Canine Red Blood Cells Research Articles

Universal peptide-based potential vaccine design against canine distemper virus (CDV) using a vaccinomic approach

Canine distemper virus (CDV) affects many domestic and wild animals. Variations among CDV genome linages could lead to vaccination failure. To date, there are several vaccine alternatives, such as a modified live virus and a recombinant vaccine; however, most of these alternatives are based on the ancestral strain Onderstepoort, which has not been circulating for years. Vaccine failures and the need to update vaccines have been widely discussed, and the development of new vaccine candidates is necessary to reduce circulation and mortality. Current vaccination alternatives cannot be used in wildlife animals due to the lack of safety data for most of the species, in addition to the insufficient immune response against circulating strains worldwide in domestic species. Computational tools, including peptide-based therapies, have become essential for developing new-generation vaccines for diverse models. In this work, a peptide-based vaccine candidate with a peptide library derived from CDV H and F protein consensus sequences was constructed employing computational tools. The molecular docking and dynamics of the selected peptides with canine MHC-I and MHC-II and with TLR-2 and TLR-4 were evaluated. In silico safety was assayed through determination of antigenicity, allergenicity, toxicity potential, and homologous canine peptides. Additionally, in vitro safety was also evaluated through cytotoxicity in cell lines and canine peripheral blood mononuclear cells (cPBMCs) and through a hemolysis potential assay using canine red blood cells. A multiepitope CDV polypeptide was constructed, synthetized, and evaluated in silico and in vitro by employing the most promising peptides for comparison with single CDV immunogenic peptides. Our findings suggest that predicting immunogenic CDV peptides derived from most antigenic CDV proteins could aid in the development of new vaccine candidates, such as multiple single CDV peptides and multiepitope CDV polypeptides, that are safe in vitro and optimized in silico. In vivo studies are being conducted to validate potential vaccines that may be effective in preventing CDV infection in domestic and wild animals.

Antifungal activity of protein hydrolysates from Thai Phatthalung Sangyod rice (Oryza sativa L.) seeds.

Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice (Oryza sativa L.) is exclusively indigenous to Thailand's Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides. Antifungal activity in four Sangyod rice seed extracts (SYPs) - namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 - was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin-Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs - SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs]). The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract - SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoformans, Talaromyces marneffei yeast phase, Trichophyton mentagrophytes, and Trichophyton rubrum. A partial inhibitory action on Candida albicans and Microsporum gypseum was possessed while showing the least activity to C. neoformans. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications. These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods.

Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.

Biologically active water complex “HALPI” effect in dermatological conditions in canine red blood cell values

This article presents an effect study of the biologically active water complex “HALPI” in dermatological conditions in canine red blood cell values. Biologically active water complex "HALPI" is produced according to the original recipe invented by the development Director of OOO "HALPI RUS", Vladimir Protasov. The prescription composition of the biologically active water complex "HALPI", in strict proportion includes components involved in the process of energy exchange in the cellular system of energy formation. The study was conducted on two groups of dogs with dermatological conditions (focal alopecia, erythema, pruritus): the experimental group was formed from dogs who were drinking biologically active water complex "HALPI" daily for 21 days and the control group, selected by the method of pairs of analogues. In the stabilized blood of both groups animals, such indicators as the number of red blood cells, hemoglobin level and hematocrit were determined. As a result of the analysis of the obtained data, it was revealed that the use of this biologically active water complex leads to normalization of the hydration status. Thus, the use of biologically active water complex "HALPI" can improve the state of metabolic processes by compensating for possible deficiencies of vitamins and trace elements, which contributes to improving the life quality of dogs with dermatological disorders.

Effect of washing units of canine red blood cells on storage lesions.

BackgroundIn humans, washing stored blood products before transfusion reduces storage lesions and incidence of transfusion reactions, but the effectiveness of washing canine blood is unknown.ObjectivesThe objective was to determine if manually washing units of stored blood would reduce storage lesions without adversely affecting erythrocytes. We hypothesized that washing stored units would reduce concentrations of storage lesions and cause minimal erythrocyte damage.AnimalsEight healthy research dogs.MethodsRepeated measure cohort study. Units of whole blood were stored for 28 days and washed 3 times with 0.9% NaCl. Blood samples were collected before and after storage, after each wash, and after being held at a simulated transfusion temperature. Variables measured included CBC variables, blood gas analysis, erythrocyte morphology, mean corpuscular fragility (MCF), and eicosanoid concentrations. A Friedman's test was used to evaluate changes in variables (P < .05 was considered significant).ResultsAfter the first wash, compared to values after storage, there was a significant decrease in potassium (4.3 mmol/L [4.0‐4.7] to 1.2 mmol/L [1‐1.6]; P < .0001, median [range]), lactate (1.45 mmol/L [1.07‐1.79] to 0.69 mmol/L [0.39‐0.93]; P = .002), and partial pressure carbon dioxide (102 mm Hg [80.2‐119.2] to 33.7 mm Hg [24.5‐44.5]; P < .0001), and increase in MCV (69.3 fL [65.7‐72.3] to 74 fL [69.6‐79.5]; P = .0003), and MCF (0.444 fL [0.279‐0.527] to 0.491 fL [0.43‐0.616]; P = .0006).Conclusions and Clinical ImportanceA single wash of stored whole blood significantly reduces most extracellular storage lesions, and additional washing might cause hemolysis.

Expression and biological functions of Ancylostoma ceylanicum saposin-like protein.

Ancylostoma ceylanicum is a common zoonotic nematode that inhabits the small intestine of humans, dogs, and cats. Saposin-like proteins (SLPs) have hemolytic and antibacterial activities and could be used as diagnostic or vaccine candidates. To explore the biological functions of Ancylostoma ceylanicum SLP (Ace-SLP-1), cDNA-encoding Ace-SLP-1 mature peptide was cloned into prokaryotic expression vector pET-28a and transformed into Escherichia coli BL21 (DE3) to induce expression. After incubation of canine red blood cell suspension with different concentrations of recombinant Ace-SLP-1, the supernatant was separated to measure OD value and calculate the hemolysis rate. The different concentrations of recombinant protein were co-cultured with E. coli and Enterococcus faecalis, and colony-forming units (CFU) were determined by the plate counting method. Peripheral blood mononuclear cells (PBMCs) from healthy dogs were incubated with different concentrations of recombinant Ace-SLP-1, and the cytokine expression was evaluated by relative quantitative PCR. Our results showed that the hemolytic activity of Ace-SLP-1 increased with the increase in protein concentration from 25 to 100μg/mL. The recombinant protein had no antibacterial activity against the two kinds of bacteria but could stimulate the secretion of cytokines (IL-4, IL-10, IL-12, and IL-13) in canine PBMCs. These data suggest that Ace-SLP-1 is involved in hookworm blood-feeding and survival and has good immunogenicity, supporting its potential as a diagnostic and vaccine target molecule.

Transfusion practice in Australia: an internet-based survey.

To describe small animal transfusion practices in Australia, including access to blood products and frequency of pre-transfusion compatibility testing and medication administration. An online survey was disseminated to target Australian veterinarians treating dogs and cats. Information collected included demographics, sources of blood products, blood storage, recipient compatibility testing and administration of medications pre-transfusion. Associations between the use of compatibility tests and premedications were assessed using the χ2 test. Significance was set at P < 0.05. A total of 199 Australian veterinarians were included; however, there was some attrition of respondents over the course of the survey. The majority of respondents were in general practice (n = 133/199). Access to fresh whole blood was commonly reported for dogs (n = 179/199) and cats (n = 131/198), whereas blood components were less commonly available (canine red blood cells [RBC], n = 52/199 and plasma, n = 157/199; feline RBC, n = 9/198 and plasma, n = 21/198). Most blood was sourced from the pets of owners affiliated with the veterinary clinic (n = 179/196). The respondents who did not blood type or crossmatch dogs were significantly more likely to use premedication than those who did these tests (both comparisons: P < 0.001). Likewise, the respondents who did not blood-type cats were significantly more likely to use premedication (P = 0.003); however, there was no association between crossmatching and using premedication in cats (P = 0.183). This is the first survey to describe transfusion practices across a variety of practice types throughout Australia. Future work is needed to determine how representative these results are of current transfusion practices across Australia, and if so, what can be done to optimise them.

Neutrophil extracellular traps in stored canine red blood cell units.

BackgroundNeutrophil extracellular traps (NETs), webs of DNA and citrullinated histones extruded from activated neutrophils cause transfusion‐related acute lung injury. Supernatants of stored red blood cell (RBC) units might promote NETosis in neutrophils from the units or from transfusion recipients.Hypotheses(1) NETs form during storage of canine RBC, (2) leukoreduction (LR) before storage of RBC reduces NETosis, and (3) supernatant from stored, nonleukoreduced (NLR) RBC units induces NETosis in healthy canine neutrophils modeling transfusion recipients.AnimalsSix healthy purpose‐bred research dogs were utilized for blood donation.MethodsProspective controlled study. RBC units were collected from each dog, aseptically divided into 2 equal subunits, 1 of which was leukoreduced, and stored for 42 days. Stored units were sampled biweekly for quantification of NET markers citrullinated histone H3 (Western blot) and cell‐free DNA (cfDNA) (DNA dye binding). Unit supernatants were applied ex vivo to canine neutrophils and extracellular DNA release representing NETosis was assessed.ResultsMarkers of NETs increased during RBC storage (cfDNA P < .0001 and citrullinated H3 P = .0002) and were higher in NLR than LR units (day 42 LR cfDNA 0.34 ± 0.82 ng/mL vs day 42 NLR 1361.07 ± 741.00 ng/mL, P < .0001; day 42 LR citrullinated H3 0.19 ± 0.13 AU vs NLR 0.57 ± 0.34 AU, P = .007). Isolated neutrophils did not form NETs when exposed to stored canine RBC supernatant.Conclusions and Clinical ImportanceNETosis occurs in stored canine NLR RBC units, and is attenuated by LR before storage. NETs might be mediators of transfusion reactions.

Semi-supervised learning with deep convolutional generative adversarial networks for canine red blood cells morphology classification

Information of Red Blood Cell (RBC) morphology, obtained by analysing RBC images, is regularly requested by veterinarians to diagnose anaemic dogs. Machine learning techniques have been exploited to speed up the image classification. Recently, many researchers used deep learning techniques for classification; however, a large quantity of labelled data is necessary to extract performance with them. A lack of annotated data, due to time and costs for pathologist and their limited numbers, has become a difficulty. This limits the amount of annotated data and leads to a large number of unannotated data, preventing traditional deep learning algorithms from being effective. We show that a semi-supervised learning method, using the Generative Adversarial Networks (GANs) for canine RBC morphology classification, can solve the lack of labelled data, when we want to train a deep learning classifier. Our semi-supervised GAN can use both labelled and unlabelled data and showed that they can achieve the same level of performance as a traditional convolutional neural network, with a smaller number of labelled images. Furthermore, we showed that augmenting the limited numbers of a labelled images enhanced the overall performance. A key benefit of our method is reduced pathologist cost and time to annotate cell images for developing a deep learning classifier.

Convolutional neural networks based focal loss for class imbalance problem: a case study of canine red blood cells morphology classification

Morphologies of red blood cells are normally interpreted by a pathologist. It is time-consuming and laborious. Furthermore, a misclassified red blood cell morphology will lead to false disease diagnosis and improper treatment. Thus, a decent pathologist must truly be an expert in classifying red blood cell morphology. In the past decade, many approaches have been proposed for classifying human red blood cell morphology. However, those approaches have not addressed the class imbalance problem in classification. A class imbalance problem---a problem where the numbers of samples in classes are very different---is one of the problems that can lead to a biased model towards the majority class. Due to the rarity of every type of abnormal blood cell morphology, the data from the collection process are usually imbalanced. In this study, we aimed to solve this problem specifically for classification of dog red blood cell morphology by using a Convolutional Neural Network (CNN)---a well-known deep learning technique---in conjunction with a focal loss function, adept at handling class imbalance problem. The proposed technique was conducted on a well-designed framework: two different CNNs were used to verify the effectiveness of the focal loss function and the optimal hyper-parameters were determined by 5-fold cross-validation. The experimental results show that both CNNs models augmented with the focal loss function achieved higher $F_{1}$-scores, compared to the models augmented with a conventional cross-entropy loss function that does not address class imbalance problem. In other words, the focal loss function truly enabled the CNNs models to be less biased towards the majority class than the cross-entropy did in the classification task of imbalanced dog red blood cell data.

Cryopreservation of feline red blood cells in liquid nitrogen using glycerol and hydroxyethyl starch.

The objective of this study was to evaluate the techniques and short-term effects of cryopreservation of feline red blood cells (RBCs) in liquid nitrogen using glycerol or hydroxyethyl starch (HES) as a cryoprotectant. Feline RBCs were manually mixed with either 20% glycerol or 12.5% HES and frozen for 24 h in liquid nitrogen. The samples were thawed and glycerolized samples were manually washed. Success of the freeze/thaw process was determined by recovery rate of RBCs and evaluation of morphological changes using scanning electron microscopy (SEM). A unit of canine packed RBCs was also subjected to the same methodology to evaluate the cryopreservation handling technique. Feline RBCs preserved with 20% glycerol had a high recovery rate (94.23 ± 1.25%) immediately after thawing. However, the majority of the cells were lost during the washing process, with a final packed cell volume of <1%. A recovery rate was unable to be assessed for samples preserved with HES owing to the high viscosity of the mixture. SEM revealed significant morphological changes after glycerol was added to the feline RBCs. Although these morphological changes were partially reversed after thawing, the majority of the RBCs were lost during the washing process. Minimal morphological changes were noted in the HES sample. Similar results were noted with the canine RBCs. The described manual technique for cryopreservation using glycerol was not able to successfully preserve feline or canine RBCs. In the present study, it was difficult to make conclusions about the efficacy of HES. Further studies evaluating HES as a cryoprotectant are warranted.

Urine sediment examination: Potential impact of red and white blood cell counts using different sediment methods.

Centrifugation is the primary method used to perform urine sediment analyses, but evaluation of other methods is required to validate centrifugation. Non-urine materials were used to examine the repeatability (precision) and effectiveness (recovery) of four sediment methodologies on red blood cell (RBC) and white blood cell (WBC) counts. Four urine sediment methods were compared using commercially available quality control material (QCM) and fresh canine RBCs in a diluent. Treatments included (a) 5mL centrifugation at 390g for 5minutes; (b) 1.5mL centrifugation at 3900g for 45seconds; (c) 60µL of neat (unspun urine) in a microtiter well; and (d) 30µL of neat on a slide with a coverslip. A within-run precision using QCM was followed by a one-run comparison test performed with a suspension of canine erythrocytes. RBC morphology was also examined. All results are listed in order of Methods A-D. Percent coefficients of variation (%CVs) for WBCs were 23.2%, 33.7%, 15.0%, and 27.2%. Red blood cells %CVs were 34.3%, 29.2%, 16.2%, and 24.4%. Average WBC counts in ten fields of view (FOV) ± 1 SD were 26.4±6.1, 14.2±4.8, 32.8±4.9, and 1.6±0.4. Average RBC counts in 10 fields of view (FOV) ± 1 SD were 45.3±15.5, 23.9±7.0, 38.4±6.2, and 2.6±0.6. The one-run comparison test reports average RBC counts per FOV at 55.2, 23.4, 92.8, and 13.8. The percentages of abnormal RBCs were 92.2%, 74.8%, 7.0%, and 55.1%. Method C had the best reproducibility, a lower frequency of cell morphology abnormalities, and similar cellular counts to those of Methods A and B.

Impact of different standard red blood cell storage temperatures on human and canine RBC hemolysis and chromium survival.

Storage temperature is a critical factor for maintaining red-blood cell (RBC) viability, especially during prolonged cold storage. The target range of 1 to 6°C was established decades ago and may no longer be optimal for current blood-banking practices. Human and canine RBCs were collected under standard conditions and stored in precision-controlled refrigerators at 2°C, 4°C, or 6°C. During 42-day storage, human and canine RBCs showed progressive increases in supernatant non-transferrin-bound iron, cell-free hemoglobin, base deficit, and lactate levels that were overall greater at 6°C and 4°C than at 2°C. Animals transfused with 7-day-old RBCs had similar plasma cell-free hemoglobin and non-transferrin-bound iron levels at 1 to 72 hours for all three temperature conditions by chromium-51 recovery analysis. However, animals transfused with 35-day-old RBCs stored at higher temperatures developed plasma elevations in non-transferrin-bound iron and cell-free hemoglobin at 24 and 72 hours. Despite apparent impaired 35-day storage at 4°C and 6°C compared to 2°C, posttransfusion chromium-51 recovery at 24 hours was superior at higher temperatures. This finding was confounded by a preparation artifact related to an interaction between temperature and storage duration that leads to removal of fragile cells with repeated washing of the radiolabeled RBC test sample and renders the test sample unrepresentative of the stored unit. RBCs stored at the lower bounds of the temperature range are less metabolically active and produce less anaerobic acidosis and hemolysis, leading to a more suitable transfusion product. The higher refrigeration temperatures are not optimal during extended RBC storage and may confound chromium viability studies.

Freezing and storage of leukodepleted erythrocyte suspensions

Studies on the frozen storage of human blood products have benefited veterinary transfusion medicine in recent years, but the long-term cryopreservation of canine red blood cells (RBCs) has not yet been thoroughly investigated. Further, no studies are available with respect to the frozen storage of leukocyte-depleted canine red blood cells (LD-RBCs). The objective of the current study was to investigate time-dependent effects of long-term frozen storage on leukocyte-depleted canine RBCs. Twelve healthy adult dogs meeting the criteria for blood transfusion were used in the study. Whole blood samples (450 ± 45 ml) collected from each dog were centrifuged for 5 min at 22 °C and 4200 × g in a cryogenic microcentrifuge and concentrated RBC (pRBC) suspensions were obtained. Leukocyte depletion was achieved by filtration (2.6 log&lt;sub&gt;10&lt;/sub&gt;). Then, the filtrated samples were prewashed three times in 0.9% NaCl solution and were allocated into three subgroups to be evaluated at three different time points (Day 0, Month 4 and Month 6). The samples for cryopreservation were subjected to glycerolisation and then stored at –80 °C for 4- and 6-month periods. At the end of this period pRBC units were thawed by manual agitation in a water bath maintained at 36–38 °C, centrifuged and then washed in a consecutive series of 12%, 1.6% and 0.9% of NaCl + 0.2 dextrose solutions. 2,3-Diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP), supernatant haemoglobin (SupHb), sodium (Na&lt;sup&gt;+&lt;/sup&gt;) and potassium (K&lt;sup&gt;+&lt;/sup&gt;) levels, residual glycerol concentrations and haemograms of thawed and deglycerolised pRBC samples were evaluated together with those of Day 0. Sterility tests were performed on all samples for bacterial contamination. No statistically significant differences were noted except for Hct and SupHb levels. No bacterial contamination was noted in any of the samples on the basis of sterility tests. It was found that the described glycerolisation procedure could be a method of choice in the cryopreservation of leukocyte-depleted pRBCs (LD-pRBCs) since no negative effect was observed on the quality of the products and long-term frozen storage did not cause RBC destruction.

The effects of freezing on long-term storage of canine erythrocytes.

Human medicine studies have so far demonstrated that erythrocytes may be preserved and stored at low temperatures for decades retaining their metabolic and biochemical properties. However, detailed studies regarding this problem are not yet available in veterinary medicine. Therefore, the objective of the current study was to investigate time-dependent effects of long-term frozen storage of canine red blood cells. Twelve healthy adult dogs meeting the criteria for blood transfusion were used in the study. Whole blood samples (450 ± 45 ml) collected from each dog were centrifuged by a cryogenic microcentrifuge and packed RBC suspensions were obtained. The samples were prewashed three times in 0.9% NaCl solution and were allocated into three groups to be evaluated at three different time points (day 0 and month 4 and 6). The samples to be frozen were subjected to glycerolization and then stored at -80°C for 4 and 6-month periods. At the end of this period the packed RBC samples were thawed, centrifuged and then washed in a consecutive series of dextrose solutions. 2,3-Diphosphoglycerate (2,3-DPG), Adenosin triphosphate (ATP), supernatant hemoglobin (SupHb), sodium (Na+) and potassium (K+) levels, residual glycerol concentrations and hemograms were evaluated and compared. Sterility tests were performed on all samples for bacterial contamination. A statistically significant decrease was noted in potassium levels, which was the natural outcome of deglycerolization process. No significant change was observed in terms of other parameters due based on different time points. In conclusion, long-term frozen storage had no negative effect on the quality parameters of canine erythrocytes.

Transfused older stored red blood cells improve the clinical course and outcome in a canine lethal hemorrhage and reperfusion model.

In canine models, transfused older stored red blood cells (RBCs) hemolyze in vivo resulting in significantly increased intravascular cell-free hemoglobin (CFH) and non-transferrin-bound iron (NTBI). During canine bacterial pneumonia with septic shock, but not in controls, older stored RBCs were associated with significantly increased lung injury and mortality. It is unknown if in shock without infection transfusion of older RBCs will result in similar adverse effects. Two-year-old purpose-bred beagles (n = 12) were transfused similar quantities of either older (42-day) or fresher (7-day) stored universal donor canine RBCs 2.5 hours after undergoing controlled hemorrhage (55 mL/kg). With older transfused RBCs, CFH (p < 0.0001) and NTBI (p = 0.004) levels increased, but lung injury (p = 0.01) and C-reactive protein levels (p = 0.002) declined and there was a trend toward lower mortality (18% vs. 50%). All three deaths after transfused fresher RBCs resulted from hepatic fractures. Lowered exogenous norepinephrine requirements (p < 0.05) and cardiac outputs (p < 0.05) after older transfused RBCs were associated with increased CFH levels that have known vasoconstrictive nitric oxide scavenging capability. In hemorrhagic shock, older RBCs altered resuscitation physiology but did not worsen clinical outcomes. Elevated CFH may lower norepinephrine requirements and cardiac outputs ameliorating reperfusion injuries. With hemorrhagic shock, NTBI levels persist in contrast to the increased clearance, lung injury, and mortality in the previously reported infection model. These preclinical data suggest that whereas iron derived from older RBCs promotes bacterial growth, worsening septic shock mortality during infection, release of CFH and NTBI during hemorrhagic shock is not necessarily harmful.

Determination of tonicity effects of ketoacids and lactate by use of two canine red blood cell assays.

To determine the tonicity effects of β-hydroxybutyrate, acetoacetate, and lactate in canine RBCs. RBCs from approximately 40 dogs. 2 in vitro methods were used to conduct 4 experiments. The modified osmotic fragility assay was used to measure the ability of ketoacid salts added to serial sucrose dilutions to protect RBCs from osmotic hemolysis. In a second assay, a handheld cell counting device was used to measure changes in RBC diameter to assess the tonicity effect of solutions of ketoacid and lactate salts. For the modified osmotic fragility assay, all ketoacid salts had an osmoprotective effect, but the effect was determined to be completely attributable to the tonicity effect of added cations (sodium and lithium) and not the ketoacid moieties. However, both the sodium and lithium lactate salts provided osmoprotection attributable to both the cation and lactate anion. For the second assay, RBC diameter was significantly increased with the addition of urea (an ineffective osmole) but did not change with the addition of glucose (an effective osmole), which established the behaviors of ineffective and effective osmoles in this assay. The RBC diameter was significantly increased over that of control samples by the addition of sodium β-hydroxybutyrate, lithium acetoacetate, and lithium lactate but was decreased by the addition of sodium lactate. For both assays, β-hydroxybutyrate and acetoacetate acted as ineffective osmoles, whereas lactate acted as an effective osmole in 3 of 4 experiments.

Monoclonal anti-A activity against the FORS1 (Forssman) antigen.

The FORS blood group system (originally recognized as the Apae phenotype) was discovered by sporadic activity against polyclonal anti-A reagents and activity against the lectin Helix pomatia. The extent of monoclonal anti-A reagent activity against the FORS1 antigen is serologically and immunochemically incomplete. In the absence of natural FORS1-positive red blood cells (RBCs), kodecytes were created with synthetic disaccharide and pentasaccharide Forssman function-spacer-lipid (FSL) constructs, Fsdi -kodecytes, and FORS1-kodecytes, respectively. FSL constructs were also applied to solid surfaces and used in solid-phase enzyme immunoassays. A range of characterized monoclonal anti-A and anti-B reagents were then serologically and immunochemically characterized against these Forssman antigens. Polyclonal human anti-A, anti-B, the lectin H. pomatia serologic reagents; and canine RBCs were used as serologic controls. None of 19 different monoclonal anti-A reagents were able to detect the pentasaccharide Forssman on FORS1-kodecytes, while three reagents were able to detect disaccharide Forssman on Fsdi -kodecytes. Most anti-A reagents were immunochemically reactive with both the di- and the pentasaccharide Forssman antigens in the solid-phase assays. Historic polyclonal human anti-A and the lectin H. pomatia reacted strongly with the FORS1-kodecytes, correlating with the discovery of the Apae phenotype and supporting the use of FORS1-kodecytes as FORS1 surrogates. Monoclonal anti-A reagents, despite showing reactivity against the FORS1 antigen in solid-phase assays are unlikely to cause the agglutination of FORS1 antigen-positive RBCs.

Xenotransfusion with canine blood in the feline species: review of the literature.

Xenotransfusion (the transfusion of blood from another species) of canine blood to cats has been historically performed commonly and is still performed nowadays in some countries. Considering the current lack of commercial availability of haemoglobin-based oxygen carrier solution (Oxyglobin), there may be rare occasions when treating an anaemic cat when compatible feline blood cannot be obtained, and where a transfusion with canine blood may need to be considered as a life-saving procedure. This article reviews the published evidence about feline xenotransfusion with canine blood and the results that can be expected with this procedure. Published evidence in a limited number of cases (62 cats) indicates that cats do not appear to have naturally-occurring antibodies against canine red blood cell antigens: compatibility tests prior to the first transfusion did not demonstrate any evidence of agglutination or haemolysis of canine red cells in feline serum or plasma. No severe acute adverse reactions have been reported in cats receiving a single transfusion with canine whole blood. Anaemic cats receiving canine blood are reported to improve clinically within hours. However, antibodies against canine red blood cells are produced rapidly and can be detected within 4-7 days of the transfusion, leading to the destruction of the transfused canine red cells in a delayed haemolytic reaction. The average lifespan of the transfused canine red cells is less than 4 days. Any repeated transfusion with canine blood later than 4-6 days after the first transfusion causes anaphylaxis, which is frequently fatal.

Production of an anticanine globulin (polyspecific) reagent for laboratory investigation

Canine allo- or autoantibodies are clinically important, but attachment of these immunoglobulin G (IgG) antibodies does not produce observable haemagglutination. Antibody to canine globulins is required to demonstrate sensitisation of red blood cells. Commercial reagents are available, but these often differ in sensitivity and specificity. Rabbit anticanine globulins (polyspecific) were produced for use in canine blood compatibility testing and in the investigation of immune-mediated haemolytic anaemia. Canine sera was pooled, IgG was purified and subsequently used to immunise rabbits. A rising titre of anticanine IgG was demonstrated by indirect enzyme-linked immunosorbent assay. Rabbit anticanine complement was isolated and investigated by agglutination of complement-coated canine red blood cells. Both antibodies were purified and checked for crossreactivity before being combined to polyspecific anticanine globulins. The obtained reagent was used to indicate sensitised canine red blood cells and free antibodies in three different types of clinical samples, including blood for compatibility testing and that for investigation of immune-mediated haemolytic anaemia and screening for post-transfusion alloantibodies and was also compared to commercial Coombs' reagent. The product provided results in accordance with those from commercial Coombs' reagent. The sensitivity for canine crossmatching was 100% and specificity for diagnosing immune-mediated haemolytic anaemia was 87%. This product is helpful for canine crossmatching purposes and in the investigation of immune-mediated haemolytic anaemia.