The effect of overnight storage of leukapheresis stem cell products on cell viability, recovery, and cost.

The cost of harvesting, processing, and freezing multiple peripheral blood stem cell (PBSC) products could easily exceed that of bone marrow harvest. To reduce costs while maintaining product viability, we examined the effect of overnight storage on PBSC products. Sixteen consecutive leukapheresis samples from 12 patients were examined prospectively. Each initial leukapheresis product was stored overnight on ice (median temperature 15 degrees C) after adding an equal amount of M199 culture medium containing heparin. After overnight storage, the product was combined with the next day PBSC harvest if required and processed/frozen per protocols. Parameters measured before and after storage include cell count and differential, viability, bacterial cultures, and colony-forming unit (CFU) assays. The results show that the median cell concentration during storage was 7.12 x 10(7)/ml and the median length of storage was 20 h. After storage, the median viability and nucleated cell recovery were 100% and 99.5%, respectively. In addition, 98% recovery of CFU-GM was achieved. No clotting or bacterial contamination was detected. All 12 patients studied engrafted promptly. In addition, 124 similarly treated patients were retrospectively analyzed. Of these, 48% required > or = 2 large-volume leukaphereses to achieve the target cell dose. As a result of overnight storage, 150 final products, instead of 224, were processed and cryopreserved. This difference is equivalent to 33% cost savings. Again, all patients were transplanted and engrafted successfully. In conclusion, overnight storage and pooling of two consecutive PBSC products are safe, reduce cost, and allow for optimum laboratory staffing.

Measurement of absolute concentration and viability of CD34+ cells in cord blood and cord blood products using fluorescent beads and cyanine nucleic acid dyes.

Conventional flow cytometric methods for CD34+ cell counting may be affected by the high number of nucleated red blood cells or nonviable cells in cord blood and its products. We developed a simple flow cytometric no-wash procedure that avoids these shortcomings because it provides absolute CD34+ cell counts and assesses cell viability. Samples were incubated with phycoerythrin (PE)-labeled anti-CD34 (Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 (BD) in bead-containing TRUCOUNT tubes (BD). After red cell lysis with a fixative-free reagent, the impermeant nucleic acid dye YO-PRO-1 (Molecular Probes, Eugene, OR) was added and samples were analyzed on a single-laser FACSCalibur (BD). A comparison with the ProCOUNT progenitor cell assay (BD) in 57 samples revealed excellent correlation of results (r = 0.98, intercept -0.2 cells/microl, slope 1.01). Precision studies conveyed coefficients of variation of 6.4 and 8.9% at concentrations of 35 and 16 CD34+ cells/microl, respectively. In untreated and leukocyte-enriched cord blood 4.5+/-3.8% of CD34+ cells were stained by YO-PRO-1, representing apoptotic or necrotic cells. In post-thawing cryopreserved samples this number increased to 10.4+/-5.5%. Isotype controls showed very low blank values of viable cells (0.1+/-0.4 cells/microl, maximum 2.4) and seemed unnecessary. We found no washing-related alteration of results in 35 samples, indicating that the method may also be performed with cell washing. Replacing YO-PRO-1 with TO-PRO-3 facilitated four-color analysis of subpopulations of viable CD34+ cells on a FACSCalibur equipped with a second (diode) laser. We found the proposed method to be a rapid, efficient, and flexible procedure that improved validity of CD34+ cell counts.

Are insulin autoantibodies markers for insulin-dependent diabetes mellitus?

Recent studies have shown that insulin autoantibodies occur in patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) before exogenous insulin treatment. Our study was designed to test the hypothesis that insulin autoantibodies, like cytoplasmic islet cell antibodies (ICAs), can identify individuals with ongoing autoimmune beta-cell destruction and increased risk of IDDM development. Insulin autoantibodies detected by use of a radioligand-binding assay were found in 1.4% of normal controls, 4% of first-degree relatives of IDDM patients, and in 37% of newly diagnosed IDDM patients. A strong positive correlation between insulin autoantibodies and ICAs was observed. HLA typing of insulin-autoantibody-positive first-degree relatives of IDDM patients, as well as in the general population, revealed a strong association with HLA-DR3 and/or-DR4, suggesting that insulin autoantibodies are restricted to persons genetically susceptible to IDDM. In an ongoing study of beta-cell function in ICA-positive nondiabetic individuals, the additional presence of insulin autoantibodies significantly increased the likelihood of beta-cell dysfunction. After intravenous glucose stimulation, insulinopenia was present in 70% of ICA and insulin-autoantibody-positive individuals in contrast to only 23% of ICA-positive, insulin-autoantibody-negative persons. These data document a significant association between insulin autoantibodies and ICAs and support the contention that insulin autoantibodies, like ICAs, are markers of ongoing beta-cell destruction.