A comparison of two techniques for the determination of serum bone-specific alkaline phosphatase activity in dogs

Bone-specific alkaline phosphatase (BALP) shows potential as a marker of bone formation in the dog. Recent studies have indicated that serum BALP may provide a useful, non-invasive indicator of skeletal health in dogs, and as a diagnostic and prognostic marker in the management of dogs with musculoskeletal or metabolic disorders. Two assay techniques (one based on wheatgerm lectin precipitation followed by a simple enzymatic reaction, the second on a specific enzyme-linked immunoassay) were used to measure serum levels of BALP in 35 dogs of different ages.As expected, BALP concentrations decreased with age. For the enzymatic assay, mean (+/-SD) serum concentrations of BALP activities were 100.3 (+/-11.6) U/liter in dogs under 1 year of age, 25.3 (+/-6.8) U/L in dogs 1 to 2 years of age, 16.5 (+/-7.3) U/L in dogs 2 to 3 years of age, 14.3 (+/-5.6) U/L in dogs 3 to 7 years of age, and 12.3 (+/-4.8) U/L in dogs aged 8 years and older. Corresponding results from the immunoassay were 56.3 (+/-9.8) U/L, 10.7 (+/-4.5) U/L, 7.0 (+/-2.5) U/L, 6.7 (+/-3.6) U/L and 7.0 (+/-2.9) U/L. There was excellent correlation between the results from the two assay techniques (r = 0. 96; P < 0.0001). The correlation between BALP and total ALP activities was poor (r = 0.20 for enzymatic BALP, r = 0.31 for immunoreactive BALP), indicating that total ALP should be considered unreliable as an indicator of BALP activity in canine serum. The immunoassay demonstrated acceptable (13 per cent) cross-reactivity with the liver isoform of ALP.The commercial immunoassay kit is simple and provides fast results. Although the wheatgerm lectin/enzymatic technique is preferred in situations where the activities of all three isoforms of ALP are required, the immunoassay should be considered whenever the activity of BALP is the focus of interest.     

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

OBJECTIVE: To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays. SAMPLE POPULATION: 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. PROCEDURE: A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay. RESULTS: The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solid-phase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays. CONCLUSIONS AND CLINICAL RELEVANCE: The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solid-phase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses.     

Treatment of canine hypoadrenocorticism with microcrystalline desoxycorticosterone pivalate

The efficacy of a microcrystalline desoxycorticosterone pivalate (DOCP) injection in the management of canine hypoadrenocorticism (CHAC) was investigated in 21 dogs. On day 0 dogs previously diagnosed with CHAC were given a physical examination and an injection (2.2 mg/kg) of DOCP. This was repeated on days 25 and 50. On day 75 of the study a final physical examination was performed and the success of therapy was evaluated. Blood samples were obtained for serum chemical analysis (Na+, K+, Cl-, BUN & creatinine) on day 0 and day 75. Body weight increased steadily from a mean (+/- SD) of 25.5 +/- 14.2 kg on day 0 to 27.1 +/- 14.8 kg on day 75. The mean serum biochemistry values on day 0 were outside normal limits for Na+ (139.3 +/- 9.2 mEq/l), K+ (5.4 +/- 0.9 mEq/l), and Na+/K+ ratio [(26.4 +/- 4.8)/l]. On day 75, after three injections of DOCP, the values for Na+ (148.2 +/- 5.2 mEq/l), K+ (4.9 +/- 0.6 mEq/l), and Na+/K+ [(30.8 +/- 4.2)/l] were normal and significantly (P less than 0.01) different from values on day 0. All dogs in the study did well on DOCP therapy. The few side effects observed resolved with concomitant administration of prednisolone and/or adjustment of the DOCP dose. All clients elected to continue DOCP therapy after the trial ended, and the dogs continue to do well.     

Estimation of reference intervals from small samples: an example using canine plasma creatinine

Background: According to international recommendations, reference intervals should be determined from at least 120 reference individuals, which often are impossible to achieve in veterinary clinical pathology, especially for wild animals. When only a small number of reference subjects is available, the possible bias cannot be known and the normality of the distribution cannot be evaluated. A comparison of reference intervals estimated by different methods could be helpful. Objective: The purpose of this study was to compare reference limits determined from a large set of canine plasma creatinine reference values, and large subsets of this data, with estimates obtained from small samples selected randomly. Methods: Twenty sets each of 120 and 27 samples were randomly selected from a set of 1439 plasma creatinine results obtained from healthy dogs in another study. Reference intervals for the whole sample and for the large samples were determined by a nonparametric method. The estimated reference limits for the small samples were minimum and maximum, mean ± 2 SD of native and Box–Cox‐transformed values, 2.5th and 97.5th percentiles by a robust method on native and Box–Cox‐transformed values, and estimates from diagrams of cumulative distribution functions. Results: The whole sample had a heavily skewed distribution, which approached Gaussian after Box–Cox transformation. The reference limits estimated from small samples were highly variable. The closest estimates to the 1439‐result reference interval for 27‐result subsamples were obtained by both parametric and robust methods after Box–Cox transformation but were grossly erroneous in some cases. Conclusion: For small samples, it is recommended that all values be reported graphically in a dot plot or histogram and that estimates of the reference limits be compared using different methods.     

Development of an indirect ELISA based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in human serum

BackgroundBrucellosis is the most common zoonotic diseases worldwide. The aim of this study was to develop and evaluate the diagnostic performance of an indirect-ELISA (I-ELISA) method based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in a human serum.Materials and methodsThe study was conducted in two species-rich endemic areas of Iran (Tehran and Lorestan provinces). Serum samples of 102 patients and 150 healthy individuals were tested by the new kit and the commercial Vircell kit for the presence of anti-Brucella IgM antibodies. The disease status was confirmed by Wright agglutination test. The difference in the mean optical densities (OD) recorded by the new and the Vircell kits for patients and healthy individuals were tested using Two-tailed Student t-test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the new kit were informed using Receiver operating curve analysis. The results were used to guide the choice of cutoff. Agreements in ODs recorded by the new and the Vircell kit was visually inspected using Bland-Altman plot.ResultsThe new I-ELISA showed excellent diagnostic performance (sensitivity and PPV = 95.7%, specificity and NPV = 97.8%) for the diagnosis of brucellosis. The cut-off area for the antibody index (AI) was determined to be 8–10, where AIs less than 8 and greater than 10 were considered Brucella-negative and -positive, respectively. AIs of 8–10 show equivocal results, requiring re-testing. The Vircell kit showed low (36.8%) sensitivity and perfect (100%) specificity on the same samples. The Bland-Altman plot showed low agreement between both tests in recording the OD values for the same individuals.ConclusionThe new I-ELISA based on whole cell Brucella abortus S99 showed a good performance for the detection of Brucella spp. Lack of agreement between the new and the Vircell kit suggest that the performance of ELISA kits might be dependent on the geographical area under study. Hence, validation of the new and the Vircell kits is recommended prior to their implementation in other geographical areas.     

Comparison of Latex Agglutination, Indirect Hemagglutination, and ELISA Techniques for the Detection of Toxoplasma gondii-specific Antibodies in the Serum of Cats

The primary purpose of this study was to determine whether commercially available latex agglutination and indirect hemagglutination kits for the detection of Toxoplasma gondii-specific antibodies were capable of detecting T. gondii-specific immunoglobulin M (IgM) in the serum of cats. Serum samples from 35 cats containing either T. gondii-specific IgM, T. gondii-specific immunoglobulin G (IgG), or both were collected. Each serum sample was assayed using a latex agglutination kit, an indirect hemagglutination kit, an enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii-specific IgG, and an ELISA for the detection of T. gondii-specific IgM. When serum samples containing only T. gondii-specific IgM as determined by ELISA were assayed, the latex agglutination kit and the indirect hemagglutination kit detected antibodies in 33.3% and 13.3%, respectively. When T. gondii-specific IgG was present in a serum sample, the results from the latex agglutination kit, the indirect hemagglutination kit, and the IgG-ELISA were similar; however, there was a wide variation in titer magnitude results between the three assays. It was concluded that the latex agglutination kit and the indirect hemagglutination kit did not adequately detect T. gondii-specific IgM in feline serum.     

Comparison of invasive and non-invasive blood pressure monitoring during clinical anaesthesia in dogs

Monitoring blood pressure during anaesthesia is widely recommended in man and animals. The accuracy of any device used to measure blood pressure is an important consideration when selecting monitoring equipment, the ANSI/AAMI SP10 standard is widely cited in this respect in recent veterinary publications. Blood pressure was monitored using invasive and non-invasive techniques during clinical anaesthesia in 19 dogs. The results were compared using Bland-Altman analysis. The bias (and limits of agreement) between invasive and non-invasive measurement was 7.1 mmHg (+/−34.7) for systolic blood pressure, −1.8 mmHg (+/−27.4) for mean blood pressure and 6.9 mmHg (+/−27.5) for diastolic blood pressure. In a clinical setting the bias between invasive and non-invasive measurement techniques was similar or smaller than laboratory reports, however the limits of agreement were considerably wider suggesting that care should be exercised when interpreting NIBP values.     

Pregnancy Diagnosis in Cats Using a Rapid, Bench-top Kit to Detect Relaxin in Urine

Relaxin is a pregnancy-specific hormone in the queen and is produced by the placenta. Both serum and urinary relaxin levels can be used to diagnose and monitor pregnancy in the cat; however, only serum levels are commonly measured in practice. The present study aimed to assess whether urine could be used for the rapid diagnosis of pregnancy at an early stage in domestic cats using a bench-top kit to detect relaxin. Paired serum and urine samples were collected during the first month of gestation in six cats. The samples were tested by applying neat serum, urine or urine diluted in non-pregnant cat serum to the Witness Relaxin kit. Relaxin concentrations in the paired samples were also measured by radioimmunoassay. All undiluted urine samples from pregnant cats tested negative using the bench-top kit; however, the kit was able to detect relaxin in urine after dilution with non-pregnant cat serum. Using this as the test sample, the kit was accurate at diagnosing pregnancy from 28 days after mating and some samples tested positive at 21 days after mating. This preliminary work could lead to the development of a home pregnancy test for cats.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

酶联免疫吸附（ELISA）法测定生猪尿液中的沙丁胺醇

将标准品、样品和和酶标记物一并加入到微孔板中孵育,样品中的游离沙丁胺醇与酶标记的沙丁胺醇竞争结合酶标板微孔中固定相化的沙丁胺醇特异性抗体,通过清洗步骤洗掉未结合的酶标记沙丁胺醇,再通过酶的专一性显色剂显色,微孔显蓝色,加入反应终止液使颜色由蓝色转为黄色。在450nm处测量吸光度（OD）值,并设置标准曲线,通过标准曲线测定样品中沙丁胺醇的含量,结果显示：OD值的大小与样品中沙丁胺醇的含量成反比。     

Evaluation of a spectrophotometric method for canine serum lipase determination

The British antilewisite butyrate-dithionitrobenzoate (BALB-DTNB) spectrophotometric serum lipase assay was evaluated for precision, accuracy, and diagnostic usefulness in analyzing canine sera. Sera samples from clinically healthy dogs, dogs with experimentally induced pancreatitis, and dogs with spontaneous pancreatitis were analyzed. A titrimetric method of serum lipase determination was used for comparison. Although the BALB-DTNB method was not found to be precise or accurate for determining the lipase activity of canine serum samples, it seemed to be at least as diagnostically useful as the titrimetric procedure. The small sample size requirement and the speed of analysis of the BALB-DTNB procedure are advantages of this method over the titrimetric method, and thus, its use in place of the titrimetric method is justified. A laboratory reference range of 3 to 37 IU/L was determined for canine serum.     

Reference Value Advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel

Abstract: International recommendations for determination of reference intervals have been recently updated, especially for small reference sample groups, and use of the robust method and Box–Cox transformation is now recommended. Unfortunately, these methods are not included in most software programs used for data analysis by clinical laboratories. We have created a set of macroinstructions, named Reference Value Advisor, for use in Microsoft Excel to calculate reference limits applying different methods. For any series of data, Reference Value Advisor calculates reference limits (with 90% confidence intervals [CI]) using a nonparametric method when n≥40 and by parametric and robust methods from native and Box–Cox transformed values; tests normality of distributions using the Anderson–Darling test and outliers using Tukey and Dixon–Reed tests; displays the distribution of values in dot plots and histograms and constructs Q–Q plots for visual inspection of normality; and provides minimal guidelines in the form of comments based on international recommendations. The critical steps in determination of reference intervals are correct selection of as many reference individuals as possible and analysis of specimens in controlled preanalytical and analytical conditions. Computing tools cannot compensate for flaws in selection and size of the reference sample group and handling and analysis of samples. However, if those steps are performed properly, Reference Value Advisor, available as freeware at http://www.biostat.envt.fr/spip/spip.php?article63 , permits rapid assessment and comparison of results calculated using different methods, including currently unavailable methods. This allows for selection of the most appropriate method, especially as the program provides the CI of limits. It should be useful in veterinary clinical pathology when only small reference sample groups are available.     

中药软膏剂体外透皮效果研究

为评价复方中药软膏剂中不同浓度的透皮剂药物氮酮对马属动物的透皮效果,首先利用中药复方水提醇沉浓缩液制备中药软膏剂,然后在软膏剂中分别加入2%、4%、5.5%、7%的透皮剂药物氮酮,以软膏剂中的淫羊藿苷成分作为标记物,利用酶标仪法测定经智能药物透皮仪处理后的马皮肤透过液中的标记物含量。结果表明,在中药软膏剂中加入5.5%的氮酮作为促渗剂,所获得的药物渗透效果最佳。该研究为马属动物中药软膏剂中透皮剂药物的选择提供了理论依据,也为复方中药透皮给药系统的研究提供了更为安全、有效、稳定的给药途径。     

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.     

A new, modern, cost-saving micro/macro method for the determination of serum fructosamine

Serum/plasma fructosamine (SeFa) concentration is a reliable indicator used in human diabetic control. Tests for monitoring the carbohydrate/energy metabolism of (farm) animals are less commonly performed in veterinary laboratories, since most of the reliable determinations, both automated and manual, are relatively expensive. The aim of this study was to develop a precise, money- (and time-) saving automated micro method for measuring SeFa. ELISA microplates (20 microL samples and 200 microL reagents) and an automatic microplate autoreader were used. The classical nitroblue tetrazolium (NBT) stain reagent solution of Johnson et al. (1982) was modified using a SIGMA reagent to render it stable for up to one year. SeFa concentrations measured by the new method in 30 human blood plasma samples were compared with values obtained by the standard (generally used) LaRoche kit procedure. Fifteen cow, 13 dog and 18 chicken plasma samples were assayed by the new automated 'micro' method as well as by the manual test tube 'macro' method commonly used earlier. The modified reagent was applied for both methods. The coefficient of correlation (r) between the results obtained by the two methods was consistently between 0.94 and 0.98 (p < 0.001).     

重组N蛋白抗原检测PPRV抗体ELISA的研究——试剂盒阴阳性临界值的测定及其与OIE参考实验室试剂盒的比较

对临床上采集的244份不同背景的羊血清样本,用纯化的重组N蛋白为包被抗原建立的检测小反刍兽疫病毒(PPRV)抗体的间接ELISA进行检测,运用统计学方法摸清了检测结果的分布规律,并同时用OIE参考实验室抗体检测试剂盒进行检测,结果表明,两种检测方法的符合率为91.73%。利用TG-ROC软件分析了ELISA抗体检测临界值,该试剂盒与国外试剂盒相比,其相对特异性和敏感性分别为98.6%和85.4%。     

Evaluation of Kits for the Detection of Fibrin(ogen) Degradation Products in Dogs

The sensitivities and specificities of 3 commercial serum fibrin(ogen) degradation product (FDP) kits and 1 plasma FDP kit for the detection of FDPs in dogs were determined. Blood was collected for measurement of serum and plasma FDP concentrations from 30 healthy dogs and from 20 dogs that fulfilled clinical and laboratory criteria for disseminated intravascular coagulation. To determine the effect of hemolysis on FDP results, blood was collected simultaneously into Bothrops atrox venom-based and thrombin-based serum collection tubes for measurement of FDPs using a single serum FDP kit. The sensitivity (80-95%) and specificity (90-100%) for a positive or negative FDP result, regardless of concentration, was similar for all kits. Kits yielded discordant results in individual dogs and FDP concentrations obtained from 1 serum FDP kit were consistently higher than those from the other kits. Serum prepared from venom-based collection tubes was significantly more hemolyzed than serum prepared from thrombin-based collection tubes or citrated plasma. Hemolysis did not affect the FDP results. On the basis of these results, we conclude that commercial latex agglutination kits for detection of FDPs in serum and plasma samples from human patients are valid for use in dogs. The plasma FDP assay is a viable alternative to currently used serum FDP assays and has the advantage of using a single (citrated plasma) sample for measuring coagulation parameters and FDP concentration.     

A comparison of commercial kit methods for assay of vitamin b(12) in ruminant blood

Five commercially available radioisotope dilution (RID) kits for assay of Vitamin B(12) were obtained for evaluation with ovine and bovine serum; also, for comparison with our laboratory's (Lab 1) RID method and the methods of two independent laboratories, one using RID and the other a microbiological method. One commercial kit was found to be suitable for both species because negligible non-specific binding occurred when extraction was performed at 100 degrees C (212 degrees F). Of the other four kits, two were found to be suitable for ovine and bovine serum provided non-specific binding of the latter was measured. This was also the case with the Lab 1 method. Two of the commercial kits were found to be unsuitable for assay of Vitamin B(12)in ruminant blood.     

Use of whole blood for spectrophotometric determination of cholinesterase activity in dogs

Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.     

Evaluation of a kinetic test kit for the determination of magnesium in bovine serum or plasma

The performance of a new kinetic assay test kit for the determination of magnesium in plasma or serum has been evaluated, using bovine plasma samples. The results obtained were compared with the results from a standard atomic absorption spectrophotometry method and a commercial colorimetric test kit. The kinetic kit compared favourably with the conventional methods and generally gave results with greater precision.