Evaluation of post-transfusion RBC alloimmunization in dogs using a gel-column crossmatch with and without anti-canine globulin enhancement.

A blood crossmatch is essential to ensure RBC compatibility for previously transfused dogs. There is no gold standard crossmatch method for dogs, although the standards used most commonly by academic institutions and reference laboratories are the tube and gel-column crossmatches. Addition of anti-canine globulin (ACG) has been suggested to increase detection of RBC incompatibilities. Our objective was to determine if there is a correlation between results of a standard and an ACG-enhanced gel-column crossmatch in detecting post-transfusion RBC alloimmunization. Pre- and post-transfusion serum or plasma samples were obtained from 33 dogs for major crossmatches to 1-6 (median: 3) blood donors. Crossmatches were performed with (n = 202) and without (n = 202) ACG, with results scored by 4 observers, 3 of whom were anonymized. Ten of 33 (30%) dogs had major crossmatch incompatibilities post-transfusion. RBC incompatibilities (2-4+ agglutination) were detected only with ACG in 4 dogs, only without ACG in 3 dogs, and with both methods in 3 dogs. There was fair correlation between crossmatch methods for determination of compatibility (ρ = 0.34; p < 0.001) and incompatibility (ρ = 0.35; p < 0.001) scores. Among 4 observers, there was near-perfect agreement in determining compatibility (κ = 0.97; p < 0.001) and substantial agreement in overall scoring of incompatibility (κ = 0.77; p < 0.001). Our results suggest that detection of RBC incompatibilities in dogs can be maximized by performing a gel-column crossmatch both with and without ACG enhancement.

Effect of donor blood storage on gel column crossmatch in dogs.

BACKGROUND: Compared with fresh blood, stored equine donor blood results in spurious tube crossmatch incompatibilities. Interpretation of blood crossmatch results is considered subjective. OBJECTIVES: We aimed to determine if the duration of canine donor blood storage impacts compatibility testing using a standard gel column crossmatch and evaluate interobserver variation in the interpretation of crossmatch results. METHODS: Observational study. Whole blood segments were obtained from 23 canine packed red blood cell (RBC) units for use in crossmatches after storage for 0, 7, 14, 21, 28, and 35 days. Major and minor crossmatches were performed using serum and RBCs, respectively, from two to three healthy "recipient" dogs per unit. All crossmatch results were interpreted by four observers, of whom three were blinded. RESULTS: All major crossmatches (n = 61) were compatible on day 0 and remained compatible through day 35 of storage. All minor crossmatches (n = 69) were compatible at all time points, except for five donor pairs with 1 to 3+ agglutination. Repeat testing of these five donor pairs confirmed crossmatch incompatibilities on days 0 through 35, with no change in the degree of incompatibility over time. There was substantial agreement among four observers in determining compatibility (κ = 0.94) and scoring incompatibility (κ = 0.76). CONCLUSIONS: The current practice of performing canine crossmatches with whole blood segments stored for up to 35 days is acceptable, with no spurious changes in compatibility expected over time. The substantial interobserver agreement suggests that the gel column is suitable for performing canine crossmatches in a laboratory setting with multiple personnel.

Clinical application of the StatSensor and StatSensor Xpress point-of-care creatinine measurement devices in dogs.

BACKGROUND: Creatinine is a universally important blood parameter used to detect and monitor acute and chronic kidney disease. Reliable measurements at the bedside remain a challenge in human and veterinary medicine. Despite its potential, a trustworthy point-of-care creatinine biosensor has yet to be established. OBJECTIVES: We aimed to determine the precision and accuracy of the StatSensor (SS) and StatSensor Xpress (SSX) handheld creatinine measurement devices in dogs. METHODS: Paired creatinine samples from dogs with normal (creatinine ≤159 µmol/L), moderate (159-354 µmol/L), and marked (>354 µmol/L) azotemia were compared with a commercial enzymatic analyzer. Within-day precision and linearity studies were performed prior to method comparison studies. Method comparison was evaluated using Bland-Altman, concordance correlation coefficient, Deming, and Passing-Bablok regression analysis. RESULTS: Seventy-eight dogs were enrolled in the study, including 28 (35%), 25 (32%), and 26 (33%) with normal, moderate, and marked azotemia. Total error surpassed recommendations for all devices, and linearity deviated from identity for the SS1 and SS2. The concordance correlation coefficients of the SS1, SS2, SSXI, and SSX2, were 0.69, 0.59, 0.82, and 0.44, respectively. Bland-Altman analyses showed a high variation in the differences, and relationships showed high heteroskedasticity with negative systemic bias among high creatinine concentrations. CONCLUSIONS: Neither the SS and SSX are considered acceptable for clinical applications in dogs. Further research is indicated for the development of a reliable, cost-effective, point-of-care creatinine analyzer to improve the rapid detection and monitoring human and veterinary patients.

Response-based modification of CHOP chemotherapy for canine B-cell lymphoma.

Despite high initial response rates, a subset of dogs with B-cell lymphoma responds less robustly to CHOP-based chemotherapy and experiences shorter survival. One hundred and four dogs with nodal B-cell lymphoma were treated with a response-based CHOP (RBCHOP) protocol modified based on response to individual drugs during the first chemotherapy cycle. Dogs achieving complete (CR) or partial response (PR) at week 3, following treatment with vincristine and cyclophosphamide, received RBCHOP 1 (n = 72), a protocol sequentially rotating vincristine, cyclophosphamide, and doxorubicin. Dogs without a detectable response at week 3 that subsequently achieved CR or PR following treatment with doxorubicin received RBCHOP 2 (n = 14), in which four doses of doxorubicin were given consecutively followed by vincristine and cyclophosphamide. Dogs that failed to respond at week 3 and then to doxorubicin at week 5 assessment were offered rescue chemotherapy (RBCHOP 3, n = 18). Median progression free survival (PFS) and overall survival time (OST) were similar between RBCHOP 1 (PFS 210 days, OST 354 days) and RBCHOP 2 (PFS 220 days, OST 456 days), but significantly shorter for RBCHOP 3 (PFS 34 days, OST 80.5 days, P < 0.001). No presenting signalment nor hematologic variable differentiated patient cohort, however, dogs in RBCHOP 2 and RBCHOP 3 were more likely to have a lymphocytosis at diagnosis (P = 0.02 and 0.04, respectively). Protocol modification based on response during the first cycle resulted in similar toxicity profiles and outcomes to previously published variants of CHOP, and prognosis remained poor for dogs failing to respond during the first treatment cycle.

Urine cortisol-creatinine and protein-creatinine ratios in urine samples from healthy dogs collected at home and in hospital.

BACKGROUND: Recently, urine protein:creatinine ratios (UPC) were shown to be lower in urine samples from dogs collected at home (AH) as compared to those collected in hospital (IH). Stress-inducing procedures and travel to the hospital have been hypothesized to cause prerenal proteinuria. OBJECTIVES: Evaluate patient stress using urine cortisol:creatinine ratios (UCCr) and correlate UCCr to UPC in urine samples obtained AH and IH. ANIMALS: Thirty-six healthy, client-owned dogs. METHODS: Prospective, non-masked study. Two voided urine samples were obtained (AH and IH). Complete urinalysis as well as UPC and UCCr were performed. Clients graded their dogs' stress level AH, in transport, and IH. RESULTS: The UCCr was significantly higher in IH samples than in AH samples (P < .0001), but UPC was not significantly different between AH and IH urine samples (P = .14). In all samples and in both collection settings, UCCr was not significantly correlated with UPC. Travel time and time IH were not correlated with change in UCCr or UPC. In 8 dogs with borderline or overt proteinuria, no significant difference was found in UPC between settings, but UCCr was significantly higher in IH samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The UPC was not higher when measured in urine samples collected IH compared to AH. Dogs had higher UCCr IH, but UCCr was not associated with UPC. Stress, as estimated by UCCr, did not affect proteinuria. Further evidence is needed to support the claim that stress may result in proteinuria in healthy dogs.

Toxicity and Efficacy Evaluation of an Adeno-Associated Virus Vector Expressing Codon-Optimized RPGR Delivered by Subretinal Injection in a Canine Model of X-linked Retinitis Pigmentosa.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated rAAV2tYF-GRK1-hRPGRco, to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (hRPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1 [GRK1]), and is packaged in an AAV2 capsid variant with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a toxicity and efficacy study of this vector administered by subretinal injection in the naturally occurring RPGR mutant (X-linked progressive retinal atrophy 2 [XLPRA2]) dog model. Sixteen RPGR mutant dogs divided into four groups of three to five animals each received either a subretinal injection of 0.07 mL of AGTC-501 at low (1.2 × 1011 vector genome [vg]/mL), mid (6 × 1011 vg/mL), or high dose (3 × 1012 vg/mL), or of vehicle control in the right eye at early-stage disease. The left eye remained untreated. Subretinal injections were well tolerated and were not associated with systemic toxicity. Electroretinography, in vivo retinal imaging, and histological analysis showed rescue of photoreceptor function and structure in the absence of ocular toxicity in the low- and mid-dose treatment groups when compared with the vehicle-treated group. The high-dose group showed evidence of both photoreceptor rescue and posterior segment toxicity. These results support the use of AGTC-501 in clinical studies with patients affected with XLRP caused by RPGR mutations and define the no-observed-adverse-effect level at 6 × 1011 vg/mL.

Prevalence of naturally occurring non-AB blood type incompatibilities in cats and influence of crossmatch on transfusion outcomes.

BACKGROUND: Recognition of the feline red blood cell (RBC) antigen Mik and the presence of naturally occurring anti-Mik antibodies resulting in acute hemolytic transfusion reactions prompted the recommendation to perform a crossmatch before a cat's first RBC transfusion, but this guideline has not yet become a standard practice. OBJECTIVE: To determine the prevalence of naturally occurring non-AB alloantibodies detectable by tube crossmatch, and to compare transfusion outcomes in cats with and without a crossmatch performed. ANIMALS: Three hundred cats that received an RBC transfusion, with or without a major crossmatch performed. METHODS: Retrospective study. RESULTS: Major crossmatch incompatibilities were documented in 23 of 154 transfusion-naive cats (14.9%) and in 15 of 55 previously transfused cats (27%; P = 0.042). Type-specific packed RBCs (pRBCs) were administered to 167 and 82 cats with and without a crossmatch, respectively. Median volume of pRBCs administered during the first transfusion was 5.3 mL/kg (range, 2.4-18 mL/kg). Median change in PCV scaled to dose of pRBCs was +0.8%/mL/kg; administration of crossmatch-compatible pRBCs was not associated with a greater increase in PCV. Febrile transfusion reactions occurred more often in cats that received non-crossmatched (10.1%) compared to crossmatched (2.5%) pRBCs (P = 0.022). Seventy-six percent of cats that received pRBC transfusions survived to hospital discharge. A crossmatch was not associated with improved survival to discharge or at 30 or 60 days posttransfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: The prevalence of naturally occurring non-AB incompatibilities is sufficiently high to justify the recommendation to perform a crossmatch before all (including the first) RBC transfusions in cats.

Higher plasma corticosterone is associated with reduced costs of infection in red-winged blackbirds.

Glucocorticoid hormones allow individuals to rapidly adjust their physiology and behavior to meet the challenges of a variable environment. An individual's baseline concentration of glucocorticoids can reflect shifts in life history stage and resource demands while mediating a suite of physiological and behavioral changes that include immune modulation and resource allocation. Thus, glucocorticoids could facilitate a response to parasites that is optimized for an individual's specific challenges and life history stage. We investigated the relationship between endogenous circulating glucocorticoids and measures of resistance and tolerance to Haemosporidian parasites (including those that cause avian malaria) in red-winged blackbirds (Agelaius phoeniceus). We found that higher endogenous concentrations of circulating glucocorticoids were associated with reduced costs of parasite infection, which is indicative of higher tolerance, but were unrelated to parasite burden in free ranging, breeding male birds. Post-breeding, both males and females with higher glucocorticoid concentrations had higher measures of tolerance to Haemosporidian infection. Our findings suggest a potentially adaptive role for glucocorticoids in shifting the response to parasites to align with an individual's current physiological state and the challenges they face.

Testosterone and Haemosporidian Parasites Along a Tropical Elevational Gradient in Rufous-Collared Sparrows (Zonotrichia capensis).

Elevation has been proposed as a dominant ecological variable shaping life history traits and subsequently their underlying hormonal mechanisms. In an earlier meta-analysis of tropical birds, elevation was positively related to testosterone levels. Furthermore, parasitism by avian haemosporidians should vary with elevation as environmental conditions affect vector abundance, and while testosterone is needed for breeding, it is hypothesized to be immunosuppressive and thus could exacerbate haemosporidian infection. Our objective in this study was to examine the relationships between elevation, testosterone levels, and parasitism by avian haemosporidians. We surveyed breeding male rufous-collared sparrows (Zonotrichia capensis) across a wide elevational range along the equator. We measured baseline testosterone levels, haemosporidian infection at four elevations spanning the species' natural range in the Ecuadorian Andes (600, 1500, 2100, 3300 m). Testosterone levels from breeding males were not related to elevation, but there was high intrapopulation variability. Testosterone levels were not related to the probability of parasitism, but our results from one population suggested that the likelihood of being infected by haemosporidian parasites was greater when in breeding condition. In conclusion, even though there is variation in life history strategies among the studied populations, wider divergence in seasonality and life history traits would probably be needed to detect an effect of elevation on testosterone if one exists. Additionally, our results show that variation in testosterone is not related to infection risk of haemosporidians, thus other factors that take a toll on energetic resources, such as reproduction, should be looked at more closely.

Effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs.

OBJECTIVE: To determine the effects of oral prednisone administration with or without ultralow-dose acetylsalicylic acid on coagulation parameters in healthy dogs and to assess intraindividual variation in thromboelastography results. ANIMALS: 14 healthy research dogs and 10 healthy client-owned dogs. PROCEDURES: In a randomized controlled trial, research dogs underwent thromboelastography twice (3 days apart), and intraindividual variation in test results was calculated. Dogs were given prednisone (2 mg/kg/d, PO) plus acetylsalicylic acid (0.5 mg/kg/d, PO) or prednisone (2 mg/kg/d, PO) plus a placebo for 14 days, after which thromboelastography and other tests were repeated. Differences from preadministration (baseline) test results between and within groups were compared. In a separate trial, client-owned dogs also underwent thromboelastography twice 2 days apart to assess intraindividual variation in untreated dogs. RESULTS: Intraindividual variation in thromboelastography results for research dogs was ≤ 10% for maximum amplitude (MA) and α angle. In the research dogs, MA and fibrinogen values significantly increased from baseline, whereas percentage lysis 30 minutes after attainment of the MA as well as antithrombin activity significantly decreased within each group. In the dogs that received prednisone plus a placebo, percentage lysis 60 minutes after attainment of the MA was significantly lower than at baseline. For all parameters for research dogs, there was no difference between groups for change from baseline. Intraindividual variation in findings for client-owned dogs was similar to the variation for research dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Prednisone administration resulted in hypercoagulability in healthy dogs as indicated by an increase in MA and plasma fibrinogen concentration and a decrease in antithrombin activity. Concurrent ultralow-dose acetylsalicylic acid use had no effect on measured thromboelastography values. The high intraindividual variation in some thromboelastography parameters may preclude routine use of this technique in clinical practice.

Simple tube centrifugation for processing platelet-rich plasma in the horse.

This study evaluated the quality and bacteriologic safety of platelet-rich plasma (PRP) produced by 3 simple, inexpensive tube centrifugation methods and a commercial system. Citrated equine blood collected from 26 normal horses was processed by 4 methods: blood collection tubes centrifuged at 1200 and 2000 × g, 50-mL conical tube, and a commercial system. White blood cell (WBC), red blood cell (RBC), and platelet counts and mean platelet volume (MPV) were determined for whole blood and PRP, and aerobic and anaerobic cultures were performed. Mean platelet concentrations ranged from 1.55- to 2.58-fold. The conical method yielded the most samples with platelet concentrations greater than 2.5-fold and within the clinically acceptable range of > 250,000 platelets/µL. White blood cell counts were lowest with the commercial system and unacceptably high with the blood collection tubes. The conical tube method may offer an economically feasible and comparatively safe alternative to commercial PRP production systems.

Centrifugation en tube pour le traitement du plasma riche en plaquettes chez le cheval. Cette étude a évalué la qualité et l'innocuité bactériologique du plasma riche en plaquettes (PRP) produit par 3 méthodes simples et économiques de centrifugation en tube et un système commercial. Du sang équin citraté prélevé de 26 chevaux normaux a été traité à l'aide de 4 méthodes : des tubes de prélèvement du sang centrifugés à 1200 et à 2000 × g, un tube conique de 50 ml et un système commercial. La numération des globules blancs, des globules rouges et des plaquettes ainsi que le volume moyen des plaquettes ont été déterminés pour le sang total et le PRP et des cultures bactériennes en aérobiose et en anaérobiose ont été réalisées. Les concentrations moyennes des plaquettes s'échelonnaient d'un ordre de grandeur variant de 1,55 à 2,58. La méthode conique a donné le plus d'échantillons avec des concentrations de plaquettes supérieures à un ordre de 2,5 et dans la fourchette cliniquement acceptable de > 250 000 plaquettes/µL. La numération des globules blancs était la plus basse avec le système commercial et était trop élevée par la méthode avec les tubes de prélèvement du sang. La méthode du tube conique peut offrir une méthode de remplacement abordable et d'innocuité comparable aux systèmes de production commerciaux de PRP.(Traduit par Isabelle Vallières).

A newly recognized blood group in domestic shorthair cats: the Mik red cell antigen.

BACKGROUND: Naturally occurring alloantibodies produced against A and B red cell antigens in cats can cause acute hemolytic transfusion reactions. Blood incompatibilities, unrelated to the AB blood group system, have also been suspected after blood transfusions through routine crossmatch testing or as a result of hemolytic transfusion reactions. HYPOTHESIS: Incompatible crossmatch results among AB compatible cats signify the presence of a naturally occurring alloantibody against a newly identified blood antigen in a group of previously never transfused blood donor cats. The associated alloantibody is clinically important based upon a hemolytic transfusion reaction after inadvertent transfusion of red cells expressing this red cell antigen in a feline renal transplant recipient that lacks this red cell antigen. METHODS: Blood donor and nonblood donor cats were evaluated for the presence of auto- and alloantibodies using direct antiglobulin and crossmatch tests, respectively, and were blood typed for AB blood group status. Both standard tube and novel gel column techniques were used. RESULTS: Plasma from 3 of 65 cats and 1 feline renal transplant recipient caused incompatible crossmatch test results with AB compatible erythrocytes indicating these cats formed an alloantibody against a red cell antigen they lack, termed Mik. The 3 donors and the renal transplant recipient were crossmatch-compatible with one another. Tube and gel column crossmatch test results were similar. CONCLUSIONS AND CLINICAL IMPORTANCE: The absence of this novel Mik red cell antigen can be associated with naturally occurring anti-Mik alloantibodies and can elicit an acute hemolytic transfusion reaction after an AB-matched blood transfusion.