Comparable bacterial growth in platelet concentrates suspended in plasma and platelet additive solution and improved detection of bacterial contamination using a new generation automated culture system.

BACKGROUND: Microbial screening of platelet concentrates (PC) with automated culture methods is widely implemented to reduce septic transfusion reactions. Herein, detection of bacterial contamination in PC was compared between units prepared in plasma and a mix of plasma and platelet additive solution (PAS) and between the BACT/ALERT 3D and next generation BACT/ALERT VIRTUO systems. STUDY DESIGN/METHODS: Double apheresis units were split into single units, diluted in either PAS (PAS-PC) or plasma (plasma-PC), and tested for in vitro quality and sterility prior to spiking with ~30 CFU/unit of Staphylococcus epidermidis, Staphylococcus aureus, Serratia marcescens, and Klebsiella pneumoniae or ~10 CFU/mL of Cutibacterium acnes. Spiked PC were sampled for BACT/ALERT testing (36 and 48 h post-spiking) and colony counts (24, 36, and 48 h post-spiking). Times to detection (TtoD) and bacterial loads were compared between PC products and BACT/ALERT systems (N = 3). RESULTS: Bacterial growth was similar in plasma-PC and PAS-PC. No significant differences in TtoD were observed between plasma-PC and PAS-PC at the 36-h sampling time except for S. epidermidis which grew faster in plasma-PC and C. acnes which was detected earlier in PAS-PC (p < .05). Detection of facultative bacteria was 1.3-2.2 h sooner in VIRTUO compared with 3D (p < .05) while TtoD for C. acnes was not significantly different between the two systems. DISCUSSION: Comparable bacterial detection was observed in plasma-PC and PAS-PC with PC sampling performed at 36-h post blood collection. PC sampling at ≤36 h could result in faster detection of facultative pathogenic organisms with the VIRTUO system and improved PC safety.

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.

Preparing small-dose red cell concentrates (RCCs) for neonatal and pediatric transfusions: Impact of RCC volume, storage, and irradiation.

BACKGROUND: Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice. MATERIALS AND METHODS: To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production. RESULTS: Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts. CONCLUSIONS: Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.

Automated closed volume reduction process for apheresis stem cell grafts: From development to clinical implementation.

BACKGROUND: Collection of HPC by apheresis (HPC-A) can sometimes result in higher collection volumes, increasing the dimethyl sulfoxide (DMSO) volume infused into patients and the space requirements in liquid nitrogen freezers. Volume reduction prior to the addition of cryoprotectant is an efficient means to reduce the DMSO load infused into patients and to optimize freezer storage space. STUDY DESIGN AND METHODS: To implement a closed semi-automated volume reduction process, a method was developed to produce leukocyte-rich mock apheresis products using buffy coats derived from whole blood collections. The mock HPC products were then used to measure the efficiency and reliability of the semi-automated process over a range of volumes and cell concentrations. The resulting data was used to support the implementation of the process with concurrent monitoring. RESULTS: A closed, semi-automated volume reduction process resulted in recoveries of over 93% and 91% of white blood cells and CD34+ cells with no significant loss of product viability or potency. Mean doses of CD34+ and CFU infused per kilogram recipient body weight were 4.0 ± 1.1 × 106 /kg and 4.2 ± 1.7 × 105 /kg, resulting in no delays in median time to neutrophil and platelet engraftment, significant increase in adverse reaction or nonconformances. DISCUSSION: The effectiveness outcomes of the first Canadian experience in the implementation of a closed semi-automated volume reduction system in the processing of HPC-A products for autologous transplant have met the predetermined acceptance criteria, supporting its use in a stem cell manufacturing laboratory compliant with good manufacturing practice regulations.

Retention of hemostatic and immunological properties of frozen plasma and COVID-19 convalescent apheresis fresh-frozen plasma produced and freeze-dried in Canada.

BACKGROUND: Randomized clinical trial data show that early plasma transfusion may save lives among trauma patients. Supplying plasma in remote environments is logistically challenging. Freeze-dried plasma (FDP) offers a possible solution. STUDY DESIGN AND METHODS: A Terumo BCT plasma freeze-drying system was evaluated. We compared pooled frozen plasma (FP) units with derived Terumo BCT FDP (TFDP) units and pooled COVID-19 convalescent apheresis fresh-frozen plasma (CC-AFFP) with derived CC-TFDP units. Parameters measured were: coagulation factors (F) II; V; VII; VIII; IX; XI; XIII; fibrinogen; Proteins C (PC) and S (PS); antithrombin (AT); α2 -antiplasmin (α2 AP); ADAMTS13; von Willebrand Factor (vWF); thrombin-antithrombin (TAT); D-dimer; activated complement factors 3 (C3a) and 5 (C5a); pH; osmolality; prothrombin time (PT); and activated partial thromboplastin time (aPTT). Antibodies to SARS-CoV-2 in CC-AFFP and CC-TFDP units were compared by plaque reduction assays and viral protein immunoassays. RESULTS: Most parameters were unchanged in TFDP versus FP or differed ≤15%. Mean aPTT, PT, C3a, and pH were elevated 5.9%, 6.9%, 64%, and 0.28 units, respectively, versus FP. CC-TFDP showed no loss of SARS-CoV-2 neutralization titer versus CC-AFFP and no mean signal loss in most pools by viral protein immunoassays. CONCLUSION: Changes in protein activities or clotting times arising from freeze-drying were <15%. Although C3a levels in TFDP were elevated, they were less than literature values for transfusable plasma. SARS-CoV-2-neutralizing antibody titers and viral protein binding levels were largely unaffected by freeze-drying. In vitro characteristics of TFDP or CC-TFDP were comparable to their originating plasma, making future clinical studies appropriate.

Challenging the 30-min rule for thawed plasma.

BACKGROUND AND OBJECTIVES: Frozen plasma (FP) is thawed prior to transfusion and stored for ≤5 days at 1-6°C. The effect of temperature excursions on the quality and safety of thawed plasma during 5-day storage was determined. MATERIALS AND METHODS: Four plasma units were pooled, split and stored at ≤-18°C for ≤90 days. Test units T30 and T60 were exposed to 20-24°C (room temperature [RT]) for 30 or 60 min, respectively, on days 0 and 2 of storage. Negative and positive control units remained refrigerated or at RT for 5 days, respectively. On Day 5, test units were exposed once to RT for 5 h. Quality assays included stability of coagulation factors FV, FVII, FVIII, fibrinogen and prothrombin time. Bacterial growth was performed in units inoculated with ~1 CFU/ml or ~100 CFU/ml of Serratia liquefaciens, Pseudomonas putida, Pseudomonas aeruginosa or Staphylococcus epidermidis on Day 0. RESULTS: Testing results of all quality parameters were comparable between T30 and T60 units (p < 0.05). Serratia liquefaciens proliferated in cold-stored plasma, while P. putida showed variable viability. Serratia epidermidis and P. aeruginosa survived but did not grow in cold-stored plasma. Positive and negative controls showed expected results. Overall, no statistical differences in bacterial concentration between T30 and T60 units were observed (p < 0.05). CONCLUSION: Multiple RT exposures for 30 or 60 min do not affect the stability of coagulation factors or promote bacterial growth in thawed plasma stored for 5 days. It is therefore safe to expose thawed plasma to uncontrolled temperatures for limited periods of 60 min.

Hypothermic storage of leukoreduced red blood cells for greater than 21 days is a safe alternative to irradiation.

BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

Transient warming affects potency of cryopreserved cord blood units.

BACKGROUND AIMS: Cryopreserved cord blood units (CBUs) can be exposed to transient warming events (TWEs) during routine banking operations, which may affect their potency. NetCord-FACT guidelines recommend removal of these CBUs from inventory. The objective of this work was to evaluate warming kinetics of frozen CBUs in different settings to determine the optimal working environment and define the impact of different TWE scenarios on CB post-thaw quality and potency. METHODS: The warming kinetics of frozen CBUs was influenced by both working surfaces and ambient working temperature, with cold plates providing better protection than vinyl or metal surfaces. Measurement of time for required operational activities revealed that CBUs are probably exposed to core temperatures greater than -150°C even when cold plates are used to reduce warming rates. RESULTS: On the basis of the warming kinetics and observed operational activities, three TWE causing scenarios (control, typical, worst case) were investigated using a pool-and-split design and cell viability, recovery and potency (colony-forming unit [CFU]) assays were performed. TWEs were found to have little impact on the recovery of total nucleated cells or on the viability of CD34+ cells. In contrast, the viability and recovery of CD45+ cells in the smaller CBU compartments were reduced by TWEs. Moreover, the worst-case TWE reduced CFU recovery from CBUs, whereas the typical-scenario TWE had little effect. CONCLUSIONS: Our results demonstrate that the distal segment underestimates the viability and potency of CBUs and that TWEs can affect the post-thaw viability and potency of CBUs. Although TWEs are almost inevitable during cord-blood banking operations, their effects must be diminished by reducing exposure time, using cold plates and strict operational protocols, to prevent worst-case TWEs.

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.

Impact of blood manufacturing and donor characteristics on membrane water permeability and in vitro quality parameters during hypothermic storage of red blood cells.

Several factors have been proposed to influence the red blood cell storage lesion including storage duration, blood component manufacturing methodology, and donor characteristics [1,18]. The objectives of this study were to determine the impact of manufacturing method and donor characteristics on water permeability and membrane quality parameters. Red blood cell units were obtained from volunteer blood donors and grouped according to the manufacturing method and donor characteristics of sex and age. Membrane water permeability and membrane quality parameters, including deformability, hemolysis, osmotic fragility, hematologic indices, supernatant potassium, and supernatant sodium, were determined on day 5 ±â€¯2, day 21, and day 42. Regression analysis was applied to evaluate the contribution of storage duration, manufacturing method, and donor characteristics on storage lesion. This study found that units processed using a whole blood filtration manufacturing method exhibited significantly higher membrane water permeability throughout storage compared to units manufactured using red cell filtration. Additionally, significant differences in hemolysis, supernatant potassium, and supernatant sodium were seen between manufacturing methods, however there were no significance differences between donor age and sex groups. Findings of this study suggest that the membrane-related storage lesion is initiated prior to the first day of storage with contributions by both blood manufacturing process and donor variability. The findings of this work highlight the importance of characterizing membrane water permeability during storage as it can be a predictor of the biophysical and chemical changes that affect the quality of stored red blood cells during hypothermic storage.

Genotyping single nucleotide polymorphisms in human genomic DNA with an automated and self-contained PCR cassette.

Point-of-care devices can lower costs through reduced reagent costs, shifting diagnostics from centralized laboratories to local clinics or hospitals, rapidly informing on the spot medical decision making, and enabling personalized treatment options. We have previously described a self-contained miniaturized device that uses an array of gel-based reaction units that can simultaneously detect multiple biomarkers and/or multiple patients in one PCR cassette and can be stored for up to 7 months. In this article, we document the ability of cassette PCR to detect single nucleotide polymorphisms (SNPs) in human genomic DNA from buccal swabs. Swab processing takes 8 minutes, and PCR is completed in just more than an hour. To demonstrate potential for genotyping, we used allele-specific PCR and melt curve analysis to detect major and minor alleles of two SNPs in the fibroblast growth factor receptor 2 gene (FGFR2) that are linked with breast cancer. After allele-specific PCR, seamless melt curve analysis and the presence or absence of melt peaks from melt curve analysis identifies the FGFR2 SNP genotypes for each patient. The near point-of-care/point-of-need genotyping methods reported here can be applied for detecting and assessing risks of diseases such as cancer and to detect SNPs that alter drug metabolism and hence response to therapy.

A lab-on-chip for malaria diagnosis and surveillance.

BACKGROUND: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges. METHODS: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection. RESULTS: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/µL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%. CONCLUSIONS: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.