Strasburg, Dustin J.

AbstractBackgroundFlow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post‐thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery.Study Design and MethodsApheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%‐Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%.ResultsFifty‐two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre‐cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%.DiscussionCryopreserved samples can be reliably prepared for flow cytometric testing using a step‐wise dilution to preserve cell integrity and robust recoveries.

AbstractBackgroundStandard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab‐induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities.Study Design and MethodsWe developed a novel method whereby thawed samples were diluted step‐wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution.ResultsTwenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter.DiscussionSlow, step‐wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

AbstractBackgroundHigh titers of cold agglutinins jeopardize the quality of an apheresis product meant for autologous or allogeneic transplant. Management of transplant patients with cold agglutinin disease (CAD) is often experience‐based and under reported, yet decisions must be made quickly to optimize product management and patient outcomes. There remains a lack of data quantifying cell recovery and viability when using various warming methodologies.Study Design and MethodsTo expand the published experimental data on this subject, our human cellular therapy lab compared cellular recoveries and viabilities after manipulation of cryopreserved apheresis products through various warming methodologies: (1) extended warming in a water bath, (2) warming via blood warmer and infusion pump, and (3) warming in a water bath followed by infusion pump as a control to assess potential shear stress effects.ResultsThe presented studies demonstrate that all methods of product warming produce the same rates of recovery of total and viable cells across vital cell types prior to patient administration. Statistically, use of an extended water bath protocol provided a marginal benefit in recovery of total nucleated cells, though this effect is diminished when products are held for an extended period to simulate a delay in administration.DiscussionThese results can inform decisions to improve patient care and minimize product manipulation and loss. Centers are encouraged to use this information to guide proactive measures to establish a standard operating procedure to manage CAD cases.