Evaluation of an automated,homogeneous enzyme immunoassay for serum thyroxine measurement in dog and cat serum

A homogenous enzyme immunoassay (EIA) for measurement of serum thyroxine (T4) concentration was evaluated for use with canine and feline serum. The EIA method was linear from 0 to 150 nmol T4/L for human serum, 0 to 94 nmol T4/L for feline serum and 10 to 60 nmol T4/L for canine serum. Intra- and interassay precision studies yielded coefficients of variation 相似文献   

Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine

Rapid, accurate detection of serum amyloid A (SAA) is needed in equine practice. We validated a patient-side point-of-care (POC) assay (Stablelab; Zoetis) compared to the turbidimetric immunoassays LZ-SAA (TIA-Hum) and VET-SAA (TIA-Vet; both Eiken Chemical). Analytical performance was assessed at 3 different concentration ranges and with interferences. Inter-method comparison using 49 equine serum samples revealed a significant difference between median SAA results (p < 0.0001), with the strongest bias between the POC and TIA-Vet (median 1,093 vs. 578 mg/L). The median SAA value obtained with the TIA-Hum method was 752 mg/L. Correlation between POC/TIA-Hum and between POC/TIA-Vet was fair (r_s = 0.77 and 0.69) and excellent between both TIAs (r_s = 0.93). Bias between POC/TIA-Hum, POC/TIA-Vet, and TIA-Hum/TIA-Vet was −56.7%, –80.9%, and −28.2%, respectively. POC intra- and inter-assay CVs (16.1–30% and 19.8–35.5%) were higher than TIA CVs (generally <12%). Bilirubin and hemoglobin had a negative bias on POC and TIA-Vet results (−16.6 to −45.6%); addition of intralipid yielded a positive bias (35.9–77.4%). The POC had good linearity of SAA concentrations up to 10,312 mg/L (R² = 0.92). A hook effect was present at SAA >3,000 mg/L for the POC assay. Equine serum SAA was stable over a median period of 2.5 y when stored at −80°C. Overall, there was excellent-to-moderate correlation between tests, but imprecision and hook effect of the POC, as well as bias between the methods, must be considered.     

Salivary testosterone measurements in growing pigs: validation of an automated chemiluminescent immunoassay and its possible use as an acute stress marker

This study aimed to validate the use of an automated chemiluminescent immunoassay analyser for salivary testosterone measurements in growing pigs and study how circadian pattern during daytime and stress can influence its values. The test method had intra- and inter-assay coefficient of variation lower than 10%. The method showed good linearity and recovery, and detection limits were low enough to detect salivary testosterone levels. No significant differences were observed in testosterone concentrations at different sampling time, and age and gender did not influence circadian pattern. In addition, this assay was used to quantify testosterone in two models of acute stress and, in both cases, significant increases (P < 0.01) in salivary testosterone were detected. Therefore, the automated assay system tested for porcine testosterone determinations would be suitable for its use in saliva samples and, furthermore, salivary testosterone levels could be used as a possible marker of acute stress in pigs.     

Practical methods of determining serum immunoglobulin M and immunoglobulin G concentrations in foals.

Serum concentrations of immunoglobulin M (IgM) and immunoglobulin G (IgG) can be determined in the horse with a satisfactory degree of accuracy, using commercially available reagents. Selected lots of anti-human IgM can be used in precipitation tests to detect and quantitate equine IgM. Commercially available anti-equine IgG tended to overestimate the amount of IgG in single radial immunodiffusion tests. Even with these limitations, commercial reagents can be used to differentiate immunodeficiency disorders of foals, including combined immunodeficiency and failure of passive transfer of colostral antibody from mare to foal.     

Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration

Background: Serum C-reactive protein (CRP) is an acute phase marker in dogs that is useful for the diagnosis and monitoring of inflammatory disease. Rapid, reliable, and automated assays are preferable for routine evaluation of canine serum CRP concentration.
Objective: The aim of this study was to evaluate whether canine serum CRP concentration could be measured reliably using an automated turbidometric immunoassay (TIA) designed for use with human serum.
Methods: A commercially available TIA for human serum CRP (Bayer, Newbury, UK) was used to measure canine serum CRP concentration. Cross-reactivity of antigen was evaluated by the Ouchterlony procedure. Intra-and interassay imprecision was investigated by multiple measurements on canine serum samples and serum pools, respectively. Assay inaccuracy was investigated by linearity under dilution and comparison of methodologies (canine CRP ELISA, Tridelta Development Ltd, Kildare, UK). Then the assay was applied to serum samples from 14 clinically healthy dogs, 11 dogs with neoplasia, 13 with infections, 8 with endocrine or metabolic diseases, and 10 with miscellaneous diseases.
Results: Cross-reactivity between canine serum CRP and the anti-human CRP antibody was found. Intra-and interassay imprecision ranged from 5.2% to 10.8% and 3.0% to 10.2%, respectively. Serum CRP concentration was measured in a linear and proportional manner. There was no significant disagreement and there was linear correlation of the results in the comparison of methodologies, except for a slight proportional discrepancy at low CRP concentrations (<10 μg/mL). Dogs with infections had a significantly higher concentration of serum CRP than did all other dogs, and dogs with neoplasia had a significantly higher concentration of serum CRP than did clinically healthy dogs.
Conclusions: Canine serum CRP concentration can be measured reliably using the commercially available TIA designed for human CRP.     

Evaluation of serum bile acid concentrations for the diagnosis of portosystemic venous anomalies in the dog and cat

The serum concentration of bile acids was measured in dogs and cats with portosystemic venous anomalies (PSVA). In 14 dogs, the mean serum bile acid concentration after 12 hours of fasting was 61.7 +/- 68.7 mumol/L (normal, 2.3 +/- 0.4 mumol/L (SEM) and when measured 2 hours after a meal in 15 dogs was 229.9 +/- 87.7 mumol/L (normal, 8.3 +/- 2.2 mumol/L). The fasting serum bile acid concentration was within the normal range in 5 of 14 dogs. The postprandial concentration was determined in 3 of the 5 and in each case increased more than tenfold above the fasting value. The mean fasting serum bile acid concentration in 4 cats was 24.4 +/- 10.1 mumol/L (normal, 1.7 +/- 0.3 mumol/L) and in 2 of the cats increased to a mean of 120.6 mumol/L (normal, 8.3 +/- 0.8 mumol/L) 2 hours after feeding. The bile acid values in patients with PSVA were correlated with values for blood ammonia content, sulfobromophthalein (BSP) retention, and results of conventional tests of hepatic function. Bile acid concentrations were more sensitive than abnormalities in serum enzyme activities or BSP retention and equal in sensitivity to the ammonia tolerance test in detecting hepatobiliary insufficiency. Bile acid measurements were accomplished with less inconvenience to the patient and clinician, than tests of BSP excretion or ammonia tolerance. Used in combination with conventional laboratory tests for hepatic disease, pre- and postprandial serum bile acid concentrations appear to be a sensitive and specific indicator of hepatobiliary dysfunction of value in the diagnosis of PSVA in the dog and cat.     

Relationships between immunoglobulin M and immunoglobulin G or A in colostrum of Japanese black multiparous cows

This study was conducted to clarify the relationships among immunoglobulin (Ig)M, IgG, IgA, β‐carotene, vitamin A and α‐tocopherol contents in colostrum of 24 Japanese Black multiparous cows in order to evaluate the role of IgM on colostral IgG and IgA production. Compared with colostral IgG, colostral IgM and IgA were very low but varied widely. There was positive correlation between colostral IgM and IgG, but colostral IgM was not related with colostral IgA. There was no relationship between colostral IgM and age of cows, although colostral IgG was increased with aging. There were positive correlations among colostral β‐carotene, vitamin A and α‐tocopherol and these vitamins were positively correlated with colostral IgM and IgG. These results indicate that fat‐soluble vitamins may affect colostral IgG and IgM in cows and colostral IgG increases with the increase of colostral IgM.     

Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves

Immunoglobulin (Ig) G and natural antibody (NAb) IgM are passively transferred to the neonatal calf through bovine colostrum. Maternal IgG provides pathogen- or vaccine-specific protection and comprises about 85% of colostral Ig. NAb-IgM is less abundant but provides broad and nonspecific reactivity, potentially contributing to protection against the dissemination of pathogens in the blood (septicemia) in a calf’s first days of life. In the dairy and beef industries, failure of passive transfer (FPT) of colostral Ig (serum total protein [STP] <5.2 g/dL) is still a common concern. The objectives of this study were to: (1) compare colostral IgG concentrations and NAb-IgM titers between dairy and beef cows; (2) assess the effect of beef breed on colostral IgG; (3) compare passive transfer of colostral Ig in dairy and beef calves; and (4) estimate the heritability of colostral IgG and NAb-IgM. Colostrum was collected from Holstein dairy (n = 282) and crossbred beef (n = 168) cows at the University of Guelph dairy and beef research centers. Colostral IgG was quantified by radial immunodiffusion and NAb-IgM was quantified by an enzyme-linked immunosorbent assay. In dairy (n = 308) and beef (n = 169) calves, STP was estimated by digital refractometry. Beef cows had significantly greater colostral IgG (146.5 ± 9.5 standard error of the mean [SEM] g/L) than dairy cows (92.4 ± 5.2 g/L, P <0.01). Beef cows with a higher proportion of Angus ancestry had significantly lower colostral IgG (125.5 ± 5.8 g/L) than cows grouped as “Other” (142.5 ± 4.9 g/L, P = 0.02). Using the FPT cutoff, 13% of dairy and 16% of beef calves had FPT; still, beef calves had a significantly larger proportion with excellent passive transfer (STP ≥6.2 g/dL, P <0.01). The heritability of colostral IgG was 0.04 (±0.14) in dairy and 0.14 (±0.32) in beef. Colostral NAb-IgM titers in dairy (12.12 ± 0.22, log₂ [reciprocal of titer]) and beef cows (12.03 ± 0.19) did not differ significantly (P = 0.71). The range of NAb-IgM titers was 9.18–14.60, equivalent to a 42-fold range in antibody concentration. The heritability of colostral NAb was 0.24 (±0.16) in dairy and 0.11 (±0.19) in beef cows. This study is the first to compare colostral NAb-IgM between dairy and beef cows. Based on the range in NAb-IgM titers and the heritability, selective breeding may improve colostrum quality and protection for neonatal calves in the early days of life.     

Preliminary evaluation of a particle-enhanced turbidimetric immunoassay (PETIA) for the determination of serum cystatin C-like immunoreactivity in dogs

Abstract: Serum cystatin C often is used in humans as a rapid and more sensitive marker than serum creatinine for glomerular filtration rate. The purpose of the present study was to evaluate whether cystatin C-like immunoreactivity (CLI) could be measured reliably in canine serum and to investigate whether dogs with clinical renal insufficiency had higher CLI levels than did clinically healthy dogs and dogs with nonrenal diseases. A commercially available particle-enhanced turbidimetric immunoassay (PETIA) for human serum cystatin C was used to measure canine serum CLI in a linear and proportional manner, with a mean recovery of 104%± 7.5% and coefficients of variation of 1.7 to 9.6%. The assay was then applied to serum samples from 17 clinically healthy dogs, 12 dogs with nonrenal diseases, and 8 dogs with renal insufficiency. Serum CLI was significantly higher in dogs with renal insufficiency (median serum CLI = 5.01 mg/L) than in clinically healthy dogs and dogs with nonrenal diseases (median serum CLI = 1.06 mg/L and 1.62 mg/L, respectively). Thus, canine serum CLI could be reliably measured using a commercially available PETIA designed for human serum cystatin C, and dogs with clinical renal insufficiency had, as expected, significantly higher serum CLI levels.     

Comparison of the VIDAS and IMMULITE‐2000 methods for cortisol measurement in canine serum

Background: Serum cortisol concentration is often measured in dogs for the diagnosis and monitoring of adrenal disease. An enzyme‐linked fluorescent assay (VIDAS method) on the MiniVidas analyzer has been validated for the measurement of cortisol concentration in human serum and could have applications for canine samples. Objective: The aim of this study was to compare canine cortisol results obtained using the VIDAS method with those obtained using the IMMULITE‐2000 immunoassay, which has previously been validated for canine serum. Methods: The concentration of cortisol in 40 canine serum samples was determined concurrently with the VIDAS and IMMULITE methods, the latter as the reference method. Pearson's correlation coefficient, linear, and Deming regression analyses and Bland–Altman analysis were used to compare the 2 methods. Acceptability of the new method was judged using a medical decision chart (MEDx chart). Results: Cortisol concentrations obtained with the IMMULITE method ranged from 23.1 to 1380 nmol/L. Correlation (r=.977) and simple linear regression (slope=1.0722, confidence interval [CI] 0.996–1.148; intercept=?4.799, CI ?42.838 to 33.240) revealed no proportional or constant error. Based on Deming regression and a Bland–Altman plot the 2 methods gave comparable results. The MEDx chart indicated that performance of the new method was good at decision limits of 40, 132, and 480 nmol/L. Conclusion: Results of the VIDAS method were comparable to those of the IMMULITE‐2000 reference method such that the VIDAS may be used as an alternative assay to evaluate serum cortisol concentration in dogs for the diagnosis of adrenal disease.     

Evaluation of a Commercially Available Human Serum Amyloid A (SAA) Turbidimetric Immunoassay for Determination of Feline SAA Concentration

Serum amyloid A (SAA) is an acute-phase protein in cats likely to be useful for diagnosing and monitoring inflammatory diseases, especially if rapid, reliable and automated assays can be made available. A commercially available automated human SAA turbidimetric immunoassay (SAA-TIA) was evaluated for determination of SAA in cats. Intra-assay and inter-assay imprecisions were in the ranges 2.1–9.9% and 7.0–12.5%, respectively, and without significant inaccuracy. Eighty-eight cats were divided into groups according to (A) the presence or absence of an acute-phase response (APR) (n = 23 and 65, respectively) and (B) clinical diagnosis (clinically healthy cats, cats diagnosed with inflammatory/infectious diseases, endocrine/metabolic diseases, neoplastic diseases, and miscellaneous disorders (n=43, 13, 8, 4 and 20, respectively)). The observed SAA concentrations were, as expected, different for (A) cats with and without an APR and (B) cats with inflammatory/infectious diseases compared to other diagnostic groups, except neoplastic diseases. In conclusion, the SAA concentration in cats could be measured reliably using the commercially available TIA designed for measuring human SAA, which should facilitate implementation of the parameter for routine diagnostic purposes. Hansen, A.E., Schaap, M.K. and Kjelgaard-Hansen, M., 2006. Evaluation of a commercially available human serum amyloid A (SAA) turbidimetric immunoassay for determination of feline SAA concentration. Veterinary Research Communications, 30(8), 863–872     

Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. I. Erythrocytes,platelets, and total leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV and the CELL‐DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results. Results: Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL‐DYN (r≥0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33–0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL‐DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts. Conclusion: The Sysmex XT‐2000iV performed as well as the CELL‐DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.     

Evaluation of an enzymatic immunoassay for free thyroxine determination in canine serum

Free thyroxine (FT4) and cholesterol were measured in 400 dogs with either suspected hypothyroidism or dermatological signs such that hypothyroidism needed to be ruled out. Hypothyroidism was diagnosed in 68 dogs from the history, physical examination and stated lower reference limit (<7 pmol/L) for FT4 in euthryoid dogs. Dogs with FT4 concentrations in the range 6–9 pmol/L were finally categorized as hypo- or euthyroid either on the basis of retesting after 2 months or on their clinical response to thyroid replacement therapy over at least 2 months.The enzyme immunoassay evaluated in this paper is considered to be of clinical value and offers many advantages compared with radioimmunoassays.Abbreviations FT4 free thyroxine fraction - MEIA microparticle enzyme immunoassay - RIA radioimmunoassays - T4 thyroxine - TBG thyroxine-binding globulin     

Evaluation of dietary stevioside supplementation on anti-human serum albumin immunoglobulin G, Alpha-1-glycoprotein, body weight and thyroid hormones in broiler chickens

Sixty male broiler chickens fed a diet supplemented with 130 mg/kg stevioside (S group) or an unsupplemented diet (C group) from day 1 of age onwards. On day 21 of age, ten birds from either the S (SH) or C (CH) group were injected subcutaneously with 100 μg human serum albumin (HSA) and ten others from either S (SP) or C (CP) group injected with 100 μl phosphate-buffered saline (PBS) in the same way. There were no significant effect of supplementation nor interaction with age on average body weights, T(3) and T(4) concentrations of non-injected chickens. After the primary immunization, α(1) -glycoprotein concentrations increased in all treatment groups except the CP group, and were significantly higher in the CH group in relation to the other groups. Fourteen and 18 days after the primary immunization, HSA injected chickens of both dietary treatments had significantly higher anti-HSA immunoglobulin G (IgG) levels than their PBS injected controls. No effect of stevioside supplementation was observed for IgG level. In conclusion, dietary stevioside inclusion can attenuate the pro-inflammatory response after stimulation of the innate immune response in broiler chickens.     

Validation of the Sysmex XT‐2000iV hematology system for dogs,cats, and horses. II. Differential leukocyte counts

Background: The Sysmex XT‐2000iV is a laser‐based, flow cytometric hematology system that stains nucleic acids in leukocytes with a fluorescent dye. A 4‐part differential is obtained using side fluorescence light and laser side scatter. Objective: The purpose of this study was to validate the Sysmex XT‐2000iV for determining differential leukocyte counts in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT‐2000iV (Auto‐diff) and the CELL‐DYN 3500. Manual differentials were obtained by counting 100 leukocytes in Wright‐stained blood smears. Results: Leukocyte populations in the Sysmex DIFF scattergram were usually well separated in equine samples, but were not as well separated in canine and feline samples. Correlation among the Sysmex XT‐2000iV, CELL‐DYN 3500, and manual counts was excellent for neutrophil counts (r ≥.97) and good for lymphocyte counts (r ≥.87) for all three species. Systematic differences between the 3 methods were seen for lymphocyte and monocyte counts. The Sysmex reported incomplete differential counts on 18% of feline, 13% of canine, and 3% of equine samples, often when a marked left shift (>10% bands) and/or toxic neutrophils were present. Eosinophils were readily identified in cytograms from all 3 species. Neither the Sysmex nor the CELL‐DYN detected basophils in the 7 dogs and 5 cats with basophilia. Conclusions: The Sysmex XT‐2000iV automated differential leukocyte count performed well with most samples from diseased dogs, cats, and horses. Basophils were not detected. Immature neutrophils or prominent toxic changes often induced errors in samples from cats and dogs.     

Evaluation of immunoglobulin G concentration in colostrum of mares by ELISA, refractometry and colostrometry

In 360 samples of colostrum and 36 samples of blood of warmblood mares, the concentration of immunoglobulin G (IgG) was evaluated in the post partal period with an ELISA and the results were compared to values obtained with 2 field methods--refractometry and colostrometry. A significant correlation (p < 0.0001) was determined between ELISA and colostrometry (r = +0.88) and between ELISA and refractometry (r = +0.93). So both field-methods seem suitable for evaluation of the colostral IgG-concentration in mares. Further the kinetic of the IgG concentration in colostrum, the volume of colostrum and the total amount of IgG was measured in the 12 hours post partum (p.p.) in each half udder of 36 mares of different parity. Immediately p.p. primiparous mares have a greater mean concentration of IgG (68 mg/ml) than multiparous mares (51 mg/ml). However, multiparous mares have a mean colostral volume of 1020 ml whereas, in primiparous mares, a mean volume of 527 ml was determined within the first three hours p.p. As a result of this the total amount of IgG was lower in primiparous (31.5 g) than in multiparous mares (48.5 g). A significant decrease of IgG concentration was measured in multiparous mares in the 1.5 hours following partum versus 3 hours in primiparous mares. The mean IgG concentration in the blood serum of the 36 mares immediately p.p. was 13.4 +/- 3.6 mg/ml. No significant correlation was observed between values of IgG concentration in the blood and in the colostrum of the mares.