Closed system processing variables affect post-thaw quality characteristics of cryopreserved red cell concentrates.

BACKGROUND: Differences in manufacturing conditions using the Haemonetics ACP 215 cell processor result in cryopreserved red cell concentrates (RCCs) of varying quality. This work studied the effect of processing method, additive solution, and storage duration on RCC quality to identify an optimal protocol for the manufacture of cryopreserved RCCs. MATERIALS AND METHODS: RCCs were pooled-and-split and stored for 7, 14, or 21 days before cryopreservation. Units were glycerolized with the ACP 215 using a single or double centrifugation method. After thawing, the RCCs were deglycerolized, suspended in AS-3, SAGM, ESOL, or SOLX/AS-7, and stored for 0, 3, 7, 14, or 21 days before quality testing. Quality assessments included hemoglobin content, hematocrit, hemolysis, adenosine triphosphate (ATP), supernatant potassium, and mean cell volume. RESULTS: Both glycerolization methods produced RCCs that met regulatory standards for blood quality. Dual centrifugation resulted in higher hemoglobin content, fewer processing alerts, and a shorter deglycerolization time than single centrifugation processing. Units processed with AS-3 and ESOL met regulatory standards when stored for up to 21 days pre-cryopreservation and 21 days post-deglycerolization. However, ESOL demonstrated superior maintenance of ATP over RBCs in AS-3. Some RCCs suspended in SAGM and SOLX exceeded acceptable hemolysis values after 7 days of post-deglycerolization storage regardless of pre-processing storage length. CONCLUSIONS: When manufacturing cryopreserved RCCs using the ACP 215, dual centrifugation processing with AS-3 or ESOL additive solutions is preferred, with storage periods of up to 21 days both pre-processing and post-deglycerolization.

The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion.

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth. Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of -5 °C/min to -20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of -5 °C/min to -20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin. The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034). The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.

Current techniques and the future of lung preservation.

Although lung transplant remains the only option for patients suffering from end-stage lung failure, donor supply is insufficient to meet demand. Static cold preservation is the most common method to preserve lungs in transport to the recipient; however, this method does not improve lung quality and only allows for 8 h of storage. This results in lungs which become available for donation but cannot be used due to failure to meet physiologic criteria or an inability to store them for a sufficient time to find a suitable recipient. Therefore, lungs lost due to failure to meet physiological or compatibility criteria may be mitigated through preservation methods which improve lung function and storage durations. Ex situ lung perfusion (ESLP) is a recently developed method which allows for longer storage times and has been demonstrated to improve lung function such that rejected lungs can be accepted for donation. Although greater use of ESLP will help to improve donor lung utilization, the ability to cryopreserve lungs would allow for organ banking to better utilize donor lungs. However, lung cryopreservation research remains underrepresented in the literature despite its unique advantages for cryopreservation over other organs. Therefore, this review will discuss the current techniques for lung preservation, static cold preservation and ESLP, and provide a review of the cryopreservation challenges and advantages unique to lungs.

High subzero cryofixation: A technique for observing ice within tissues.

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above -80 °C without sample temperature alteration. Rat liver sections (1 cm2) were frozen at a rate of -1 °C/min to -20 °C, stored for 1 h at -20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to -80 °C methanol. After 1, 3, or 5 days of -80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at -20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at -20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.

Evaluating the quality of red blood cell concentrates irradiated before or after cryopreservation.

BACKGROUND: Cryopreserved red blood cell concentrates (RCCs) are often required for patients with rare blood groups. Although transfusions from blood relatives are irradiated before transfusion, research has yet to make clear if this is necessary in cryopreserved RCCs. Given insufficient evidence to the contrary, irradiation of cryopreserved RCCs has been recommended, but the effect of irradiation timing is unknown. Therefore, this study was performed to assess the effect of RCC irradiation pre- and postcryopreservation on RCC quality. STUDY DESIGN AND METHODS: Nine whole blood units from healthy donors were processed into RCCs using the buffy coat method. ABO- and Rh-matched units were pooled and split into three groups: precryopreservation irradiation (pre-CIG), postcryopreservation irradiation (post-CIG), and nonirradiated controls. Hemoglobin, hematocrit, white blood cell (WBC) count, extracellular potassium, mean cell volume, red blood cell (RBC) morphology, and RBC deformability were measured. RESULTS: Extracellular potassium was greater in the irradiated conditions when compared to the nonirradiated controls and was greater in the post-CIG group when compared to the pre-CIG group (p < 0.05). WBC counts decreased after cryopreservation in all groups to values lower than the sensitivity of the assay. RBC deformability was greater in the post-CIG group when compared to the pre-CIG group and control group. No other significant differences were observed between groups. CONCLUSION: Irradiation of RCCs can be performed pre- or postcryopreservation with little effect on the RCC product, as both irradiated groups resulted in RCCs that were comparable to the nonirradiated cryopreserved RCCs.