other methods for at least 95% of the samples. RESULTS: Fresh products' mean viability difference between NC-200 and TB or 7AAD CD45+ method was 4.9% (95%CI 4.6-5.4) and 2.8% (95%CI 2.2-3.4), respectively. Thawed products' mean viability difference between NC-200 and TB was 3.0% (95%CI 0.4-5.6). CONCLUSION: The NC-200 automated fluorescent-based method can be used effectively to determine HPC's viability for both fresh and cryopreserved products. It can help eliminate human bias and provide consistent data and operational ease.

Lys 43 and Asp 46 in alpha-helix 3 of uteroglobin are essential for its phospholipase A2 inhibitory activity.

Uteroglobin (UG) is an anti-inflammatory, secreted protein with soluble phospholipase A2 (sPLA2)-inhibitory activity. However, the mechanism by which UG inhibits sPLA2 activity is unknown. UG is a homodimer in which each of the 70-amino acid subunits forms four alpha-helices. We previously reported that sPLA2-inhibitory activity of UG may reside in a segment of alpha-helix 3 that is exposed to the solvent. In addition, it has been suggested that UG may inhibit sPLA2 activity by binding and sequestering Ca++, essential for sPLA2 activation. By site-specific mutation, we demonstrate here that Lys 43 Glu, Asp 46 Lys or a combination of the two mutations in the full-length, recombinant human UG (rhUG) abrogates its sPLA2-inhibitory activity. We demonstrate further that recombinant UG does not bind Ca++ although when it is expressed with histidine-tag (H-tag) it is capable of binding Ca++. Taken together our results show that: (i) Lys 43 and Asp 46 in rhUG are critical residues for the sPLA2-inhibitory activity of UG and (ii) Ca++-sequestration by rhUG is not likely to be one of the mechanisms responsible for its sPLA2-inhibitory activity.