Investigation into factors influencing performance of the canine antiglobulin test

Antiglobulin (Coombs') reagents were assessed for their ability to detect immunoglobulin and complement attached to red cells. Polyspecific and monospecific reagents were prepared using a number of immunisation protocols. Performance of these antisera against control red cells was compared, in a direct Coombs' test, with samples from cases of canine autoimmune haemolytic anaemia (AIHA). A combined reagent containing two monospecific antisera (anti-IgG + anti-C3) gave optimum results. Positive control red cells were required to standardise canine Coombs' reagents for the laboratory diagnosis of AIHA. The optimum incubation temperature for the canine Coombs' test was shown to be 37 degrees C.     

Production, characterization, and applications of a murine monoclonal antibody to dog erythrocyte antigen 1.1.

A murine IgM monoclonal antibody, which recognizes dog erythrocyte antigen (DEA) 1.1, has been produced. The antibody correctly identified canine RBC possessing DEA 1.1 in a panel of RBC typed by an independent laboratory. Reactivity of the monoclonal antibody was compared with canine anti-DEA 1.1 antiserum with 163 RBC samples from 145 dogs. Results of agglutination tests with the 2 reagents were in agreement for all samples. A card agglutination test that uses the monoclonal antibody with blood is described. A monoclonal antibody-based test should facilitate blood typing for DEA 1.1 in clinical practice.     

Pattern of Coombs' test reactivity has diagnostic significance in dogs with immune-mediated haemolytic anaemia

OBJECTIVES: To investigate the clinical significance of the pattern of Coombs' test reactivity in dogs with immune-mediated haemolytic anaemia. METHODS: Sixty-five anaemic dogs with a positive Coombs' test were included. Coombs' testing was performed at 4 and 37 degrees C with polyvalent canine Coombs' reagent and antisera specific for each of canine immunoglobulin G, immunoglobulin M and complement factor C3. The impact of performing testing with only polyvalent antiserum at 37 degrees C was assessed. Chi-squared tests were used to compare Coombs' test reactivity in dogs with primary immune-mediated haemolytic anaemia (group A) and in dogs with concurrent/underlying disease (group B). Following Bonferroni correction, significance was set at P < or = 0.003. RESULTS: Eleven dogs would have been regarded as Coombs' negative had they been tested with polyvalent antiserum at 37 degrees C alone. Group A dogs were significantly more likely to be positive with polyvalent antiserum and/or anti-dog immunoglobulin G at 4 and/or 37 degrees C (P < or = 0.001) and tended to be less likely to be positive with anti-dog immunoglobulin M at 4 degrees C (P=0.040). CLINICAL SIGNIFICANCE: Testing of anaemic dogs with polyvalent Coombs' reagent at 37 degrees C was less sensitive than testing with monovalent reagents at 4 and 37 degrees C. The pattern of Coombs' test reactivity differed significantly between dogs with primary immune-mediated haemolytic anaemia and those with concurrent/underlying disease.     

Evaluation of a rapid agglutination method for detection of equine red cell surface antigens (Ca and Aa) as part of pretransfusion testing

BACKGROUND: Blood typing before transfusion minimizes the risk of transfusion reactions and prevents immunization of the recipient against incompatible RBC antigens. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. Standard blood-typing protocols are time-consuming (2.5-3.0 hours) and impractical in emergency settings. OBJECTIVES: The purpose of this study was to determine whether equine RBCs could be typed for Ca and Aa antigens using sera from horses with RBC antibodies in a modified rapid (15 minute) blood-typing protocol. METHODS: Serum was obtained from a horse with anti-Ca antibodies and from another horse with anti-Aa antibodies. The presence of agglutinating antibodies was confirmed with antibody screening. Venous blood samples, collected in citrate-phosphate-dextrose, were obtained from 21 horses of various breeds. Samples were blood typed in the Veterinary Medical Teaching Hospital Hematology Laboratory using standard methodology. Washed RBCs from each of the 21 horses were incubated individually with anti-Ca and anti-Aa sera at dilutions of 1:4, 1:8, and 1:16 for 15 and 30 minutes at room temperature and 37 degrees C. RESULTS: Of the 21 horses, 13 were identified as Aa+/Ca+, four were Aa+/Ca-, two were Aa-/Ca+, and two were Aa-/Ca-. All 17 Aa-positive horses had a positive agglutination reaction at all dilutions of anti-Aa serum, incubation times, and temperatures, while all Aa-negative horses were negative. Each Ca-positive horse had a positive agglutination reaction at all incubation time points and temperatures up to the 1:16 dilution of the anti-Ca serum. All Ca-negative horses were negative at all times, temperatures, and dilutions of anti-Ca serum. Use of the modified protocol on 26 hospitalized horses resulted in accurate typing, based on complete antibody screens. CONCLUSIONS: These results support the hypothesis that equine RBCs can be blood typed using a rapid (15 minute) protocol, at room temperature, for the presence of Ca and Aa antigens using equine-derived antisera. This technique may be beneficial for pretransfusion testing of equine patients in an emergency setting.     

Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs

BACKGROUND: Flow cytometry has been used to detect anti-red blood cell (RBC) antibodies in dogs with immune-mediated hemolytic anemia (IMHA), but the prevalence of anti-RBC antibodies in anemic and nonanemic dogs with a variety of different diseases has not been assessed previously. HYPOTHESIS: We hypothesized that anti-RBC antibodies would be more common in anemic dogs and in dogs with immune-mediated disorders and cancer. ANIMALS: Blood samples from 292 dogs were analyzed prospectively by flow cytometry for anti-RBC antibodies. METHODS: Blood samples from 147 anemic and 145 nonanemic dogs were evaluated by flow cytometry to detect surface-bound immunoglobulin (Ig) G and IgM antibodies on RBC. Disease associations with RBC antibodies were determined, as was the correlation between disease status and the percentage of Ig(+) RBC. The specificity and sensitivity of flow cytometry and clinical variables for the diagnosis of IMHA were compared by Bayesian analysis. RESULTS: Anemic dogs were significantly more likely to be positive for anti-RBC antibodies (IgG, IgM, or both) than nonanemic dogs. Anemic dogs also had significantly higher percentages of Ig(+) RBC than nonanemic dogs, whereas dogs with IMHA had significantly higher percentages of Ig(+) RBC than dogs with all other diseases. Dogs with IMHA, infectious diseases, and immune-mediated thrombocytopenia were significantly more likely to have anti-RBC antibodies than dogs with other medical or surgical diseases. CONCLUSIONS: Anemic dogs with immune-mediated diseases and infectious diseases were at the highest risk for the development of anti-RBC antibodies, and flow cytometry for the detection of IgG on RBC was highly sensitive and specific for the diagnosis of IMHA.     

Performance of 2 microtiter canine Coombs' tests

BACKGROUND: The Coombs' test, which detects immunoglobulin or complement on RBC surfaces, has long been the standard for laboratory confirmation of immune-mediated hemolytic anemia (IMHA), a common cause of hemolytic anemia in the dog. This test, however, suffers from relatively low sensitivity. Optimization of test sensitivity would lead to fewer discrepancies between laboratory results and clinical diagnoses, and in some cases institution of appropriate therapy in a timely manner. OBJECTIVES: The objectives of this study were to 1) characterize the sensitivity and specificity of 2 canine Coombs' tests for the detection of IMHA, 2) document the efficacy of using multiple antisera dilutions beyond what is directed by manufacturers, and 3) evaluate the necessity of monovalent antisera in the test protocol. METHODS: Sixty-five canine whole-blood samples submitted for Coombs' testing were evaluated. Patients were classified as IMHA positive or negative based on a set of predetermined criteria. IMHA classification was compared to Coombs' test results from 2 different Coombs' tests adapted to a microtiter-plate format. One Coombs' test (VMRD Coombs' test) utilized a single polyvalent antiserum (VMRD, Inc, Pullman, WA, USA), while a second Coombs' test (University of Minnesota [U of MN] Coombs' test) used both polyvalent and monovalent antisera. RESULTS: Sensitivity and specificity were 61% and 100% for the VMRD Coombs' test, and 82% and 95% for the U of MN Coombs' test. The use of multiple antisera dilutions resulted in 6 additional Coombs' positive test results. All positive Coombs' test results were positive by polyvalent antisera. CONCLUSIONS: When used in a microtiter-plate format, the U of MN Coombs' test was a more sensitive test for the detection of IMHA in canine patients when compared to the VMRD Coombs' test. The use of multiple antisera dilutions increased test sensitivity. Sensitivity, however, was not increased by the use of monovalent antisera in the Coombs' test protocol.     

Serological diagnosis of the species Haemophilus equigenitalis using the rapid agglutination and coagglutination method

The method of rapid slide agglutination and coagglutination was tested in the detection of Haemophilus equigenitalis (Taylorella equigenitalis)--the causal agent of contagious equine metritis (CEM). It was demonstrated that both methods were suitable for the serological diagnosis of the species under study. The antisera obtained from rabbits immunized with Haemophilus equigenitalis strains treated in different ways were specific, but with different antibody titres. When cross reactions with other species of microorganisms were verified, the antisera did not react with any of the strains, even after binding them to protein A of the positive strain Staphylococcus aureus--Cowan I. Coagglutination was much more rapid and pronounced than the ordinary rapid agglutination test. It was characterized by a low consumption of specific antiserum. The specific antibodies bound to staphylococci were kept at the temperature of 4 degrees C for several months without losing agglutinin activity.     

Measurement of canine blood microparticles by flow cytometry: effect of anticoagulants and staining reagents

Microparticles (MPs) are released from budding plasma membranes into body fluids. The use of flow cytometry for the measurement of MP in canines has not been standardized. In this fundamental study, we compared the effect of anticoagulant agents, such as acid-citrate-dextrose (ACD) and heparin on the measurement of canine MPs in platelet-free plasma (PFP) using flow cytometry. In addition, we used annexin V, carboxyfluorescein succinimidyl ester (CFSE), or calcein tetraacetoxymethyl ester (calcein-AM), and explored the characteristics of the staining reagents in MP detection using flow cytometry. We were able to measure canine MPs in PFP prepared from ACD-anticoagulated blood using flow cytometry, in which the highest positive rate for fluorescent staining was observed when CFSE was used.     

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.     

Clinical, pathological and serological features of spontaneous canine leptospirosis. An evaluation of the IgM- and IgG-specific ELISA

The clinical, pathomorphological and serological features of acute canine leptospirosis are evaluated and the IgM- and IgG-specific ELISA for leptospirosis serology in dogs is assessed. The clinical syndrome of acute canine leptospirosis was characterized by depression, anorexia, vomiting and often haemorrhagic diarrhoea. In addition, jaundice, uraemia, elevated creatinine and alkaline phosphatase were observed in the majority of the dogs. In pups invagination of the intestines was a noteworthy finding. The clinical signs and the post-mortem findings were rather non-specific so that the clinical and post-mortem diagnosis had to be confirmed serologically. In acute clinical cases of canine leptospirosis a high anti-leptospiral IgM titre, ranging from 160 in pups to 10240 in adults, was always present, whereas the anti-leptospiral IgG titre and the agglutination titre usually were negative or low. Dogs died from leptospirosis in spite of a high anti-leptospiral IgM titre. Only two dogs having, at the first examination, a high IgM titre in conjunction with a high IgG titre survived an acute infection. The possible role of IgM and IgG in the pathogenesis of an acute leptospiral infection is discussed. Different serological patterns in reference dogs, which were not suffering from acute leptospirosis, are presented.     

Comparison of various canine blood-typing methods

OBJECTIVE: To compare canine blood-typing results determined by use of the card (CARD), gel (GEL), Michigan State University (MSU), and tube (TUBE) tests. SAMPLE POPULATION: Blood samples from 23 healthy dogs. PROCEDURES: Blood samples anticoagulated with EDTA were screened by use of each blood-typing method according to manufacturers' protocols. RESULTS: Strong RBC agglutination reactions were observed with dog erythrocyte antigen (DEA) 1.1 reagents of the CARD and GEL tests as well as MSU test (only after adding Coombs' reagent) in 9 blood samples. By use of the CARD test, RBCs from 4 additional dogs agglutinated weakly; on the basis of MSU test results, these 4 dogs were classified as DEA 1.2 positive. All blood samples agglutinated with the B antigen reagent of the TUBE test. All but 2 blood samples had strong positive reactions with the DEA 4 reagent of the MSU test. All but 3 blood samples reacted with the E antigen reagent of the TUBE test. Three blood samples agglutinated with the DEA 3 reagent of the MSU test and A antigen reagent of the TUBE test. Five blood samples had strong agglutination reactions with the DEA 5 reagent of the MSU test. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CARD test allows for rapid identification of DEA 1.1 but may produce weak reactions with blood from DEA 1.2-positive dogs. The GEL test is a reliable and rapid clinical laboratory method for identification of DEA 1.1. The MSU test requires Coombs' reagent for identification of DEA 1.1 and 1.2.     

Canine serum C-reactive protein detected by means of a near-patient test for human C-reactive protein

OBJECTIVES: The aim of the study was to evaluate the reliability of a rapid human C-reactive protein near-patient slide reversed passive latex agglutination test (Randox) for the semi-quantitative determination of canine serum C-reactive protein. METHODS: The concentration of C-reactive protein was determined in 244 canine serum samples by an established automated immunoturbidimetric method and in various predilutions by a commercially available reversed passive latex agglutination test for human C-reactive protein. The results were compared to assess if the reversed passive latex agglutination test reflected the results of the established method with special emphasis on the reversed passive latex agglutination test's ability to identify samples characterised as positive or negative by the established method. RESULTS: The reversed passive latex agglutination test reflected the C-reactive protein concentration in canine serum samples at all the tested predilutions (undiluted, 1:4, 1:8 and 1:16). When applying a predilution of 1:8, the positive and negative analytical predictive values for discriminating between positive and negative samples (according to the established quantitative method) were high (0.94 [0.82 to 0.99] and 0.97 [0.93 to 0.99], respectively). CLINICAL SIGNIFICANCE: In conclusion, this near-patient test was able to reflect the serum C-reactive protein concentration in canine samples in a reliable and clinically useful manner and could be applicable for general practice for evaluating C-reactive protein levels in canine serum.     

Interaction of bovine immunoglobulins with complement

Whereas complement (C) in rabbit serum (CR) was bound by bovine antibodies in seven different IgG1 preparations, only two IgG1 preparations could bind the C in guinea pig serum (CGP). Addition of the Clq component of CR to CGP was alone sufficient to render the C-cascade in CGP activable in the presence of bovine erythrocytes sensitized with specific antisera, i.e. reagents. Normal bovine serum was also capable of restoring the haemolytic activity of CGP. However, the bovine serum was much more temperature sensitive than was CR and, as was observed in the sera from MZ twins, it showed considerable variation both in titre values and in prozones when added to CGP.     

Cold agglutinin activity in 2 dogs

A 5‐year‐old neutered male Mastiff and an 8‐year‐old spayed female Labrador Retriever were presented to the University of Minnesota Veterinary Medical Center. The Mastiff was presented for evaluation of lameness and pyoderma one month prior in Missouri, where he tested positive for Ehrlichia canis by serum ELISA test, treated with doxycycline. PCR for Ehrlichia sp, Anaplasma sp, Babesia sp, and Bartonella sp, and PCR for antigen receptor rearrangement were negative, serum protein electrophoresis (SPE) revealed polyclonal gammopathy, and mildly reactive lymphoid cells were seen cytologically. The Labrador presented with a proliferative rostral mandibular gingival mass and lipomas for further presurgical evaluation of cold agglutinin activity documented by a commercial laboratory 2 years earlier prior to removal of a grade II mast cell tumor. This dog had a negative SNAP4Dx, normal SPE, and persistently increased serum ALP activity and polyuria/polydipsia suggestive for hyperadrenocorticism. Both dogs had markedly agglutinated RBC in the EDTA samples that dispersed with warming, and normal plasma color. Cold agglutinin activity was demonstrated by direct saline agglutination testing using whole blood and washed erythrocytes demonstrating agglutination at 30°C, 25°C, 15°C, and 4°C, but not at 37°C . CBC results (ADVIA 2120i) from the Mastiff revealed no significant differences in the RBC results obtained at room temperature (RT) and at 37°C; however, the RT run demonstrated negative bias in neutrophil and platelet concentrations attributed to rapid RBC settling. This uncommon hematologic condition may cause artifacts on the automated leukogram and platelet count, and may be subclinical for long periods.     

Alterations in normal canine platelets during storage in EDTA anticoagulated blood

Abstract: Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24–72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4°C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6-to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8-to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.     

Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

Background

The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT) for the diagnosis of immune-mediated haemolytic anaemia (IMHA).

Methods

Canine (n = 247) and feline (n = 74) blood samples were submitted for DAT testing to two laboratories. A subset of canine samples was categorized as having idiopathic IMHA, secondary IMHA, or no IMHA.

Results

The kappa values for agreement between the tests were in one laboratory 0.86 for canine and 0.58 for feline samples, and in the other 0.48 for canine samples. The lower agreement in the second laboratory was caused by a high number of positive canine DATs for which the gel test was negative. This group included significantly more dogs with secondary IMHA.

Conclusions

The gel test might be used as a screening test for idiopathic IMHA and is less often positive in secondary IMHA than the DAT.     

Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.     

Diagnosis of bovine coronaviruses using mouse erythrocyte suspensions stabilized with formalin

Bovine coronavirus isolated from calf faeces diseased with gastroenteritis and passaged to colostrum-free calves agglutinated mouse and rat erythrocytes. The agglutination reaction depended on temperature and took place only at a temperature of 4 degrees C. At a temperature of 37 degrees C the agglutinate broke down within 15 minutes. The coronavirus could be detected by the haemagglutination test in the contents of the small and large intestines and in the faeces of experimentally and naturally infected calves. The agglutination capacity of mouse erythrocytes was not affected by careful fixation of these erythrocytes with formalin and subsequent lyophilization and remained unchanged for as long as 52 weeks of storage at a temperature of 4 degrees C. It was demonstrated by a comparative examination of 182 samples of the faeces of calves suffering from diarrhoea that haemagglutination test was as sensitive as electron microscopy.     

Pitfalls in immunofluorescence testing in canine dermatology

Indirect immunofluorescence testing for pemphigus-like antibodies was performed on 100 dogs: 50 dogs with various nonpemphigus dermatologic diseases, 25 dogs with various nondermatologic diseases, and 25 normal dogs. One dog (generalized demodicosis) was positive for pemphigus-like antibodies at a titer of 1:10. It was concluded that canine pemphigus-like antibodies are a potential source of misinterpretation and misdiagnosis in indirect immunofluorescence testing. Direct immunofluorescence testing for IgG, IgA, IgM, and C3 was performed on the footpads of 11 normal dogs. Granular deposition of IgM at the basement membrane zone was demonstrated in 5 of the 11 dogs. It was concluded that direct immunofluorescence testing of canine footpads using only polyvalent immunoglobulin antisera or anti-IgM antisera may lead to misinterpretation and misdiagnosis in up to 45% of all dogs tested.     

A latex test for canine rheumatoid factor

Canine rheumatoid factor (RF) has been reported in several canine diseases, particularly in arthritis. Although RF can be assayed using IgG sensitized erythrocytes, the test has a number of disadvantages. As an alternative, latex sensitized with canine IgG was investigated as an assay of canine RF. The canine IgG-latex could be easily produced, was stable, and could be standardized with commercial antisera. The reagent detected RF of the IgM anti-canine-aggregated-IgG type. A comparison of the titres obtained using the canine IgG-latex reagent with those obtained using a rabbit IgG-erythrocyte reagent showed no correlation, suggesting that the two assays may detect RF of different specificities.