Effects of pre-freeze pathogen reduction with riboflavin and UV light on red cells stored post-thaw in AS-3 additive solution.

BACKGROUND: Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. STUDY DESIGN AND METHODS: 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. RESULTS: PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. CONCLUSIONS: Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.

Cryopreservation of apheresis platelets treated with riboflavin and UV light.

BACKGROUND: Pathogen reduction technology (PRT) is increasingly used in the preparation of platelets for therapeutic transfusion. As the Czech Republic considers PRT, we asked what effects PRT may have on the recovery and function of platelets after cryopreservation (CP), which we use in both military and civilian blood settings. STUDY DESIGN AND METHODS: 16 Group O apheresis platelets units were treated with PRT (Mirasol, Terumo BCT, USA) before freezing; 15 similarly collected units were frozen without PRT as controls. All units were processed with 5-6% DMSO, frozen at - 80 °C, stored > 14 days, and reconstituted in thawed AB plasma. After reconstitution, all units were assessed for: platelet count, mean platelet volume (MPV), platelet recovery, thromboelastography, thrombin generation time, endogenous thrombin potential (ETP), glucose, lactate, pH, pO2, pCO2, HCO3, CD41, CD42b, CD62, Annexin V, CCL5, CD62P, and aggregates > 2 mm and selected units for Kunicki score. RESULTS: PRT treated platelet units had lower platelet number (247 vs 278 ×109/U), reduced thromboelastographic MA (38 vs 62 mm) and demonstrated aggregates compared to untreated platelets. Plasma coagulation functions were largely unchanged. CONCLUSIONS: Samples from PRT units showed reduced platelet number, reduced function greater than the reduced number would cause, and aggregates. While the platelet numbers are sufficient to meet the European standard, marked platelets activation with weak clot strength suggest reduced effectiveness.

The use of cryopreserved platelets in the treatment of polytraumatic patients and patients with massive bleeding.

BACKGROUND: The short shelf-life of fresh platelets limits their efficient inventory management and availability during a massive transfusion protocol. Risk of insufficient availability can be mitigated by building an inventory of cryopreserved platelets (CPs). METHODS: A comparative study of fresh apheresis platelets (FAPs) and CPs was performed. Type-O CPs were processed with DMSO frozen at -80°C and reconstituted in thawed AB plasma. All patients enrolled in the study had the following parameters evaluated on admission: vital signs (body temperature, heart rate, mean arterial pressure), blood count, prothrombin time, activated partial thromboplastin time, fibrinogen level, and, in trauma patients, international severity score. Several outcomes were evaluated: 30-day survival, adverse events, quantity of administered blood products, fibrinogen concentrate and thromboxane (TXA), and laboratory parameters after transfusion (blood count, prothrombin time, activated partial thromboplastin time, fibrinogen level). RESULTS: Twenty-five (25) patients in the study group received transfusions totaling 81 units of CPs. Twenty-one (21) patients in the control group received a total of 67 units of FAPs. There were no significant differences in patient characteristics (p > 0.05) between groups. Both groups were comparable in clinical outcomes (30-day survival, administered blood products, fibrinogen concentrate, TXA, and adverse events). Among posttransfusion laboratory parameters, platelet count was higher in the group transfused with FAPs (97.0 ×109 /L) than in the group transfused with CPs (41.5 ×109 /L), p = 0.02025. Other parameters were comparable in both groups. CONCLUSION: The study suggests that CPs are tolerable and a feasible alternative to FAPs. However, larger randomized studies are needed to draw definitive conclusions.

Comparison of titer results obtained using immediate spin one-dilution techniques to a reference method.

BACKGROUND: Many transfusion services determine the titer of potentially incompatible plasma-containing products by performing a one-dilution titer at their selected titer threshold. This study compared the results of immediate spin (IS) one-dilution titers determined by three methods with a reference standard method. METHODS: Plasma-containing products from group A and O donors were titered using the participant's routine IS one-dilution titer method. No time or temperature incubations were performed, and antihuman globulin reagent was not used. The samples were then tested using a reference method, which was a saline tube test with a 1-hour room temperature incubation; antihuman globulin was not used in the reference method. The results of the one-dilution titer were then compared to that obtained in the reference method. RESULTS: Nine centers participated in this study. There were 698 antibodies from 374 units tested by the manual IS tube one-dilution titer method; sensitivity was 0.88 (95% confidence interval [CI], 0.83-0.92), and specificity was 1.00 (95% CI, 0.98-1.00). There were 412 antibodies from 206 units tested by the manual and automated IS buffered gel card one-dilution titer method; sensitivity was 0.95 (95% CI, 0.91-0.98), and specificity was 0.87 (95% CI, 0.81-0.91). There were 98 antibodies from 49 units tested by an automated microplate IS one-dilution titer method; sensitivity was 0.76 (95% CI, 0.71-0.93), and specificity was 0.96 (95% CI, 0.92-0.99). All three methods had an accuracy rate of 90% or greater. CONCLUSION: The manual and automated one-dilution titer methods are suitable for screening plasma-containing units, although more evaluation of the automated microplate method might be required.

Increased oxidative/nitrosative stress markers measured non- invasively in patients with high 2,3,7,8-tetrachloro-dibenzo-p-dioxin plasma level.

OBJECTIVES: 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) is a highly toxic persistent environmental contaminant, classified as a human carcinogen affecting any target organ. The mechanism of carcinogenesis by TCDD is unclear as TCDD shows a lack of direct genotoxicity. Experimental studies also support the role of oxidative stress in TCDD neurotoxicity and vascular dysfunction. The aim was to investigate markers of oxidative/nitrosative stress and inflammation using non-invasive methods in subjects who got ill due to severe occupational exposure to TCDD in the years 1965-1968. METHODS: In 11 TCDD-exposed patients, and 16 controls, the analysis of following oxidative products of lipids, proteins and nucleic acids in plasma, urine and exhaled breath condensate (EBC) was performed: 8-iso-prostaglandin F2α (8-isoprostane), 4-hydroxy-trans-2-nonenale (HNE), malondialdehyde (MDA), o-tyrosine (o-Tyr), 8-hydroxyguanosine (8-OHG), 8-hydroxy-2´-deoxy-guanosine (8-OHdG), 5-hydroxymethyluracil (5-OHMeU). In addition, nitric-oxide-tyrosine (NO-Tyr) and leukotriene (LT) B4, C4, D4, and E4 were detected by liquid chromatography-mass spectrometry/mass spectrometry (LC-ESI-MS/MS). TCDD was measured by HRGC/HRMS, body lipid content by densitometry. Single-photon emission spectrometry (SPECT) of the brain was performed and compared with the findings of the patients in 2008. RESULTS: Mean TCDD plasma level in 2010 was 175 ± 162 pg/g lipids (population level about 2 pg/g), total TCDD content in the body 5.16 ± 4.62 mg. Reduction of cerebral blood flow in SPECT progressed in 8 patients, finding was stable in 2 subjects, and improvement occurred in 1 patient. In the EBC, 10 from 12 markers (all except LT D4 and LT E4), were significantly increased in the patients (p<0.05). In the urine, 7 markers were significantly higher than in the controls (p<0.05): 8-isoprostane, MDA, HNE, LT C4, LT E4, o-Tyr and NO-Tyr. In plasma, only NO-Tyr and 8-OHG were elevated (p<0.05). CONCLUSION: NO-Tyr was increased in all matrices in dioxin-exposed patients. EBC is not limited to lung disorders as the markers of oxidative stress and inflammation were elevated in EBC of patients with normal lung functions. TCDD-induced oxidative stress and inflammation markers can be detected non-invasively in the EBC and urine in the follow-up of the highly-exposed patients. Their prognostic value, however, needs to be elucidated.