Single-Pass System to Characterize the Effects of Blood Species and Hematocrit on Flow-Induced Hemolysis.

One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10−4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way.

The nonphysiological mechanical shear stress in blood-contacting medical devices is one major factor to device-induced blood damage. Animal blood is often used to test device-induced blood damage potential of these devices due to its easy accessibility and low cost. However, the differences in shear-induced blood damage between animals and human have not been well characterized. The purpose of this study was to investigate shear-induced hemolysis of human and three commonly used preclinical evaluation animal species (ovine, porcine, and bovine) under shear conditions encountered in blood-contacting medical devices. Shear-induced hemolysis experiments were conducted using two single-pass blood-shearing devices. Driven by an externally pressurized reservoir, blood single-passes through a small annular gap in the shearing devices where the blood was exposed to a uniform high shear stress. Shear-induced hemolysis at different conditions of exposure time (0.04 to 1.5 s) and shear stress (25 to 320 Pa) was quantified for ovine, porcine, bovine, and human blood, respectively. Within these ranges of shear stress and exposure time, shear-induced hemolysis was less than 2% for the four species. The results showed that the ovine blood was more susceptible to shear-induced injury than the bovine, porcine, and human blood. The response of the porcine and bovine blood to shear was similar to the human blood. The dependence of hemolysis on shear stress level and exposure time was found to fit well the power law functional form for the four species. The coefficients of the power law models for the ovine, porcine, bovine, and human blood were derived.

The common complications in heart failure patients with implanted ventricular assist devices (VADs) include hemolysis, thrombosis, and bleeding. These are linked to shear stress-induced trauma to erythrocytes, platelets, and von Willebrand factor (vWF). Novel device designs are being developed to reduce the blood trauma, which will need to undergo in vitro and in vivo preclinical testing in large animal models such as cattle, sheep, and pig. To fully understand the impact of device design and enable translation of preclinical results, it is important to identify any potential species-specific differences in the VAD-associated common complications. Therefore, the purpose of this study was to evaluate the effects of shear stress on cells and proteins in bovine, ovine, and porcine blood compared to human. Blood from different species was subjected to various shear rates (0-8000/s) using a rheometer. It was then analyzed for complete blood counts, hemolysis by the Harboe assay, platelet activation by flow cytometry, vWF structure by immunoblotting, and function by collagen binding activity ELISA (vWF:CBA). Overall, increasing shear rate caused increased total blood trauma in all tested species. This analysis revealed species-specific differences in shear-induced hemolysis, platelet activation, and vWF structure and function. Compared to human blood, porcine blood was the most resilient and showed less hemolysis, similar blood counts, but less platelet activation and less vWF damage in response to shear. Compared to human blood, sheared bovine blood showed less hemolysis, similar blood cell counts, greater platelet activation, and similar degradation of vWF structure, but less impact on its activity in response to shear. The shear-induced effect on ovine blood depended on whether the blood was collected via gravity at the abattoir or by venepuncture from live sheep. Overall, ovine abattoir blood was the least resilient in response to shear and bovine blood was the most similar to human blood. These results lay the foundations for developing blood trauma evaluation standards to enable the extrapolation of in vitro and in vivo animal data to predict safety and biocompatibility of blood-handling medical devices in humans. We advise using ovine venepuncture blood instead of ovine abattoir blood due to the greater overall damage in the latter. We propose using bovine blood for total blood damage in vitro device evaluation but multiple species could be used to create a full understanding of the complication risk profile of new devices. Further, this study highlights that choice of antibody clone for evaluating platelet activation in bovine blood can influence the interpretation of results from different studies.

We investigated the applicability of measuring the viscoelasticity of bovine, ovine, and porcine whole blood for the evaluation of sublethal damage to red blood cells (RBCs). An increase in blood viscosity and elasticity without changes in hematocrit and plasma viscosity would signify a decrease in RBC deformability. Blood viscoelasticity was assessed using a Vilastic Scientific viscoelastometer. Due to the natural absence of RBC aggregation and small RBC size in normal bovine and ovine blood, viscoelastic properties are less readily detected. However, we found that adjustment of blood hematocrit to a standard level of 40-50% allows for sensitive assessment of viscoelasticity in these blood types demonstrating a marked non-Newtonian behavior mostly related to RBC deformability. Porcine blood showed a pronounced non-Newtonian behavior at all tested hematocrit values, which makes it rheologically comparable to human blood. Both viscosity and elasticity were elevated after blood exposure to a uniform mechanical stress. RBCs rigidified by heat exposure demonstrated a loss of viscoelasticity dependence on shear rate. Measurements of blood viscoelasticity can be meaningful in bovine, ovine, and, especially, porcine blood, and can be used for evaluation of sublethal blood damage during in vitro and animal trials of heart-assist devices.

The reproductive performances were compared of adult stable flies, Stomoxys Calcitrans (L.), fed fresh porcine or bovine blood or reconstituted, freeze-dried porcine or bovine blood. Adult stable flies fed fresh or reconstituted, freeze-dried porcine blood oviposited significantly more eggs than adults fed fresh or freeze-dried bovine blood. No decline in fecundity was observed for adults maintained for 10 successive generations on fresh porcine blood or on reconstituted, freeze-dried porcine or bovine blood. However, fecundity of adults maintained on fresh, heparinized bovine blood declined in later generations. The data provide evidence that stable fly adults can be maintained successfully on reconstituted, freeze-dried blood, and that porcine blood is nutritionally superior to the blood of cattle.

Changes in porcine, ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

To determine suitable alternatives to human blood for in vitro dynamic thrombogenicity testing of biomaterials, four different animal blood sources (ovine, bovine, and porcine blood from live donors, and abattoir porcine blood) were compared to fresh human blood. To account for blood coagulability differences between individual donors and species, each blood pool was heparinized to a donor-specific concentration immediately before testing in a dynamic flow loop system. The target heparin level was established using a static thrombosis pre-test. For dynamic testing, whole blood was recirculated at room temperature for 1h at 200 ml/min through a flow loop containing a single test material. Four materials with varying thrombotic potentials were investigated: latex (positive control), polytetrafluoroethylene (PTFE) (negative control), silicone (intermediate thrombotic potential), and high-density polyethylene (HDPE) (historically thromboresistant). Thrombus weight and surface area coverage on the test materials were quantified, along with platelet count reduction in the blood. While donor-specific heparin levels varied substantially from 0.6U/ml to 7.0U/ml among the different blood sources, each source was able to differentiate between the thrombogenic latex and the thromboresistant PTFE and HDPE materials (p < 0.05). However, only donor ovine and bovine blood were sensitive enough to differentiate an increased response for the intermediate thrombotic silicone material compared to PTFE and HDPE. These results demonstrated that multiple animal blood sources (particularly donor ovine and bovine blood) may be suitable alternatives to fresh human blood for dynamic thrombogenicity testing when appropriate control materials and donor-specific anticoagulation levels are used.

Competitive Enzyme-Linked Immunosorbent Assay for Quantitative Detection of Bovine Blood in Heat-Processed Meat and Feed

Ornithodoros moubata: Breeding in vitro

An amino acid analysis of freeze-dried bovine and porcine blood used for In vitro rearing of Glossina palpalis palpalis (ROB.-DESV.)

Background: Since sourcing porcine blood donors for emergent transfusions to porcine patients is difficult, bovine or canine blood donors might represent alternative sources. The primary objective of this study was to determine the frequency of incompatible major (CMMa) and minor (CMMi) crossmatches by the standard saline agglutination tube method (SSA) between bovine whole blood (bWB) and whole blood from commercial pigs (pWB), and canine universal donor whole blood or commercially-prepared packed red blood cells (pRBCs) with whole blood from companion pigs. A secondary objective was determining the agreement between the reference method (SSA) and a quick slide (QS) method. Methods: Blood was collected from 12 heifers, seven companion pigs, and eight commercial-cross pigs. A0 blood typing was performed for all porcine samples. Bovine blood was pooled into eight bags each containing three crossmatch-compatible individuals. Canine blood included whole blood from three canine blood donors (DEA 1.1, 5, 7 negative, and DEA 4 positive), and three bags each of DEA 1.1 negative and DEA 1.1 positive pRBCs. Crossmatch pairs were performed for bovine-to-porcine (n = 64) and canine-to-porcine (n = 63) samples. Incompatibility was defined as any agglutination or hemolysis on either CMMa and CMMi and reported separately. Complete incompatibility was defined as incompatibility of both CMMa and CMMi on the same pair. Kappa statistics tested the agreement between SSA and QS (significance at P < 0.05). Results: For bWB and pWB, agglutination was observed in 9.4% of CMMa and 100% of CMMi via SSA. Incompatibility on CMMa of bWB was more frequent with porcine blood type "0" (P = 0.0107) than with type "A", whereas porcine blood group had no effect on CMMi results. All canine-to-porcine CMMa were incompatible with SSA and showed hemolysis severe enough to prevent evaluation of agglutination. The accuracy of QS at detecting incompatibilities was 87.5% in CMMa and 98.4% in CMMi in bovine-to-porcine samples. Agreement between SSA and QS methods was fair (k = 0.36) for bovine-to-porcine CMMa but could not be calculated for CMMi due to lack of compatible matches. Because all canine-to-porcine CMMa were incompatible, the effects of the porcine blood group on incompatibility, accuracy of QS, and agreement between SSA and QS could not be calculated for CMMa. For CMMi, the agreement between tests was poor (k = 0). Discussion: When a xenotransfusion to a pig is indicated, bWB appears to be suitable based on in vitro CMMa testing, whereas canine blood products are contraindicated for in vivo administration to swine based on absolute CMMa incompatibility and incidence of hemolysis. In vivo studies are needed to elucidate the clinical significance of CMMi incompatibilities. Based on these results, QS cannot be accurately used as a surrogate of SSA in pretransfusion testing for porcine patients due to the increased risk of false compatible results as QS can only be identified as agglutination, not hemolysis.

An improved freeze-dried blood diet for membrane feeding of glossina palpalis palpalis

Whole bovine blood use in forensic research: Sample preparation and storage considerations

Background: This study was conducted to confirm the reliability of an in vitro mechanically induced hemolysis test (ISO 10993-4:2017), which is essential for ensuring the safety of blood pumps. Methods: For appropriate anticoagulant selection, porcine blood was prepared in anticoagulant citrate dextrose solution A (ACD-A), heparin, and citrate phosphate dextrose adenine (CPDA-1), respectively, according to the ASTM F1830 standard. Anticoagulant-treated porcine and bovine blood were circulated in a mock circulatory loop (MCL) for 6 h to observe the rate of plasma-free hemoglobin (pfHb) and RBCs with morphological integrity. Results: A morphological loss of red blood cells (RBCs) was observed over time. While there were differences in morphological loss depending on the anticoagulant, no consistent trend could be identified. The pfHb concentration was significantly higher in bovine than in porcine blood. Conversely, the number of RBCs with morphological integrity decreased over time in both, but the ratio of RBCs with morphological integrity was similar across all timepoints. Conclusions: The percentage of RBCs with morphological integrity can be used as a reliable indicator for the interpretation of mechanically induced hemolysis results in different blood types. Furthermore, the reliability of the in vitro mechanically induced hemolysis test (ISO 10993-4:2017) was assessed.

Hematological, chemical and functional characteristics of porcine, chicken and duck blood were evaluated. A porcine blood sample showed the most abundant red blood cell, hemoglobin concentration, packed cell volume and plasma protein content as well as its freeze-dried blood possessed the highest contents of protein, fat, Cu and Cr with the highest percentage of heme iron (p<0.05). Unlike porcine blood, chicken blood showed a well balance in some essential amino acids, specifically for a higher isoleucine content (p<0.05). Furthermore, it possessed the highest contents of carbohydrate, Zn and non-heme iron (p<0.05). The most rapid response to form a strong gel, especially at 70°C and 80°C, was found in chicken blood, followed by duck and porcine blood, respectively. The result of emulsion activity index (EAI) and emulsion stability index (ESI) at the low protein concentration indicated that chicken blood had the most superior emulsion properties (p<0.05). Regarding duck blood, it exhibited the highest content of Mg and Mn (p<0.05). Moreover, duck blood had similar foaming properties to porcine blood in which they showed higher values than chicken blood (p<0.05). Specific characteristics of blood were therefore diminished by animal species in which this information could be used as food supplementation or product development based on their potential applications.