Effect of leukoreduction on the omics phenotypes of canine packed red blood cells during refrigerated storage.

BACKGROUND: Red blood cell (RBC) storage promotes biochemical and morphological alterations, collectively referred to as storage lesions (SLs). Studies in humans have identified leukoreduction (LR) as a critical processing step that mitigates SLs. To date no study has evaluated the impact of LR on metabolic SLs in canine blood units using omics technologies. OBJECTIVE: Compare the lipid and metabolic profiles of canine packed RBC (pRBC) units as a function of LR in fresh and stored refrigerated (up to 42 days) units. ANIMALS: Packed RBC units were obtained from 8 donor dogs enrolled at 2 different Italian veterinary blood banks. STUDY DESIGN AND METHODS: Observational study. A volume of 450 mL of whole blood was collected using Citrate-Phosphate-Dextrose-Saline-Adenine-Glucose-Mannitol (CPD-SAGM) transfusion bags with a LR filter to produce 2 pRBC units for each donor, without (nLR-pRBC) and with (LR-pRBC) LR. Units were stored in the blood bank at 4 ± 2°C. Sterile weekly samples were obtained from each unit for omics analyses. RESULTS: A significant effect of LR on fresh and stored RBC metabolic phenotypes was observed. The nLR-pRBC were characterized by higher concentrations of free short and medium-chain fatty acids, carboxylic acids (pyruvate, lactate), and amino acids (arginine, cystine). The LR-pRBC had higher concentrations of glycolytic metabolites, high energy phosphate compounds (adenosine triphosphate [ATP]), and antioxidant metabolites (pentose phosphate, total glutathione). CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction decreases the metabolic SLs of canine pRBC by preserving energy metabolism and preventing oxidative lesions.

Effect of leukoreduction on the metabolism of equine packed red blood cells during refrigerated storage.

BACKGROUND: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited. OBJECTIVE: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC). ANIMALS: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks. METHODS: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses. RESULTS: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC. CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC.

Influence of a cell salvage washing system and leukocyte reduction filtration on bacterial contamination of canine whole blood ex vivo.

OBJECTIVE: To determine the ability of cell salvage washing and leukoreduction filtration to remove bacterial contamination from canine whole blood. STUDY DESIGN: Ex vivo nested cohort study. SAMPLE POPULATION: Commercially purchased fresh canine whole blood (n = 33 units). METHODS: Commercially obtained canine whole blood was inoculated with known concentrations of one of three species of bacteria, Escherichia coli (ATCC 25922), Staphylococcus pseudintermedius (quality control strain; Texas A&M University), or Pseudomonas aeruginosa (ATCC 27853). Negative controls were inoculated with sterile saline. The inoculated blood was processed through a cell salvage system and filtered through a series of two leukocyte reduction filters. Samples were aseptically collected at five points during processing (inoculum, prewash, postwash, post-first filtration, and post-second filtration) for bacterial enumeration. RESULTS: Bacterial concentrations were reduced by 85.2%, 91.5%, and 93.9% for E coli, S pseudintermedius, and P aeruginosa, respectively, after washing (P < .0001), and bacterial concentrations were reduced by 99.9%, 100%, and 100%, respectively, after the first filtration (P < .0001). After the second filtration, none of the three species of bacteria could be isolated (100% reduction). No bacterial growth was obtained from negative controls throughout the study. The type of bacteria (P = .29) did not allow prediction of bacterial reduction. CONCLUSION: Cell salvage washing combined with leukoreduction filtration eliminated bacterial contamination of whole dog blood (P < .0001). CLINICAL SIGNIFICANCE: Cell salvage washing and leukoreduction filtration could be applied to intraoperative autotransfusion in clinical animals, especially those treated for trauma or hemorrhage with concurrent bacterial contamination.

Performance of a Nageotte hemocytometer method and a flow cytometric assay for residual leukocyte quantification in leukoreduced canine packed red blood cells.

OBJECTIVE: To evaluate the performances of a manual Nageotte hemocytometer method and commercial fluorescent bead-based flow cytometric assay for quantifying [rWBC] in leukoreduced canine packed red blood cell (pRBC) units. DESIGN: Prospective study. Five, commercially purchased, double leukoreduced canine pRBC units were spiked with canine leukocytes to create 6 pRBC standards with the following [rWBC]: < 0.1, 0.375, 1.5, 3.0, 6.0, and 24.0 WBC/µL. [rWBC] of each pRBC standard was measured with the Nageotte hemocytometer and flow cytometric techniques. Limit of detection (LoD), linearity, and bias were determined for each method. For each standard, accuracy and precision were calculated; the cumulative accuracy and mean precision for measurements between the LoD and 24.0 WBC/µL were also determined. SETTING: University veterinary blood bank and clinical pathology laboratory. MEASUREMENTS AND MAIN RESULTS: The Nageotte hemocytometer method had an LoD = 1.48 WBC/µL, inadequate linearity (R2  = 0.92), and a significant negative proportional bias (slope best-fit line = 0.52 ± 0.03). Between [rWBC] 1.5-24 WBC/µL, the technique demonstrated poor cumulative accuracy (6.7%) but acceptable mean precision (17.3%). Relative to a 2 rWBC/µL threshold, at 1.5 WBC/µL the method was inaccurate (6.7%) with acceptable precision (16.6%). The flow cytometric assay had an LoD = 1.3 WBC/µL, acceptable linearity (R2  = 0.99), and a mild positive proportional bias (slope best-fit line = 1.11 ± 0.01). The technique had acceptable cumulative accuracy (80%) and mean precision (10.7%) for measuring [rWBC] between 1.5 and 24 WBC/µL. At 1.5 WBC/µL, this method was acceptably accurate (86.7%) and precise (16.0%). CONCLUSIONS: The flow cytometric assay demonstrated acceptable performance for quantification of [rWBC] in leukoreduced canine pRBC units. The Nageotte hemocytometer method should be used cautiously due to poor accuracy and significant negative bias.

Effects of irradiation and leukoreduction on down-regulation of CXCL-8 and storage lesion in stored canine whole blood.

White blood cells (WBCs) and storage period are the main factors of transfusion reactions. In the present study, cytokine/chemokine concentrations after leukoreduction (LR) and irradiation (IR) in stored canine whole blood were measured. Red blood cell storage lesion caused by IR and LR were also compared. Blood samples from 10 healthy Beagles were divided into four groups (no treatment, LR-, IR-, and LR + IR-treated). Leukocytes were removed by filtration in the LR group and gamma radiation (25 Gy) was applied in the IR group. Immunologic factors (WBCs, interleukin-6 [IL-6], C-X-C motif chemokine ligand 8 [CXCL-8], and tumor necrosis factor-alpha) and storage lesion factors (blood pH, potassium, and hemolysis) were evaluated on storage days 0, 7, 14, 21, and 28. Compared to the treated groups, IL-6 and CXCL-8 concentrations during storage were significantly higher in the control (no treatment) group. LR did not show changes in cytokine/chemokine concentrations, and storage lesion presence was relatively mild. IR significantly increased CXCL-8 after 14 days of storage, but IR of leukoreduced blood did not increase CXCL-8 during 28 days of storage. Storage lesions such as hemolysis, increased potassium, and low pH were observed 7 days after IR and storage of blood, regardless of LR. IR of leukoreduced blood is beneficial to avoid immune reactions; however, storage lesions should be considered upon storage.

Removal of hemangiosarcoma cells from canine blood with a cell salvage system and leukocyte reduction filter.

OBJECTIVE: To determine the ability of an intraoperative cell salvage (IOCS) system and a leukocyte reduction filter (LRF) to remove hemangiosarcoma (HSA) cells from canine blood. STUDY DESIGN: Cultured HSA cells were added to canine blood to simulate intraoperative hemorrhage and address hemoabdomen from ruptured splenic HSA. The blood/HSA cell mixture was processed through an IOCS, followed by LRF processing. SAMPLE POPULATION: Whole blood from 3 healthy dogs combined with cultured HSA cells. METHODS: The ability of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), multiparameter flow cytometry, and cytologic examination to detect 50 HSA cells per milliliter of culture media was confirmed. RT-PCR, multiparameter flow cytometry, and cytologic examination were used to determine the presence of cultured HSA cells at 4 points during processing. RESULTS: HSA cells were found in all control samples and in all samples after IOCS but prior to LRF processing with all 3 cell detection methods. HSA cells were not found after IOCS/LRF processing with all 3 cell detection methods. CONCLUSION: IOCS combined with LRF processing is able to remove cultured HSA cells from canine blood. The addition of LRF to IOCS may allow application of IOCS in dogs with HSA. CLINICAL SIGNIFICANCE: A combination of IOCS and LRF processing may provide an alternative to allogeneic blood transfusion in dogs with hemoabdomen due to HSA.

Effects of Leukoreduction and Storage on Erythrocyte Phosphatidylserine Expression and Eicosanoid Concentrations in Units of Canine Packed Red Blood Cells.

BACKGROUND: Storage of canine packed red blood cells (pRBCs) can increase erythrocyte phosphatidylserine (PS) expression and eicosanoid concentrations. HYPOTHESIS/OBJECTIVES: To determine the effects of leukoreduction on erythrocyte PS expression and eicosanoid concentrations in stored units of canine pRBCs. Our hypothesis was that leukoreduction would decrease PS expression and eicosanoid concentrations. ANIMALS: Eight healthy dogs. METHODS: In a cross-over study, units of whole blood were leukoreduced (LR) or non-LR and stored (10 and 21 days) as pRBCs. Samples were collected at donation, and before and after a simulated transfusion. PS expression was measured by flow cytometry, and concentrations of arachidonic acid (AA), prostaglandin F2α (PGF2α ), prostaglandin E2 (PGE2 ), prostaglandin D2 (PGD2 ), thromboxane B2 (TXB2 ), 6-keto-prostaglandin F1α (6-keto-PGF1α ), and leukotriene B4 (LTB4 ) were quantified by liquid chromatography-mass spectrometry. RESULTS: There was no change in PS expression during leukoreduction, storage, and simulated transfusion for non-LR and LR units. Immediately after leukoreduction, there was a significant increase in TXB2 and PGF2α concentrations, but during storage, these eicosanoids decreased to non-LR concentrations. In both LR and non-LR units, 6-keto-PGF1α concentrations increased during storage and simulated transfusion, but there was no difference between unit type. There was no difference in AA, LTB4 , PGE2 , and PGD2 concentrations between unit types. CONCLUSIONS AND CLINICAL IMPORTANCE: Leukoreduction, storage, and simulated transfusion do not alter erythrocyte PS expression. Leukoreduction causes an immediate increase in concentrations of TXB2 and PGF2α , but concentrations decrease to non-LR concentrations with storage. Leukoreduction does not decrease the accumulation of 6-keto-PGF1α during storage.

Procoagulant phospholipid concentration in canine erythrocyte concentrates stored with or without prestorage leukoreduction.

OBJECTIVE: To evaluate canine erythrocyte concentrates (ECs) for the presence of procoagulant phospholipid (PPL), determine whether PPL concentration changes during the course of storage of ECs, and ascertain whether prestorage leukoreduction (removal of leukocytes via gravity filtration) reduces the development of PPL. SAMPLE: 10 whole blood units (420 g each) collected from 10 random-source, clinically normal dogs (1 U/dog). PROCEDURES: The dogs were randomized to 1 of 2 groups. Of the 10 whole blood units collected, 5 were processed through a standard method, and 5 underwent leukoreduction. Whole blood units were processed to generate ECs, from which aliquots were aseptically collected from each unit weekly for 5 weeks. Supernatants from the concentrates were evaluated for procoagulant activity, which was converted to PPL concentration, by use of an automated assay and by measurement of real-time thrombin generation. RESULTS: Supernatants from stored canine ECs contained procoagulant activity as measured by both assays. In general, the PPL concentration gradually increased during the storage period, but leukoreduction reduced the development of increased procoagulant activity over time. CONCLUSIONS AND CLINICAL RELEVANCE: The presence of PPL in canine ECs may be associated with procoagulant and proinflammatory effects in vivo, which could have adverse consequences for dogs treated with ECs.

Effects of four additive solutions on canine leukoreduced red cell concentrate quality during storage.

BACKGROUND: Additive solutions (AS) and prestorage leukoreduction (LR) are important tools used to maintain erythrocyte viability during storage and avoid transfusion reactions in recipients, respectively. OBJECTIVES: The purpose of the study was to determine the efficacy of a WBC filter (Immugard IIIRC) and compare the effect of 4 AS (phosphate-adenine-glucose-guanosine-gluconate-mannitol [PAGGGM], saline-adenine-glucose-mannitol [SAGM], Adsol, Optisol) on the in vitro quality of canine leukoreduced packed RBC units (pRBC) stored for 41 days. METHODS: Five hundred milliliters of blood were collected from 8 healthy dogs each into 70 mL of citrate-phosphate-dextrose (CPD) solution, and were leukoreduced by a polyurethane filter. pRBC of each dog were divided equally into 4 bags containing a different AS. Bags were stored for 41 days at 4°C and evaluated every 10 days. Variables analyzed included pH, PCV, and% hemolysis, and lactate, glucose, potassium, sodium, ATP, and 2,3-diphosphoglycerate (2,3-DPG) concentrations. RESULTS: The LR resulted in residual WBC counts comparable to human standards. During storage, pH, and glucose, 2,3-DPG, and ATP concentrations decreased, and hemolysis, and lactate, sodium, and potassium concentrations increased (P < .05). Significant differences between AS were seen in the glucose and sodium concentrations, due to the composition of AS. Also, the pH maintained by PAGGGM at day 21 was significantly higher than that seen with SAGM or Adsol. CONCLUSIONS: All AS used gave satisfactory results during the first 21 days of storage based on the degree of hemolysis, and on ATP and 2,3-DPG concentrations. When compared with day 1 values, significant changes were seen in these variables by day 31 with all AS.

Microparticles in stored canine RBC concentrates.

BACKGROUND: Transfusion of RBC concentrates may cause adverse effects in the recipient, particularly when stored > 2 weeks. Prestorage removal of WBCs and platelets (leukoreduction, LR) improves clinical outcome in the human recipient. As blood ages during storage, progressive alterations in the structure and function of the cells occur. Changes in cell membranes may lead to formation of microparticles (MPs) in stored blood. OBJECTIVES: The aim of the study was to quantify MP concentration in supernatants from canine RBC concentrates from 11 clinically healthy dogs. METHODS: Whole blood units (n = 11) were collected and randomized either to be stored without LR (n = 5), or to be subject to prestorage LR (n = 6). Whole blood was processed for the generation of RBC concentrates, from which aliquots were aseptically collected weekly for 5 weeks. Supernatants from the concentrates were evaluated for phosphatidylserine-expressing MPs by flow cytometry using staining with Annexin-V-phycoerythrin. RESULTS: Microparticle counts were similar between non-LR and LR units on storage days 0 and 7, but were significantly higher in non-LR units on days 14, 21, 28, and 35. MPs increased during the 35-day storage by a mean (SD) of 1.8 (1.4)-fold in LR units and 5.5 (3.1)-fold in non-LR units. CONCLUSIONS: There was marked formation of phosphatidylserine-expressing MPs during storage beyond 7 days in canine RBC concentrates. Prestorage LR attenuated the generation of MPs.

Effect of leukoreduction treatment on vascular endothelial growth factor concentration in stored canine blood transfusion products.

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) concentrations in canine blood products treated with or without a leukoreduction filter. SAMPLE: 10 canine blood donors. PROCEDURES: Dogs underwent blood collection. Five of 10 units were leukoreduced prior to separation into packed RBCs and fresh frozen plasma (FFP). Concentrations of VEGF were measured by ELISA in plasma supernatants from aliquots of packed RBCs obtained immediately after separation and on days 7, 14, and 21 of storage. Fresh frozen plasma samples of 2 filtered and 2 nonfiltered units were examined after storage. RESULTS: RBC counts in whole blood before and after leukoreduction did not differ significantly, but WBCs and platelets were removed effectively. The VEGF concentration was lower than the detection limit (9 pg/mL) in 9 of 10 plasma samples and in all packed RBC and FFP units immediately after separation. The median VEGF concentrations in 5 nonfiltered packed RBC units were 37, 164, and 110 pg/mL on days 7, 14, and 21 of storage, respectively. In 5 filtered packed RBC and all FFP units, VEGF concentrations remained lower than the detection limit. CONCLUSIONS AND CLINICAL RELEVANCE: Leukoreduction filters were effective in preventing the release of VEGF during storage of canine RBC products.

Effect of leukoreduction on transfusion-induced inflammation in dogs.

BACKGROUND: Removal of leukocytes (LR) has been shown to eliminate or attenuate many of the adverse effects of transfusion in experimental animals and humans. HYPOTHESIS/OBJECTIVES: Transfusion of stored packed red blood cells (pRBCs) is associated with an inflammatory response in dogs and prestorage LR attenuates the inflammatory response. ANIMALS: Thirteen random-source, clinically healthy, medium and large breed dogs. METHODS: Experimental study. On day 0, animals were examined and baseline blood samples were collected for analysis. Whole blood was then collected for processing with and without LR, and stored as pRBC. Twenty-one days later, stored pRBCs were transfused back to the donor. Blood samples were collected before and 1 and 3 days after transfusion. RESULTS: In the dogs that received non-LR pRBCs (n = 6) there was a significant increase from baseline in white blood cell count from a mean (SD) of 8.20 (2.74) to 13.95 (4.60) × 10(3) cells/µL (P < .001) and in segmented neutrophil count from a mean (SD) of 5.76 (2.70) to 11.91 (4.71) × 10(3) cells/µL (P < .001). There were also significant increases in fibrinogen from a mean (SD) of 129.7 (24.2) to 268.6 (46.7) mg/dL (P < .001) and C-reactive protein from a mean (SD) of 1.9 (2.1) to 78.3 (39.3) µg/mL (P < .001). There was no significant increase from baseline in any of the markers in the dogs that received LR pRBC (n = 5). CONCLUSIONS AND CLINICAL IMPORTANCE: There is a profound inflammatory response to transfusion in normal dogs, which is eliminated by LR of the pRBC units.